Otoacoustic emissions analysers for monitoring aminoglycosides ototoxicity

ROMANIAN JOURNAL OF INTERNAL MEDICINE Volume 47 No. 3, 2009 CONTENTS REVIEWS CORINA LICHIARDOPOL, MARIA MOŢA, The thyroid and autoimmunity ......................................................................... OLGA ORĂŞAN, ANGELA COZMA, N. REDNIC, D. SÂMPELEAN, ANDRADA PÂRVU, L. PETROV, Anemia – a complication of antiviral treatment in chronic viral hepatitis C ..................................................................................... 207 217 ORIGINAL ARTICLES D. ZDRENGHEA, DANA POP, OANA PENCIU, M. ZDRENGHEA, Drug treatment of heart failure patients in a general Romanian hospital ............................................................................................................................................................ TOMINA POPESCU, MARIA MOŢA, Dyslipidemia and hypertension in patients with type 2 diabetes and retinopathy ........ D.L. DUMITRAŞCU, TEODORA SURDEA-BLAGA, LILIANA DAVID, Esophageal achalasia – manometric patterns ...... DUMITRA BĂLAN, P.A. BABEŞ, Incidence and type of stroke in patients with diabetes. Comparison between diabetics and nondiabetics ...................................................................................................................................................................... MARIOARA DANIELA BRAICU, CRISTINA PRIŢULESCU, D. ALEXANDRU, MARIA MOŢA, The assessment of subclinic atherosclerosis objected through IMT in normal and dyslipidemic patients with various degrees of glucose tolerance ........................................................................................................................................................................... INIMIOARA MIHAELA COJOCARU, M. COJOCARU, ADINA NICOLETA POPESCU, L. POPESCU, R. TĂNĂSESCU, Study of antiphospholipid antibodies in type 2 diabetes mellitus with and without diabetic retinopathy ......................... RUXANDRA MOROTI CONSTANTINESCU, MĂDĂLINA GEORGESCU, A. PASCU, ADRIANA HRISTEA, VICTORIA ARAMĂ, C. BĂICUŞ, RUXANDRA OANA CĂLIN, EUGENIA KOVACS, Otoacoustic emissions analysers for monitoring aminoglycosides ototoxicity ..................................................................................................... BOGDANA VÎRGOLICI, IRINA STOIAN, CORINA MUSCUREL, MAGDA MĂRĂCINE, LAURA POPESCU, C. MORARU, VERONICA DINU, Systemic redox modifications in senile cataract ...................................................... MARILENA GÎLCĂ, IRINA STOIAN, DANIELA LIXANDRU, LAURA GĂMAN, BOGDANA VÎRGOLICI, V. ATANASIU, Protection of erythrocyte membrane against oxidative damage by Geriforte in healthy human subjects 227 235 243 249 257 267 273 279 289 CASE REPORTS C. JURCUŢ, CRINA FILIŞAN, CLAUDIA POPOVICI, LIANA TOMA, B. PANAITE, O. NICODIN, I. COPACI, Young woman with polyserositis, ovarian cystic mass and increased level of CA-125. Case report of peritoneal and pleural tuberculosis ...................................................................................................................................................................... DIANA ZANFIR, SABINA ZURAC, FLORICA STĂNICEANU, B. ANDREESCU, A. REBOŞAPCĂ, Incidental findings during ENT routine examination for head and neck trauma ............................................................................................ ROM. J. INTERN. MED., 2009, 47, 3, 205–306 297 301 206 REVIEWS The Thyroid and Autoimmunity CORINA LICHIARDOPOL1, MARIA MO Aβ 1 β Department of Endocrinology, Department of Diabetes, University of Medicine and Pharmacy, Craiova, Romania Autoimmune thyroid diseases (Hashimoto thyroiditis, Graves’ disease, postpartum thyroiditis, atrophic thyroiditis and drug induced thyroiditis) are prevalent disorders worldwide, especially in women (related to the millieu of sex steroids and X chromosome effects on the thyroid and the immune system). Disruption of thyroid self tolerance, usually induced by an infection, generates abnormal thyroid – immune interactions, implicating an array of cytokines and their receptors. Thyrocytes achieve antigen presenting cell properties which stimulate effector immune cells (Th1, Thβ, Th17), in the context of defective immunomodulatory T regulatory cells, resulting in thyroid lymphocytic infiltration and activation of B cells, with production of antibodies against thyroid antigens, thyroid destruction or stimulation, depending on the Th1-Thβ balance. During pregnancy there is a Thβ predominance sustained by the increased glucocorticoid, estrogen and progesteron levels, which allows tolerance versus the histoincompatible fetoplacental unit. In the postpartum period, the return shift Thβ to Th1 favors the occurrence of postpartum thyroiditis. Altered thyroid hormone levels can influence the immune system, and, on the other side, some immune cells secrete TSH, which exerts endocrine and paracrine, cytokine-like effects. Understanding the complex pathogenesis of autoimmune thyroid disorders is crucial for prevention and management. Key words: thyroid autoimmunity, Th cells, interleukins, chemokines. THYROID AUTOIMMUNITY: PREVALENCE AND CLINICAL SPECTRUM Autoimmune thyroid diseases (AITD), which affect approximately 5% of the population (with a larger incidence in women), result from disruption of self tolerance induced by environmental factors in genetically susceptible individuals [1], and are characterized by lymphocytic infiltration of the thyroid, antibodies against thyroid antigens and thyroid dysfunction. The true prevalence of AITD may be higher, at least in certain areas, since studies showed a variable percentage of antithyroid antibodies positivity in disease free populations: 18% in the United States – NHANES III cohort; [β], 1β.4% in Australia – The Busselton Thyroid Study [γ], 18.8% in Denmark [4], 7% in Germany [5]. In the Wickham Survey autoimmune thyroiditis was identified in 10% of subjects with a 4.γ% annual rate progression to overt hypothyroidism [6]. The clinical spectrum of autoimmune diseases encompasses Hashimoto thyroiditis (the most frequent form), Graves’ disease, postpartum thyroiditis, drugROM. J. INTERN. MED., β009, 47, 3, β07–β15 induced thyroiditis, thyroiditis within polyglandular autoimmune syndromes and atrophic thyroiditis [7]. Phenotypic expression of thyroid autoimmunity depends on the pattern of immune response that predominates at a given time. Abnormal interactions between thyrocytes and immunocompetent cells (macrophages, dendritic cells, T cells) as well as between immune cells generate autoimmunity. Hashimoto thyroiditis is a typical T-cell-mediated autoimmune disease, characterized by formation of tertiary lymphoid follicles within the thyroid (containing T cells, mainly Th1, and B cells), with destruction of thyroid follicular cells generating hypofunction, and the presence of antiTPO and/or antiTg antibodies in the serum. Graves’ disease is a Thβ mediated disease with mild lymphocytic infiltration of the thyroid and production of antibodies that stimulate TSHR, thus generating hyperthyroidism. Postpartum thyroiditis occurs in 5–9% of women due to Thβ to Th1 immune response return shift. Atrophic thyroiditis is caused by antibodies blocking TSH receptor (TSHR) [1]. β08 Corina Lichiardopol and Maria Mo a INITIATION OF THYROID AUTOIMMUNITY Probably the trigger of thyroid autoimmunity is represented by infection, the most important environmental factor. Epstein Barr virus infection has been associated with many chronic autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren syndrome, autoimmune thyroiditis, autoimmune hepatitis, Kawasaki disease and polymyositis. Infectious agents reported to trigger AITD include: Yersinia enterocolitica, Borrelia burgdorferi, Helicobacter pylori, Escherichia coli, foamy virus, rubella virus, retroviruses, human T cell leukemia virus 1, hepatitis B and C viruses, enteroviruses. There are six mechanisms by which infection generates autoimmunity: 1. molecular mimicry: the infecting agent contains an epitope similar to that of a self antigen; β. epitope spreading; overprocessing and overpresentation of antigens by presenting cells causes activation of large numbers of T cells with broad specificities; γ. polyclonal activation; infection of B cells with B cell proliferation, enhanced antibody production and generation of circulating immune complexes which may damage self tissues; β 4. bystander activation; enhanced cytokine production with expansion of autoreactive T cells; 5. binding of infectious superantigens to the variable domain of the T cell receptor beta chain and major histocompatibility complex class β molecules allows binding to a wide variety of T cells, irrespective of their specificity and, thus, induces autoimmunity [8]. 6. Viruses can induce expression of genes necessary for antigen presentation in thyroid cells (Fig. 1); thyrocytes of patients with Hashimoto thyroiditis express costimulatory molecules (ICAM, B7-1), increased MHC class I molecules, CXCL-10 (a chemokine ligand that exerts chemotactic activity on lymphoid cells), Fas which interacts with Fas ligand on other thyrocytes surface inducing apoptosis, and toll like receptor γ (surface receptor usually present on dendritic cells, critical for the development of antigen specific adaptive immunity, both hormonal and cell mediated) which activates NF-k and interferon regulatory factor γ (IRF-γ) pathways and production of TNFα and interferon , respectively. ARN viruses activate IRF-γ causing, also, aberrant expression of MHC class II on thyrocytes along with other molecules implicated in antigen presentation [9]. Fig. 1. – Initiation of thyroid autoimmunity. γ β09 The thyroid and autoimmunity The lack of infections has also detrimental effects by diluting the capacity of the immune system to avoid autoimmune responses [10]. Production of thyroid autoantibodies precedes the development of clinical AITD and is also an epiphenomenon secondary to the release of thyroid antigens by thyrocytes undergoing either stimulation or apoptosis [11]. CD40 is a member of the TNF-R receptor family expressed mainly on B cells and antigen presenting cells, implicated in B cell proliferation and antibody secretion.Fig. 1. Enhanced CD40 expression in thyroid follicular cells induces antigen presenting properties by overexpression of IL-6 and expression of MHC class II molecules [1β]. Thyroid autoantigens are represented by TSH receptor, thyroid peroxidase (TPO), thyroglobulin, sodium/iodide symporter and pendrin. TPO and pendrin are expressed at the apical pole of the thyrocyte and are not accessible to circulating immune cells (cryptic antigens). It is generally accepted that a cryptic antigen, once exposed, activates immune cells better than dominant antigens [1γ]. Anti Tg antibodies do not fix complement but ATPO antibodies (especially IgG1 subclass) damage thyroid cells, once follicular structure has been disrupted, by antibody dependent cell cytotoxicity (ADCC) and complement mediated cytotoxicity. Moreover, ATPO can damage the thyroid by ADCC when associated with monocytes, the process being triggered by FC RI (CDγβ) and FC RII (CD64) expressed on monocytes [14]. IMMUNE CELLS Initiation of thyroid autoimmunity is poorly understood. An early event is represented by an increased appearance of intrathyroidal antigen presenting cells (APC) which take up and present thyroid autoantigens along with MHC class II molecules and costimulatory molecules to T helper cells [15]. Dendritic cells secrete IL-1β, IL-βγ, IL-6 and TGF 1. There is a polarization of macrophages: a M1 polarization when stimulated by IFN , LPS and GM-GSF, increasing antibacterial proinflammatory and antiangiogenic functions, and a Mβ polarization under the influence of IL-10, IL-4, IL-1γ (proangiogenic). M1 macrophages produce IL-1β, IL-βγ, IL-6, TNFα and low levels of IL-10 while Mβ macrophages produce IL-10 and low levels of IL-6 and TNFα [16]. Effector Th cells evolve from naive CD4+ T cells (CD4 is a cell surface glycoprotein that acts as a coreceptor for the T cell receptor) stimulated by APC with a distinct TCR engagement (CD80/86 proteins on APC surface bind CDβ8 on T cell surface) (Fig. β). Fig. β. – Interactions between immune cells; differentiation of effector T cells. β10 Corina Lichiardopol and Maria Mo a Th cells polarization dictates the type of immune response: 1. Th1 cells develop under the influence of IL-1β and secrete IL-β, TNF and IFN providing immunity against intracellular pathogens by macrophage activation, cell mediated immunity and phagocyte-dependent protective responses. Th1 responses induce B cells to produce IgG1 and IgGγ (IgG1 are complement fixing and opsonizing Ab). β. Thβ cells develop under the influence of IL-4, secrete IL-4, IL-5, IL-1γ assuring immunity against extracellular pathogens by antibody production (mainly IgE and IgG4), eosinophil activation, inhibition of several macrophage functions (phagocyte independent protective response). Th1 and Thβ cells do not represent distinct cell lineages, but extreme polarized forms; Th1 response, when it becomes dangerous, can be shifted to a less polarized profile – Th0 or even Thβ – process called immune deviation. γ. Th17 cells appear as a result of TGF action in the presence of IL-6 (IL-βγ promoting their maintenance) and assure host defense against bacteria and fungi. Th17 cells produce IL-17 with proinflammatory effects by induction of proinflammatory cytokines (IL-6, TNFα) and chemokines (KC, MCP-1 and MCP-β) and also, proliferation, maturation and chemotaxis of neutrophils, that mediate tissue infiltration and destruction. Autoreactive Th17 cells have an important role in autoimmunity. 4. Tregs are a heterogeneous family of T cells which inhibit autoimmunity and protect against tissue injury. Self tolerance is maintained through clonal deletion, clonal anergy, normal level of lymphogenesis and activation induced cell death [17]. The primary mechanism leading to self tolerance is recessive tolerance induced by thymic deletion of autoreactive cells, but, even in healthy individuals, some of them escape, and intervention of another mechanism – peripheral dominant tolerance, mediated by iTregs, is required [18]. Tregs are classified in naturally occurring Tregs, CD4+ CDβ5+ FOXPγ+ cells (nTregs), which mature early within the thymus and act cytokine – independently by cell to cell contact and induced or adaptive Tregs (iTregs), which are generated in the periphery from mature T cells in 4 response to particular antigenic stimulation, concomitant with dominant CTLA-4 engagement and a certain cytokine milieu. Adaptive Tregs are more efficient than nTregs in suppressing antigen specific effector cell responses; although hyporesponsive in term of proliferation, they are activated upon specific suboptimal antigen stimulation to produce IL-10 and TGF , which suppress effector T cell function. There are several subsets of iTregs [19]:TR1, Thγ cells, CD8+ cells, NK Tcells, δ Tcells, CD4-CD8-Tcells. In the case of a microbial infection nTregs class convert to Th17 cells [β0], when stimulated with IL-6, with subsequent neutrophil mobilization and proinflammatory effects; later appearing TGF induced Tregs terminate antigen specific response and prevent the emergence of nonspecifically stimulated or cross reactive self reactive Tcells. Tregs decrease CD80/86 expression on dendritic cells, inhibit dendritic cell accumulation and maturation in the spleen and lymph nodes, and suppress Th1 immune response [19]. Also, Tregs suppress immune response by preferential consumption of IL-β (which stimulates proliferation of T cells), induction of effector T cell apoptosis via CDγ0/CDγ0L interactions or perforin/ granzyme B, production of immuno-suppressive cytokines such as IL-10 and TGF and production of immunosuppressive metabolites as a result of upregulation of indoleamine-β, γ-dioxygenase (IDO) in dendritic cells [β1]. In patients with autoimmune thyroid disease different Tregs subsets are detected in the peripheral blood and in the thyroid; in peripheral blood there is an increased number of CD4+ lymphocytes expressing GITR (glucocorticoid inducible TNF receptor), FOXPγ, IL-10, TGF and CD69 while the thyroid infiltrate contains an increased number of CD69+, CDβ5+, GITR+ cells with moderate FOXPγ expression. However, the suppressive function of these Tregs seems to be defective and they are not able to modify the clinical course of inflammation; GITR ligand from the inflamed thyroid binds GITR on Tregs and acts as a costimulatory molecule, favoring their proliferation but inhibits their regulatory activity. On the other side, effector T cells may be resistant to Tregs, a phenomenon that could be related to defects in intracellular signaling [ββ][βγ]. 5 β11 The thyroid and autoimmunity PROGRESSION OF AUTOIMMUNE PROCESS Once the tolerance to thyroid antigens is broken, endothelial cells of regional postcapillary venulae are activated, allowing the extravasation of blood leucocytes attracted by chemokines. The recruitment and arrest of lymphocytes in the thyroid is only partially understood. Immune cells adhere to the endothelium, then migrate across it, then through the interstitium and move toward thyroid follicular cells, sometimes organizing into germinal centers, resembling lymph node germinal centers. In autoimmune thyroid diseases there is an enhanced expression of adhesion molecules (selectins and integrins) on lymphocytes and endothelial cells (lymphocyte function associated antigen 1-LFA1, interstitial cell adhesion molecule 1-ICAM 1, very late antigen 4-VLA4) as well as selectin ligands. CXC chemokines are secreted by thyrocytes stimulated by TNFα, IFN and IL1β; CXCL10, CXCL9 are responsible for recruitment of activated lymphocytes in the thyroid. CXCL10 promotes differentiation of Th0 cells into Th1 cells, while CCLβ- the differentiation of Th0 cells into Thβ cells. CXCL10 binding to CXCRγA generates an angiostatic effect, while binding to CXCRγB exerts chemotactic and immune effects. CXCRγ is expressed on immune cells (T cells, especially Th1, B cells, NK cells), vascular pericytes, microvascular endothelial cells. Immune cells expressing CXCRγ are attracted in the inflamed thyroid. Th1 cells secrete IFN , which stimulates production of chemokines by thyroid follicular cells, thus maintaining and expanding the autoimmune process [β4]. Predominance of Th1 a immune response promotes thyrocytes apoptosis [β5] mediated by Fas and TRAIL (TNF – related apoptosis inducing ligand), leading to Hashimoto thyroiditis. Predominance of a Thβ immune response induces antigen-specific B cells to produce antithyroid antibodies; stimulatory antiTSH receptor antibodies are responsible for Graves’ disease and blocking antiTSH receptor antibodies are responsible for atrophic thyroiditis. Thus, the clinical expression of thyroid autoimmunity depends on the Th1-Thβ balance [15]. In Hashimoto thyroiditis hypothyroidism is not only the result of thyrocytes destruction, but also of the thyroid function impairment induced by Th1 cytokines. IL-1 and IFN down regulate the expression of TG, TPO, Na/I symporter and HβOβ generating enzymes (DUOX), effects partially mediated by nitric oxide and antagonized by increased TSH concentrations. IL-4, a Thβ cytokine, blocks Th1 induced alterations in DUOX, TPO and Tg secretion. Thγ cytokines-TGF and IL-10-repress thyrocytes function but their effects can also be overturned by Thβ cytokines [βγ]. PREGNANCY AND THYROIDITIS During pregnancy there is a physiologic shift of Th1 to Thβ immune responses aimed to avoid rejection of the histoincompatible fetoplacental unit by a maternal cell-mediated immune attack. The increase of glucocorticoids, estrogen and progesterone levels suppresses cell mediated immunity and enhances humoral immunity, explaining the remission of Th1 mediated diseases (rheumatoid arthritis, multiple sclerosis, type 1 diabetes and autoimmune thyroiditis) and aggravation of Thβ mediated diseases (systemic lupus erythematosus). During the postpartum period, abrupt decrease of glucocorticoids, estrogens and progesterone to subnormal levels allows the “return shift” of Thβ to Th1 immune response favoring the occurrence of Th1 mediated diseases such as postpartum thyroiditis [15]. INTERFERON INDUCED THYROIDITIS Autoimmune thyroid diseases appear in 15% of interferon α treated patients (75–80% Hashimoto thyroiditis and β0–β5% Graves’ disease). Interferon α induces a transient increase in Th1 response but, also, an increased production of Thβ cytokines. Thβ activation is higher in patients with interferon induced thyroiditis who remain euthyroid, whereas in patients with thyroid dysfunction there is a decreased Thβ response and an increased Th1 response. The innate response is crucial in determining whether an autoimmune reaction will occur; a type 1 bias of NK and NKT cells was demonstrated in experimental models of autoimmune diabetes and encephalomyelitis. In interferon treated patients both type 1 and type β activation of peripheral NK and NKT CD56cells was found, type 1 occurring in patients with or without thyroid dysfunction and type β in patients who remained euthyroid. Type 1 activation of CD56+ cells was earlier than that observed in CD4+ cells, but it is not capable alone to induce β1β Corina Lichiardopol and Maria Mo a destruction of the thyroid; by contrast, the type 1 activation of CD4+ and CD8+ cells is critical [β7]. Type I interferons (α and ) activate interferon regulatory factors and upregulate TLRγ in nonimmune cells in an autocrine/ paracrine manner [9]. IMMUNE REGULATION OF THE THYROID AXIS TSH is produced by splenic dendritic cells, bone marrow CD11b+ monocytes/ macrophages and granulocyte precursors, intestinal epithelial cells from subvillus crypt regions, T cells, B cells. This extrapituitary TSH may act on immune cells either directly, as a cytokine like regulatory molecule or indirectly, by secretion of thyroid hormones. TSH has been shown to stimulate antibody production, lymphocytes proliferation, NK cells activity, phagocytic stimuli, secretion of TNFα by CD11b- bone marrow cells. Thyroid hormones influence the functional or developmental activity of cells in bone marrow and secondary lymphoid tissues. In euthyroid sick syndrome (a basic host mechanism aimed to conserve energy during fasting, sepsis, trauma, acute severe diseases, bone marrow transplantation) a failure in T4 to Tγ conversion occurs. Proinflammatory cytokines IL-1, IL-6 and TNFα decrease TSH level. An increased conversion of T4 to Tγ within the tanicytes of the third ventricle generates a localized Tγ feedback mechanism that suppresses TRH and TSH. In the recovery phase, the immune system is responsible for increasing thyroid hormone output; bone marrow CD11b+ cells, which secrete TSH, migrate into the thyroid and operate in a paracrine manner [β8]. Whether this TSH-producing immune cells with potential to traffick into the thyroid play a role in thyroid autoimmunity is not known. THYROID DYSFUNCTION AND THE IMMUNE SYSTEM Thyroid hormones are not essential for the development of the immune system but are involved in the maintenance of immune homeostasis. Hypothyroidism decreases thymic activity, causes spleen and lymph nodes involution and thus, represses both cell mediated and humoral immune responses. In hyperthyroidism both stimulatory and suppressing effects on the immune system have been described. 6 It was demonstrated that thyroid hormones modulate lymphocytic activity through protein kinase C signaling pathway [1]. GENDER AND AUTOIMMUNE THYROID DISEASES It is well known that autoimmune thyroid diseases are more prevalent in females and this phenomenon was initially ascribed to hormonal differences, since autoimmune thyroid disease are rare prepubertally; estrogens exacerbate and androgens inhibit immune responses. Gonadotropin-releasing hormone and sex steroids are strongly implicated in the development and function of the immune system. GnRH and its type I receptor are both expressed in primary lymphoid organs and peripheral immune cells and exacerbate autoimmune diseases, independent of gonadal steroids, by increasing the number of Th cells, the level of interferon gamma, the number of IL-β receptors with proliferation and activation of T and B cells. Receptors for estrogens and androgens are expressed in primary lymphoid organs and are implicated in immune cell development in the thymus (negative regulators of different immune cells subsets), and bone marrow. There is a gender difference in immune responsiveness. Peripheral T and B cells possess only estrogen receptors which are responsible for the increased number of Th cells and autoantibody production. Although androgen receptors were not documented in peripheral T and B cells, androgens have suppressor effects by increasing the number of suppressor T cells and decreasing the number of B cells [β9]. Autoimmune thyroid disease appearance after menopause suggests that X chromosome may be more important than estrogens [β0]. A potential mechanism is skewed X-chromosome inactivation, a pattern where 80% or more of the cells inactivate the same X-chromosome. Thus, self antigens on one X-chromosome are insufficiently expressed in the thymus and in peripheral sites involved in tolerance induction, but may yet be highly expressed in peripheral tissues and blood cells. Recently it was demonstrated that skewed XCI is associated with an increased risk of developing AITD with an odds ratio of 9 (1.64–49.4) [γ1]. Another mechanism important for the pathogenesis of AITD may be represented by haplo-insufficiency 7 β1γ The thyroid and autoimmunity of X linked genes consequent to chromosome instability, generating a high rate of monosomy X in the peripheral white blood cells [γ0]. The increased prevalence of AITD in patients with Turner syndrome (hypoestrogenic state) stands also for haploinsufficiency of X chromosome genes implicated in thyroid function (TBG-codes for thyroglobulin, MCT8-codes for a thyroid hormone transporter into cells, MNG2-related to susceptibility to multinodular goiter, GRDX-related to susceptibility to Graves’ disease) or in the immune response (FOXP3-essential for the development of self tolerance, genes coding for many interleukin receptors, factors that stimulate lymphocytes proliferation and production of immunoglobulins, CD40 ligand – a glycoprotein expressed on T cells and epithelial cells which stimulates B cell proliferation, adhesion and differentiation) (www.pubmed.gov/OMIM). CHRONIC THYROIDITIS AND HEPATITIS C VIRUS INFECTION According to WHO data, the highest prevalence of HCV hepatitis in Europe is registered in Romania (4.56%), so a special attention should be paid to the association between HCV infection and chronic thyroiditis. AITD (ORţ1.6) and hypothyroidism (ORţβ.9) are more prevalent in HCV positive patients than in HBV infected patients or healthy controls. Also epidemiologic studies showed an association between HCV infection and thyroid cancer that needs to be confirmed [γβ]. The high prevalence of AITD in HCV infection is independent of alpha-interferon therapy, which suggests the role of HCV in breakdown of immune tolerance and emphasizes the necessity of screening all AITD patients for HCV and, also, monitoring all HCV infected patients for AITD [γγ]. It was recently shown that HCV envelope glycoprotein Eβ can bind to thyroid CD81 receptors leading to IL-8 release without inducing thyrocytes apoptosis, thus triggering thyroiditis in genetically susceptible individuals by a bystander activation mechanism [γ4]. CONCLUSIONS Thyroid autoimmunity results from an imbalance in Th1-Thβ immune responses, usually triggered by infections in genetically susceptible individuals. Predominance of the Th1 phenotype induces cell-mediated thyrocytes’ apoptosis with subsequent Hashimoto thyroiditis or postpartum thyroiditis. On the contrary, predominance of the Thβ phenotype induces production of antibodies against the TSH receptor either stimulatory causing Graves’ disease or inhibitory, causing atrophic thyroiditis. Autoimmune thyroid diseases may also be drug induced (interferon induced thyroiditis). This spectrum of thyroid autoimmunity reflects the complexity of immune-thyroid interactions, and clarifying their pathogenic roles will allow a better clinical management of thyroid autoimmune diseases. Bolile tiroidiene autoimune (tiroidita Hashimoto, boala Graves, tiroidita postpartum, atrofică şi medicamentoasă) sunt frecvent întâlnite, în special la femei (legat de efectele steroizilor sexuali şi a cromozomului X asupra tiroidei şi sistemului imun). Pierderea toleranţei faţă de antigenele tiroidiene, de obicei indusă de o infecţie, generează interacţiuni anormale cu sistemul imun, ce implică numeroase citokine şi receptorii lor. Tirocitele dobândesc proprietăţi de prezentare a antigenului, stimulează celulele imune efectoare (Th1, Th2, Th17) în contextul deficienţei celulelor T reglatoare, determinând infiltrare limfocitară tiroidiană, activarea celulelor B cu producere de anticorpi antitiroidieni, distrugere sau stimulare tiroidiană, în funcţie de balanţa Th1-Th2. În timpul sarcinii, datorită creşterii glucocorticoizilor, estrogenilor şi progesteronului, predomină răspunsul imun Th2, asigurând toleranţa faţă de unitatea fetoplacentară. După naştere, restabilirea Th1 favorizează apariţia tiroiditei postpartum. Nivelul hormonilor tiroidieni influenţează activitatea sistemului imun, β14 Corina Lichiardopol and Maria Mo a 8 iar pe de altă parte, unele celule imune secretă TSH, ce acţionează endocrin, paracrin şi asemănător citokinelor. Descifrarea patogeniei complexe a bolilor autoimune tiroidiene este deosebit de importantă pentru profilaxia şi managementul acestora. Corresponding author: Corina Lichiardopol, MD Department of Endocrinology, University of Medicine and Pharmacy, 4, Petru Rares, Craiova, Romania E-mail: [email protected] REFERENCES 1. β. γ. 4. 5. 6. 7. 8. 9. 10. 11. 1β. 1γ. 14. 15. 16. 17. 18. 19. β0. β1. 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Immunology, β006, 117, β89–γ00. ZHENG, S.G., The Critical Role of TGF-beta1 in the Development of Induced Foxp3+ Regulatory T Cells. Int. J. Clin. Exp. Med., β008, 1, 19β–β0β. KORN, T., MITSDOERFFER, M., CROXFORD, A.L., AWASTHI, A., DARDALHON, V.A. et al., IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+ regulatory T cells. Proc. Natl. Acad. Sci. U.S. A., β008, 105, 18460–18465. VAN DER VLIET H.J.J., NIEUWENHUIS, E.E., IPEX as a result of mutations in FOXP3. Clin. Dev. Immunol., β007, 2007, 89017. 9 The thyroid and autoimmunity β15 ββ. MARAZUELA, M., GARCIA-LOPEZ, M.A., FIGUEROA-VEGA, N., DE LA, F.H., VARADO-SANCHEZ, B. et al., Regulatory T cells in human autoimmune thyroid disease. J. Clin. Endocrinol. Metab., β006, 91, γ6γ9–γ646. βγ. MCLACHLAN, S.M., NAGAYAMA, Y., PICHURIN, P.N., MIZUTORI, Y., CHEN, C.R. et al., The link between Graves’ disease and Hashimoto's thyroiditis: a role for regulatory T cells. Endocrinology, β007, 148, 57β4–57γγ. β4. ROTONDI, M., CHIOVATO, L., ROMAGNANI, S., SERIO, M., ROMAGNANI, P., Role of chemokines in endocrine autoimmune diseases. Endocr. Rev., β007, 28, 49β–5β0. β5. CORONA, G., BIAGINI, C., ROTONDI, M., BONAMANO, A., CREMONINI, N. et al., Correlation between, clinical, biochemical, color Doppler ultrasound thyroid parameters, and CXCL-10 in autoimmune thyroid diseases. Endocr. J., β008, 55, γ45–γ50. β6. PONCIN, S., LENGELE, B., COLIN, I.M., GERARD, A.C., Differential interactions between Th1/Th2, Th1/Th3, and Th2/Th3 cytokines in the regulation of thyroperoxidase and dual oxidase expression, and of thyroglobulin secretion in thyrocytes in vitro. Endocrinology, β008, 149, 15γ4–154β. β7. MAZZIOTTI, G., SORVILLO, F., PISCOPO, M., MORISCO, F., CIOFFI, M. et al., Innate and acquired immune system in patients developing interferon-alpha-related autoimmune thyroiditis: a prospective study. J. Clin. Endocrinol. Metab., β005, 90, 41γ8–4144. β8. KLEIN, J.R., The immune system as a regulator of thyroid hormone activity. Exp. Biol. Med. (Maywood.), β006, 231, ββ9–βγ6. β9. TANRIVERDI, F., SILVEIRA, L.F., MACCOLL, G.S., BOULOUX, P.M., The hypothalamic-pituitary-gonadal axis: immune function and autoimmunity. J. Endocrinol., β00γ, 176, β9γ–γ04. γ0. GUARNERI, F., BENVENGA, S., Environmental factors and genetic background that interact to cause autoimmune thyroid disease. Curr. Opin. Endocrinol. Diabetes Obes., β007, 14, γ98–409. γ1. BRIX, T.H., KNUDSEN, G.P., KRISTIANSEN, M., KYVIK, K.O. et al., High frequency of skewed X-chromosome inactivation in females with autoimmune thyroid disease: a possible explanation for the female predisposition to thyroid autoimmunity. J. Clin. Endocrinol. Metab., β005, 90, 5949–595γ. γβ. ANTONELLI A., FERRY C., FALLAHI P., FERRARI S.M., GHINOI A. et al., Thyroid disorders in chronic hepatitis C virus infection. Thyroid, β006, 16(6), 56γ–7β. γγ. TESTA A., CASTALDI P., FANT V., FIORE G.F., GRIECO V. et al., Prevalence of HCV antibodies in autoimmune thyroid disease. Eur. Rev. Med. Pharmacol. Sci., β006, 10(4), 18γ–6. γ4. AKENO N., BLACKARD J.T., TOMER Y., HCV E2 protein binds directly to thyroid cells and induces IL-8 production: a new mechanism for HCV induced thyroid autoimmunity. J. Autoimmun., β008, 31(4), γγ9–44. Received May 10, β009 β16 Corina Lichiardopol and Maria Mo a 10 Anemia – A Complication of Antiviral Treatment in Chronic Viral Hepatitis C OLGAăăOR ŞAN1,ăăANGELAăăCOZMA1,ăăN.ăREDNIC1,ăăD.ăSÂMPELEAN1,ăăANDRADAăăPÂRVU2,ăăL.ăPETROV2ă 1ă 4thăMedicalăClinic,ăUniversityăofăMedicineăandăPharmacy,ăCluj-Napoca,ăRomaniaă HematologyăClinic,ăUniversityăofăMedicineăandăPharmacy,ăCluj-Napoca,ăRomaniaă 2ă Anemiaă isă ană importantă andă frequentă secondaryă effectă ofă theă treatmentă withă pegylatedă interferonă andă ribavirină ină patientsă withă chronică virală Că hepatitis.ă Ribavirină producesă moreă oftenă hemolytică anemia,ă whileă pegylatedă interferonă mayă determineă medullaryă suppression.ă Theă levelă ofă hemoglobină beneathă 10ă g/dLăisă consideredă byă mostă authorsă asă beingă theă referenceă levelă foră anemiaă secondaryătoătheăantiviralătreatment.ăBeneathăthisăhemoglobinăvalueăităisărecommendedătoăreduceăorătoă stopă theă treatmentă withă ribavirin,ă toă administeră recombinantă humană erythropoietină oră bloodă transfusion,ăbasedăonătheăseverityăofătheăanemia.ăă Theăgrowthărateăofătheăserumăerythropoietinăinătheăfirstăfewăweeksăofătreatmentăisăcorrelatedăwithă theănecessityăofădecreasingătheădosesăorăevenătoăstopătheătreatmentăwithăribavirin.ăTheăSVRă(sustainedă viralăresponse)ărateăofătheăpatientsăisăreducedăwhenătheăribavirinădosesăareăreducedădueătoăanemia.ă Key words: hepatitis,ăCăvirusăinfection,ăanemia. HepatitisăCăvirusă(HCV)ăinfectionăisăaămajoră causeăofăchronicăhepaticădiseaseăaffectingă170ămillionă peopleăthroughoutătheăworld.ăChronicăHCVăinfectionă causesăprogressiveăhepaticăfibrosisăandăcirrhosisăină 20%ăofăpatients,ăandă10–20%ăofăpatientsăwithăcirrhosisă developăhepatocarcinomaăwithină5ăyearsă[1–4].ă Theăgoldăstandardăinătheătreatmentăofăchronică virală hepatitisă Că isă pegylatedă interferonă alphaă 2aă (Pegasysă–ăHoffmannălaăRoche)ăorăpegylatedăinterferonă alphaă2bă(Pegintronă–ăScheringăPlough)ăandăribavirină (Copegusă–ăHoffmannălaăRoche,ăRebetolă–ăScheringă Plough,ă respectively).ă Combinedă treatmentă increasesă theă efficacyă ofă therapy,ă i.e.ă ită increasesă theă rateă ofă sustainedăviralăresponseă(SVR)ăcomparedătoăpegylatedă interferonă monotherapy.ă Theă combinedă pegylatedă interferonă andă ribavirină antivirală treatmentă isă superioră toă standardă interferonă treatmentă oră standardă interferonă andă ribavirină treatment,ă SVRă beingă obtainedăină54–56%ăofăpatientsă[1–6].ă Antivirală treatmentă hasă numerousă adverseă effects;ăităcausesăanemia,ăneutropenia,ăandăthrombocytopeniaăinăparticular.ăAnemiaăisăassociatedăwithăaă decreaseăinătheăqualityăofălife,ăwithătheăneedăforătheă reductionăofăribavirinădosesăorăevenătheăcessationăofă treatment,ăwhichămostăfrequentlyăresultsăinăaăweakeră responseătoăantiviralătreatmentă[1].ă Anemiaă isă veryă commonă ină patientsă withă chronică virală hepatitisă Că underă antivirală treatment.ă Aăretrospectiveăstudyăonă677ăpatientsăshowsăthatăină ROM.ăJ.ăINTERN.ăMED.,ă2009,ă47,ă3,ă217–225ă 56%ă ofă patients,ă Hbă decreasedă byă 3ă g/dL,ă ină 10%ă Hbăhavingăvaluesăofălessăthană10ăg/dLă[7].ă Anemiaă ină patientsă withă virală hepatitisă Că treatedăwithăpegylatedăinterferon/ribavirinăhasăseverală causes:ăhemolyticăanemia,ăperniciousăanemia,ăaplastică anemiaă secondaryă toă ribavirină treatment;ă pegylatedă interferon-inducedă boneă marrowă suppressionă andă chronică posthemorrhagică anemia,ă secondaryă toă nutritională deficits,ă areă presentă ină variableă degreesă inăallăchronicădiseasesă[8].ă Hemoglobină (Hb)ă concentrationsă decreasedă toălessăthană12ăg/dLă(meanădecreaseă=ă3.7ăg/dL)ăină 52%ăofăpatientsăwhoăreceivedăcombinedăpegylatedă interferonăalphaă2aăandăribavirinătherapy,ăandădoseă reductionăwasărequiredăină22%ăofăpatientsă[8][9].ă DEFINITION OF ANEMIA IN PATIENTS WITH CHRONIC VIRAL HEPATITIS C TREATED WITH PEGYLATED INTERFERON AND RIBAVIRIN Ină1968,ătheăWorldăHealthăOrganizationă(WHO)ă setă theă loweră limită ofă theă normală hemoglobină (Hb)ă valueăată12ăg/dLăforăwomenăandă13ăg/dLăforămen.ăTheă NationalăCancerăInstituteă(NCI)ăandăCommonăToxicityă Criteriaă foră Adverseă Eventsă (CTCAE)ă defineă anemiaă ofăfirstădegreeăofăseverityăasăHbălevelsăbetweenă10ăg/dLă andă theă loweră limită foră menă andă womenă [8][10].ă ThisăHbăvalueăwasăconsideredăasătheăreferenceăforă theăestablishmentăofăclinicallyăsignificantăanemiaăină clinicalătrialsăusingăribavirină[8][11].ă 218ă ă OlgaăOr şanăet al.ă PATHOGENIC MECHANISMS INVOLVED IN ANEMIA IN PATIENTS WITH CHRONIC VIRAL HEPATITIS C TREATED WITH PEGYLATED INTERFERON AND RIBAVIRIN ANEMIAăăINăăPATIENTSăăWITHăăCHRONICăăVIRALăă HEPATITISăăCă Anemiaăsecondaryătoăchronicădiseasesăisăalsoă foundă ină patientsă withă chronică virală hepatitisă C.ă Thisă hasă aă complexă etiologyă includingă deficientă ironăuse,ăaăreducedădegreeăofăhemolysis,ăaădecreasedă lifeă durationă ofă erythrocytes,ă lowă erythropoietină secretion,ă alongă withă aă reducedă tissueă responseă toă erythropoietin.ă Theseă mechanismsă areă consideredă toă beădueătoătheăinflammatoryăactionăofăcytokines,ăwhichă amongă otheră effectsă increaseă hepcidină productionă [12].ă Hepcidină preventsă ironă fromă leavingă theă cellsă andă tendsă toă increaseă intracellulară ironă levels,ă whichă inhibitsă erythropoietină productionă [13].ă Ină addition,ă ironă boundă toă hepcidină isă moreă difficultă toă useă forăă Hbăsynthesisă[8].ă Aplasticăanemiaăisărarelyăfoundă(approximatelyă 2%)ăinăchronicăhepatitisăCăvirusăinfection.ăOnlyă0.3– 0.5%ă ofă aplastică anemiaă casesă occură followingă virală hepatitis,ăusuallyăaăhepatitisăCăvirusăinfection.ăSeverală peculiaritiesă areă described:ă mostă ofă theă patientsă areă youngămen;ătheăsignsăofăboneămarrowăaplasiaăappeară aboută 2ă monthsă afterătheă acuteă episode;ăprognosisăisă generallyă unfavorable.ă 25%ă ofă patientsă withă aplastică anemiaăhaveăfunctionalăhepaticăalterationsăatătheătimeă ofă diagnosisă (subclinicală infections).ă Theă alterationsă ofă theă hepatică functionă referă toă aă decreaseă ină theă numberăofăTălymphocytes,ăanăincreaseăinătheănumberă ofă Tă suppressoră lymphocytesă resultingă ină gammainterferonăproductionăwhichădepressesăhematopoiesis.ă ANEMIAăăSECONDARYăăTOăăRIBAVIRINăăTREATMENTăă INăăPATIENTSăăWITHăăCHRONICăăVIRALăăHEPATITISăăCă Hemolytică anemiaă inducedă byă ribavirină treatmentă isă knownă toă beă theă mostă importantă adverseăeffectăofăantiviralătreatmentăinăpatientsăwithă chronicăviralăhepatitisăCă[15–18].ă Ribavirină entersă theă erythrocytesă withă theă helpă ofă aă nucleosideă transporteră andă isă initiallyă convertedă toăribavirinămonophosphate,ăsubsequentlyătoăribavirină diphosphateă andă triphosphate.ă Theă accumulationăă ofă ribavirină phosphatesă alongă withă theă relativeă adenosineă triphosphateă deficiencyă increasesă theă susceptibilityă toă oxidativeă processes,ă causingă ană increaseă ină cellulară toxicityă andă subsequentlyă extravascularăhemolysisă[8][16].ă ă 2ă Theăincreaseăinăironă(Fe)ădepositsăinătheăliveră isăknownăinăhemolysis.ăHemolysisădueătoăribavirină treatmentăisăaccompaniedăbyăFeăstorageăinătheăliver,ă approximatelyă 1500ă µg/year.ă Theă ironă storedă afteră 6–12ă monthsă ofă ribavirină treatmentă mayă causeă theă developmentă ofă hepatică fibrosis.ă Ferritină levelsă doă notăsignificantlyăchangeăinătheseăpatientsă[19].ă Someă patientsă developă symptomsă ofă anemia,ă includingă fatigue,ă superficială breathing,ă palpitations,ă cephalea.ă Theă suddenă decreaseă ină hemoglobină cană precipitateă anginaă pectorisă ină susceptibleă patients.ă Deathsăfromăacuteămyocardialăinfarctionăandăcerebrovascularăaccidentsăhaveăbeenăreportedăinăpatientsăwithă chronică hepatitisă Că receivingă combinedă therapy.ă Ribavirinăshouldănotăbeăadministeredătoăpatientsăwithă preexistingă anemiaă andă coronaryă diseasesă oră cerebrovascularădiseases.ăRibavirinăisăeliminatedăfromă theă organismă byă theă kidneys.ă Patientsă withă renală diseasesă cană developă severeă life-threateningă hemolysis.ăPatientsăwithăcreatinineălevelsăhigherăthană 2ăm/dLă(whichărevealsărenalădysfunction)ăshouldănotă beătreatedăwithăribavirin.ăInătheăcaseăofătheseăpatientsă withăhepatitisăCăwhoăneedătreatment,ămonotherapyăisă recommendedă[8].ă Ribavirină causesă hemolysisă dependingă onă theă administeredă dosesă [16][20].ă Theă decreaseă ină Hbă startsă betweenă theă firstă andă theă fourthă weeksă ofă therapyă andă mayă beă sudden.ă Hemolytică anemiaă isă reversibleăapproximatelyă4–8ăweeksăafterătheăcessationă ofă treatmentă [7].ă Ită improvesă withă theă reductionă ofă dosesă (10–15%ă ofă patients)/cessationă ofă treatment/ă discontinuousătreatment.ăErythropoietinăalphaăisăusefulă inăpatientsăwithăprogressiveăanemia,ătheămajorăbenefită beingătheămaintenanceăofăribavirinădosesă[21–23].ă Tsubotaăandăco-workersăreportedăanăincreaseăină plasmaă ribavirină levelsă betweenă weeksă 4ă andă 14ă ofă treatmentă [24].ă Theă SVRă rateă wasă higheră inăpatientsă withă aă higheră serumă ribavirină concentration.ă Dueăă toă theă factă thată theă determinationă ofă ribavirină concentrationsă isă expensive,ă ită isă notă cost-effectiveă forăallăpatients.ăInăcontrast,ăHbădeterminationăisăeasyă toăperform,ărapidăandăinexpensive;ăso,ătheăuseăofăthisă parameterăasăaăprognosticăfactorăforătheădevelopmentă ofăanemiaăbyăitsărepeatedădeterminationăeveryă2ăweeksă becomesăextremelyăimportantă[27].ăă Inăaăstudyăofă2001ăonă244ăpatientsăchronicallyă infectedă withă hepatitisă Că virusă (Devineă EBă et al.),ă threeă factorsă thată significantlyă influenceă ribavirininducedă hemolysisă areă described:ă theă pretreatmentă thrombocyteăcountă(P–0.01),ătheăadministeredăinterferonă alphaădoseă(P–0.001)ăandătheăhaptoglobinăphenotypeă (P–0.01).ăPatientsăwhoăhaveăstartedătreatmentăwithăaă 3ă lowerăthrombocyteăcountăhaveăaămoreămarkedădecreaseă ofă Hbă levels.ă Interferonă alpha,ă whichă isă myelosuppressiveă ină highă doses,ă alsoă contributesă toă theă developmentăofăanemia.ăTheăthirdăfactorăisăhaptoglobin,ă whichă hasă threeă phenotypes.ă Haptoglobină bindsă toă erythrocytes,ăandăthereăareădifferencesăbetweenătheă threeă phenotypesă regardingă theiră bindingă properties.ă Theă presentedă studyă suggestsă thată ribavirină uptakeă differsădependingăonătheăhaptoglobinăphenotypeă[25].ă Ină2004,ăNomuraăH.ăandăco-workersăconsideredă femaleă sex,ă ageă >ă 60ă years,ă aă ribavirină dose/bodyă weightă>ă12ămg/kg,ătheăpretreatmentăHbăvalue,ăaăHbă decreaseă ată 2ă weeksă ofă treatmentă byă >ă 2ă g/dLă asă predictiveăfactorsăforătheădevelopmentăofăanemiaă[27].ă AdvancedăageăandătheăpretreatmentăHbăvalueă areă independentă factorsă associatedă withă theă importantă decreaseă ină Hbă levelsă duringă theă courseă ofătreatmentă[25–27].ă Recently,ă ribavirină clearanceă (Tableă I)ă hasă beenă usedă asă aă predictiveă factoră foră ribavirininducedăhemolyticăanemiaă[28–30].ă Table I Definitionăofăribavirinăclearance [17]ă ă CL/F (L/h) = 32.3×G ×(1 – 0.0094×age)×(1 – 0.42×sex)/ Scr Gă–ăweightă(kg)ă Scră–ăserumăcreatinineă ă Naokiă Hiramitsuă andă co-workersă establishedă CL/Fă<ă15ăasăaăreferenceălevelăforătheăcessationăofă ribavirină treatment,ă becauseă ită wasă foundă thată ată thisăvalue,ă6%ăofăpatientsăhadăHbălevelsălowerăthană 8.5ă g/dL,ă aă valueă ată whichă treatmentă shouldă beă stopped.ă Noă patientă withă CL/Fă >ă 15ă presentedă Hbă valuesălowerăthană8.5ăg/dL.ăTheămeanăHbădecreaseă isă moreă sensitiveă ină theă evaluationă ofă theă riskă ofă discontinuousătreatmentăthanăCL/F.ăTheăassessmentă ofă anemiaă everyă 2ă weeksă ină patientsă withă CL/Fă <ă 15ăisătheămostăsensitiveămodalityăforătheăevaluationă ofătheăprogressionăofăanemiaă[20].ă ANEMIAăăSECONDARYăăTOăăINTERFERONăă TREATMENTăăINăăPATIENTSăăWITHăăCHRONICăăVIRALăă HEPATITISăăCă Theăliteratureădescribesărareăcasesăofăperniciousă anemiaă secondaryă toă longă durationă interferonă therapyă [31],ă bută interferonă causesă boneă marrowă suppressionă withă theă decreaseă ofă Hbă concentrations,ă neutropeniaă andă thrombocytopeniaă [6][10][32].ă ă 219 AnemiaăandăchronicăviralăhepatitisăCă Deficientă erythropoietină productionă alsoă contributesă toătheăpathophysiologyăofăanemiaă[33],ăFig.ă1.ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă Fig.ă1.ă–ăSideăeffectsăofăinterferonăduringătheăfirstă12ăweeksăofă treatmentă(upătoădatedă2004).ă SERUM ERYTHROPOIETIN AND ANEMIA IN PATIENTS WITH CHRONIC VIRAL HEPATITIS C TREATED WITH PEGYLATED INTERFERON AND RIBAVIRIN Erythropoietină isă ană endogenousă hormoneă thatăcausesăanăincreaseăinătheănumberăofăprogenitoră cellsăofătheăerythrocyticăseriesăinătheăblood-formingă marrowă[34].ă TheădiminutionăofătheăHbălevelăisăphysiologicallyă accompaniedă byă ană increaseă ină theă serumă erythropoietinălevel,ăwhichăsubsequentlyănormalizesătheăHbă levelă[35].ăTheăHbă–ăserumăerythropoietinărelationshipă isă lessă obviousă ină patientsă withă chronică disordersă (suchă asă neoplasiasă [36],ă humană immunodeficiencyă virusă (HIV)ă infectionă [37])ă thană ină patientsă withă chronicăVHCăunderăcombinedăantiviralătreatment.ă Ină theă studyă ofă Balană V.ă andă co-workers,ă serumă erythropoietină levelsă increasedă duringă theă firstă 8ă weeksă ofă treatmentă ină allă theă monitoredă groupsă ofă patients:ă patientsă whoă completedă theă studyă withoută requiringă ribavirină doseă reduction,ă patientsă whoă hadă ribavirină doseă reductionă duringă theă8ăweeksăofăstudy,ăandăpatientsăwhoăinterruptedă treatmentă beforeă theă completionă ofă theă study.ă Theă greatestă increaseă wasă foundă ină patientsă whoă receivedă treatmentă foră 8ă weeksă andă whoă neededă ribavirinădoseăreductionă[33],ăFig.ă2.ă Inăpatientsăwhoăcompletedă8ăweeksăofăcombinedă antivirală treatmentă requiringă theă adjustmentă ofă ribavirină doses,ă theă meană ribavirină doseă reductionă wasă225ămg/dayăatătheăendăofătheăstudy.ăTheămeană 220ă ă OlgaăOr şanăet al.ă 4ă Fig.ă 2.ă –ă Meană serumă erythropoietină studiedă weeklyă duringă theă firstă 8ă weeksă ofă treatmentă withă pegylatedă interferonă andă ribavirin.ă SEMă ăăăăăăăăăăăăstandardăerrorăofătheămeană[33].ă ribavirină doseă remainedă relativelyă constantă duringă theă firstă threeă weeksă ină patientsă whoă interruptedă treatmentăatăanăearlyăstageă[33].ă Aă tendencyă ofă meană Hbă toă decreaseă andă ofă meană serumă erythropoietină toă increaseă isă notedă untilă weeksă 5–6ă ofă treatment,ă afteră whichă theiră levelsăbecomeărelativelyăconstant. Theă erythropoietică responseă ină patientsă withă HCVăinfectionăseemsălowăcomparedătoăpatientsăwithă ironă deficiencyă anemiaă andă cancer,ă ironă deficiencyă anemiaă andă HIVă infection,ă respectivelyă [36][37].ă Foră theă sameă Hbă decrease,ă theă increaseă ină serumă erythropoietină wasă lessă markedă ină patientsă withă HCVătreatedăwithăpegylatedăinterferonăandăribavirină comparedătoăpatientsăwithăironădeficiencyăanemia.ă Theă resultsă ofă recentă clinicală trialsă showă thată anemiaă ină HCVă infectedă patientsă respondsă toă recombinantă humană erythropoietină (erythropoietină alpha)ă treatmentă [21][23].ă Itsă earlyă administrationă preventsă theă reductionă ofă ribavirină doses,ă maintainsă highăHbălevelsăandăimprovesătheăqualityăofălifeă[33].ă EFFECT OF ANEMIA AND OF THE REDUCTION OF PEGYLATED INTERFERON/RIBAVIRIN DOSES ON VIROLOGICAL RESPONSE IN PATIENTS WITH CHRONIC VIRAL HEPATITIS C Theă HCVă genotypeă andă viremiaă areă importantă predictorsă foră theă sustainedă virologicală responseăalongăwithăotherăfactorsăsuchăasăage,ăsex,ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă race,ăhepaticăhistologicalăchangesăandătransaminaseă levelsă[38–42].ă Recentă studiesă suggestă thată highă serumă ribavirină levelsă causeă ană increaseă ină theă SVRă rate.ă However,ă theă increaseă ofă ribavirină dosesă shouldă alsoă takeă intoă accountă theă adverseă effectsă ofă therapy.ă Theă lowă Hbă levelă ată theă onsetă ofă therapyă andătheăhighăserumăribavirinălevelăareăpredictorsăofă anemiaă ină patientsă treatedă withă ribavirină [38][43– 45].ă22%ăofătheăpatientsărequiredătheăadjustmentăofă ribavirinădosesădueătoăanemiaă[6].ă Theăprobabilityăofăanemiaăincreasesădependingă onătheăribavirinădoseăusedă(particularlyăinătheăcaseăofă dosesă higheră thană 15ă mg/kg)ă ină patientsă treatedă withă pegylatedă interferonă alphaă 2aă andă ribavirin,ă whileă theă obtainingă ofă theă SVRă dependingă onă theă ribavirină doseă usedă isă influencedă byă theă HCVă genotype.ă Lowă ribavirină dosesă (800ă mg/day)ă areă sufficientă toă obtaină SVRă ină patientsă withă HCVă genotypesă2ăandă3.ăHCVăgenotypeă1ărequiresătheăuseăofă theă standardă ribavirină doseă (1000ă oră 1200ă mg/day)ă withă ană increasedă riskă foră theă developmentă ofă anemiaă[38],ăFig.ă3.ă Viremiaă ată theă onsetă ofă treatment,ă age,ă baselineă ALATă levels,ă theă ribavirină dose/kgă bodyă weight,ă cirrhotică statusă andă ribavirină clearanceă influenceă theă sustainedă virologicală response,ă whileă theă probabilityă ofă appearanceă ofă anemiaă isă influencedă byă sex,ă theă ribavirină dose/kgă bodyă weight,ă baselineă hemoglobină levels,ă age,ă baselineă ALATălevelsăandăcirrhoticăstatusă[38],ăFig.ă4. ă 5ă AnemiaăandăchronicăviralăhepatitisăCă 221 Fig.ă3.ă–ăMeanăprobabilityăofăobtainingăSVRăinăpatientsăwithăchronicăviralăhepatitisăCăgenotypeă1ătreatedăforă48ăweeksădependingăonă theăprognosticăfactorsăofăSVRă(confidenceăintervală95%)ă[38].ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă Fig.ă4.ă–ăMeanăprobabilityăofăanemiaăinăpatientsăwithăchronicăviralăhepatitisăCăgenotypeă1ătreatedăforă48ăweeksădependingăonătheă prognosticăfactorsăofăanemiaă(confidenceăintervală95%)ă[38].ă ă 222ă ă OlgaăOr şanăet al.ă TREATMENT OF ANEMIA IN PATIENTS WITH CHRONIC VIRAL HEPATITIS C TREATED WITH PEGYLATED INTERFERON AND RIBAVIRIN Theă treatmentă ofă anemiaă ină patientsă withă chronică hepatitisă Că virusă infectionă treatedă withă combinedă antivirală therapyă consistsă ofă ribavirină doseă reduction/cessationă ofă ribavirină treatment,ă administrationă ofă recombinantă humană erythropoietină orătransfusion.ă A.ăREDUCTION/CESSATIONăăOFăăRIBAVIRINăă TREATMENTă Theă specifică recommendationsă ină theă caseă ofă anemiaă occurringă afteră treatmentă areă asă follows:ă theă ribavirinădoseăshouldăbeădiminishedătoă600ămilligramsă peră dayă (200ă milligramsă ină theă morningă andăă 400ă milligramsă ină theă evening),ă ină anyă ofă theă followingă cases:ă 1)ă patientă withoută aă significantă cardiovasculară disease,ă showingă aă decreaseă ină hemoglobinăvaluesăofăupătoă<ă10ăg/dLăandă>ă8.5ăg/dLă oră 2)ă patientă withă aă stableă cardiovasculară disorder,ă showingă duringă anyă 4ă weekă periodă ofă treatmentă aă decreaseă ină hemoglobină valuesă >ă 2ă g/dL.ă Ină theseă cases,ă theă readministrationă ofă theă initială doseă isă notă recommended.ă Theă administrationă ofă ribavirină shouldă beă interruptedăinăanyăofătheăfollowingăcases:ă1)ăpatientă withoutăaăsignificantăcardiovascularădiseaseăpresentingă aă confirmedă decreaseă ină hemoglobină valuesă toăă <ă 8.5ă g/dL;ă2)ă patientă withă aă stableă cardiovasculară disease,ăwithăhemoglobinăvaluesă<ă12ăg/dL,ăinăspiteă ofădoseăreductionăforă4ăweeks.ă Ifăanemiaăremits,ătheăadministrationăofăribavirină canăbeăreinitiatedăinăaădoseăofă600ămilligramsăperăday,ă whichăcanăbeăsubsequentlyăincreasedătoă800ămilligramsă perăday,ădependingăonătheătherapist’sădecision.ăInătheseă cases,ă theă readministrationă ofă theă initială doseă isă notă recommendedă[46–48].ă B.ăRECOMBINANTăăHUMANăăERYTHROPOIETINă Erythropoietinăalphaăisăaărecombinantăhumană erythropoietică growthă factor.ă Itsă administrationă stimulatesă erythrocyteă productionă andă increasesă hemoglobinăconcentration.ăAăsignificantăincreaseăină hemoglobină isă foundă afteră 2ă weeks,ă sometimesă upă toă6ăweeksăofătreatment,ăbecauseătheămaturationăofă erythroidă progenitorsă andă theiră releaseă intoă circulationătakesătimeă[8][50].ă Antivirală treatmentă ină patientsă withă chronică hepatitisăCăvirusăinfectionăsignificantlyăchangesătheă ă 6ă qualityă ofă lifeă [51][52].ă Theă useă ofă erythropoietină alphaăincreasesătreatmentăcomplianceăandăimprovesă theăqualityăofălifeă[8][49].ă Erythropoietină alphaă isă indicatedă ină patientsă withă aă significantă decreaseă ină hemoglobină levelsă duringă theă firstă weeksă ofă ribavirină treatment.ă Ină thisă case,ătwoăstrategiesăcanăbeăused:ăeitherăaăconservativeă approachă oră aă prophylactică one.ă Ină theă conservativeă approach,ă patientsă areă carefullyă monitoredă andă erythropoietină treatmentă isă indicatedă whenă hemoglobină levelsădecreaseătoălessăthană10ăg/dL.ăErythropoietină isă administeredă prophylacticallyă toă patientsă withă aă significantăcardiovascularăriskă[8].ăă C.ăINDICATIONSăăOFăăTRANSFUSIONă Transfusionă isă rarelyă necessaryă ină patientsă withă chronică virală hepatitisă Că underă antivirală treatment.ăForă aă longătimeă transfusionă wasă indicatedă ată aă Hbă valueă ofă 10ă g/dLă (Hematocrită –ă Htă 30%).ă However,ă dueă toă theă risksă ofă transfusionă ină theseă patientsă (transmissionă ofă infectionsă –ă e.g.ă HIV;ă adverseă immuneă reactionsă toă transfusion)ă alongă withă theă risk/benefită ratio,ă transfusionă isă recommendedă ată Hbă valuesă rangingă betweenă 7ă andăă 10ăg/dL:ă7ăg/dLăinăpatientsăwithoutăotherăcomorbiditiesă (myocardială oră cerebrală ischemia,ă hemorrhage)ă [54].ă Accordingă toă NCIă andă CTCAE,ă transfusionă isă indicatedăatăHbăvaluesălowerăthană8ăg/dLă[53]. CONCLUSIONS Anemiaăisăaăfrequentăandăredoubtableăadverseă reactionă ofă pegylatedă interferonă andă ribavirină treatmentă ină patientsă withă chronică virală hepatitisă C.ă Ribavirinăcausesăhemolyticăanemiaăinăparticular,ăandă pegylatedăinterferonăinducesăboneămarrowăsuppression.ă Aă Hbă valueă loweră thană 10ă g/dLă isă consideredă byă mostăauthorsăasătheăreferenceăforăanemiaăsecondaryă toăantiviralătreatment.ăBelowăthisăhemoglobinăvalue,ă theăreductionăorăcessationăofăribavirinătreatment,ătheă administrationăofărecombinantăhumanăerythropoietină oră transfusionă areă recommendedă dependingă onă theă severityăofăanemia.ă Theă rateă ofă increaseă ofă serumă erythropoietină duringă theă firstă weeksă ofă treatmentă isă correlatedă withătheăneedăforăribavirinădoseăreduction/cessationă ofă ribavirină treatment.ă Theă SVRă rateă isă loweră ină patientsă who,ă dueă toă anemia,ă receiveă reducedă ribavirinădoses.ă 7ă AnemiaăandăchronicăviralăhepatitisăCă 223 Anemia este o reacţie adversă redutabilă şi frecventă a tratamentului cu pegilatinterferon şi ribavirină la pacienţii cu hepatită cronică virală C. Ribavirina produce îndeosebi anemie hemolitică iar pegilatinterferonul induce supresie medulară. Valoarea hemoglobinei sub 10 g/dL este considerată de cei mai mulţi autori ca fiind de referinţă pentru anemia secundară tratamentului antiviral. Sub această valoare a hemoglobinei se recomandă reducerea sau întreruperea tratamentului cu ribavirină, administrarea eritropoetinei umane recombinate sau tranfuzia în funcţie de gravitatea anemiei. Rata creşterii eritropoetinei serice în primele săptămâni de tratament se corelează cu necesitatea scăderii dozelor/ întreruperii tratamentului cu ribavirină. 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NIGGă L.,ă SCHANZă U.,ă AMBUHLă P.M.,ă FEHRă J.,ă BACHLIă E.B.,ă Prolonged course of pure red cell aplasia after erythropoietin therapy.ăEur.ăJ.ăHaematol.,ă2004,ă73,ă376–9.ă ReceivedăFebruaryă9,ă2009ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă ă 226ă ă ă ă ă ă ă OlgaăOr şanăet al.ă 10ă ORIGINAL ARTICLES Drug Treatment of Heart Failure Patients in a General Romanian Hospital D. ZDRENGHEA1, DANA POP1, OANA PENCIU1, M. ZDRENGHEA2 1 University of Medicine and Pharmacy “Iuliu Haţieganu”, Cluj-Napoca, Romania, Rehabilitation Hospital–Cardiology Department 2 University of Medicine and Pharmacy “Iuliu Haţieganu”, Cluj-Napoca, “Ion Chiricuţă” Oncology Institute, Hematology Department Objective. It is very well known that in clinical practice, according to the published guidelines, the heart failure patients are undertreated. There are striking differences in applying the guidelines in various countries and, up to the present, there are no consistent data for Romania. Material and Methods. There were studied 459 heart failure patients admitted in the Cardiology Department of a general hospital in Cluj-Napoca, Romania. They were evaluated from the point of view of the drugs used during hospitalization and recommended to be taken after discharge. Results. The patients, 393 males and 66 females, aged 61±9 years (lower in comparison with other countries) were included in NYHA III and IV class, except 3%, in NYHA II class. The ischemic etiology was registered in 56% of the patients. Drug treatment was as follows: diuretics–86.98%, ACEI–77.77%, beta blockers–55.95, ARB’s–7%, digoxin–51.63%, aldosterone antagonists–49%, nitrates–61.28%, statins–24%, antithrombotic therapy–60% (antiplatelet–45.75%, anticoagulants– 28.5%), antiarrythmics–27.91% (mainly amiodarone). In comparison with other European countries, the use of RAAS antagonists is similar, there are more beta blockers and nitrates used, but there is less use of antithrombotic therapy (especially anticoagulation) and of statins. Conclusion. Even though Romania still belongs to the group of developing countries, the drug treatment of heart failure patients does not much differ from the treatment applied in developed countries. Key words: heart failure, drug treatment, guidelines. The drug treatment of chronic heart failure patients is now very well established and available guidelines offer very concrete data about the usefulness of some medications [1–5]. The control of congestive syndrome is usually assured by diuretics and RAAS antagonists together with rate control in patients with rapid rate atrial fibrillation-digoxin, beta blockers [1–4]. In turn, the evolution, quality of life and survival are significantly improved by RAAS antagonists and beta blockers, but also by aldosterone antagonists [3–7]. The relief of pulmonary congestion, leading to a rapid resolution of breathlessness, can be easier obtained by using intravenous diuretics and nitrates [2][4]. Arrhythmias are controlled by amiodarone (supraventricular and ventricular arrhythmias), and digitalis and beta blockers are used for rate control of atrial flutter and fibrillation [4–6][8–11]. Long term anticoagulation is indicated in patients with atrial fibrillation, previous thrombembolic events, or much enlarged cardiac chambers [11][12]. ROM. J. INTERN. MED., 2009, 47, 3, 227–233 Statins are recommended in ischemic heart failure patients, but also in nonischaemic ones, to improve the endothelial function, even if the recent CORONA trial reported benefits for hospitalization, but not for survival [13][14]. Unfortunately, there is a significant gap between guidelines recommendations and their implementation in clinical practice, generally all categories of drugs being underused, or their doses being lower than those recommended and demonstrated as being efficient [18]. This is true mainly for the general practice, but also for the internal medicine or even cardiology units [19–21]. There are some explanations of the phenomenon. First, during clinical practice, the heart failure patients are somewhat different from those studied during clinical trials which represent, even in case of very accurate randomization, a selected population [22]. Second, in daily practice many heart failure patients have preserved LVEF and consequently drug treatment is not necessary to be D. Zdrenghea et al. 228 as aggressive as for systolic heart failure patients. On the other hand, during daily clinical practice it is often difficult to reach the recommended doses for RAAS antagonists and beta blockers because of the severe adverse effects, and consequently, the excellent results reported by the very large trials are much less [23][24]. Also, many doctors do not know the optimal treatment recommended by guidelines or the patients are not compliant with long term treatment [25–27]. Thus, it is necessary to optimize the implementation of guidelines for all the countries, including the developing ones. This is once more important for former communist countries because of the lower economic status [18–25]. In Romania, the aspects of guidelines implementation were less studied, this being the reason of the present clinical study. MATERIAL AND METHODS There were studied 459 consequently admitted patients in the Cardiology Department of a General Hospital (Rehabilitation Hospital), in Cluj-Napoca Romania, between January 2006 and December 2006, being discharged with the diagnosis of chronic heart failure. The patients were evaluated using the hospital files, from the point of view of the drugs used during hospitalization, and recommended to be taken at discharge. The study included both patients with systolic heart failure (LVEF< 40%) and with preserved LVEF (> 40%). 393 patients were males and 66 females, aged 61±9 years. The ischemic etiology was registered in 56% of the patients, idiopathic in 12%, toxic in 10% and others in 22%. We also registered the cardiovascular risk factors known to intervene as underlying and/or precipitating factors of the heart failure: hypertension, diabetes mellitus, arrhythmias, etc. STATISTICAL ANALYSIS The data were analyzed using SPSS 8.0 for Windows. We calculated mean and standard deviation for normal distributed quantitative variables. Differences between quantitative variables were examined using Student test (independentsample T test), and for qualitative variables we used χ2 test. A p value less than 0.05 was considered significant from the statistical point of view. 2 RESULTS The great majority of the patients were included in III and IV NYHA class (97%) and only 3% in II NYHA class. Hypertension was registered in 33% of the patients, valvulopathies (primary or secondary to LV systolic dysfunction) in 45.5%, diabetes mellitus in 14.38%, and arrhythmias in 76.47%. Atrial fibrillation was registered in 47.37% of the patients, and severe ventricular arrhythmias in 15.38%. Drug treatment during hospitalization and/or recommended at discharge was as follows: diuretics 86.98%, RAAS antagonists 84.77% (77.77% ACEI, 7% ARB’s), beta blockers 55.95%, nitrates 61.28%, statins 24%, antithrombotic therapy 60% (antiplatelet drugs 44.75% anticoagulants 28.5%), antiarrhythmics 27.91% (amiodarone 25%), calcium channel blockers 8%. Very interesting results were obtained from the analysis of readmissions, registered in 52% of the patients. If we considered the drugs excluded or introduced in the treatment schedule during readmission, beta blockers and antiarrhythmics were less excluded and most introduced (in more than 40% of the cases), followed by nitrates, probably in relationship with associated angina pectoris. In turn, ACEI and digoxin were excluded and introduced in low, and almost equal proportions (Fig. 1). After treatment adjustment the evolution was good in 62%, stable in 15% and worse in 23% of the patients. DISCUSSION The mean age of our patients (61±9 years) was about ten years less than that registered in the developed countries, where the mean age of heart failure patients is about 74 years [4]. This is in relationship with the increased percent and the early onset of cardiovascular diseases in our population, the primary preventive measures still lacking for the great majority of the population. In comparison with the results of Euro Heart Failure Survey [28], published in 2003, but analyzing 2000–2001 treated patients, our data are similar or even better (Fig. 2). Thus, ACEI are used in 77.77% vs. 61.8%; ARBs in 7% vs. 4.5%, beta blockers in 55.95% vs. 36.95, diuretics in 86.58 vs. 89.95, aldosterone antagonists in 49% vs. 20%, aspirin 45.75% vs. 21.1%. Antithrombotic therapy was less used (60% vs. 77.6%), but digoxin (51.65% vs. 35.7%) and nitrates (61.25% vs. 32.1%) were 3 Drug treatment of heart failure 229 Fig. 1. – Drugs excluded or introduced in the treatment schedule during readmission for decompensated heart failure. Fig. 2. – Comparison between drugs used in heart failure patients in Cluj-Napoca Study and Euro Heart Failure Survey. used significantly more. There are no comparative data about antiarrhythmic therapy, but calcium channel blockers were less used (8%) than in Euro Heart Failure Survey (21.2%). Also, our data are much closer to the published guidelines [2][4–6] than those recently reported about heart failure treatment in Scotland: the use of diuretics was similar – 89%, ARBs were 230 D. Zdrenghea et al. more used – 9% but ACEI, beta blockers and spironolactone were used in only 63%, 34% and 15% of the patients [29].. The results suggest that even in developing countries, to which Romania belongs, the drug treatment of heart failure patients is now similar to that applied in developed countries. The explanation could be the availability of information (internet, medical meetings, etc.), but also the availability of modern cardiovascular drugs, even if not always the last generation. Unfortunately, because the number of the studied patients was not very large, we could not analyze each component of every category of drugs. Probably this is not so important because even “old drugs” were demonstrated, by large and very well-known clinical trials, to significantly improve the clinical picture, evolution and survival in heart failure patients [1–6]. Considering the medication used, 40% of the patients received the recommended triple association: diuretics, ACEI, beta blockers, the percent being more than double with respect to that reported in the Euro Heart Failure Survey [28]. The high use of ACEI can be explained not only throughout a correct applying of the guidelines, but also throughout the high percent (33%) of the hypertensive patients [2][4] [5]. It is also important that 97% of the patients were included in III or IV NYHA class, the ACEI being imposed by the severe clinical picture, a situation somehow different from that noted in general medical practice. The high percent of diuretics is explained by the congestive syndrome, almost all our patients being on III or IV NYHA class [2]. The higher use of beta blockers in comparison with other countries (Euro Heart Failure Survey) is explained not only by the dominant ischemic etiology of the heart failure (56%) and associated hypertension (33%), but it reflects a good knowledge of the guidelines [4–7], [28][30]. Unfortunately, the general use of beta blockers remained less than recommended by guidelines. This cannot be explained only by the severe clinical picture, 23% of the patients being included in IV NYHA class, and with severe congestive syndrome, which can be initially worsened by the administration of beta blockers [30]. Spironolactone was also used in a very high percent, the dose being usually 50–100mg/day, higher than that generally reported. The explanation 4 is that of the severe congestive syndrome which imposed high dose spironolactone to preserve and increase the diuretic effect of furosemide [2][4][5]. The higher use of digoxin can be explained, not only by a traditional approach to the heart failure treatment, but also by the high percent of atrial fibrillation patients (48%), digoxin being recommended as an AV blocker to obtain an appropriate heart rate control [3–5]. Calcium channel blockers are surprisingly less used, even if almost 60% of the patients were ischemic, but this is according to the current guidelines, which recommended calcium antagonists to be used only in uncontrolled or vasospastic angina, or associated hypertension [4][5][31]. Antithrombotics, and especially anticoagulant therapy, were much less used than recommended or than registered in other studies [11][12]. We do not have a clear explanation for the less than recommended use of aspirin (50%). For anticoagulants, the very low percent could be explained by a traditional approach, even in patients with atrial fibrillation, but also by the difficulties to follow up the coagulation for a large part of the heart failure patients. A final “minus” is represented by the low use of antiarrhythmic therapy, even if the implantable cardioverter defibrillators (ICD) are very rarely available and used in Romania [2][32]. The arrhythmogenic risk in heart failure patients is continuously increasing and it is necessary, together with cardiac resynchronization therapy (CRT) and ICD implantation, to promote the antiarrhythmic therapy as a practically temporary solution [33], [34], [35]. CRT with or without ICD seems to improve survival and morbidity in selected patients with chronic heart failure who are optimally treated with pharmacologic agents according to current guidelines [2][4][33][34]. LIMITS OF THE STUDY The data used in this analysis were collected from hospital files, from the point of view of the drugs used during hospitalization, and recommended to be taken at discharge (but not sure to be taken at home). We did not always investigate the reason for not taking the proper drugs (side effects, absolute or relative contraindication). Study was performed mainly on an urban sample (university hospital) and, probably, in a rural sample the situation is even worse (lower percentage of patients taking proper medication). 5 Drug treatment of heart failure CONCLUSION Even though Romania still belongs to the group of the developing countries, the drug treatment of the heart failure patients does not much differ, or is even better than the treatment 231 applied in developed countries. At the same time, almost all the category of drugs are underused in relationship with the guidelines recommendations, being necessary to improve their use in current clinical practice. Obiective. Inhibiţia farmacologică a sistemelor neurohormonale cu IECA, sartani, antagonişti aldosteronici sau betablocante a demonstrat o reducere semnificativă a mortalităţii şi a numărului internărilor la bolnavii cu insuficienţă cardiacă. Ghidurile de diagnostic şi tratament oferă o sinteză a acestor rezultate, însă recomandările acestora nu sunt aplicate la toţi bolnavii în practica clinică, rezultatele obţinute fiind inferioare celor raportate de marile trialuri. Până la ora actuală nu există date în ceea ce priveşte această situaţie în România. Material şi metodă. Am luat în studiu 459 de pacienţi care au fost internaţi în secţia de Cardiologie a Spitalului Clinic de Recuperare – Cluj-Napoca cu diagnosticul de insuficienţă cardiacă, şi la care s-a urmărit tratamentul recomandat, atât în timpul internării cât şi la externare. Rezultate. Pacienţii, 393 bărbaţi şi 66 femei, cu o vârstă medie de 61±9 ani (mai scăzută comparativ cu alte ţări europene) au fost incluşi în clasele funcţionale NYHA III şi IV cu excepţia a 3%, în clasa NYHA II. Etiologia ischemică a fost înregistrată la 56% dintre pacienţi. În ceea ce priveşte tratamentul medicamentos administrat, rezultatele au fost după cum urmează: diuretice–86.98%, IECA–77.77%, beta blocante–55.95%, sartani–7%, digoxin–51.63%, antagonişti aldosteronici–49%, nitraţi–61.28%, statine–24%, medicaţie antitrombotică–60% (antiagregante plachetare– 45.75%, anticoagulante–28.5%), antiaritmice–27.91% (in special amiodaronă). Comparativ cu alte ţări europene, utilizarea antagoniştilor sistemului renină–angiotensinăaldosteron este similară, betablocantele şi nitraţii sunt utilizate mai frecvent, însă terapia antitrombotică (în special anticoagulantele) şi statinele sunt mai rar recomandate. Concluzie. Cu toate că România face parte din grupul ţărilor în curs de dezvoltare, tratamentul medicamentos recomandat pacienţilor cu insuficienţă cardiacă nu diferă semnificativ faţă de cel aplicat în ţările europene dezvoltate. Corresponding author: D. Zdrenghea. 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SWEDBERG K., CLELAND J., COWIE M.R. et al., Successful treatment of heart failure with devices requires collaboration. Eur. J. Heart Fail, 2008; 10(12):1229–35. 34. GASPARINI M., Indications to implantable cardioverter defibrillator in heart failure patients: a comment on the recently released ESC Guidelines on treatment and diagnosis of heart failure. Eur. Heart J., 2005; 26(22):2472–2473. Received May 25, 2009 Dyslipidemia and Hypertension in Patients with Type 2 Diabetes and Retinopathy TOMINA POPESCU1, MARIA MO A2 2 1 Clinical Hospital CF Craiova Clinic of Diabetes, Nutrition and Metabolic Diseases, University of Medicine and Pharmacy, Craiova Aim. Type 2 diabetes is associated with dyslipidemia and higher cardiovascular risk and also generates multiple microvascular complications. Diabetic retinal changes, diabetic maculopathy and proliferative retinopathy are major causes of vision loss among the people of working age worldwide. We searched the association between diabetic retinopathy (as one of the most important long time microvascular complications in diabetes), lipids disorder and blood pressure in subjects with T2DM without known cardiovascular diseases. Material and methods. We examined 100 patients with type 2 diabetes, without clinical evidence of coronary, cerebrovascular or peripheral artery disease; 48 (48%) were women (mean age 61.23±8.49) and 52 (52%) were men (mean age 60.75±8.43). Diabetic retinopathy was graded from retinal photograph according to Early Treatment for Diabetic Retinopathy Study severity scale. In each patient we measured total cholesterol, HDL, LDL, triglycerides. Statistical analysis was performed using Mann-Whitney U-test, Kruskal-Wallis and Oneway Anova tests. Results. Diabetic retinopathy was found in 59.5% men and 40.5% women. The patients who presented diabetic retinopathy had significantly higher values of total cholesterol (223.05±42.39 mg/dL vs 199.84±45.73 mg/dL) and LDL-C (152.29±42.46 mg/dL vs 117.33±45.35 mg/dL) and smaller HDLC (38.95±12.46 mg/dL vs 48.64±14.70 mg/dL) as compared to the patients without retinopathy. Although values of triglycerides were higher in the group with retinopathy than in the control one (179.71±112.60 mg/dL vs 172.53±104.53 mg/dL) there were no statistically significant differences. Conclusions. We found a statistically significant correlation between retinopathy and dyslipidemia, which suggested that the use of systemic lipid lowering therapy in type 2 diabetes is of benefit in patients with retinal changes, in order to prevent visual loss. Key words: diabetic retinopathy, type 2 diabetes, dyslipidemia, blood pressure. Diabetic retinopathy is a major microvascular complication of diabetes, and is the commonest cause of blindness in people of working age in the Western world. It can be defined as a damage of retina’s microvascular system due the prolonged hyperglycaemia [1]. The appearance of retinopathy is directly related to the time of evolution of the disease and metabolic control. It is estimated that diabetes mellitus affects 4 per cent of the world’s population, almost half of whom have some degree of diabetic retinopathy at any given time [2]. Diabetic retinopathy occurs both in type 1 and type 2 diabetes mellitus and has been shown that nearly all type 1 and 75 per cent of type 2 diabetes will develop DR after 15 years duration of diabetes as shown in earlier epidemiological studies [3]. The individual risk can be significantly reduced by early blood glucose control and treatment of vascular risk factors [4]. It was found, ROM. J. INTERN. MED., 2009, 47, 3, 235–241 in some studies [5][6], that the risk for diabetic retinopathy is directly correlated with serum lipidis, but it was not found an association between the lowering lipids therapy and retinopathy. Early intensive intervention to control cardiovascular risk factors is essential in clinical management. Atherogenic dyslipidemia characterized by elevated triglycerides, a low level of high-density lipoprotein cholesterol (HDL-C), and an increase in the preponderance of small, dense low-density lipoprotein (LDL) particles, is a key modifiable risk factor for macrovascular diabetic complications [7], but it is still unclear to what extent it can influence the appearance and evolution of diabetic retinopathy. The potential for systemic lipid modulation to prevent visual loss still remains unclear. Recently, in the Fenofibrate Intervention in Event Lowering in Diabetes (FIELD) study fenofibrate treatment demonstrated a significant 30% reduction in the 236 Tomina Popescu and Maria Mo a need for laser therapy in patients with and without known diabetic retinopathy, and more particularly, in the first course of laser treatment for both macular edema and proliferative retinopathy [8]. Regarding hypertension, epidemiologic studies have not found blood pressure to be a consistent risk factor for diabetic retinopathy incidence and progression [9][10]. However, other trials indicate that tight control of blood pressure is a major modifiable factor for the incidence and progression of diabetic retinopathy. The beneficial effects of tight controlling blood pressure still remain unclear. Two large Randomized Controlled Trials are currently ongoing. The Action in Diabetes and Vascular Disease (ADVANCE) study will evaluate the effect of a perindopril-indapamide combination on the incidence of DR [11], while the Diabetic Retinopathy Candesartan Trial (DIRECT) will evaluate the angiotensin II receptor blocker candesartan [12]. In this article we examined the relation between serum lipids, blood pressure and retinopathy in a group of patients with type 2 diabetes. MATERIAL AND METHODS We used the data of 100 patients with type 2 diabetes recorded at Clinical Hospital C.F. Craiova. Exclusion criteria – clinical evidence of macrovascular complication (peripheral arterial disease, previous stroke or myocardial infarction, abnormal EKG), active liver disease. Diabetic retinopathy was graded from retinal photograph according to Early Treatment for Diabetic Retinopathy Study severity scale. In each patient we measured total cholesterol, HDL-C, triglycerides and calculated LDL-C using Fiedwald formula LDL-Cţtotal cholesterol-(HDL-C+TG/5) mg/dL, when TG were under 400 mg/dL. We also questioned about diabetes duration, smoking and measured blood pressure, BMI and abdominal circumference. Blood pressure was recorded as the mean of three consecutive measurements in the sitting position taken 5 min apart. Hypertension was defined according to the current guidelines [13] as BP levels ≥ 140/90 mmHg or the use of anti-hypertensive drugs. STATISTICAL ANALYSIS Statistical analysis was performed using programs available in the SPSS 17.0 statistical 2 package. All variables were tested for normal distribution of the data. Data are presented as means ± standard deviation or percentages. Differences between the studied groups examined used t-test or the Mann-Whitney U-test for parametric and nonparametric data, respectively, while a chi-square test was used for categorical data. P-values Ţ 0.05 were considered statistically significant. RESULTS General characteristics: Total number of subjects 100 Men 52 (52%) mean age at examination 60.75±8.43 Women 48 (48%) mean age at examination 61.23±8.49 Classification of the groups – we found diabetic retinopathy in 42 (42%) subjects. Among these, 25 (59.5%) were men and 17 (40.5%) were women. The control group (patients without retinopathy) has 58 subjects, with 27 (46.6%) men and 31 (53.4%) women. Clinical and laboratory characteristics of the groups are shown in the table below (Table I). In patients with diabetic retinopathy we found, as we expected, a higher diabetes duration (13.86 years in proliferative retinopathy and 8.31 years in nonproliferative retinopathy vs 6.53 years in patients without retinopathy), with pţ0.020 (Fig. 1). Bars show means and error bars show means –/+ 1.0 SE. We did not find correlations between the presence of retinopathy and sex, although the percentage of nonproliferative retinopathy had slightly increased in men versus women (36.54% vs 33.33%) and increased net in proliferative retinopathy (11.54% vs 2.08%), but without statistical significance (Fig. 2). Smoking was strongly correlated with retinopathy, 31.35% of non-smokers presented retinopathy compared to 63.6% of smokers (pţ0.003) (Fig. 3). Systolic blood pressure correlates significantly with the presence of retinopathy, with pţ0.031, but does not correlate with the severity of retinopathy; diastolic blood pressure correlates with both presence (pţ0.003) and severity of retinopathy (pţ0.004) (Fig. 4). Dyslipidemia correlates well with the presence and severity of retinopathy, especially HDL-C and LDL-C. Although triglycerides were higher in patients with retinopathy, we did not find statistical significance (Table II). The graphics represent mean values of lipidis with standard deviation (Fig. 5). 3 Dyslipidemia and hypertension 237 Table I Clinic and laboratory characteristics of the groups Variable Without retinopathy With retinopathy p (2 tailed) 27 (46.6%)/31(53.4%) 25 (59.5%)/17(40.5%) 0,228 60.69±8.79 61.38±7.99 0.690 6.00 (3.00-9.00) 10.00 (4.00-12.25) 0.020 Smoker (%) 12 (20.7%) 21 (50.0%) 0.003 Hypertension (%) 40 (69.0%) 41 (97.6%) 0.000 Total cholesterol (mg/dL) 199.84±45.73 223.05±42.39 0.011 Triglycerides (mg/dL) 172.53±104,53 179.71±112,60 0.574 HDL-C (mg/dL) 48.64±14.70 38.95±12.46 0.001 LDL-C (mg/dL) 117.33±45.35 152.29±42.46 0.000 138.33±16.74 145.76±16.74 0.031 83.64±9.13 89.12±8.70 0.003 30.33±4.48 29.20±3.65 0.183 100.17±10.79 98.19±12.69 0.402 Male/female (%) Mean age Diabetes duration (years) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Body mass index (kg/m2) Abdominal circumference (cm) Fig. 1. – Relation between diabetic retinopathy and diabetes duration. Fig. 2. – Relation between diabetic retinopathy and sex. Fig. 3. – Relation between smoking habit and retinopathy. Fig. 4. – Blood pressure and retinopathy. 5 Dyslipidemia and hypertension 239 Table II Lipids and diabetic retinopathy Variable Diabetic retinopathy triglycerides (mg/dL)One-Way ANOVA HDL-C (mg/dL) LDL-C (mg/dL) p (2 tailed) test 221.89±42.39 228.86±45.26 0.038 188.14±111.66 0.855 Kruskal-Wallis Absent Nonproliferative Proliferative Total cholesterol (mg/dL) 199.84±45.73 172.53±104.53 178.03±114.33 48.64±14.70 38.14±12.54 43.00±12.08 0.001 Kruskal-Wallis 117.33±45.35 153.44±41.64 146.71±49.40 0.001 One-Way ANOVA Fig. 5. – Lipids and diabetic retinopathy. CONCLUSION 1. In our data, diabetic retinopathy is correlated with diabetes duration, hypertension (especially diastolic value) and smoking and it is sustained that blood pressure control can reduce the incidence of diabetic retinopathy and it must be obtained as early as possible. 2. Dyslipidemia was significantly correlated with the presence of diabetic retinopathy, but this significance decreases with the severity of retinopathy. We suggest that lipid lowering therapy must be initiated as soon as possible in order to prevent the development of diabetic retinopathy. We did not find correlations between triglycerides and retinopathy, but most of the patients with retinopathy in our study were on fibrates therapy. 3. We could not find associations between diabetic retinopathy and BMI or waist circumference, probably because patients with poor glycaemic control and smokers have a better BMI but not a “protection” for microvascular complications. Diabetul zaharat de tip 2 este asociat cu dislipidemie şi cu risc cardiovascular crescut şi generează, de asemenea, multiple complicaţii microvasculare. Modificările 240 Tomina Popescu and Maria Mo a 6 retinei în diabet, maculopatia diabetică şi retinopatia proliferativă reprezintă principalele cauze de orbire la populaţia adultă la nivel mondial. Am studiat asocierea între retinopatia diabetică (una dintre cele mai importante complicaţii microvasculare pe termen lung în diabet), dislipidemie şi valorile tensiunii arteriale la un grup de subiecţi cu diabet zaharat tip 2 fără boli cardiovasculare manifeste. Material şi metodă. Am examinat 100 de pacienţi cu diabet tip 2 fără boli cardiovasculare clinic manifeste. 48 (48%) au fost femei (vârsta medie la examinare 61.23±8.49 ani) şi 52 (52%) au fost bărbaţi (vârsta medie 60.75±8.43). Retinopatia diabetică a fost diagnosticată conform protocolului oferit de Early Treatment for Diabetic Retinopathy Study. Fiecărui pacient i-au fost măsurate colesterolul total, trigliceridele, HDL-C, LDL-C. Analiza statistică a fost efectuată utilizând testele Mann-Whitney U-test, Kruskal-Wallis şi Oneway Anova. Rezultate. Retinopatia diabetică a fost diagnosticată la 59.5% dintre bărbaţi şi la 40.5% dintre femei. Pacienţii cu retinopatie au prezentat valori semnificativ crescute ale colesterolului total (223,05±42,39 mg/dL vs 199,84±45,73 mg/dL) şi ale LDL-C (152,29±42,46 mg/dL vs 117,33±45,35 mg/dL) precum şi valori mai mici ale HDL-C (38,95±12,46 mg/dL vs 48,64±14,70 mg/dL) comparativ cu cei fără retinopatie. Deşi valorile trigliceridelor au fost mai mari în grupul cu retinopatie decât în cel de control (179,71±112,60 mg/dL vs 172,53±104,53 mg/dL), acest fapt nu a prezentat semnificaţie statistică. Concluzii. Am constatat o corelaţie semnificativă statistic între retinopatie şi dislipidemie, ceea ce sugerează că utilizarea tratamentului hipolipemiant la pacientul cu diabet tip 2 şi complicaţii retiniene este benefică pentru prevenirea pierderii vederii. Corresponding author: Tomina Popescu, MD Clinical Hospital C.F. 6, Ştirbei Vodă, Craiova, Romania E-mail: [email protected] REFERENCES OSHITARI T., ROY S., Common therapeutic strategies for diabetic retinopathy and glaucoma. Curr. Drug Ther., 2007; 2:224–32. AIELLO L.P., GARDNER T.W., KING G.L., BLANKENSHIP G., CAVALLERANO J.D., FERRIS F.L. 3rd et al., Diabetic retinopathy. Diabetes Care, 1998; 21: 143–56. 3. KLEIN R., KLEIN B.E.K., MOSS S.E., DAVIS M.D., DEMETS D.L., The Wisconsin epidemiologic study of diabetic retinopathy III. Prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Arch. Ophthalmol., 1984; 102: 527–32. 4. REMA M., PRADEEPA R., Diabetic Retinopathy: An Indian perspective. Indian J. Med. Res., 125, March 2007, pp. 297–310. 5. VAN LEIDEN H.A., DEKKER J.M., MOLL A.C. et al., Blood pressure, lipids, and obesity are associated with retinopathy: the hoorn study. Diabetes Care, 2002; 25(8):1320–1325. 6. KLEIN R., SHARRETT A.R., KLEIN B.E. et al., ARIC Group. The association of atherosclerosis, vascular risk factors, and retinopathy in adults with diabetes: the atherosclerosis risk in communities study. Ophthalmology, 2002; 109(7):1225–1234. 7. Steinmetz A., Lipid-lowering therapy in patients with type 2 diabetes: the case for early intervention. Diabetes Metab. Res. Rev., 2008 May–Jun.; 24(4):286–93. 8. ANSQUER J.C., FOUCHER C., AUBONNET P., LE MALICOT K., Fibrates and microvascular complications in diabetes – insight from the FIELD study. Curr. Pharm. Des., 2009; 15(5):537–52. 9. KLEIN B.E., KLEIN R., MOSS S.E., PALTA M., A cohort study of the relationship of diabetic retinopathy to blood pressure. Arch. Ophthalmol., 1995; 113(5): 601–6061. 10. KLEIN R., SHARRETT A.R., KLEIN B.E. et al., ARIC Group. The association of atherosclerosis, vascular risk factors, and retinopathy in adults with diabetes: the atherosclerosis risk in communities study. Ophthalmology. 2002; 109(7):1225–1234. 1. 2. 7 Dyslipidemia and hypertension 241 11. ADVANCE Collaborative Group. ADVANCE – Action in Diabetes and Vascular Disease: patient recruitment and characteristics of the study population at baseline. Diabet. Med., 2005; 22(7):882–888. 12. SJØLIE A.K., PORTA M., PARVING H.H., BILOUS R., KLEIN R., DIRECT Programme Study Group. The DIabetic REtinopathy Candesartan Trials (DIRECT) Programme: baseline characteristics. J. Renin Angiotensin Aldosterone Syst., 2005; 6(1):25–32. 13. CHOBANIAN A.V., BAKRIS G.L., BLACK H.R., CUSHMAN W.C., GREEN L.A., IZZO J.L., Jr. et al., Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. National Heart, Lung, and Blood Institute; National High Blood Pressure Education Program Coordinating Committee: Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension, 2003; 42:1206–1252. doi: 10.1161/01.HYP.0000107251.49515.c2. Received May 30, 2009 242 Tomina Popescu and Maria Mo a 8 Esophageal Achalasia – Manometric Patterns D.L. DUMITRAŞCU, TEODORA SURDEA-BLAGA, LILIANA DAVID “Iuliu Haţieganu” University of Medicine and Pharmacy, 2nd Department of Internal Medicine, Cluj-Napoca, Romania Achalasia is a primary esophageal motility disorder involving the body of the esophagus and lower esophageal sphincter. Esophageal manometry is the gold standard to diagnose achalasia. Two cardinal features are encountered in achalasia: incomplete LES relaxation and absent peristalsis in the lower esophagus. In a group of 94 patients with dysphagia we looked for the manometric changes, trying to identify clinical and manometric particularities of the cases with achalasia. 35 cases had manometric changes compatible with the diagnostic of achalasia. A number of 41 patients had other esophageal motility disorders and 8 patients had normal esophageal manometry. The majority of patients (80%) had an increase basal pressure in the lower esophageal sphincter (LES) and the rest of them had a normal pressure of the LES. Absence of the lower esophageal sphincter relaxation or incomplete relaxation was observed in all cases. Aperistalsis was found in 33 patients (94.3 %) and 2 patients (5.7%) presented hyperperistaltism of the tubular esophagus. We followed the manometric changes pre and post treatment in 10 cases, all having similar manometric modifications, before and after treatment; only the basal pressure of the lower esophageal sphincter was modified after treatment. Key words: lower esophageal sphincter, achalasia, manometry. Achalasia is a primary esophageal motility disorder involving the body of the esophagus and lower esophageal sphincter, affecting equally both genders and all ages [1]. The disease seems to be caused by denervation of the esophagus resulting from loss of nitric oxide-producing inhibitory neurons in the myenteric plexus [2][3]. In terms of diagnosis, esophageal manometry is the gold standard to diagnose achalasia [1]. Achalasia is characterised manometrically by two cardinal features: incomplete LES relaxation and absent peristalsis in the lower esophagus. Additional features that are often encountered but that are not in themselves diagnostic are an increased basal intraesophageal pressure that is higher than intragastric pressure, and an increased basal LES pressure [4]. Laparoscopic myotomy and pneumatic balloon dilatations of the lower esophageal sphincter are considered definitive treatments for achalasia. The role of manometry in post-treatment surveillance remains controversial [1]. In our study we looked for the manometric changes in patients with achalasia, trying to identify clinical and manometric particularities of these cases. In a small group of patients we also assessed the manometric changes in esophagus before and after surgical or dilation treatment. ROM. J. INTERN. MED., 2009, 47, 3, 243–247 MATERIAL AND METHODS A total of 94 patients with dysphagia were enrolled in the study. The patients were admitted in our hospital in a period of 3 years – between January 2005 and December 2007; the majority of them were referred to manometry department after endoscopic and radiologic evaluation by other clinicians. All patients signed the informed consent form. Clinical symptoms and signs, endoscopic changes and radiologic findings were also recorded for all the patients. Patients with no signs of organic esophageal stenosis were submitted to manometric studies. Esophageal motility was measured using a perfused manometric system (Medtronic) with 8 channels (8 recording points). The patients were evaluated in the lateral recumbent position. We recorded the following manometric parameters: the pressure in the upper esophageal sphincter, the peristalsis of the esophagus (the amplitude, shape, duration and progression velocity of contractions), basal lower esophageal sphincter pressure and residual LES pressure after swallowing. For the assessment of LES we used the pull-through technique. Peristalsis and LES relaxation were assessed in response to 5 ml of water swallows, using the pull-through technique; a minimum of 10 swallows (spaced at least 15 seconds apart) were tested. D.L. Dumitraşcu et al. 244 RESULTS From the 94 patients, 35 cases proved to have manometric changes compatible with the diagnostic of achalasia. A number of 41 patients had other esophageal motility disorders (diffuse esophageal spasm, hyperperistaltic esophagus, nonspecific motility disorders) and 8 patients had normal esophageal manometry (Table I). Table I 2 We followed the manometric changes pre and post treatment in 10 cases. 8 patients were treated through balloon dilation and 2 patients underwent surgical treatment (myotomy through laparoscopy). All these cases had similar manometric modifications, before and after treatment (as listed in Table III); as expected, only basal pressure of the lower esophageal sphincter was modified after treatment. The severity of the symptoms decreased in all cases, all patients reporting the amelioration of dysphagia. Manometric diagnostic for the patients with dysphagia Disorder Achalasia 94 patients 35 (37.3 %) Diffuse esophageal spasm 10 (10.6%) Hyperperistaltic esophagus 9 (9.6%) Nonspecific motility disorders 32 (34.0%) 8 (8.5%) Normal manometry The majority of patients (80%) had an increased basal pressure in the lower esophageal sphincter (LES) and the rest of them had a normal pressure of the LES. None of these patients had complete LES relaxation during deglutition. Absence of the lower esophageal sphincter relaxation or incomplete relaxation were observed in all cases. Regarding the peristalsis of the esophageal body, aperistalsis was found in 33 patients and 2 patients presented hyperperistalsis of the tubular esophagus. So, from 35 cases of achalasia, 94.3% had a typical form, while 5.7% corresponded to vigorous, atypical achalasia (Table II). Table II Manometric changes in patients with achalasia MANOMETRIC CHANGES N (%) LES pressure LES hypertonia 28 (80%) LES normotonia 7 (20%) LES relaxation during deglutition time Lack of LES relaxation 35 (100%) LES relaxation 0 Peristalsis of the tubular esophagus Normal peristalsis of tubular esophagus 0 Aperistalsis of tubular esophagus 33 (94.28%) Hyperperistalsis of tubular esophagus 2 (5.71%) We also evaluated the pressure in the upper esophageal sphincter (UES). We identified 18 patients with UES hypertonia (51.42%) and 17 patients (48.58%) with normal pressure in the UES. Table III Manometric changes before and after treatment Manometric modifications Pre-treatment Post-treatment LES hypertonia present reduced LES relaxation absent absent Tubular peristalsis absent absent UES hypertonia present present DISCUSSION Achalasia, a primary esophageal motor disorder, is characterized by the absence of esophageal peristalsis especially in the distal two-thirds (smooth muscle) and impaired lower esophageal sphincter (LES) relaxation due to damage to the myenteric plexus. It is a rare disease affecting both genders with a prevalence of < 1/10.000 and an incidence of 1/100 000 [5]. In addition to the typical symptom of dysphagia for both liquids and solids, patients may initially present with chest pain and regurgitation. Diagnosis is typically delayed 2–3 years from the beginning of symptoms [1]. Esophageal manometry is indicated in patients whose symptoms and other investigations, for example, endoscopy or radiologic studies, suggest a motor disorder. Manometric studies are usually second- or third-line investigations following initial assessment by radiology and endoscopy [4]. Until now, esophageal manometry remains the gold standard in diagnosing achalasia [1]. Motor function can be assessed by a variety of recording techniques including radiology, scintigraphy manometry, and most recently intraluminal electrical impedance monitoring. Some of these are complementary. Manometric measurement of esophageal pressure is the most direct method for assessment of motor function. Only manometry can give information on the strength of contractions. However, when the diagnosis requires information about intraluminal 3 Esophageal achalasia flow, this can be obtained by complementary measurement of transit by radiology, scintigraphy, or intraluminal impedance monitoring [4]. The typical manometric features in achalasia are as follows: 1. Incomplete lower esophageal sphincteric relaxation with swallowing. Whereas the normal sphincter relaxes by over 90%, relaxation with most swallows in patients with achalasia is less than 50% [4][6][7]. 2. Complete absence of peristalsis; swallowing results in simultaneous waves that are usually of low amplitude. Additional features that are often encountered but that are not in themselves diagnostic are an increased basal intraesophageal pressure that is higher than intragastric pressure, and an increased basal LES pressure (in about 50% of patients) [4]. In cases of achalasia that are established by all available clinical criteria, the defining manometric features (aperistalsis and incomplete LES relaxation) are present in >90% of patients. Other manometric features (increased intraesophageal baseline pressure or isobaric waveforms) provide supportive evidence to improve the suspicion rate [8]. Variations in the manometric features occur between patients, which makes manometric diagnosis difficult, and manometric findings should always be considered in the context of the clinical radiologic and endoscopic findings. Impaired LES relaxation is frequently observed. It may be difficult to pass the manometric assembly through the esophagogastric junction. The nadir pressure threshold that defines incomplete relaxation depends on the method of analysis of LES pressure, and whether end-expiration, midexpiration, or average LES pressure is used. Typically there is partial relaxation [4]. Normal LES relaxation is commonly believed to be “complete”. However, it is usual for there to be a small nadir pressure of up to 7 mmHg even at maximal relaxation [6][7][9]. With a sleeve sensor, the duration of LES relaxation is usually <8 seconds [9]. Our results are similar with data from literature; in our study all patients with achalasia had impaired lower esophageal sphincter relaxation. Complete relaxation was not observed in any of the cases. In achalasia, occasionally relaxation may appear to be complete [10][11]. However, in such instances careful inspection of the LES recording usually reveals subtle abnormalities such as a 245 delayed onset and shortened duration of relaxation, and relaxation appears to be functionally inadequate [12][13]. Aperistalsis is the other cardinal feature of achalasia; it is considered a “sine qua non” condition in order to diagnose achalasia. Classically, the esophageal body is characterized by only lowamplitude (<30 mmHg) simultaneous isobaric pressure waves [1]. In our study more than 94% of patients with achalasia showed no peristalsis in tubular esophagus and they were diagnosed with a typical form of achalasia. A few patients have wave amplitudes within or above the normal range. When the average contraction amplitude is above 37 mmHg, the term “vigorous” achalasia is used [14][15]. Reported frequencies about vigorous achalasia in literature varies from 1.5% (in studies using high resolution manometry) [16] to 31% [17]. From our 35 patients with achalasia, only 2 patients (less than 6%) had atypical, vigorous form of achalasia. The relevance of this sub classification remains unclear as the prognostic and therapeutic value of this separation are controversial [17]. Occasionally, the pattern of esophageal motility may mimic a diffuse spasm, and overlap between achalasia and diffuse spasm has been reported [18]. The aperistaltic segment usually involves the entire esophageal body. However, partial preservation of peristalsis may be present in the proximal esophagus [4]. A hypertensive LES (i.e. basal LES pressure >45 mmHg) may be present and occasionally increased intraesophageal basal pressure above the intraabdominal pressure is observed [8][19]. This is a consequence of esophageal retention and it often disappears if the patient regurgitates retained esophageal contents or excess fluid is aspirated from the esophagus during the manometry. Up to 40% of the patients with achalasia have normal LES pressure (10–40 mm Hg); however, low pressure LES is not seen in untreated achalasia patients [20]. The majority of our patients with achalasia (80%) had an increased basal LES pressure, while 7 out of 35 patients had a normal basal LES pressure. Our results regarding LES resting pressure are similar with data from literature. Differential diagnosis of manometrical changes in achalasia includes pseudoachalasia. Manometrically the features are the same and the diagnosis depends on clinical, radiologic, and endoscopic features. Severe peristaltic failure and complete aperistalsis 246 D.L. Dumitraşcu et al. may also be seen in patients with connective tissue diseases such as scleroderma, amyloidosis, and diabetes mellitus, and in reflux disease. However, in such patients, basal LES pressure is typically very low, LES relaxation is complete, and there is no increase in basal intraesophageal pressure [21][22]. Laparoscopic myotomy of and pneumatic balloon dilatations of the lower esophageal sphincter are considered definitive treatments for achalasia. Both treatment options offer sustained clinical responses, but are in fact palliative treatments. Botulinum toxin injection in the lower esophageal sphincter is considered an acceptable alternative in patients who are not candidates for surgery or balloon dilatation. Pharmacologic therapies for achalasia offer mild, transient improvement at best [1]. After treatment, patients should be followed up regularly (yearly) in order to identify disease progression early and avoid the development of the end-stage disease when esophagectomy may become the only treatment option. In order to monitor the success of the therapeutic intervention and to detect disease recurrence prior to the development of symptoms, Vaezi et al. proposed the “timed barium swallow”. It consists of the ingestion of 50-100 ml of barium with plain thoracic radiographs taken at 1, 2 and 5 minutes. Measuring the height of the barium column at these time intervals allows quantifying the degree of esophageal bolus retention [23]. 4 The role of manometry in post-treatment surveillance remains controversial. Curing achalasia would mean restoring esophageal peristalsis and restoring the neurons of the myenteric plexus [1]. We repeated manometry in 10 patients one month after treatment and we did not find significant manometric changes compared to pre-treatment manometric studies. Only the basal LES pressure decreased, secondary to the rupture of muscle fibers. We did not evaluate these patients a year after treatment. The patients included in our study were referred to manometry department so they were previously selected (using endoscopic and radiologic studies). All of the patients had dysphagia, so the high number of patients diagnosed with achalasia and other esophageal motility disorders in this study cannot be used to determine the prevalence and incidence of these diseases in our region. In conclusion our results showed that in achalasia the majority of patients have incomplete or no relaxation of LES; the peristalsis in esophageal body is usually absent; only in rare cases the amplitude of waves in esophagus are increased (vigorous achalasia); the basal LES pressure is higher than normal. It seems that therapy does not modify the pattern of esophageal motility at the level of the esophageal body. Achalasia este o tulburare de motilitate esofagiană primară implicând corpul şi sfincterul esofagian inferior (SEI). Manometria esofagiană este standardul de aur în diagnosticul achalasiei. Două caracteristici cardinale apar în achalasie: relaxare incompletă a SEI şi absenţa peristaltismului în corpul esofagian. Într-un grup de 94 pacienţi cu disfagie s-au urmărit modificările manometrice pentru identificarea particularităţilor clinice şi manometrice ale achalasiei. 35 de cazuri au prezentat modificări manometrice compatibile cu achalasia. Alţi 41 pacienţi au prezentat alte tulburări de motilitate esofagiană şi 8 au prezentat manometrie esofagiană normală. Majoritatea pacienţilor (80%) au prezentat creşterea presiunii bazale a SEI, restul au avut presiunea SEI normală. Absenţa relaxării SEI sau relaxarea incompletă s-a observat în toate cazurile. Aperistaltismul a fost identificat la 33 pacienţi (94,3%) şi 2 (5,7%) au prezentat hiperperistaltism al esofagului tubular. În 10 cazuri s-a efectuat manometrie postterapeutic: presiunea bazală a SEI a fost modificată de tratament, motilitatea pe esofagul tubular nu a fost modificată. Corresponding author: D.L. Dumitraşcu, Professor 2nd Department of Internal Medicine 2–4, Clinicilor Str., 4000 Cluj-Napoca, Romania E-mail: [email protected] 5 Esophageal achalasia 247 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. POHL D., TUTUIAN R., Achalasia: an overview of diagnosis and treatment. J. Gastrointestin. Liver Dis., 2007; 16(3):297–303. DUNTEMANN T.J., DRESNER D.M., Achalasia-like syndrome presenting after highly selective vagotomy. Dig. Dis. Sci., 1995; 40(9):2081–3. CASSELLA R.R., BROWN A.L., SAYRE G.P., ELLIS F.H., Achalasia of the esophagus: pathologic and etiologic considerations. Ann. Surg., 1964 Sep., 160:474–87. 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VANTRAPPEN G., VANGOIDSENHOVEN G.E., VERBEKE S., VANDENBERGHE G., VANDERBROUCKE J., Manometric studies in achalasia of the cardia, before and after pneumatic dilations. Gastroenterology, 1963 Sep., 45:317–25. KAHRILAS P.J., KISHK S.M., HELM J.F., DODDS W.J., HARIG J.M., HOGAN W.J., Comparison of pseudoachalasia and achalasia. Am. J. Med., 1987; 82(3):439–46. TRACEY J.P., TRAUBE M., Difficulties in the diagnosis of pseudoachalasia. Am. J. Gastroenterol., 1994; 89(11):2014–18. VAEZI M.F., BAKER M.E., ACHKAR E., RICHTER J.E., Timed barium oesophagram: better predictor of long term success after pneumatic dilation in achalasia than symptom assessment. Gut, 2002; 50(6):765–70. Received June 12, 2009 248 D.L. Dumitraşcu et al. 6 Incidence and Type of Stroke in Patients with Diabetes. Comparison Between Diabetics and Nondiabetics DUMITRAăăB LAN¹,ăăP.A.ăBABE޲ă 1 ăEmergencyăCountyăHospital,ăVâlcea,ăRomaniaă ăFacultyăofăMedicine,ăOradeaăUniversity,ăRomaniaă 2 Background. Strokeă isă theă thirdă causeă ofă death,ă afteră heartă diseaseă andă cancer.ă Diabetesă mellitusăisăoneăofătheăriskăfactorsăforăstrokeăandăitsăincidenceăisăincreasing,ăleadingătoăanăaccentuationă ofătheăriskăofăstroke.ăWeăexploredătheărelationshipăbetweenădiabetesăandătypesăofăstroke,ăriskăfactorsă forătheălatterăone,ăbothăinăsubjectsăwithădiabetesăandăinăthoseăwithoutădiabetes.ă Methods.ă Theă studyă wasă conductedă onă twoă groupsă thată includedă 228ă subjects;ă groupă 1–136ă diabetică patientsă andă strokeă recentlyă installed;ă groupă 2–92ă patientsă withoută diabetesă bută strokeă recentlyă installed,ă hospitalizedă ină theă Neurologică Departmentă ofă theă Countyă Emergencyă Hospital,ă Vâlceaăduringă2006–2008.ă Onăeveryăbatchăweăhaveămadeăbiochemicalămeasurements:ăglycemică profile,ălipidăprofile,ăweă haveăestablishedănormalăvaluesăforăeveryăgroupă(nondiabeticsăandădiabeticăsubjects),ăweădeterminedă theătypeăandălocationăofăstrokeăasătheăresultăofăCT.ă Statisticală analysisă wasă performedă usingă SPSSă 15ă foră Windowsă application,ă adaptedă toă processingă medicală statistics.ă Theyă calculatedă theă averageă ofă theă parameters,ă standardă deviation,ă standardădeviationăofătheăaverage,ăfrequencyăranges,ătablesăfrequencyătestsăofăstatisticalăsignificanceă byăStudentămethodă(tătest),ăandăcalculationăofăHi2ăscore.ăWeăcalculatedăPearsonăcorrelationăcoefficientsăthată weăhaveăinterpretedăusingătheărulesăofăColton.ă Results.ă Ină bothă groupsă ischaemică strokeă predominatedă –ă 116ă (85%)ă casesă ină diabeticsă andăă 68ă (74%)ă casesă ină nondiabetics;ă haemorrhagică strokeă wasă approximatelyă equală ină diabeticsă andă nondiabeticsă20ă(15%)ăcasesăvs.ă24ă(26%)ăcases.ăTheăaverageăageăwasăsignificantlyăhigherăinăgroupă2ă versusăgroupă1ă(pă=ă0.026*).ăMostăofătheă2ndăgroupăsubjectsăareăfromăruralăareas,ăwhileăină1stăgroupă theyă areă fromă theă urbană areas.ă Hypertensionă wasă mostlyă aă pre-existingă riskă factoră ină bothă groups.ă Serumă lipidă wasă overă theă valuesă recommendedă ină bothă groups.ă Theă indexă ofă insulinoresistance,ă materializedăbyăHOMA-IR,ăwasăpresentăină80%ăofăsubjectsăofă1stăgroupăandăină52%ăofăsubjectsăofătheă 2ndă group.ă Theă incidenceă rateă ofă strokeă wasă muchă higheră ină diabetică populationă thană ină theă nondiabeticsăbetweenă2006ăandă2008.ăă Conclusions.ăStrokeăisăinstalledăatăaăyoungerăageăinădiabeticăsubjectsăthanăinănondiabeticăonesă andăităisămoreăfrequentăinănondiabeticămales;ăinsulinoresistanceăisăaăriskăfactorăpresentăinăbothăgroups,ă butăweăfoundăaăstatisticalăsignificanceăonlyăinăgroupă2.ă Key words:ădiabetes,ăischaemicăstroke,ăhaemorrhagicăstroke,ăriskăfactor.ă Diabetesă mellitusă isă associatedă withă ană increasedă riskă ofă atherosclerotică vasculară diseases,ă includingă cerebrovasculară disease.ă Thisă increaseă isă probablyătheărelationshipăbetweenătheăsideăeffectsăofă hyperglycaemiaă ină associationă withă otheră riskă factors,ăsuchăas:ăhypertension,ădyslipidaemia,ăobesity,ă etc.ăMorbidityăandămortalityăinăatheroscleroticădiseaseă appearătoăbeăhigherăinădiabeticăpatientsăasăcomparedă toă nondiabetics.ă WHOă estimatesă thată 16.6ă millionă peopleăworldwideădieăofăcardiovascularădiseaseăeveryă year,ăwhichăisătheăthirdăcauseăofădeath.ăă Otheră 15ă millionă persons/yeară haveă aă minoră strokeăandă600ămillionăpeopleăwithăhypertensionăhaveă ROM.ăJ.ăINTERN.ăMED.,ă2009,ă47,ă3,ă249–255ă ă aăriskăforăheartăfailureăorăstroke.ăCardiovascularădiseasesă areă responsibleă foră 76%ă ofă mortalityă ină diabetes,ă 55%ă ofă themă areă dueă toă ischaemică heartă disease,ă andă12%ătoăstrokeăandăcerebrovascularădisease.ă OBJECTIVESă Startingă fromă theseă facts,ă weă establishedă theă followingăobjectives:ă • toă studyă theă incidenceă andă typeă ofă strokeă ină patientsăwithădiabetesăasăcomparedătoănondiabetică subjects,ă fromă theă sameă geographicală areaă –ă aă comparativeăstudyăinăpatientsăwithădiabetesăandă 250ă ă • DumitraăB lanăandăP.A.ăBabeşă strokeă recentlyă installedă (groupă 1)ă andă patientsă withoutădiabetesăbutăwhoăhaveărecentlyăinstalledă strokeă(groupă2);ă toă compareă theă incidenceă rateă ofă theă stroke,ă duringă3ăyearsă(2006–2008)ăinădiabeticăpopulationă andăgeneralăpopulation.ăă MATERIAL AND METHODS Theăstudyăwasăconductedăonătwoăgroupsăthată includedă228ăsubjects:ăgroupă1–136ădiabeticăpatientsă andăstrokeărecentlyăinstalledăandăgroupă2–92ăpatientsă withoută diabetes,ă bută havingă recentlyă installedă stroke,ă hospitalizedă ină theă Neurologică Departmentă ofă theă Countyă Emergencyă Hospitală Vâlcea,ă ină theă periodă2006–2008.ă Theă diagnosisă ofă strokeă wasă madeă basedă onă clinicală data,ă andă cerebrală CT.ă Groupă 1ă includedă 136ă patientsă withă diabetesă andă strokeă recentlyă installed;ă thereă wereă notă includedă ină theă studyă patientsăwithăhistoryăofătransitoryăischaemicăattackă oră recurrentă stroke.ă Theă dataă wereă collectedă fromă theăclinicalădataăsheet:ăCT,ălipidăprofile,ăglycaemică profile,ăHbA1c,ăinsulinemia,ăCăpeptide.ă Groupă 2ă includedă 92ă nondiabetică patients,ă admittedă ină theă sameă periodă ină theă Departmentă ofă Neurologyă –ă Countyă Emergencyă Hospitală Vâlcea.ă Groupă 2ă wasă formedă afteră groupă 1,ă bută wasă notă orderedăbyăage,ăsexăandăhomeăenvironment,ăasăweă consideredă necessaryă toă determineă theă impactă ofă diabetesămellitusăasăriskăfactorăinădiabeticăpopulationă thană ină nondiabeticsă andă theă presenceă ofă otheră riskă factorsă ină nondiabetică population.ă Patientsă withă IFGă orăIGTăhistory,ăhistoryăofăischaemicătransitoryăstrokeă orărecurrentăstroke,ăwereănotăincludedăinătheăstudy.ăInă theăstudyăprotocol,ăpatientsăhaveăreportedădataărelatedă to:ă age,ă gender,ă homeă environment,ă BMI,ă personală historyă ofă pathologyă (hypertension,ă heartă disease),ă regulară submissionă toă theă specialistă (periodică visits),ă theătypeăofădiabetesăandăantidiabeticătherapyăused,ătheă systolică andă diastolică bloodă pressure.ă Computerizedă tomographyă (CT)ă wasă performedă withină theă firstă 36–48ă hoursă afteră admission.ă Onlyă seriousă cases,ă whichă usuallyă haveă haemorrhagică stroke,ă wereă examinedă withină 24ă hours.ă Weă determinedă theă followingăbiochemicalăparameters:ăfastingăandăpostprandială glycaemia,ă totală cholesterol,ă LDL-chol.,ă HDL-chol.,ă Col/ă HDLă ratio,ă triglycerides.ă Weă determinedă HbA1c,ă theă fastingă plasmaă insulin,ă weă calculatedăHOMA-IRăindex.ăWeămadeăaăcomparisonă betweenă typesă ofă strokeă ină theă twoă groupsă andă ă 2ă calculatedă theă riskă factorsă foră eachă typeă ofă stroke.ă Theă statisticsă testsă wereă calculatedă usingă statisticală softwareăSPSSă15ăforăWindows.ăForăsomeăcasesăweă usedă Epiinfoă 2000,ă epidemiologicală software.ă Confidenceă intervalsă associatedă toă theă variablesă ină theă studyă wereă calculatedă foră aă thresholdă ofă significanceă ofă 5%ă basedă onă aă methodă usingă bionominalădistribution.ă RESULTS AND DISCUSSION Inătheăischemicăstrokeăweăfoundăaăstatisticallyă significantă differenceă ă betweenă theă ageă ofă subjectsă fromă groupsă 1ă andă 2ă (pă <0.05*)(Tableă I),ă ină theă diabeticăgroupăofăsubjectsăităwasăfoundătheăaverageă ageă ofă 67.53 ± 8.1ă years,ă andă ină nondiabetică subjectsă 70.50 ± 9.45ă yearsă (Tableă II).ă Theă sameă statisticallyăsignificantădifferenceă(pă<0.05)(TableăIII)ă wasă foundă ină theă caseă ofă haemorrhagică stroke,ă theă averageăageăofădiabeticăsubjectsăwasă63ă±ă7.2ăyears,ă andăthatăofănondiabeticsă69.5ă±ă10.8ăyearsă(TableăIV).ă Inătheădiabeticăpopulation,ăstrokeăwasăinstalledăearlier,ă andă asă thisă isă well-known,ă diabetesă acceleratesă theă atherosclerosis.ăComparingătheăvaluesăofăfastingăandă postprandială glycaemiaă ofă theă twoă groups,ă foră bothă typesă ofă stroke,ă weă foundă statisticallyă significantă differencesă(pă<0.05)ă(TablesăIăandăIII).ă Ină theă firstă group,ă glycosylatedă haemoglobină hadăapproximatelyătheăsameăvalue,ăinăsubjectsăwithă ischemicăandăhaemorrhagicăstrokeă(8.6%ă±ă1.3ăvs.ă8,ă 5ă±ă1.5%);ăinătheăsecondăgroup,ăallătheăsubjectsăhadă normală valuesă ofă theă HbA1c.ă Statisticală analysisă showedă aă statisticallyă significantă differenceă betweenă groups,ă ină bothă ischemică andă haemorrhagică strokeăă (pă<0.05*)ă(TablesăIăandăIII).ăFastingăinsulinemiaăhadă approximatelyătheăsameăvaluesăinăsubjectsăwithăstrokeă inăbothăgroups,ălackingăinăanyăstatisticalăsignificance.ă Inăsubjectsăwithăhaemorrhagicăstroke,ăinsulinemiaă wasăhigherăinătheăgroupă1ăasăcomparedăwithăgroupă2ă (19.37ă ±ă 16.21ă µUI/mLă vs.ă 10.43ă ±ă 4.06ă µUI/mL)ă (Tableă IV),ă aă statisticallyă significantă differenceă (p=0.013*)ă(TableăIII).ăTheăaverageăplasmaăcholesterolă wasă significantlyă increasedă ină subjectsă withă diabetesă andă ischaemică strokeă asă comparedă withă nondiabeticsă (233.28ă ±ă 47.81ă mg/dLă vs.ă 215.74ă ±ă 36.79ă mg/dL,ă p=0.01*)ă (Tablesă Iă andă II).ă Aă statisticallyă significantă differenceă wasă foundă ină subjectsă withă haemorrhagică stroke,ă comparingă theă twoă groups,ă bută hereă theă averageă plasmaă cholesterolă wasă higheră ină groupă 2ă (214.83ă±ă33.01ămg/dLăvs.ă180.40ă±ă66.91ămg/dL,ă p=0.046*)(Tableă III).ă Hypertriglyceridemia,ă theă dys- 3ă 251 Strokeăandădiabetesă lipidemiaăcommonăofădiabetesămellitus,ăwasăfoundăină diabetică subjectsă withă ischemică strokeă (199.07ă ±ă 124.03ă mg/dL),ă bută notă ină nondiabetică subjectsă withă ischemicăstrokeă(129.21ă±ă64.07ămg/dL);ăbetweenătheă twoă groupsă thereă wasă aă statisticallyă significantă differenceă(pă<0.05)ă(TablesăIăandăII).ă Table I TheăStudentătestă(comparisonăbetweenăgroupsăwithăischemicăstroke)ă Data Ageă FastingăGlucoseă PostprandialăGlucoseă HbA1că Insulinemiaă Totalăcholesterolă Triglyceridesă LDL-cholesterolă HDL-cholesterolă Systolicăbloodăpressureă Diastolicăbloodăpressureă BMIă p value Confidence interval 95% 0.026*ă <<0.05*ă <<0.05*ă <<0.05*ă 0.25ă 0.01*ă <<0.05*ă 0.026*ă 372ă 731ă 0.925ă 0.557ă [–5.56;ă–0.36]ă [85.47;115.67]ă [123.30;156.87]ă [318.49;382.31]ă [–[110.33;420.52]ă [4.32;ă30.75]ă [37.86;ă101.86]ă [1.26;ă19.80]ă [–3.2;ă1.20]ă [–6.06;ă8.62]ă [–3.98;ă3.61]ă [–0.83;ă1.54]ă ăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăăp<0.05*ă Table II ăComparisonăbetweenăgroupă1ăandăgroupă2ăwithăischemicăstrokeă Group Statisticsă Age Fasting Glucose Postprandial Glycaemia HbA1c Insulinemia Cholesterolemia Triglycerides LDL-chol HDL-chol Systolic BP Diastolic BP BMI Group N Mean 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 116ă 68ă 67.53ă 70.50ă 193.98ă 93.41ă 245.62ă 105.53ă 8.5776ă 5.0735ă 13.7833ă 12.2324ă 233.28ă 215.74ă 199.07ă 129.21ă 131.91ă 121.38ă 40.21ă 41.21ă 159.22ă 157.94ă 87.76ă 87.94ă 28.41ă 28.06ă Std. Deviation Std. Error Mean 8.100ă 9.455ă 80.889ă 11.017ă 89.606ă 13.477ă 1.52453ă .64336ă 10.83365ă 7.36347ă 47.481ă 36.790ă 124.300ă 64.078ă 35.240ă 20.941ă 8.723ă 6.324ă 26.732ă 19.666ă 11.765ă 13.936ă 4.261ă 3.341ă .752ă 1.147ă 7.510ă 1.336ă 8.320ă 1.634ă .14155ă .07802ă 1.00588ă .89295ă 4.408ă 4.461ă 11.541ă 7.771ă 3.272ă 2.539ă .810ă .767ă 2.482ă 2.385ă 1.092ă 1.690ă .396ă .405ă Standardădeviationă=ă±ă2SDă ă 252ă ă DumitraăB lanăandăP.A.ăBabeşă 4ă Table III TheăStudentătestă(comparisonăbetweenăgroupsăwithăhaemorrhagicăstroke)ă Data Ageă FastingăGlucoseă PostprandialăGlucoseă HbA1că Insulinemiaă Totalăcholesterolă p value 0.026*ă <<0.05*ă <<0.05*ă <<0.05*ă 0.25ă 0.01*ă Confidence interval 95% [–5,56;ă–0,36]ă [85,47;115,67]ă [123,30;156,87]ă [318,49;382,31]ă [–[110,33;420,52]ă [4,32;ă30,75]ă Triglyceridesă LDL-cholesterolă HDL-cholesterolă Systolicăbloodăpressureă Diastolicăbloodăpressureă BMIă <<0.05*ă 0.026*ă 0.372ă 0.731ă 0.925ă 0.557ă [37,86;ă101,86]ă [1,26;ă19,80]ă [–3,2;ă1,20]ă [–6,06;ă8,62]ă [–3,98;ă3,61]ă [–0,83;ă1,54]ă p<0.05*ă Ină subjectsă withă haemorrhagică stroke,ă theă triglycerideă values,ă ină theă comparisonă ofă twoă groups,ă didă notă reveală anyă statisticallyă significantă difference.ă Plasmaă triglycerideă valuesă wereă higheră ină subjectsă withă ischaemică strokeă thană ină subjectsă withăhaemorrhagicăstroke.ă TheăaverageăvaluesăofăLDL-chol.ăwereăincreasedă ină subjectsă withă diabetesă andă ischemică strokeă vs.ă nondiabeticsă (131.91ă ±ă 35.24ă mg/dLă vs.ă 121.38ă ±ă 20.94ămg/dL)ăwithăaăstatisticallyăsignificantădifferenceă (pă =ă 0.026*)ă (Tableă I).ă Weă foundă aă statisticallyă significantădifferenceă(pă=ă0.026*)ăwhenăweăcomparedă subjectsă withă haemorrhagică strokeă fromă theă twoă groups,ă bută LDL-chol.ă wasă increasedă ină nondiabeticsă subjectsă (119.50ă ±ă 19.37ă mg/dLă vs.ă 104.20ă±ă23.61ămg/dL)ă(TableăIV).ăă Systolicăbloodăpressureăvaluesăwereăapproximatelyă equalăinăsubjectsăwithăischemicăstrokeăinăbothăgroups,ă bută muchă loweră asă comparedă toă subjectsă withă hemorrhagică stroke.ă Thus,ă ină theă diabetică subjectsăă andăischaemicăstroke,ăaverageăsystolicăbloodăpressureă wasă159.22ă±ă26.73ămmHg,ăandăinădiabeticăsubjectsă withă haemorrhagică stroke,ă theă averageă wasă 200ă ±ăă 29.73ă mmHg;ă ină nondiabetică subjectsă weă foundă theă sameă difference:ă 157.94ă ±ă 19.66ă mmHgă ină patientsă withă ischemică strokeă andă 195.83ă ±ă 27.80ă mmHgă ină patientsă withă hemorrhagică stroke.ă Thereă wereă noă statisticallyă significantă differencesă betweenă subjectsă withăischaemicăorăhaemorrhagicăstrokeăinăbothăgroups.ă Diastolicăbloodăpressureăvaluesăwereăapproximatelyă equală ină bothă groupsă ofă subjects.ă Weă haveă notă foundă significantădifferencesăbetweenăgroupsăbyătypeăofăstroke,ă asă regardsă theă distributionă ofă BMI;ă subjectsă withă diabetesă andă ischemică strokeă hadă aă meană ofă BMIă –ă 28.41ă±ă4.26ăkg/m2,ăandănondiabeticăsubjectsă–ă28.06ă ±ă3.34ăkg/m2ă(TableăII).ă ă Thereăwereănoăstatisticallyăsignificantădifferencesă ină subjectsă ofă theă twoă groupsă withă haemorrhagică stroke,ă bută diabetică subjectsă hadă aă meană ofă BMIă greateră (31.9ă ±ă 4.75ă kgă /ă m2)ă thană nondiabeticsă subjectsă(30.25ă±ă3.33ăkg/m2).ă Theă riskă factorsă foră ischaemică strokeă ină theă diabeticăgroupăăwere:ătheăruralăareaă(RRă=ă1.160,ăORă=ă 2.87,ă pă =ă 0.038),ă plasmaă totală cholesterolă (RRă =ă 1.561,ăORă=ă7.28,ăp<0.05*),ăplasmaătriglyceridesă(RRă=ă 1.193,ăORă=ă3.08ăpă=ă0.02*);ăchol.total/HDL-chol.ratioă (RRă =ă 3.653,ă ORă =ă 24.43,ă p<0.05*);ă systolică bloodă pressureă(RRă=ă1.631,ăORă=ă7.67,ăpă<ă0.05*);ădiastolică bloodă pressureă (RRă =ă 1.253,ă ORă =ă 3.56,ă pă <0.05*);ă postprandială glycaemiaă (RRă =ă 1.098,ă ORă =ă 2.59,ăă p <<0.05*).ăForăhaemorrhagicăstrokeănoăriskăfactoră withăstatisticalăsignificanceăwasăfound.ă Riskă factorsă foră hemorrhagică strokeă ină nondiabeticăsubjectsăwere:ăfemaleă(RRă=ă3.412,ăORă=ă 5.556,ăp <<0.05*)ăandădiastolicăbloodăpressureă(RRă=ă 2.250,ăORă=ă3.375,ăpă=ă0.018*)ăandăischemicăstroke:ă maleă(RRă=ă1.63,ăORă=ă5.56,ă pă<0.05*)ăandăinsulină resistanceă determinedă byă HOMA-IRă indexă (RRă =ă 1.310,ă ORă =ă 2.86, pă =ă 0.032*).ă Weă calculatedă theă incidenceărateăofăstrokeăinătheădiabeticăpopulationăandă ină generală populationă onă theă overallă studyă periodă (2006–2008)ă(Fig.ă1).ă In conclusion ină bothă groups,ă theă ischemică strokeă hasă predominated,ă bută riskă factorsă wereă notă common,ă exceptingă foră diastolică bloodă pressure.ă Ină group 1,ă theă riskă factorsă foră theă ischaemică strokeă were:ăpostprandialăglycaemia,ăserumătotalăcholesterol,ă LDL-cholesterol,ătotalăchol./HDL-chol.ăratio,ăsystolică andădiastolicăbloodăpressureă andătriglycerides;ăinătheă group 2, theă riskă factorsă foră theă ischaemică strokeă were:ămaleăandăinsulinoresistanceă(byăHOMA-IR).ăă 5ă 253 Strokeăandădiabetesă Table IV Comparisonăbetweenăgroupă1ăandăgroupă2ăwithăhaemorrhagicăstrokeă ă Group Statisticsăă group N Mean 1ă 20ă 2ă 24ă 1ă Ageă Fastingăglycaemiaă Postprandialăglycaemiaă HbA1că Insulinemiaă PeptideăCă Cholesterolă Triglyceridesă LDL-Chol.ă HDL-Chol.ă SystolicăBPă DiastolicăBPă BMIă ă ă ă ă ă ă ă ă ă ă Fig.ă1.ă–ăComparisonăofăătwoăgroups.ă ă Std. Deviation Std. Error Mean 63.00ă 7.226ă 1.616ă 69.50ă 10.863ă 2.217ă 20ă 200.80ă 61.704ă 13.797ă 2ă 24ă 92.50ă 10.571ă 2.158ă 1ă 20ă 253.60ă 88.040ă 19.686ă 2ă 24ă 118.75ă 26.742ă 5.459ă 1ă 20ă 8.6400ă 1.36667ă .30560ă 2ă 24ă 5.4917ă .76039ă .15521ă 1ă 20ă 19.3700ă 16.21624ă 3.62606ă 2ă 24ă 10.4333ă 4.06733ă .83024ă 1ă 20ă 3.5800ă 2.01667ă .45094ă 2ă 24ă 3.1833ă .39746ă .08113ă 1ă 20ă 180.40ă 66.917ă 14.963ă 2ă 24ă 214.83ă 33.011ă 6.738ă 1ă 20ă 139.00ă 70.990ă 15.874ă 2ă 24ă 113.83ă 37.657ă 7.687ă 1ă 20ă 104.20ă 23.610ă 5.279ă 2ă 24ă 119.50ă 19.371ă 3.954ă 1ă 20ă 44.50ă 12.433ă 2.780ă 2ă 24ă 39.25ă 8.456ă 1.726ă 1ă 20ă 200.00ă 29.736ă 6.649ă 2ă 24ă 195.83ă 27.806ă 5.676ă 1ă 20ă 111.00ă 15.355ă 3.434ă 2ă 24ă 103.33ă 17.362ă 3.544ă 1ă 20ă 31.90ă 4.756ă 1.064ă 2ă 24ă 30.25ă 3.339ă .682ă 254ă ă DumitraăB lanăandăP.A.ăBabeşă Haemorrhagicăstrokeăwasămoreăfrequentăinăgroupă2,ăină personsăoveră70ăyearsăandăriskăfactorsăwereăfemaleăandă diastolică bloodă pressure.ă Subjectsă withă haemorrhagică strokeă hadă aă greateră BMI,ă thană thoseă withă ischaemică stroke.ăAverageăageăofănondiabeticăsubjectsăwithăstrokeă wasăsignificantlyăhigherăthanăofădiabeticăsubjects.ăTheă incidenceă rateă ofă stroke,ă calculatedă ină theă diabetică populationăină2006–2008,ăwasămuchăhigherăthanăinătheă generală populationă duringă theă sameă periodă (2006ă –ă 18.81‰ăvs.ă5.21‰;ă2007ă–ă27.15‰ăvs.ă6.9‰;ă2008ă–ă 16.64‰ăvs.ă6.85‰).ă ă Accidentul vascular cerebral este a treia cauză de deces, după bolile cardiace şi cancer. Diabetul zaharat este unul dintre factorii de risc pentru accidentul vascular cerebral şi incidenţa lui este în continuă creştere, ceea ce duce la o accentuare a riscului de accident vascular cerebral. Studiul de faţă şi-a propus să exploreze relaţia dintre diabetul zaharat, frecvenţa şi tipul de accident vascular cerebral; factorii de risc pentru acesta, atât la subiecţii cu diabet zaharat, cât şi la cei fără diabet. Material şi metode. Studiul a fost efectuat pe două loturi, care au inclus 228 subiecţi; lotul 1–136 pacienţi cu diabet şi accident vascular cerebral recent instalat şi lotul 2–92 pacienţi fără diabet zaharat şi accident vascular cerebral recent instalat, internaţi în Secţia de Neurologie a Spitalului Judeţean de Urgenţă Vâlcea în perioada 2006–2008. La fiecare lot am efectuat determinări biochimice pentru a obiectiva profilul glicemic, profilul lipidic, insulinorezistenţa; am stabilit valorile normale pentru lotul subiecţilor diabetici şi nediabetici, am stabilit tipul şi localizarea accidentului vascular cerebral după rezultatul tomografiei computerizate. Analiza statistică s-a efectuat cu ajutorul aplicaţiei SPSS 15 for Windows, adaptată prelucrărilor statisticii medicale. S-au calculat medii ale parametrilor, deviaţii standard, abaterea standard a mediei, intervale de frecvenţă, tabele de frecvenţă, teste de semnificaţie statistică prin metoda Student (testul t), şi calcularea scorului Hi pătrat. S-au calculat coeficienţi de corelaţie Pearson, care s-au interpretat folosind regulile lui Colton. Rezultate. În ambele loturi a predominat accidental vascular ischemic – 116 (85%) cazuri la subiecţii diabetici şi 68 (74%) cazuri la nediabetici, faţă de cel hemoragic – 20 (15%) cazuri în lotul 1 şi 24 (26%) cazuri în lotul 2. Sexul masculin a fost majoritar în lotul 2, în lotul 1 distribuţia fiind aproximativ egală. Vârsta medie a fost semnificativ mai mare în lotul 2 faţă de lotul 1(p=0,026*). În lotul 2 au predominat subiecţii din mediul rural, iar în lotul 1 cei din mediul urban. HTA a fost majoritară ca factor de risc preexistent în ambele loturi. Valorile lipidelor plasmatice au fost peste cele recomandate în ambele loturi. Insulinorezistenţa obiectivată prin indicele HOMA-IR a fost prezentă la 80% dintre subiecţii lotului 1 şi la 52% din subiecţii lotului 2. Incidenţa accidentului vascular cerebral a fost mult mai mare în populaţia diabetică decât în cea nediabetică în perioada 2006–2008. Concluzii. Accidentul vascular cerebral se instalează la vârste mai tinere la pacienţii cu diabet comparativ cu cei fără diabet, este mai frecvent la sexul masculin la nediabetici, insulinorezistenţa este un factor de risc prezent în ambele loturi, dar cu semnificaţie statistică doar în lotul 2. ă ă ă Corresponding author: DumitraăB lan,ăMDă EmergencyăCountyăHospitalăVâlceaă E-mail:ă[email protected]ă 6ă 7ă Strokeăandădiabetesă 255 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. WOLFă P.A.,ă D’AGOSTINOă R.B.,ă BELANGERă A.J.ă et al.,ă Probability of stroke: a risk profile from the Framingham study.ă Stroke,ă1991; 22:313–318.ă DEăFRONZOăR.A.,ăFERANNINIăE.,ăKEENăH.,ăZIMMETăP.,ăInternational Text Book of Diabetes Mellitus, thirdăed.ă2004,ăTHEă WORLDăHEALTHăREPORTă–ă2002.ă ABBOTă R.D.,ă DONAHUEă R.P.,ă MACMAHONă S.W.ă et al.,ă Diabetes and the risk of stroke: The Honolulu Heart Program. 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Ann.ăEpidemiol.,ă2005;ă10:ă81–87.ă INTERNATIONALăDIABETESăFEDERATION,ăGuideline for the management of post-meal glucose.ă2007.ă ReceivedăJuneă1,ă2009ă ă ă ă ă ă ă ă ă ă ă ă ă ă 256ă ă ă ă ă ă DumitraăB lanăandăP.A.ăBabeşă 8ă The Assessment of Subclinic Atherosclerosis Objected through IMT in Normal and Dyslipidemic Patients with Various Degrees of Glucose Tolerance MARIOARA DANIELA BRAICU1, CRISTINA PRI ULESCU2, D. ALEXANDRU3, MARIA MO A3 Clinical Emergency Hospital Craiova, Clinic of Diabetes, Nutrition & Metabolic Diseases, 1 CF Hospital, Craiova 2 University of Medicine and Pharmacy Craiova – Medical Informatics 3 University of Medicine and Pharmacy Craiova, Clinic of Diabetes, Nutrition & Metabolic Diseases Aim. The evaluation of atherosclerosis, by the measurement of intima media thickness (IMT) at the level of right common carotid arteries (CCA Right) and left (CCA Left), for normolipidemic patients (NL) and dislipidemic patients (DLP) with various degrees of alteration to glucose tolerance. Design and methods of research. We have analyzed a group of 120 NL patients and a group of 120 DLP patients. The test of orally provoked hyperglycemia was performed to assess the disturbance of tolerance to glucose. IMT was determined by the method of ultrasonography in model 2B at the level of right and left common carotid arteries, measured at 2 cm from the bifurcation of common carotids. Results. For patients with DLP the IMT medium was statistically higher than the group of NL patients. The normoglycemic normolipidemic patients (NG_NL) presented a lower IMT value than the normoglycemic dislipidemic patients (NG_DLP). For the group of DLP patients, IMT was statistically higher for patients with pre-diabetes (ALT) – impaired fasting glycaemia (IFG) and the impaired glucose tolerance (IGT) – compared to NG patients (with statistical signification). Conclusions. The presence of sub-clinic atherosclerosis objected through IMT was higher for DLP patients compared to NL patients demonstrating the role of dyslipidemia in the production of atherosclerosis. For patients with diabetes (DM), of both groups, the value of IMT was higher as compared to NG patients, demonstrating the influence of hyperglycemia in the production of atherosclerosis. Key words: diabetes, dyslipidemia, intima media thickness, atherosclerosis. The cardiovascular diseases are responsible for approximately 50–80% of the decease causes for DM persons, while the mortality of coronary causes is two times higher for DM males and 4–5 times higher for DM females compared to persons without DM [1]. The atherogene mechanisms are complex, plurifactorial and dynamic and they usually assume the synergic action of non-lipidic and lipidic factors. Hypercholesterolemia, hypertension, smoking and the association of these three factors of cardiovascular risk represent approximately 75% of the cardiovascular diseases worldwide [2]. The endothelial dysfunction plays a main role both in the initiation as well as in the progression of atherosclerosis, numerous studies showing its role in the pathogeny of atherosclerosis [3][4]. The endothelial cells are sensible to modifications of glucose molecules, fat acids, lipoproteins so that any injuries lead to the supra-production of ROS which determine inflammation and annul the protective role of endothelium [5]. ROM. J. INTERN. MED., 2009, 47, 3, 257–265 One of the main consequences of the increase of superoxide anion production, as a response to the action of different stimuli (hypercholesterolemia, HTA, smoking, etc.), is the inactivation of nitric oxide and the modification of the function of metaloproteins, important enzymes in the process of vascular remodeling [6][7]. Presently, it is considered that the chronic hyperglycemia has a complex and possibly secondary role in the accession of atherogenesis, a role also moderated by the effects of other cardiovascular risk factors [8]. The ways through which hyperglycemia leads to the appearance of atherogenesis lesions are not fully elucidated, the endothelial dysfunction representing the starting point. The full knowledge of the atherosclerotic process is highly important for early interventions in the prevention of atherosclerosis as well as in the case in which the process of reduction of progression rate was initiated [9]. 258 Marioara Daniela Braicu et al. MATERIAL AND METHODS In 2008 we studied two lots of patients (a lot of 120 patients with dyslipidemia and a control lot of 120 patients with normal values of sanguine lipids) from the registers of family doctors in Craiova, patients who had not been diagnosed with DZ and who did not follow a dyslipidemia treatment, according to inclusion criteria (age between 20 and 80 and a jeun glycemia under 126 mg/dL) and to exclusion criteria (diagnosed DM, pregnancy, gastrointestinal resections, acute stress: recent trauma, surgery, inflammatory processes, AVC, IMA; treatment with glucocorticoids, thiazide diuretics, synthesis estrogens, salicylates, thyroid hormones, IRC, renal transplant, chronic nephropathy, neoplasia, cirrhosis, LES, and psychical diseases). We recorded anamnestic data and anthropometric parameters: weight, height, waist and hip. We 2 calculated the waist/hip indexes, IMC, the indexes of insulin resistance (HOMA-IR). We determined the plasmatic values of glycemia using the enzymatic method, insulinemia through the electrochemiluminescence method on ELECSYS 1010, total cholesterol and triglycerides using enzymatic methods, HDL cholesterol through precipitation method and LDL cholesterol through Friedwald formula (when the value of triglycerides was Ţ 400 mg/dL). We made TTOG with 75 g of pulvis glucose, determining à jeun glycemia two hours after the administration of glucose. Blood pressure was also measured and EKG was made. The IMT was determined at the level of right and left common carotid arteries through Doppler ultrasonography in model 2B. Presentation of characteristics of NL groups (Table I) and DLP group (Table II). Table I Characteristics of NL group Characteristics Age[years] Waist [cm] Body indexes [kg/m2] Glycemia à Jeun [mg/dL] Glycemia in 2 hours [mg/dL] Total cholesterol [mg/dL] LDL cholesterol [mg/dL] HDL cholesterol [mg/dL] Triglycerides [mg/dL] Insulinemia [µU/ml] HOMA IR Systolic blood pressure [mmHg] Diastolic blood pressure [mmHg] Intima Media Thickness – ACC right [mm] Intima Media Thickness – ACC left [mm] Average 51.74 94.43 27.72 105.53 131.41 168.18 93.68 52.60 110.80 9.80 2.54 128.79 78.63 0.74 0.75 Standard deviation 13.84 12.89 4.94 17.18 43.77 17.54 17.43 7.77 29.56 5.04 1.37 19.78 14.39 0.09 0.09 Variation factor 26.74 13.65 17.83 16.28 33.31 10.43 18.61 14.77 26.68 51.39 54.04 15.36 18.30 11.68 11.79 Table II Characteristics of DLP group Characteristics Age[years] Waist [cm] Body indexes [kg/m2] Glycemia à Jeun [mg/dL] Glycemia in 2 hours [mg/dL] Total cholesterol [mg/dL] LDL cholesterol [mg/dL] HDL cholesterol [mg/dL] Triglycerides [mg/dL] Insulinemia [µU/ml] HOMA IR Systolic blood pressure [mmHg] Diastolic blood pressure [mmHg] Intima Media Thickness – ACC right [mm] Intima Media Thickness – ACC left [mm] Average 54.03 98.71 29.61 112.34 158.31 229.99 144.03 46.37 186.13 11.11 3.08 134.25 80.11 0.77 0.78 Standard deviation 10.84 10.95 4.97 18.86 67.71 48.05 49.73 13.24 74.10 4.66 1.35 17.78 12.18 0.08 0.08 Variation factor 20.06 11.09 16.79 16.78 42.77 20.89 34.53 28.54 39.81 41.97 43.71 13.25 15.21 9.90 9.91 3 259 Atherosclerosis in normal and dyslipidemic patients STATISTICAL ANALYSIS RESULTS For data processing there have been used the EPI2000 software, distributed by OMS, SPSS, specialized in scientific calculations, produced by SPSS. The recording of data about patients using the EXCEL program produced the initial data base from where the significant aspects of this study have been taken. Analyzing IMT at the level of ACC Right (Table III, Fig. 1), and at the level of ACC Left (Table IV, Fig. 2) for the NL patients lot and DLP patients lot and on the various degrees of tolerance to glucose (NG, IFG, IGT and DM), we have obtained the following results. Table III Analysis IMT to ACC Right to NL and DLP groups with sub-groups NG, IFG, IGT, DZ ACC R T T T Avg Std CV NL 0.74 0.09 11.68 NL_NG 0.71 0.08 11.36 NL_IFG 0.76 0.08 10.85 NL_IGT 0.76 0.09 11.20 NL_DZ 0.79 0.08 10.36 DLP 0.77 0.08 9.90 DLP_NG 0.77 0.08 10.17 DLP_IFG 0.74 0.08 10.36 DLP_IGT 0.77 0.07 9.44 DLP_DZ 0.80 0.07 9.11 Comparing ACC Right average between groups 0.80 0.74 0.77 0.71 0.77 0.76 0.74 0.76 0.77 0.79 0.80 Average 0.70 0.60 0.50 0.40 NL DLP 0.30 0.20 0.10 0.00 NG Lot IFG IGT DZ Sub-group Fig.1. – Comparing ACC Right average between groups. Table IV Analysis IMT to ACC Left to Nl and DLP groups with sub-groups NG, IFG, IGT, DZ Avg Std CV NL 0.75 0.09 11.79 NL_NG 0.72 0.08 11.59 NL_IFG 0.77 0.08 10.52 NL_IGT 0.78 0.09 11.06 NL_DZ 0.80 0.08 10.10 DLP 0.78 0.08 9.91 DLP_NG 0.78 0.08 10.24 DLP_IFG 0.75 0.07 9.85 DLP_IGT 0.78 0.08 9.75 Comparing ACC Left average between groups 0.90 Average ACC R T T T 0.80 0.75 0.78 0.72 0.78 0.77 0.75 0.78 0.78 0.80 0.80 0.70 0.60 0.50 0.40 NL 0.30 DLP 0.20 0.10 0.00 Lot NG IFG IGT DZ Sub-group Fig. 2. – Comparing ACC Left average between groups. DLP_DZ 0.80 0.07 9.13 260 Marioara Daniela Braicu et al. We have analyzed the value of IMT to the studied lots according to sex and we have obtained the following results: 4 1. For male IMT at the level ACC Right (Table V, Fig. 3) at the level ACC Left (Table VI, Fig. 4). Table V Analysis IMT to ACC Right to NL and DLP groups with sub-groups NG, IFG, IGT, DZ – male ACC R M M M Avg Std CV NL NL_NG NL_IFG NL_IGT NL_DZ DLP DLP_NG DLP_IFG DLP_IGT DLP_DZ 0.74 0.08 11.35 0.71 0.07 10.13 0.77 0.09 11.81 0.77 0.09 11.08 0.81 0.06 7.97 0.78 0.08 9.81 0.79 0.07 8.55 0.73 0.09 12.49 0.78 0.08 10.16 0.79 0.06 8.04 Comparing ACC Right average between groups, for male 0.90 Average 0.80 0.74 0.78 0.71 0.79 0.77 0.73 0.77 0.78 0.81 0.79 0.70 0.60 0.50 0.40 NL 0.30 DLP 0.20 0.10 0.00 NG Lot IFG IGT DZ Sub-group Fig. 3. – Comparing ACC Right average between groups, for male. Table VI Analysis IMT to ACC Left to NL and DLP groups with sub-groups NG, IFG, IGT, DZ – male M M M Avg Std CV NL NL_NG NL_IFG NL_IGT NL_DZ DLP DLP_NG DLP_IFG DLP_IGT DLP_DZ 0.75 0.09 11.42 0.71 0.07 10.30 0.77 0.09 11.20 0.79 0.09 10.83 0.81 0.08 9.31 0.79 0.08 9.76 0.80 0.07 8.41 0.74 0.09 11.52 0.79 0.08 10.51 0.80 0.07 8.42 Comparing ACC Left average between groups, for male 0.90 0.80 Average ACC R 0.75 0.79 0.71 0.80 0.77 0.74 0.79 0.79 0.81 0.80 0.70 0.60 0.50 0.40 NL 0.30 DLP 0.20 0.10 0.00 Lot NG IFG IGT DZ Sub-group Fig. 4. – Comparing ACC Left average between groups, for male. 5 261 Atherosclerosis in normal and dyslipidemic patients 2. For female IMT for the level ACC Right (Table VII, Fig. 5) for the level ACC Left (Table VIII, Fig. 6). Comparing the average ACC Right for patients in NL and DLP lots using Student test, we found pţ0.01, therefore a lower value than the limit of 0.05 (that is 5%), which shows us that there is a statistically significant difference between the average of the values ACC Right for the two groups (Table IX). Table VII Analysis IMT to ACC Right to NL and DLP groups with sub-groups NG, IFG, IGT, DZ – female ACC R NL NL_NG NL_IFG NL_IGT NL_DZ DLP DLP_NG DLP_IFG DLP_IGT DLP_DZ F F F 0.74 0.09 12.03 0.72 0.09 12.28 0.76 0.08 10.53 0.75 0.09 11.68 0.79 0.09 11.32 0.77 0.08 10.03 0.75 0.09 11.40 0.76 0.06 7.87 0.76 0.06 8.57 0.80 0.08 10.06 Comparing ACC Right average between groups, for female Average 0.90 0.80 0.74 0.77 0.72 0.75 0.76 0.76 0.75 0.79 0.80 0.76 0.70 0.60 0.50 0.40 NL 0.30 DLP 0.20 0.10 0.00 NG Lot IFG IGT DZ Sub-group Fig. 5. – Comparing ACC Right average between groups, for female. Table VIII Analysis IMT to ACC Left to NL and DLP groups with sub-groups NG, IFG, IGT, DZ – female ACC R NL NL_NG NL_IFG NL_IGT NL_DZ DLP DLP_NG DLP_IFG DLP_IGT DLP_DZ F F F 0.75 0.09 12.17 0.72 0.09 12.55 0.77 0.08 10.48 0.77 0.09 11.68 0.80 0.09 10.78 0.77 0.08 10.06 0.75 0.09 11.37 0.76 0.06 8.13 0.76 0.07 8.97 0.81 0.08 9.91 Comparing ACC Left average between groups, for female 0.90 Average 0.80 0.75 0.77 0.72 0.75 0.77 0.76 0.77 0.76 0.80 0.81 0.70 0.60 0.50 0.40 NL 0.30 DLP 0.20 0.10 0.00 Lot NG IFG IGT DZ Sub-group Fig. 6. – Comparing ACC Left average between groups, for female. 262 Marioara Daniela Braicu et al. Comparing NL and DLP groups on various degrees of tolerance to glucose, we find for NG patients’ lots: Comparing the average ACC Right for patients in groups NL_NG and DLP_NG using Student Test, we found p~ţ0, so a lower value than the limit of 0.05 (that is 5%), which shows that there is a statistically significant difference between the average of the values ACC Right for the two groups. Analyzing ACC Right for IFG patients’ groups with IGT and DZ it is observed that no matter the degree of glycemia alteration, the presence or not of DLP does not influence significantly IMT (Table X). Table IX Comparing IMT to NL and DLP groups Average Dispersion Standard deviation Variation factor NL ACC Right 0.74 0.01 0.09 P(TŢţt) two-tail 11.68 6 DLP ACC Right 0.77 0.01 0.08 0.01 9.90 For ACC Left we found pţ0.01, showing the existence of a statistically significant difference in favour of DLP. Table X Comparing IMT to NL_NG and DLP_NG; NL_IFG and DLP_IFG; NL_IGT and DLP_IGT; NL_DZ and DLP_DZ groups Average Dispersion Standard deviation Variation factor NL_NG ACC Right 0.71 0.01 DLP_NG ACC Right 0.77 0.01 NL_IFG ACC Right 0.76 0.01 DLP_IFG ACC Right 0.74 0.01 NL_IGT ACC Right 0.76 0.01 DLP_IGT ACC Right 0.77 0.01 NL_DZ ACC Right 0.79 0.01 DLP_DZ ACC Right 0.80 0.01 0.08 0.08 0.08 0.08 0.09 0.07 0.08 0.07 P(TŢţt) Two-tail 00 P(TŢţt) Two-tail 0.41 P(TŢţt) Two-tail 0.83 P(TŢţt) Two-tail 0.78 11.36 10.17 10.36 11.20 9.44 10.36 9.11 For ACC Left we found p~ţ0, showing the existence of a statistically significant difference in favour of DLP (for NL_NG and DLP_NG). Analyzing ACC Left, for groups of patients with IFG, IGT and DM, we observe that no matter the degree of glycemia alteration, the presence or not of DLP does not influence IMT significantly. Studying the average value of IMT of ACC Right for the NL group of patients and comparing the average between the sub-groups of patients NG, IFG, IGT and DZ using Student test, we have obtained pţ0.02, respectively pţ0.01 and pţ0.01 lower values than the limit of 0.05 (that is 5%), which shows a statistically significant difference between the average of ACC Right for the groups (Table XI). Table XI Comparing IMT to NL_NG and NL_ IFG; NL_NG and NL_ IGT; NL_NG and NL_ DZ groups NL_NG ACC Right 0.71 NL_IFG ACC Right 0.76 NL_NG ACC Right 0.71 NL_IGT ACC Right 0.76 NL_NG ACC Right 0.71 NL_DZ ACC Right 0.79 Dispersion 0.01 0.01 0.01 0.01 0.01 0.01 Standard deviation 0.08 0.08 0.08 0.09 0.08 0.08 P(TŢţt) Two-tail 11.36 0.02 P(TŢţt) Two-tail 11.36 0.01 P(TŢţt) Two-tail 11.36 0.01 Average Variation factor 10.85 For ACC Left, the value p~ţ0 with statistical significance in favour of IFG, IGT and DZ. Comparing the average ACC Right for patients in groups NL_IFG and NL_IGT, in groups NL_IFG with NL_DM, from groups NL-IGT with NL-DM, 11.20 10.36 there were not statistically significant modifications between the groups compared (Table XII). Comparing the average ACC Left for patients in groups NL_IFG with NL_IGT, from groups NL_IFG with NL_DM, from groups NL_IGT with 7 263 Atherosclerosis in normal and dyslipidemic patients NL_DM, there were not statistically significant modifications between the groups compared. Comparing the average ACC Right for patients in groups NL_DM and NL_ALT using Student test, we found pţ0.31, so a higher value than the limit of 0.05 (that is 5%), which shows there is not a statistically significant difference between the average of values ACC Right for the two groups (Table XIII). Table XII Comparing IMT to NL_IFG and NL_ IGT; NL_IFG and NL_ DZ; NL_IGT and NL_ DZ groups Average Dispersion Standard deviation Variation factor NL_NG ACC Right 0.76 0.01 0.08 P(TŢţt) two-tail 10.85 NL_IFG ACC Right 0.76 0.01 0.09 NL_NG ACC Right 0.76 0.01 0.08 P(TŢţt) two-tail 10.85 0.99 11.20 NL_IGT ACC Right 0.79 0.01 0.08 0.38 10.36 NL_NG ACC Right 0.76 0.01 0.09 P(TŢţt) two-tail 11.20 NL_DZ ACC Right 0.79 0.01 0.08 0.34 10.36 Table XIII Comparing IMT to NL_ DZ and NL_ALT groups Average Dispersion Standard deviation Variation factor NL_DZ ACC Right 0.79 0.01 0.08 P(TŢţt) two-tail 10.36 Comparing the average ACC Left for patients in lots NL_DM and NL_ALT, there were not statistically significant modifications between the groups compared. Studying the value IMT of ACC Right to the group of DLP patients and comparing the average between the sub-groups NG, with IFG, with IGT and with DZ, we obtained the following results: • NL_ALT ACC Right 0.76 0.01 0.08 0.31 10.95 Comparing the average ACC Right for patients in groups DLP_NG with DLP_IFG, from groups DLP_NG with DLP_IGT, from groups DLP_NG with DLP_DM (Table XIV), from groups DLP_IFG with DLP_IGT, from groups DLP_IGT with DLP_DZ (Table XV), there were not statistically significant modifications between the groups compared. Table XIV Comparing IMT to DLP_NG and DLP_IFG; DLP_NG and DLP_IGT; DLP_NG and DLP_DZ groups Average Dispersion Standard deviation Variation factor DLP_NG ACC Right 0.77 0.01 0.08 P(TŢţt) two-tail 10.17 DLP_IFG ACC Right 0.74 0.01 0.08 0.20 10.36 DLP_NG ACC Right 0.77 0.01 0.08 P(TŢţt) two-tail 10.17 DLP_IGT ACC Right 0.77 0.01 0.07 0.89 9.44 DLP_NG ACC Right 0.77 0.01 0.08 P(TŢţt) two-tail 10.17 Table XV Comparing IMT to DLP_IFG and DLP_IGT; DLP_IGT and DLP_DZ groups Average Dispersion Standard deviation Variation factor DLP_IFG ACC Right 0.74 0.01 0.08 P(TŢţt) two-tail 10.36 DLP_IGT ACC Right 0.77 0.01 0.07 0.24 9.44 DLP_IGT ACC Right 0.77 0.01 0.07 P(TŢţt) two-tail 9.44 DLP_DZ ACC Right 0.80 0.01 0.07 0.11 9.11 DLP_DZ ACC Right 0.80 0.01 0.07 0.14 9.11 264 Marioara Daniela Braicu et al. 8 groups DLP_DM with DLP_ALT (Table XVII) through Student test, we found pţ0.01, respectively pţ0.02, so a lower value than the limit of 0.05 (that is 5%), which shows there is a statistically significant difference between the groups. There were not any statistically significant modifications even when we analyzed the average ACC Left. • Comparing the average ACC Right for patients in groups DLP_IFG with DLP_DM (Table XVI) and Table XVI Comparing IMT to DLP_IFG and DLP_DZ groups Average Dispersion Standard deviation Variation factor DLP_IFG ACC Right 0.74 0.01 0.08 P(TŢţt) two-tail 10.36 DLP_DZ ACC Right 0.80 0.01 0.07 0.01 9.11 Table XVII Comparing IMT to DLP_DZ and DLP_ALT GROUPS Average Dispersion Standard deviation Variation factor DLP_DZ ACC Right 0.80 0.01 0.07 P(TŢţt) two-tail 9.11 Comparing the average ACC Left for patients in groups DLP_IFG with DLP_DM and groups DLP_DM with DLP_ALT we found pţ0.01, respectively pţ0.03, with a statistically significant difference between the groups. CONCLUSIONS Analyzing the average value of IMT at the level of ACC Right and ACC Left, it has been observed that there is a statistically significant difference of the NL group of patients compared to DLP group. Studying the average of IMT based on sex, it has been observed that at the level of both ACC the value is higher for males than for females, which confirms the literature data. Comparing the average value of IMT for the two groups of patients: NL and DLP, according to the tolerance degree to glucose, it has been noticed that only for NG patients there is an IMT value statistically higher than for DLP patients, which underlines the importance of DLP in the production of atherogenesis; for patients with IFG, with IGT and DM there have not been statistically significant differences between groups. DLP_ALT ACC Right 0.76 0.01 0.07 0.02 9.84 Studying the average value of IMT to the NL lot of patients on tolerance degrees on glucose, it has been noted that there is a statistically significant difference between the NL_NG group compared to the patients NL-IFG, NL_IGT, NL_DM, at the level of both ACC, this thing demonstrating the contribution of hyperglycemia in the development of atherosclerosis. There has not been a statistically significant correlation through the comparison of sub-groups IGT_IFG, IFG_DM, IGT_DM to NL patients, results also sustained by literature data, which show that the lower the atherogene risk is, the lower the glycemia values are. For DLP group of patients, comparing the DLP_NG patients with DLP_IFG patients, DLP_IGT, DLP_DM, there have not been statistically significant differences, at the level of the two carotid arteries, between the compared groups. Comparing the subgroup of patients DLP-IFG and DLP_DM patients, both at the level of ACC Right as well as at the level of ACC Left, there have been statistically significant differences. Comparing the DLP_DM group of patients with the group of patients DLP_ALT and with the group DLP_IFG, at the level of both carotids there have been higher statistically significant differences, in favour of DM, demonstrating the contribution of glycemia alteration degree in the production of atherogenesis. 9 Atherosclerosis in normal and dyslipidemic patients 265 Obiectiv. Evaluarea prezenţei aterosclerozei, prin măsurarea grosimii intimă medie (IMT) la nivelul arterei carotide comune dreaptă (ACC Dr) şi stângă (ACC St), la pacienţi normolipidici (NL) şi dislipidemici (DLP) cu grade variate ale alterării toleranţei la glucoză. Designul şi metodele cercetării. Am analizat un lot de 120 pacienţi NL şi un lot de 120 pacienţi cu DLP. A fost efectuat testul de hiperglicemie provocată oral pentru evaluarea perturbărilor toleranţei la glucoză. IMT a fost determinată prin metoda ultrasonografiei în model 2B la nivelul arterei carotide comune drepte şi stângi, măsurat la 2 cm de bifurcaţia carotidelor comune. Rezultate. La pacienţii DLP, media IMT a fost statistic semnificativ mai mare comparativ cu lotul de pacienţi NL. Pacienţii normoglicemici normolipidemici (NG_NL) au prezentat o valoare a IMT mai mică decât pacienţii normoglicemici dislipidemici (NG_DLP). La lotul de pacienţi DLP, IMT a fost statistic semnificativ mai mare la pacienţii ce prezentau prediabet (ALT) – alterarea glicemiei à jeun (IFG) şi scăderea toleranţei la glucoză (IGT) – comparativ cu pacienţii NG. Pacienţii cu diabet din lotul DLP au prezentat o IMT mai mare comparativ cu pacienţii NG (cu semnificaţie statistică). Concluzii. Prezenţa aterosclerozei subclinice obiectivată prin IMT a fost mai mare la pacienţii DLP faţă de pacienţii NL, demonstrând rolul dislipidemiei în producerea aterosclerozei. La pacienţii cu diabet (DZ), din ambele loturi, valoarea IMT a fost mai mare comparativ cu pacienţii NG, demonstrând influenţa hiperglicemiei în producerea aterosclerozei. Corresponding author: Prof. Maria Mo a, MD, PhD Clinical Centre of Diabetes, Nutrition & Metabolic Diseases 1, Tabaci Str., Craiova, Romania E-mail: [email protected] REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IDF e – Atlas, 2007, Available at http://www.eatlas.Idf.org/ American Heart Association. Statistical Fact Sheet – Populations. International Disease Statistics. Dallas, Tex: American Heart Association; 2003. Available online at: http://www.americanheart.org/presenter.jhtml?identifierţ3001001. LUSIS A., Atherosclerosis. Nature, 2000; 407:233–241. WIDLANSKY M.E., GOKC N., KEANEY J.F. JR. et al., The clinical implications of endothelial dysfunction. J. Am. Col. Cardiol., 2003; 42:1149–1160. DEFRONZO R.A., FERRANNINI E., KEEN H., ZIMMET P., International Textbook of Diabetes Mellitus, Third Edition, Volume Two, 2004. JACKSON C., Matrix metalloproteinases and angiogenesis. Curr. Oprin. Nephrol. Hypertens., 2002; 11: 295–299. WASSMANN S., NICKENIG G., Improvement of endothelial function by HMG-CoA reductase inhibitors, Drug News Perspect, 2002; 15 (2): 85–92. PICKUP J.C., WILLIAMS G., Text book of Diabetes, third ed., 2003. BRUNZELL J.D., MICHAEL DAVIDSON M., FURBERG C.D., GOLDBERG R.B, HOWARD B.V. et al., Lipoprotein Management in Patients with Cardiometabolic Risk. Consensus statement from the American Diabetes Association and the American College of Cardiology Foundation. Diabetes Care, 2008; 31: 811–822. Received May 25, 2009 266 Marioara Daniela Braicu et al. 10 Study of Antiphospholipid Antibodies in Type 2 Diabetes Mellitus with and without Diabetic Retinopathy INIMIOARA MIHAELA COJOCARU1, M. COJOCARU2, ADINA NICOLETA POPESCU3, L. POPESCU4, R. TĂNĂSESCU1 1 “Carol Davila” University of Medicine and Pharmacy, Department of Neurology, “Colentina” Clinical Hospital 2 “Titu Maiorescu” University, Faculty of Medicine, Department of Physiology 3 Department of Neurology, “Colentina” Clinical Hospital 4 Clinical Hospital of Ophthalmologic Emergencies Antiphospholipid antibodies (aPL) are considered to be contributory factors in the development of thrombotic events. The objective of the study was to determine if aPL are involved in the pathogenesis of diabetic retinopathy (DR) in patients with type 2 diabetes mellitus. IgG anticardiolipin antibodies (IgG aCL), lupus anticoagulant (LA), and anti-IgG β2-glycoprotein I antibodies (anti-β2-GPI) were prospectively tested in 34 patients with DR (group 1), 20 males and 14 females, range of age 52–79 years, mean age 57±4.6 years, duration of diabetes 8–15 years, as compared to 29 type 2 diabetic patients without DR (group 2), 19 males and 10 females, range of age 54–77 years, mean age 58±4.8 years, duration of diabetes 10–13 years, and to 31 controls matched for age and sex (group 3). IgG aCL and anti-β2-GPI were detected by enzyme-linked immunosorbent assay (ELISA) and LA was detected by activated partial thromboplastin time, kaolin clotting time, dilute Russell’s viper venom time, dilute prothrombin time. Comparison between patients and controls and patients group were expressed as relative risk with its 95% confidence interval (RR [95%/CI], where a lower limit>1.0 was considered significant. All values were determined by Fischer’s exact test. A value of p<0.05 was considered statistically significant. The incidence of IgG aCL positive (low 4–15 GPL U IgG aCL titers) in group 1 was 21/34 (62%) vs. 12/29 (41%) in group 2 (RR 1.460 95% CI [0.9052 to 2.382]), p=0.1330. The incidence of LA positive in group 1 was 27/34 (79%) vs. 8/29 (28%) in group 2 (RR 3.086 95% CI [1.584 to 6.010]), p<0.0001. The incidence of anti-IgG β2-GPI positive in group 1 was 29/34 (85%) vs. 6/29 (21%) in group 2 (RR 4.640 95% CI [2.067–10.418]), p<0.0001. The results suggest the possible participation of anti-β2-GPI and LA in the pathogenesis of DR, shifting the hemostatic balance toward a pro-thrombotic state increasing the risk of developing thrombosis. Key words: Antiphospholipid antibodies (aPL), diabetic retinopathy (DR), type 2 diabetes mellitus. Diabetic retinopathy (DR) is a major complication in patients with diabetes mellitus and a cause of disability, its prevalence being related to the duration of diabetes and the glycemic control. Diabetic retinopathy is a multifactorial process in which genetic, metabolic, haemostatic and growth factors play an important role. In the pathogenesis of diabetic microangiopathy the immunological mechanism might play a role. Antiphospholipid antibodies (aPL) are considered to be contributory factors in the development of thrombotic events [1]. APL have been linked to vascular damage in diabetes mellitus (DM). The objective of the study was to determine if aPL are involved in the pathogenesis of DR in patients with type 2 DM. ROM. J. INTERN. MED., 2009, 47, 3, 267–271 MATERIAL AND METHODS IgG anti-cardiolipin antibodies (IgG aCL), lupus anticoagulant (LA) and anti-IgG β2-glycoprotein I antibodies (anti-β2-GPI) were prospectively tested in 34 patients with DR (group 1), 20 males and 14 females, ranges of age 52–79 years, mean age 57±4.6 years, duration of diabetes 8–23 years as compared to 29 type 2 diabetic patients without DR (group 2) 19 males and 10 females range of age 43– 78 years, mean age 58±4.8 years, duration of diabetes 10–13 years, and to 31 controls matched for age and sex. The type 2 DM patients were classified according to National Diabetes data group criteria [2]. Diabetic retinopathy was assessed by 7 field stereo fundus photograph and was classified according to the ETDRS-Airlie House Classification [3]. Inimioara Mihaela Cojocaru et al. 268 Exclusion criteria were: autoimmune disorders, chronic hepatitis, poor glycemic control (HbA1c>7%), borderline hypertension (>140/90), hypertriglyceridemia (>1.9 mol/L) or hypercholesterolemia (>5.6 mol/L). Both diabetics and controls were non-smokers and had not taken drugs which could affect haemostasis for at least 4 weeks before the study. Informed consent was obtained from all subjects after explanation of the nature of the study and the investigation was approved by the bioethics committee of the hospital. IgG aCL and anti-IgG β2-GPI were detected by enzyme-linked immunosorbent assay (ELISA). The concentration of aCL was measured in GPL intenational units (a unit being equivalent to the binding activity of 1 mg of aCL/mL). Positivity of aCL was defined as titers of IgG aCL higher than 15 GPL units. LA was detected by activated partial thromboplastin time, kaolin clotting time, dilute Russell’s viper venom time, dilute prothrombin time. The concentration of anti-IgG-β2-GPI was measured in U/mL (normal cut off 20 U/mL). Comparison between patients and controls and patients group were expressed as relative risk with its 95% confidence interval (RR [95%/CI]), where a lower limit>1.0 was considered significant. All values were determined by Fischer’s exact test. A value of p<0.05 was considered statistically significant. RESULTS The incidence of IgG aCL positive (low 4–15 GPL U IgG aCL titers) in group 1 was 21/34 (62%) vs. 12/29 (41%) in group 2 (RR 1.460 95% CI [0.9052 to 2.382]), p=0.1330. The incidence of LA positive in group 1 was 27/34 (79%) vs. 8/29 (28%) in group 2 (RR 3.086 95% CI [1.584 to 6.010]), p<0.0001. The incidence of anti-IgG β2-GPI positive in group 1 was 29/34 (85%) vs. 6/29 (21%) in group 2 (RR 4.640 95% CI [2.067–10.418]), p<0.0001. DISCUSSION It has been hypothesized that aPL directed against endothelial antigens could be responsible for initiating vascular injury. Vascular damage and disturbed endothelial function occur early in the course of diabetic 2 microangiopathy converting the endothelium from a thromboresistant to a thrombogenic surface [4][5]. It has been suggested that immunological mechanisms have a role in the pathogenesis of diabetic microangiopathy via immune complex deposition [6]. Antibodies directed against endothelial antigens could be responsible for initiating vascular injury, but could also be marker of endothelial dysfunction. Potential synergism between generation of autoantibodies, haemostatic alterations and endothelial stress has been also suggested. Moderate high aCL titers must be exceptional in patients with type 2 DM, low aCL titers may occur in patients with type 2 DM, these low titers do not seem to be associated with complicated DM, cardiovascular disease, nephropathy or retinopathy [7]. Some authors find low IgG aCL titers in about one third of their patients [7]. Other study has described that low (5– 20 GPL U) IgG aCL titers were significantly more frequent in patients with diabetes with and without cardiovascular disease than in controls [8]. In a series of 205 patients with type 1 and 2 DM, no patient had moderate-high (>20 GPL U) IgG aCL titers and only one had moderate high IgM aCL titers [8]. In a series of 46 patients with type 2 DM, no case had an IgG aCL titer higher than 20 GPL units [9]. On the contrary, in a series of 21 patients with type 2 DM, a IgG aCL titer equal or higher than 15 GPL U was present in 9.5% of cases [10]. The immunosenescence theory suggests that immune dysfunction with aging is associated with increased autoantibodies production; these autoantibodies may be “innocent bystanders” or contribute to the pathogenesis of diseases such as atherosclerosis [11]. The patients of authors’ series were mostly elderly, and the frequency of low aCL titers might be explained by the immunosenescence theory. It has been reported a significantly higher frequency of aPL (IgG or IgM aCL and/or LA) in patients with type 1 and 2 diabetes with macroangiopathy than in patients with uncomplicated diabetes or controls, the results suggesting a potential role of PL-binding antibodies in the progression of vascular complication in DM [10]. These authors did not find statistical differences in the frequencies of a low IgM or IgG aCL titer in patients with and without nephropathy or with and without retinopathy. Other studies describe no association between aCL and retinopathy and nephropathy [8][9]. 3 Antiphospholipid antibodies in type 2 diabetes mellitus Vascular damage and disturbed endothelial function occur early in the course of diabetic microangiopathy converting the endothelium from a thromboresistant to thrombogenic surface [4][5]. The immunological mechanism could have a role in the pathogenesis of diabetic microangiopathy via immune complexes deposition [6]. Antibodies directed against endothelial antigens could be responsible for initiating vascular-injury, but could also be a marker of endothelial dysfunction. It has been suggested a potential synergism between generation of autoantibodies, haemostatic alterations and endothelial stress. LA is though to be involved in this endothelial alteration and is frequently associated with diabetic vascular complications (overt nephropathy or macroangiopathy) and retinal occlusive vasculopathies [10]. The presence of LA was detected in 32.2% diabetic patients, aCL was found in 80% of LA positive and only in 9% of LA negative patients [12]. Clinically manifest microangiopathy (retinopathy and/or nephropathy) was found in 60% LA positive and in 18% LA negative diabetics [12]. A high prevalence of proliferative DR was observed in LA positive patients (66.6%). The clinical association between LA positivity and thrombotic events is thought to be due to an LA induced endothelial cell dysfunction; in DM a similar dysfunction of the coagulopathy and anticoagulant pathways, as a result of endothelial damage, has been described [13–16]. Some authors had observed decreased APC (activated protein C) concentrations and increased F1+2 (prothrombin degradation products) plasma levels in LA positive patients, as evident expression of prothrombotic condition and endothelial dysfunction [12]. LA positivity might be considered an additional risk factor in the pathogenesis of microangiopathy (retinopathy and/or nephropathy) in DM patients, representing an intersection point between immune and haemostatic alteration [17]. 269 The presence of aPL (81%) [18] appears to be greater than the prevalence of aPL reported in patients with other autoimmune disorders [19–22]. In addition, studies of such autoimmune diseases have revealed that aPL are not always directed against PL alone, for purified IgG from certain aCL positive patients does not react with CL unless β2-GPI or prothrombin are present [23][24]; the β2-GPI-phospholipid complex is a novel epitope that stimulates the immune system to produce aPL [23–25]. The same type of novel epitopes occur for the kininogens when they are combined with phosphatidylethanolamine (PE) [26]. Experimental evidence suggests that hyperglycemia triggers a cascade of biochemical events that leads to vascular dysfunction and early structural changes of vessels [27][28]. This is confirmed by increased concentrations of von Willebrand factor [29], reduced endotheliumderived relaxing factor and prostacyclin [28], decreased concentrations of tissue plasminogen activator [13], increased plasma concentrations of fibrinogen and activated factor VII [28], and down-regulation of natural anticoagulant pathways [30][31]. The net effect of these changes is the conversion of endothelium from a thromboresistant to a thrombogenic surface [30][31]. Detection of aPl in patients with DR may help recommend prophylactic treatment [32]. CONCLUSION The results of our study suggest the possible participation of anti-β2-GPI and LA in the pathogenesis of DR, shifting the haemostatic balance toward a pro-thrombotic state increasing the risk of developing thrombosis. Se consideră că anticorpii antifosfolipidici sunt factori care contribuie la producerea fenomenelor trombotice. Obiectivul acestei cercetări a fost determinarea anticorpilor antifosfolipidici posibil implicaţi în patogenia retinopatiei diabetice la pacienţi cu diabet tip 2. Anticorpii anticardiolipinici (IgG aCL), anticoagulantul lupic (LA) şi anti IgG-β2-glicoproteina I (anti-β2-GPI) au fost testaţi prospectiv la 34 pacienţi cu retinopatie diabetică (grupul 1), 20 bărbaţi şi 14 femei cu vârste între 52–79 ani, vârsta medie 57±4,6 ani, durata diabetului 8–15 ani, comparativ cu 29 pacienţi cu diabet tip 2 fără retinopatie diabetică (grupul 2), 19 bărbaţi şi 10 femei cu vârsta între 54–77 ani, vârsta medie 58±4,8 ani, durata diabetului 10–13 270 Inimioara Mihaela Cojocaru et al. 4 ani, şi cu 31 martori asemănători ca vârstă şi sex (grupul 3). IgG aCL şi anti-IgG β2-GPI au fost determinaţi folosind tehnica ELISA, iar LA a fost determinat folosind timpul de tromboplastină parţial activată, timpul de coagulare cu caolin, timpul Russel cu venin de viperă diluat, timpul cu protrombină. Comparaţia între pacienţi şi martori şi grupurile de pacienţi a fost exprimată ca risc relativ cu intervalul de confidenţă 95% (RR[95% CI] la care o limită mai mică de >1.0 a fost considerată semnificativă. Toate valorile au fost determinate prin testul de exactitate Fischer. O valoare p<0.05 a fost considerată semnificativă statistic. Incidenţa pozitivităţii IgG aCL (titruri reduse de 4–15 GPL U IgG aCL) a fost la grupul 1 de 21/34 (62%) vs. 12/29 (41%) la grupul 2 (RR 1.460 95%/CI [0.9052– 2.382]), p=0.1330. Incidenţa pozitivităţii LA la grupul 1 a fost de 27/34 (79%) vs. 8/29 (28%) la grupul 2 (RR 3.086 95% CI [1.584–6.010]), p<0.0001. Incidenţa pozitivităţii anti-IgG β2-GPI la grupul 1 a fost de 9/34 (85%) vs. 6/29 (21%) la grupul 2 (RR 4.640 95% CI [2.067–10.418]), p<0.0001. În concluzie, rezultatele indică participarea posiblă a anticorpilor anti-β2-GPI şi LA la patogenia retinopatiei diabetice. Corresponding author: Inimioara Mihaela Cojocaru MD, PhD, Senior lecturer “Colentina” Clinical Hospital, Clinic of Neurology 19–21, Şos. Ştefan cel Mare 020125, Bucharest Romania E-mail: [email protected] REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. BERTOLACCINI L.M., HUGES G.R., Antiphospholipid antibody testing: which are most useful for diagnosis? Rheum. Dis. Clin. North Am., 2006; 32: 455–63. NATIONAL DIABETES DATA GROUP, Classification of diabetes mellitus and other categories of glucose intolerance. Diabetes, 1979; 28: 1039–57. DIABETIC RETINOPATHY STUDY RESEARCH GROUP, Report 7. A modification of the Arilie House classification of diabetic retinopathy. Invest. Ophthalmol. Vis. Sci., 1981; 21: 210–26. HAEFLIGER I.O., MEYER P., FLAMMER J. et al., The vascular endothelium as a regulator of the ocular circulation: a new concept in ophthalmology? Surv. Ophthalmol., 1994; 39: 123–32. KOHNER B.M., PATEL V., RASSMAN S.M.B., Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy. Diabetes, 1995; 44: 603–7. AHMED E., NITYANAND S., MUSTAFA A. et al., Anti-cardiolipin antibodies and circulating immune complexes in type 1 diabetes mellitus: increased prevalence and relation to vascular complications. Clin. Exp. Immunol., 1999; 115(2): 255–9. CALVO-ROMERO J.M., LIMA-RODRIGUEZ M.L., Anticardiolipin antibodies in patients with type 2 diabetes mellitus. Clin. Med. Res., 2009; Feb. 26 (published online). HENDRA T.J., BAGULEY E., HARRIS E.N. et al., Anticardiolipin antibody levels in diabetic subjects with and without coronary artery disease. Postgrad. Med. J., 1989; 65: 140–3. TARKUN J., HACIHANEFIOGLU A., TARKUN P. et al., Anticardiolipin and anti-beta 2 glycoprotein I antibody concentrations in patients with type 2 diabetes mellitus. Diabetes Res. Clin. Pract., 2005; 68: 181–7. GALTIER-DEREURE F., BIRON C., VIES M. et al., Vascular complications of diabetes mellitus: what role for phospholipidbinding antibodies? Lupus, 1998; 7: 469–74. LING K.L., GABRIEL S.E., Autoantibodies; innocent bystander or key player in immunosenescence and atherosclerosis. J. Rheumatol., 2007; 34: 1203–7. GIUSTI C., SCHIAFFINI R., BOSCO D. et al., Lupus anticoagulant positivity in insulin dependent diabetic patients: an additional risk factor in the pathogenesis of diabetic retinopathy. Br. J. Ophthalmol., 2000; 84: 531–3. WALMSLEY D., HAMPTON K.K., GRANT P.F., Contrasting fibrinolytic responses in type 1 (insulin-dependent) and type 2 (non-insulin-dependent diabetes). Diabetic Med., 1991; 8: 954–9. BRENNER B.M., TROY J.L., BALLERMANN B.J., Endothelium-dependent vascular responses: mediator and mechanism. J. Clin. Invest., 1989; 84: 1373–8. GARCIA-FRADE L.J., DE LA CALLE H., TORADO M.C. et al., Hypofibrinolysis associated with vasculopathy in noninsulin-dependent diabetes mellitus. Thromb. Res., 1990; 59: 51–9. MANSFIELD M.W., GRANT P.J., Fibrinolysis and diabetic retinopathy in NIDDM. Diabetes Care, 1995; 18: 1577–89. 5 Antiphospholipid antibodies in type 2 diabetes mellitus 271 17. CARR M.E., Diabetes mellitus: a hypercoagulate state. J. Diabetes Complications, 2001; 15: 44–54. 18. GARGIULO P., GOLDBERG J., ROMANI B. et al., Qualitative and quantitative studies of autoantibodies to phospholipids in diabetes mellitus. Clin. Exp. Immunol., 1999; 118: 30–4. 19. FIELDS R.A., TOUBLOCH H., SCOULES R.P. et al., The prevalence of anticardiolipin antibodies in a healthy elderly population and in association with antinuclear antibodies. J. Rheumatol., 1989; 16: 623–5. 20. LECERF V., ATHENE-GELAS M., LAUZIAN C. et al., Antiphospholipid antibodies in atherosclerosis. Am. J. Med., 1992; 92: 576. 21. PALOMO I.J., MUJICA V.E., ALARCON M.L. et al., Prevalence of antiphospholipid antibodies is not different in Chilean diabetic patients and normal individuals. J. Diabetes Complications, 2005; 19: 133–7. 22. GIN H., VERGNOT V., DIAKOU V. et al., Antiphospholipid antibodies in diabetes mellitus. Clinical Chemistry and Laboratory Medicine, 2002; 40: 604–8. 23. HUGHES G.R.V., Antiphospholipid syndrome: ten years on Lancet, 1993; 343: 341–4. 24. McINTYRE J.A, WOGENKNECHT D.R., SUGI T., Phospholipid binding plasma proteins required for antiphospholipid antibody detection. An overview, Am. J. Reprod. Immunol., 1997; 37: 101–10. 25. MATSUURA E., IGARASHI I., FUJIMOTO M. et al., Heterogeneity of anticardiolipin antibodies defined by anticardiolipin cofactor. J. Immunol., 1992; 248: 3885–91. 26. SAGI T., McINTYRE J.A., Phosphatidylethanolamine induces specific conformational changes in the kininogens recognizable by antiphosphatidylethanolamine antibodies. Thromb. Haemost., 1996; 76: 354–60. 27. ANDREANI D., The labyrinth of diabetic vascular disease: crossroads and waysout. Diab. Nutr. Metab., 1995; 8: 54–64. 28. CERIELLO A., Coagulation activation in diabetes mellitus: the role of hyperglycemia and therapeutic prospects. Diabetologia, 1993; 36: 1119–25. 29. STEHOUER C.D.A., ZELLENRATH P., POLACK B.C.P. et al., Von Willebrand factor and early diabetic retinopathy; no evidence for a relationship in patients with type 1 (insulin-dependent) diabetes mellitus and normal urinary albumin excretion. Diabetologia, 1992; 35: 555–9. 30. GARGIULO P., SCHIAFFINI R., BOSCO D. et al., Diabetic microangiopathy: lupus anticoagulant dependent thrombotic tendency in type 1 diabetes mellitus. Diab. Med., 1997; 14: 132–7. 31. LORENZI M., CAGLIERO E., Pathophysiology of endothelial and other vascular cells in diabetes mellitus: call for data. Diabetes, 1991; 40: 653–9. 32. CARBONE J., SANCHEZ-RAMON S., COBO-SORIANO R. et al., Antiphospholipid antibodies: a risk factor for occlusive retinal vascular disorders. J. Rheum., 2001; 28: 2437–41. Received June 19, 2009 272 Inimioara Mihaela Cojocaru et al. 6 Otoacoustic Emissions Analysers for Monitoring Aminoglycosides Ototoxicity RUXANDRA MOROTI CONSTANTINESCU1,4, MĂDĂLINA GEORGESCU2,4, A. PASCU2, ADRIANA HRISTEA1,4, VICTORIA ARAMĂ1,4, C. BĂICUŞ3,4, RUXANDRA OANA CĂLIN1,4, EUGENIA KOVACS4 2 1 “Matei Balş” National Institute for Infectious Diseases, Bucharest, Romania “Dorin Hociota” Phono-Audiology and ENT Functional Surgery Institute, Bucharest, Romania 3 “Colentina” Clinical Hospital, Bucharest, Romania 4 “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Objectives. Aminoglycosides (AG) are widely prescribed despite their notorious toxicity. These antibiotics cause irreversible hearing loss, starting with high frequencies and progressing toward conversational frequencies (0.5–2 kHz), by destroying the acoustic hair cells in the inner ear. The integrity of these cells could be analysed by recording faint sounds that they produce – otoacoustic emissions (OAE). The aim of the present study was to monitor and to characterise the acoustic toxicity of the AG using an OAE analyser. Method. We performed a prospective study of 49 patients receiving gentamicin (G), during 2007–2008. We made serial OAE recordings with an ILO 92 analyser (1–8 kHz) on at least 3 occasions: at the start, during treatment and after the cessation of G therapy (1–6 months). The recordings were performed at the patient’s bedside and did not require the active participation of the patient. The method is fast, non-invasive, accurate and does not request an ENT specialist. We included patients presenting OAE. Ototoxicity was defined using adapted ASHA (American Speech Hearing Association) criteria. Results. We studied 49 patients (24/25 female/male), mean age of 37.24 (3–70 years old), who received G for 4–42 days: less than 10 days – 24 patients and more than 10 days –25 patients. Hearing impairment was observed in 10 patients (20.4%) and appeared during the therapy or in the next 3 months, with a loss of one or 2 high frequencies that occurred unilaterally in 9 out of 10 cases. The acoustic damage correlates with the length of G treatment. We did not find a statistical correlation between the degree of impairment and the age, regimen or addition of another ototoxic drug, although the hearing loss was slightly higher in those with concomitant exposure. Conclusion. OAE monitoring of AG treatment is a very useful way for detecting and preventing acoustic toxicity, because it could warn about hearing loss before damage of the conversational frequencies. The accuracy is similar to the classical methods, but it is faster and easier to perform. Key words: aminoglycosides, acoustic toxicity, otoacoustic emissions. Aminoglycosides are proven ototoxic and nephrotoxic drugs, probably because of concentration in, and retention by sensory hair cells of the cochleovestibular and renal proximal tubule cells. AGs are rapidly taken up by these cells and damaged them by initiating cellular apoptosis [1–6]. The incidence of AG’s ototoxicity in patients varied from 2% to 41% in different studies [3][7–10]. The classical method for testing the cochlear function is pure tone audiometry (PTA), which presumes an active participation of the patient, so it is not applicable to newborns, young children and unconscious patients; besides, PTA imply a phonoisolated special room and could not be applicable at the patient’s bedside. ROM. J. INTERN. MED., 2009, 47, 3, 273–278 The Corti stimulation by pressure waves of sounds takes place tonotopically, depending on reaching the travelling wave’s peak: the high frequencies sounds are perceived at the base of the cochlea and the lower frequencies sounds are perceived closer and closer to the apex [11]. A clear perception of a sound (very good sensitivity and frequency selectivity) depends on the anatomical integrity and functioning of the cochlear amplifier, represented by the outer hair cells (OHC), parts of the Corti organ [12]. Cochlear injury starts from the basal region of the cochlea and extends progressively to the apex, so the first loss in hearing begins with the acute sounds and then the low sounds, till the conversational frequencies (0.5–2 kHz) are reached [3][13–14]. Ruxandra Moroti Constantinescu et al. 274 Patients frequently develop symptoms usually during the ototoxic therapy, but these can appear by a little extent in the next months, because of the AG persistence in the cochlear organ of Corti (animal studies have shown the presence of AG molecules in hair cells up to 6 months after administration) [1][15]. OHC are the most susceptible structure in the inner ear to the ototoxic drugs and OHC damage can be monitored by testing for otoacoustic emissions (OAE). Otoacoustic emissions are low intensity sounds of cochlear origin that can be recorded by a microphone fitted into the ear canal. They are generated by the motion of the OHC when the sound is processed [12][16]. 95% of normal people present evoked OAE, but these tend to decrease in amplitude with ageing: infants have large OAE, but many adults with normal hearing have low-level OAE [12]. Recording OAE represents a simple, noninvasive, sensitive and specific tool for screening cochlear function. Distortion Product OAE (DPOAE) are the most frequency-specific indicator, especially for higher frequencies (>4kHz) [17]. That can be used to monitor the effect of ototoxic treatment and enables early diagnosis when acoustic impairment occurs [12][17]. METHOD This is a prospective study involving 49 patients that received G during 2007–2008, in “Matei Balş” National Institute of Infectious Diseases, Bucharest, Romania. We used an OAE analyser ILO 92, a portable device, able to record OAE in a range of 1–8 kHz in DP-OAE mode. The recordings took place in a silent room or even at the patient’s bedside, in a non-invasive manner. The method is rapid (several minutes) and does not require an active participation of the patient. The inclusion criterion was the presence of evoked OAE. We excluded patients with middle ear pathology (because OAE’s recording depends on the integrity of the way through the middle and external ear). The patients provided an informed consent and completed a questionnaire (which included questions about previous exposure to ototoxic agents, noise or pre-existing hearing impairment). 2 Any concomitant medication during G treatment was also noted. We made serial recordings of DP-OAE for each patient, in at least 3 moments: prior to G therapy, during and after the end of therapy (1–6 months). The initial recording was considered to be the baseline DP-OAE record. We checked the presence of the DP-OAE signal at high frequencies (6, 8 kHz), bilaterally. The following measurements focused on these higher frequencies which are first implicated when the G ototoxicity emerged. Ototoxicity was defined adapting ASHA 1994 criteria, regarding the highest patient’s frequencies: as a 20dB or greater decrease at one frequency, 10dB or greater decrease at 2 adjacent frequencies or loss of 3 consecutive frequencies. RESULTS Of the 49 patients studied, 24 were female and 25 male; the mean age was 37.24 (3–70 years old); the G treatment had a length between 4 and 42 days: 24 patients were treated for less than 10 days and 25 patients had longer courses. The pathological conditions for AG therapy were diverse: endocarditis, sepsis, nosocomial urinary tract infections, pneumonia, listerial meningitis and miscellaneous ENT (ear-nose-throat) acute infections. Hearing impairment (Fig. 1) was observed in 10 patients (20.4%) and appeared during the therapy or in the next 3 months, with the loss of one or two high frequencies (Fig. 2), that occurred unilaterally in 9 out of 10 cases. The acoustic damage tends to correlate with the length of AG treatment (25% for long versus 16% for short courses) (Fig. 3). We did not find a statistical correlation between the degree of impairment and the age (one patient out of 6 under 16 years old was affected versus 9 out of 43 patients beyond 16 years old), administration rhythm (among 40 patients those received multiple daily doses regimen, 8 were affected versus 2 out of 9 which received oncedaily G dose) or association of another ototoxic drug (glycopeptides or loop diuretics), although the hearing loss was slightly higher in those with concomitant exposure: 5 out of 15 (33.3%) versus 5 out of 34 non-exposed (14.7%) (Fig. 4). 3 275 Otoacoustic emissions Fig. 1. – The hearing impairment due to G treatment. 10.1 16.6 12.1 10.4 15.1 –2.6 –6.5 20.6dBspl 4.4 –3.8 –11.1 –11.3 –12.9 –15.2 –18.5 5.7 20.4 23.2 21.7 28.0 12.6 12.0 12.9dBspl 11.2 19.9 13.2 12.3 10.2 –26.2 –30.0 22.0dBspl 5.4 –2.2 –11.6 –13.5 –14.5 –14.2 –17.0 5.8 22.1 24.8 25.8 24.7 –12.0 –13.0 14.2dBspl Fig. 2. – DP-OAE at the beginning of G treatment (left), with the presence of OAE at every recorded frequency (from 1 to 8 kHz – appearing in light grey columns) and after 3 months from the cessation of the treatment (right), with 6 and 8 kHz frequencies lost (arrows). Fig. 3. – The hearing impairment related to the length of the treatment. Ruxandra Moroti Constantinescu et al. 276 4 Fig. 4. – The hearing impairment related to the simultaneous exposure to G and another ototoxic agent. DISCUSSION Although AG’s toxicity is well recognised, some medical conditions require this therapy. So, it is useful to identify the patients who are at risk to develop ototoxicity and to observe any auditory impairment that can occur. In our study, one fifth of patients receiving AG develop ototoxicity, similar with those obtained with classical methods. G-induced hearing loss was moderate in its effect: none of the patients lost the conversational frequencies. The acoustic impairment slightly correlates with the length of G administration. Simultaneous use of another ototoxic medication also seems to enhance the harmful effect. Because the patient usually does not recognize a hearing loss until it has progressed to the conversational frequencies (0.5–2 kHz), the test of high frequencies (6–10 kHz) appears to be the proper indicator of early toxicity. This could be done by OAE analysers or by classical PTA method (the last is not always applicable and necessitates special conditions). OAE examination is a simple, fast and noninvasive method for detecting the hearing injury, has similar results to the classical methods, but it is easier to perform (at the patient’s bedside and does not require an active participation of the patient). A screening with OAE analysers could be implemented as a monitoring tool of any potential ototoxic therapies. Acknowledgements. Dr. Peter Steyger – Associate Professor of Otolaryngology – Head & Neck Surgery, Oregon Hearing Research Center, USA – for his help in providing articles related to our work. Aminoglicozidele sunt antibiotice larg prescrise, în pofida toxicităţii lor binecunoscute. Sunt cohleotoxice ireversibile, afectând iniţial frecvenţele înalte şi apoi progresiv frecvenţe din ce în ce mai joase, până la atingerea frecvenţelor conversaţionale (0,5–2 kHz), prin distrugerea anatomică a celulelor ciliate acustice din urechea internă. Funcţionalitatea acestor celule poate fi analizată prin înregistrarea unor sunete de mică intensitate produse de acestea – otoemisiuni acustice (OAE). Scopul studiului actual este de monitorizare şi caracterizare a ototoxicităţii cohleare a gentamicinei (G) folosind un analizor OAE. Metodă. Am desfăşurat în 2007–2008 un studiu prospectiv implicând pacienţi care au primit tratament cu G, folosind un otoanalizor acustic (ILO 92), cu baleiaj în frecvenţe de la 1–8 kHz. S-au realizat înregistrări seriate ale OAE: la startul terapiei (considerat baseline), pe parcursul acesteia şi la 3–6 luni postterapie. Au fost incluşi în studiu pacienţi care aveau OAE prezente la 5 Otoacoustic emissions 277 înregistrarea iniţială. Ototoxicitatea acustică a fost definită folosind criteriile ASHA (American Speech Hearing Association) adaptate. Rezultate. Lotul studiat a fost constituit din 49 pacienţi, 24 femei şi 25 bărbaţi, cu media de vârstă 37,24 ani (3–70 ani), trataţi cu G între 4 şi 42 zile: sub 10 zile – 24 pacienţi şi peste 10 zile – 25 pacienţi. Afectarea de auz s-a produs la 10 pacienţi (20,4%), a apărut în timpul terapiei sau pe parcursul următoarelor 3 luni şi a implicat una sau două dintre frecvenţele cele mai înalte recepţionate la baseline; pierderea a fost unilaterală în 9 din 10 cazuri. A existat o corelaţie între pierderile de auz şi durata tratamentului aminoglicozidic. Nu am găsit corelaţii semnificative între pierderile de auz şi vârstă, regim de administrare sau asocierea unui al doilea ototoxic în medicaţie, deşi a existat o diferenţă în acest sens (afectare mai mare la cei dublu expuşi). Concluzii. Monitorizarea tratamentului aminoglicozidic cu analizoare acustice aduce date importante şi precoce despre apariţia disfuncţiei auditive pe parcursul administrării terapiei, prin detectarea afectării iniţial a frecvenţelor înalte, care ar putea progresa ulterior spre atingerea frecvenţeler conversaţionale. Acurateţea metodei este similară cu a metodelor clasice însă este mai rapidă, simplă şi universal aplicabilă. Correspondence author: Ruxandra Moroti Constantinescu “Matei Balş” National Institute for Infectious Diseases, 1, Grozovici str, sect 2, Bucharest, Romania, E-mail: [email protected], phone: +40722550335, +40213186100/5204 fax: +40213186094 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. DAY C.F., MANGIARDI D., COTANCHE D.A., STEYGER P.S., Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo. Hear Res., 2009, 213:64–78. NAKASHIMA T., TERANISHI M., HIBI T., KOBAYASHI M., UMEMURA M., Vestibular and cochlear toxicity of aminoglycosides – A review. Acta Otolaryngol., 2000, 120: 904–911. ROTSTEIN C., MANDELL L., Clinical aminoglycoside ototoxicity. In: Roland P.S. & Rutka J.A. (eds.) Ototoxicity, Hamilton, Ontario, Decker B.C., 2004, pp. 82–92. SONG B.B., SCHACHT J., Variable efficacy of radical scavengers and iron chelators to attenuate gentamicin ototoxicity in guinea pigs in vivo. Hear Res., 1996, 94: 987–993. DENAMUR S., VAN BAMBEKE F., MINGEOT-LECLERCQ M.P., TULKENS P.M. (2008), Apoptosis induced by aminoglycosides in LLC-PK1 Cells: comparative study of neomycin, gentamicin, amikacin, and isepamicin using electroporation. Antimicrob. Agents Chemother., Jun. 52(6):2236–8. Epub., 2008 Apr. 7. DEHNE N., RAUEN U., DE GROOT H., LAUTERMANN J., Involvement of the mitochondrial permeability transition in gentamycin ototoxicity. Hear Res., 2002, 169: 47–55. SMYTH A.R., BHATT J., Once-daily versus multiple-daily dosing with intravenous aminoglycosides for cystic fibrosis. Cochrane Database Systematic Reviews, 2006, Issue 3. Art. No.: CD002009. DOI: 10.1002/14651858.CD002009.pub2. SATALOFF J., WAGNER S., MENDUKE H., Kanamycin ototoxicity in healthy men. Arch. Otolaryngol., 1964; 80(4):413–417. MOORE R.D., LERNER S.A., LEVINE D.P., Nephrotoxicity and ototoxicity of aztreonam versus aminoglycosides therapy in seriously ill non-neutropenic patients. J. Infect. Dis., 1992, 165:683–688. SHA S.H., SCHACHT J., QIU J.H., Aspirin to prevent gentamicin-induced hearing loss. N. Engl. J. Med., 2006, April: 1856–1857. TUPER G., AHMAD N., SEIDMAN M., Mechamisms of ototoxicity. Perspectives on Hearing and Hearing Disorders; Research and Diagnostics, 2005; 9: 2–10. DOI:10.1044/hhd9.1.2 PRIEVE B.A., FITZGERALD T.S., Otoacoustic Emissions. In: Katz J. (ed.) Handbook of Clinical Audiology, 5th edn. Lippincott Williams and Wilkins, Philadelphia, 2001, pp. 440–463. BROCKENBROUGH J.M., RYBAK L.P., MATZ G.J., Ototoxicity. In: Bailey B.J. (ed.) Head and Neck surgeryotolaryngology, Lippincott Williams and Wilkins, Philadelphia, pp. 1893–1898. 278 Ruxandra Moroti Constantinescu et al. 6 14. BRUMMET R.E., FOX K.E., Aminoglycosides-induced hearing loss in humans. Antimicrob. Agents Chemother., 1989, 33: 797–800. 15. DULON D., HIEL H., AUROUSSEAU C., ERRE J.P., ARAN J.M., Pharmacokinetics of Gentamicin in the sensory hair cells of the organ of Corti: rapid uptake and long term persistence. C.R. Acad. Sci. III, 1993; 316: 682–687. 16. KEMP D.T. (2002), Otoacoustic emissions, their origin in cochlear function, and use. Br. Med. Bull., 2002, 63:223–241. 17. ILO O.A.E., Instrument User Manual Otodynamics, Ltd., 1997. Received June 10, 2009 Systemic Redox Modifications in Senile Cataract BOGDANA VÎRGOLICI1, IRINA STOIAN1, CORINA MUSCUREL1, MAGDA MĂRĂCINE1, LAURA POPESCU1, C. MORARU2, VERONICA DINU1 1 “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2 Ophthalmic Surgery Department of “Oculus” Clinic, Bucharest Recent studies on cataract formation focus on a primary role of systemic oxidative stress, generated outside the lens. Plasma inflammatory markers are associated with senile cataract. Objective. The aim of this study was to find correlations between blood oxidative stress markers and some inflammatory plasma markers in cataractous patients. Design and Methods. The blood samples were collected from 38 patients (aged 50 to 80). Patients were subdivided according to two criteria. Considering age criteria, presenile and senile cataract groups were formed. According to the absence or presence of other ocular comorbidities (age–related macular degeneration, glaucoma), pure cataract and nonpure cataract groups were constituted. Fifteen age and sex matched healthy subjects were selected for the control group. Results. In our study, for all groups of patients, the measured markers of oxidative stress were modified vs. control values. Plasma antioxidant capacity, plasma antioxidant “gap”, cholesterol and albumin/globulin levels were significantly decreased while RBC SOD activity, RBC catalase activity and plasma ceruloplasmin were significantly increased. Inflammatory markers, ceruloplasmin and albumin/globulins were correlated with different parameters of oxidative stress. Conclusion. The blood redox values and the level of some inflammatory markers demonstrate that senile cataract is a systemic disease with an inflammatory component. Key words: senile cataract, oxidative stress, inflammation, systemic disease. Cataract is one of the major causes of blindness throughout the world [1]. Understanding the mechanisms of cataractogenesis will lead to a better therapy. It is estimated that the need for cataract extractions would be diminished by half if onset of cataract would be delayed by only ten years [2]. Oxidative stress continues to be the leading mechanism of cataractogenesis. The lens exists in an environment that is rich in endogenous sources of reactive oxygen species (ROS), which are produced by the high local oxygen concentration, the chronic exposure to light, and the pathogenic activities of lens epithelial cells. Although multiple physiologic defenses exist to protect the lens from the toxic effects of light and oxidative damage, mounting evidence suggests that chronic exposure to oxidative stress over the long-term may damage the lens and predispose it to cataract development [3]. J.J. Harding proposes a variety of factors implicated in maturity onset cataractogenesis: a low antioxidant defense capacity, a high lipid peroxidation, augmented nonenzymic glycosylation, a reduced ROM. J. INTERN. MED., 2009, 47, 3, 279–287 chaperone function of alpha-crystallins and an increased permeability of lens membrane. There are a few arguments for these statements: low levels of reduced glutathione and reduced activity of antioxidant enzymes, high content of sugar phosphates in lens which are reactive glycating molecules, altered structure of alpha-crystallins with diminished chaperone function, membranes lesions with loss of gap junctions and an increased flux through nonspecific cation channels [4]. Some theories of oxidative stress in senile cataract are based on the generation of reactive oxygen species and reactive molecules within the nucleus itself. Truscott considers that the lens barrier, which becomes apparent in middle age, acts to impede the flow of small molecules between the cortex and the nucleus. The barrier may contribute to the lowered concentration of reduced glutathione (GSH) in the lens nucleus after middle age. By extending the residence time within the lens centre, the barrier also facilitates the decomposition of intrinsically unstable metabolites and may exacerbate the formation of H(2)O(2) in the nucleus [5]. 280 Bogdana Vîrgolici et al. However, many studies on cataract formation focus on a primary role of systemic oxidative stress, generated outside the lens. We mention some of their conclusions: – – – subjects with early cataract are under increased systemic oxidative stress, which can be identified by a sensitive biomarker of lipid peroxidation, such as isomers of total hydroxyoctadecadienoic acid (HODE) [6]. patients with cataract have lower values of serum investigated parameters of antioxidative defense system (ascrobate, dehydroascorbate, vitamin E, glutathione, peroxidase and catalase activity) and higher level of plasma lipid peroxidation product [7]. cataractous patients had significantly lower whole blood GSH values, erythrocyte superoxide dismutase (SOD) activity and higher plasma thiobarbituric acid reactive substances (TBARS) than those in the control group [8]. In order to underline the importance of redox blood modification on the lens, we mention two studies: Street et al. found an association between atherosclerosis and cataract, which was strongest for people aged 65–69 years; they raised the question of whether a deficiency in the natural defenses against free radicals contributes to the development of both cataract and atherosclerosis [9]. Stahl et al. demonstrated that the regulation of the glutathione system in the eye, at least in aqueous humor, is dependent on plasma glutathione levels; this correlation reflects the importance of sufficient glutathione levels in venous plasma and suggests the possibility of modulating the glutathione system in the eye via manipulation of plasma glutathione levels [10]. Within the strategy to maintain redox balance against oxidant conditions, blood has a central role because it transports and redistributes antioxidants to each part of the body. Nonenzymatic antioxidants such as: albumin, uric acid, bilirubin, glutathione (GSH), ascorbic acid, α-tocopherol, β-carotene, and flavonoids constitute the first line of antioxidant defense systems of the blood. Among the red blood cells (RBC) enzyme systems (the second antioxidant defense systems), superoxide dismutase (SOD), catalase (CAT) are very important. Plasma antioxidant capacity (AC) is modulated either by radical overload or by intake of dietary 2 antioxidants and can therefore be regarded as more representative of the in vivo balance between oxidizing species and antioxidant compounds than the concentration of single, selected low molecular antioxidant. The aim of our study was to measure some plasma and red blood cell oxidative stress markers in cataractous patients. Correlations between these markers and some inflammatory ones were calculated. MATERIAL AND METHODS The patients were recruited from the ophthalmic surgery department of “Oculus” Clinic, Bucharest. The blood samples were collected from 38 patients (aged 50 to 80) with cataract, without metabolic or severe somatic diseases and from 15 age and sex matched healthy subjects. Smokers and alcohol consumers were excluded from the study. Because of the advanced age, most of the patients had different associated illnesses: high blood pressure, hearing loss, other ocular age related diseases, etc. Patients were subdivided according two criteria. Considering age criteria, presenile and senile cataract groups were formed. According to the absence or presence of other ocular comorbidities (age–related macular degeneration, glaucoma), pure cataract and nonpure cataract (cataract associated with other ocular diseases) groups were constituted. Blood samples were taken into 10 ml vacutaine tubes, containing heparin, from a peripheral vein after 12 hours of fasting and drugs break. The study protocol was approved by the Ethical Commission of “Carol Davila” University of Medicine and Pharmacy Bucharest and a written informed consent was obtained from each study participant. Plasma total cholesterol, uric acid, bilirubin and glucose were analyzed by enzymatic methods, using kits (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden Germany). For total proteins and albumin we used kits from the same source. Protein thiols in plasma were measured by a colorimetric method. We measured the absorption at 412nm of acids formed in the reaction between the -SH groups and 5-5’-dithiobis-nitrobenzoic acid [11]. The plasma thiobarbituric acid-reacting substances (TBARS) were determined by thiobarbituric acid reaction at 540 nm [12]. The globulins were calculated by the difference between total plasma proteins and albumin. 3 281 Systemic redox modifications in cataract Superoxide dismutase (SOD) activity was measured in erythrocytes by the spectrophotometric method described by Marklund S., [13]. The inhibition of pyrogallol oxidation by SOD was monitored at 420 nm, and the amount of enzyme producing 50% inhibition is defined as one unit of enzyme activity. Erythrocyte catalase activity was determined by using hydrogen peroxide as a substrate, at 240nm [14]. Plasma total antioxidant capacity (AC) was determined by Miller et al. method and the antioxidant gap was calculated according to the equation: (GAP) = AC (mmol/l) – [albumin (mmol/l) X 0.69 + uric acid (mmol/l) X 1)] [15]. The plasma ceruloplasmin level determination was based on the o-dianisidine dihydrochloride endpoint method [16]. The hemoglobin quantity was measured using the Drabkin method. The results were analyzed statistically using Student test. Data are expressed as mean ± s.d. Two-tailed p-values <0.05 were considered statistically significant. The relationship between the various parameters was assessed by correlation (multiple regression). RESULTS The characteristics of the groups are shown in Table I. The assessed parameters of oxidative stress are shown in Table II. Table I Parameter Age (years) Sex (male/female) Type of cataract: SC/NC/NC+PSC/CC/MC Healthy subjects Presenile cataract group (n=15) (n=15 ) 57.18±6.6 57.27±5.9 5/10 – 5/10 Senile cataract Pure cataract Cataract associated with group (n=23) group (n=25) other ocular illnesses (n=13) 73.12±4.6 68.94±8.17 70.5±9,06 p<0.02 vs. 1 8/15 9/16 4/9 1/6/6/1/1 2/5/8/1/7 2/7/9/2/5 1/4/5/0/3 SC/NC/NC+PSC/CC/MC = Subcapsular cataract/Nuclear cataract/nuclear and posterior subcapsular cataract/cortical cataract/mixt cataract Table II Plasma parameter Healthy controls n=15 (group M) Presenile cataract group n=15 (group 1) Senile cataract group n=23 (group2) Pure cataract group n=25 (group B) Plasma thiols (µmol/g protein) Plasma TBARS (µmol/l) 7.37±0.7 7.55±0.54 6.98±0.55 2.18±0.59 3.42±0.63 p<0.01 vs. M 200.68±33.49 p<0.04 vs. M 6.71±0.63 p<0.02 vs. 1 2.39±0.67 p<0.02 vs. group1 224.04±57 Cataract associated with other ocular illnesses n=13 (Group C) 7.05±0.73 3.03±0.65 3.01±0.62 232.35±56.69 192.53±35.95 p<0.05 vs. Group B p<0.001 vs. M 4.33±0.29 Plasma cholesterol [mg/dl] 243.45±46.85 Plasma albumin [g/dl] 4.52±0.33 4.11±0.27 p<0.002 vs. M 4.25±0.22 p<0.01 vs. M Plasma proteins [g/dl] 7.51±0.49 7.55±0.20 Plasma bilirubin [mg/dl] 0.47±0.17 0.49±0.13 8.01±0.39 p<0.01 vs. group1 p<0.025 vs. M 0.54±0.15 Plasma uric acid [mg/dl] 4.05±0.44 3.77±0.59 Total antioxidant capacity [mM Trolox] Plasma residual antioxidant capacity [mM] 0.87±0.12 0.74±0.08 p<0.01 vs. M 0.093±0.03 p<0.005 vs. M 0.21±0.04 4.41±0.48 p<0.02 vs. group1 p<0.03 vs. M 0.75±0.07 p<0.01 vs. M 0.087±0.03 p<0.001 vs. M 4.04±0.42 p<0.004 vs. M p<0.04 vs. groupC 7.88±0.33 0.46±0.16 4.16±0.89 0.74±0.09 p<0.01 vs. M 0.12±0.03 p<0.04 vs. M 7.74±0.49 0.62±0.19 p<0.05 vs. group B 4.13±0.62 0.75±0.05 p<0.01 vs. M 0.06±0.01 p<0.01 vs. Group B p<0.001 vs. M Bogdana Vîrgolici et al. 282 4 Plasma globulins [g/dl] 3.30±0.64 3.44±0.29 3.76±0.37 p<0.03 vs. M Albumin/globulins 1.42±0.26 1.21±0.17 1.13±0.11 p<0.026 vs. M Plasma ceruloplasmin [UI/l] RBC SOD [UI/g Hb] 276.69±107.43 375.88±168.81 p<0.03 vs. M 470.07±111.75 p<0.05 vs. M 396.57±128.59 p<0.01 vs. M 469.46±107.56 p<0.05 vs. M RBC Catalase [kat/g Hb] 146.95±68.2 Plasma glucose [mg/dl] 95.61±12.27 235.82±89 p<0.02 vs. M 98.49±10.46 253.13±137.54 p<0.01 vs. M 96.18±10.15 409.82±58.84 3.82±0.17 p<0.03 vs. M p<0.04 vs. groupC 1.09±0.16 p<0.01 vs. groupC p<0.004 vs. M 390.31±117.66 p<0.01 vs. M 398.33±93.77 263.99±129.2 p<0.01 vs. M 95.93±12.3 3.41±0.23 1.26±0.12 p<0.05 vs. M 386.98±134.99 p<0.02 vs. M 514.04±104.26 p<0.001 vs.group B p<0.005 vs. M 251.08±89.13 p<0.01 vs. M 96.11±9.55 The correlations between blood oxidative markers and inflammatory markers are shown in Tables III and IV. Table III Correlated parameters Plasma thiols [µmol/g protein] Plasma TBARS [nmols/ml] Plasma globulins [mg/dl] Plasma globulins [mg/dl] Plasma albumin [mg/dl] Plasma albumin [mg/dl} Plasma cholesterol [mg/dl] Plasma ceruloplasmin [UI/l] Plasma ceruloplasmin [UI/l] Plasma TBARS [nmol/ml RBC SOD [UI/l] RBC Catalase [kat/g Hb] Plasma cholesterol [mg/dl] Plasma TBARS [nmols/ml] Plasma TBARS [nmols/ml] Plasma globulins [mg/dl] Albumin/globulins [kat/g Hb] Plasma globulins [mg/dl] Plasma globulins [mg/dl] r value Presenile cataract Senile cataract group group (n=15) (n=23) –0.76 –0.47 +0.53 – +0.58 –0.72 –0.65 +0.74 –0.68 +0.58 – – – – – – – +0.52 +0.55 +0.80 Table IV Correlated parameters Plasma globulins [mg/dl] Plasma cholesterol [mg/dl] Plasma albumin [mg/dl] Plasma albumin [mg/dl] Plasma albumin [mg/dl] Plasma thiols [µmol/gprotein] Plasma TBARS [nmol/ml] Plasma Bilirubin [mg/dl] RBC SOD [UI/l] Plasma cholesterol [mg/dl] DISCUSSION The recognition of cataract in a growing number of systemic diseases and syndromes has necessarily prompted investigation into unifying mechanisms [17]. Oxidative stress may be one of them. Also, human cataract in older age groups seems to be due to an accumulation of risk factors, r value Pure cataract group Non-pure cataract (n=22) group (n=16) – –0.48 +0.48 –0.56 – +0.47 – –0.63 +0.64 – even if individual mean concentrations are well within normal limits but, of course, differing significantly from the corresponding means in control population [18]. Case-control studies showed that nuclear cataract was significantly associated with raised serum bilirubin and that all forms of cataract are significantly associated with raised plasma albumin and reduced levels of cholesterol [18–20]. 5 Systemic redox modifications in cataract Conflicting results concerning different levels for plasma antioxidant capacity and erythrocyte antioxidant systems in cataractous patients can be found in literature [21–24]. In our study, for all groups of patients, the measured markers of oxidative stress were modified vs. control values. Plasma AC, plasma antioxidant “gap”, cholesterol and albumin/globulin levels were significantly decreased and RBC SOD activity, RBC catalase activity and plasma ceruloplasmin were significantly increased. Each of these parameters was to be discussed. Human lens membranes have the highest cholesterol content of any known biological membrane. Exogenous sources of cholesterol are essential for the lens. Hockwin et al., working on bovine eye, demonstrated a direct relation between the plasma and aqueous humor cholesterol concentrations [25]. Disturbances in cholesterol homeostasis may result in cell damage by a variety of mechanisms: membrane ionic pumps, electrolytic imbalance and calcium homeostasis. The low serum levels of cholesterol together with the high plasma levels of TBARS, obtained in patients aged 50–65 years, may be responsible for the early onset of senile cataract. Knowing that cholesterol rises with age and despite the age difference between the control group and the group with cataract associated with ocular comorbidities, it is interesting to notice that plasma cholesterol was significantly lower in the patient group. High statistical power studies have demonstrated that all forms of cataract are associated with low plasma cholesterol while patients with age-related maculopathy have no significant differences of plasma cholesterol level vs. control subjects [26]. Positive correlations between plasma levels of albumin and cholesterol and negative correlations between TBARS either with albumin or cholesterol were calculated. Albumin and cholesterol may be oxidized in conditions of increased oxidative stress reflected by high levels of TBARS. It is known that oxidation of the protein thiol groups, especially the ones from serum albumin, is a sensible and specific oxidative stress marker in the vascular compartment [27]. Albumin inhibits oxidative damage by binding copper ions and it can react with H2O2 and HOCl. In this study, for all four groups of patients, the low levels for albumin, the high values for globulins, and the low albumin/globulin ratio can 283 be explained in an inflammatory context. Senile cataract can be considered an illness with a low grade inflammatory status. It is known that inflammation is characterized by oxidative reactions and generation of high oxygen reactive species. Also, the negative correlations between plasma total thiols and globulins represent an argument for a relation between inflammation (high globulins) and the plasma antioxidant defense (consumption of plasma thiols). The association of systemic inflammation and senile cataract has been demonstrated in recent studies. Elevated levels of CRP are associated with future risk of cataract in apparently healthy men [28]) and two serum markers of systemic inflammation and vascular endothelial dysfunction (interleukin-6 and intracellular adhesion molecule-1) are associated with nuclear cataract [29]. Donnelly et al. measured plasma constituents in a population of 1000 pairs (healthy and cataractous patients), with strict control of age in matched pair protocol. The investigators found high levels of albumin and of total protein in the cataract group. The significant lower albumin/ (total protein-albumin) ratio, (an approximation for albumin/globulin ratio) may imply an increase in globulin, suggestive of possible (chronic) infection in patients with senile cataract [14, 30]. Leske et al. and Schoenfeld et al. obtained similar results and both research teams reported that a high albumin/globulin ratio decreased risk for mixed cataract [31][32]. The positive correlation, calculated in this study, between globulins and plasma ceruloplasmin is not surprising in an inflammatory status. Maybe the raised globulin level is due to the high α2 globuline fraction (including ceruloplasmin). Human plasma normally contains 200–400 mg/l of caeruloplasmin, accounting for at least 90% of total plasma copper. The remaining plasma copper is often claimed to consist of copper ions bound to albumin, histidine or small peptides. Caeruloplasmin is increased greatly in the acute phase reaction and it has also a ferrroxidase activity: it oxidizes Fe2+ to Fe3+ and can facilitate iron loading on to transferrin [33]. Increased plasma level of caeruloplasmin was found in patients with senile cataract and in galactosaemic children with cataract and without cataract [34][35]. Maybe this enzyme is induced by reactive oxidative species and its high level is in accordance with the raised PCR level observed in cataractous patients. We emphasize that the high ceruloplasmin values obtained in this study may 284 Bogdana Vîrgolici et al. reflect that senile cataract is a systemic disease with inflammatory status. Peroxidation is associated with inflammation. Lipid peroxidation, measured as TBARS concentration was the highest in presenile cataract and these modification may be linked to the premature development of senile cataract [36]. The low molecular weight antioxidants in plasma are “sacrificial” compounds. They rapidly get consumed during the scavenging of reactive oxygen species and need to be regenerated or replaced by new dietary-derived compounds. It is essential to measure these antioxidants in assessing in vivo antioxidant status. However, the number of different antioxidants in plasma or other biological samples makes it difficult to measure each antioxidant separately. The possible interaction among different antioxidants in vivo could also make the measurement of any individual antioxidant less representative of the overall antioxidant status [37]. Recent literature indicates that antioxidants may ameliorate the risk for age related cataract. As an example, higher intakes of vitamin C or the combined intake of antioxidants had long-term protective associations against development of nuclear cataract in a group of older population [38]. The results of observational studies suggest that a healthy lifestyle with a diet containing food rich in antioxidants, particularly lutein and zeaxanthin, as well as n-3 fatty acids, appears beneficial in age macular degeneration and possibly cataract [39]. While data from the observational studies generally demonstrate a protective role for antioxidants in foods or supplements, results from intervention trials are less encouraging with respect to limiting risk for age related cataract/ age related maculopathy prevalence or progress through antioxidant supplementations, or maintaining higher levels of antioxidants either in diet or blood [40]. In our study all groups of patients had low levels of plasma AC, reflecting a reduced plasma antioxidant defense system. The lowest plasma residual antioxidant capacity was calculated for cataractous patients with ocular comorbidities. This result underlines the importance of vitamins and other low weight antioxidant defence compounds in age-related macular degeneration and glaucoma. Antioxidant capacity of plasma for 96 patients with senile cataract was determined by Tissie et al. [41]. Irrespective of the previous studies that demonstrated a relationship between decreased levels of antioxidant components in blood and the 6 occurrence of lens opacities, the investigators couldn’t find any significant difference between the subjects with and without senile cataract. Regarding the antioxidant gap, it can be noticed that the lowest value is present in cataractous patients with ocular comorbidities. This value is another strong argument for the importance of plasma antioxidant defense in age related ocular diseases and can justify the benefits of antioxidant therapy for specific groups of cataractous patients. Uric acid is an important antioxidant. The contribution of uric acid to total antioxidative status (Randox test) amounted to 38% in tear fluid, 10% in aqueous humour and 37% in serum. Positive correlations for uric acid levels in tear fluid, aqueous humour and serum were demonstrated [42]. This is another argument for a link between plasma parameters and ocular ones, or between systemic status and the lens one. We obtained the highest significant uric acid plasma levels in senile cataract group, characterized by nuclear and posterior subcapsular cataract forms. Our result is in accordance with those from literature, as it follows. It was demonstrated that higher uric acid levels increased the risk for mixed opacities (OR=1.74) Leske et al. [31] or for posterior subcapsular cataract (OR = 1.62) [the Italian-American Cataract Study Group [43]. Bilirubin is a scavenger of O2.–, HO.. In both Edinburgh studies its serum level was significantly raised in cataractous patients compared to control subjects [18][19]. The authors of the studies considered that the significant rise in three plasma constituents: alkaline phosphates, bilirubin, and γ-glutamyl transpeptidase with the lack of significant increase in alanine aminotransferase, would be consistent with an association between mild intrahepatic cholestasis and cataract. They mentioned that the mean values estimated in both groups of subjects (cataractous and normal ones) are within the generally accepted normal reference range, which are 2 standard deviations from the mean of a large parent population [18][19]. In our study, cataractous patients with associated ocular comorbidities had significant high plasma bilirubin values compared to pure cataract group. According to many studies, the antioxidant enzymes (catalase, SOD, GPx, GR) in cataract lens have lower activity then the enzymes from the normal lenses [44–48]. However, the results of the measurement of the activity of the RBC antioxidant enzymes from the cataractous patients diverge, as is illustrated in the following studies: 7 – Systemic redox modifications in cataract a small size case-control study did not show any difference in the levels of erythrocyte SOD, GPx and glucose 6-phosphate dehydrogenase (G6PD) in cataractous patients [21]; – Chinese researchers found a lower activity of several RBC antioxidant enzymes (SOD, catalase) and a significantly decreased erythrocyte GPx level in subjects with senile lens changes [49]; – the POLA study showed a strong association of high levels of erythrocyte SOD with increased risk of nuclear cataract [50]. In a relatively recent published study, the erythrocyte SOD activity was significantly increased in the cataractous group compared with healthy matched subjects and catalase activity was not modified [51]. 285 In this study, made in 38 patients with predominant nuclear and posterior subcapsular senile cataract, the erythrocyte SOD and catalase activities were significantly raised in all studied groups versus control group. The highest value was determined in the group with cataract and ocular comorbidities. These values may reflect an adaptive protective response, which is indicative of mild oxidative stress. In conclusion, taking into account all the modified blood parameters, senile cataract may be considered an illness with systemic echo, not only a local disease. But, it is still hard to say if the modified oxidative stress parameters are cause or effect in senile cataract. Studii recente asupra cataractogenezei atrag atenţia asupra rolului primar al stresului oxidativ sistemic, generat înafara cristalinului. Markeri plasmatici ai inflamaţiei sunt asociaţi cu cataracta senilă. Obiective. Scopul acestui studiu este de a determina corelaţii între markerii sanguini de stres oxidativ şi câţiva markeri de inflamaţie, la pacienţii cu cataractă senilă. Materiale şi metode. Probele de sânge au fost recoltate de la 38 pacienţi, cu vârste între 50–80 ani. Pacienţii au fost împărţiţi după două criterii. Considerând criteriul vârstei, s-au contituit două loturi, cu cataractă presenilă şi respectiv senilă. Folosind criteriul comorbidităţilor oculare asociate (degenerescenţa maculară, glaucom), s-au format lotul cataractei pure şi cel al cataractei ce asociază alte afecţiuni oculare. Cincisprezece subiecţi control, corespunzători ca vârstă şi sex au fost recrutaţi. Resultate. În acest studiu, pentru toate loturile cu pacienţi, toţi parametrii de stres oxidativ au fost modificaţi faţă de cei din lotul control. Astfel, la pacienţi, capacitatea antioxidantă plasmatică, gap-ul antioxidant plasmatic, colesterolul, raportul albumine/globuline au avut valori semnificativ reduse, în timp ce activitatea eritrocitară a SOD, a catalazei, precum şi a ceruloplasminei plasmatice au avut valori semnificativ crescute. Markerii inflamatorii, ceruloplasmina şi raportul albumine/globuline s-au corelat cu diferiţi markeri de stres oxidativ. Concluzie. Cataracta senilă este o boală sistemică cu componentă inflamatorie, conform valorilor sanguine redox şi concentraţiei unor markeri plasmatici ai inflamaţiei. Corresponding author: Bogdana Vîrgolici, MD Biochemistry Department, Faculty of Medicine “Carol Davila” University, 8, Bd. Eroilor Sanitari, Bucharest, 050471, Romania REFERENCES 1. 2. 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Received May 23, 2009 288 Bogdana Vîrgolici et al. 10 Protection of Erythrocyte Membrane Against Oxidative Damage by Geriforte in Healthy Human Subjects MARILENAăăGÎLC 1 ,ăăIRINAăăSTOIAN1,2,ăăDANIELAăăLIXANDRU1,ăăLAURAăăG MAN1,ăă BOGDANAăăVÎRGOLICI1,ăăV.ăATANASIU1ă 1ă BiochemistryăDepartment,ăFacultyăofăMedicine,ă“CarolăDavila”ăUniversity,ăBucharest,ăRomaniaă 2ă R&DăIRISTăLABMEDăSRL,ăBucharest,ăRomaniaă Theă influenceă ofă Geriforte,ă ană Ayurvedică naturală supplement,ă onă theă antioxidantă defenseă systemsă ină humană erythrocytesă andă plasmaă wasă investigatedă ină ană openă humană clinicală study.ă Theă abilityăofăGeriforteătoăinhibitătheăazo-bisă2-amidinopropaneădihydrochlorideă(AAPH)ădependentălysisă ofă erythrocytesă wasă alsoă evaluated.ă Geriforteă supplementationă increasedă theă activityă ofă erythrocyteă catalaseă (265.745ă ±ă 15.768ă vsă 352,329ă ±ă 18.480ă K/gă Hb/mL,ă p<0.01ă )ă andă reducedă theă freeă radicală mediatedă cytotoxicityă inducedă byă azo-bisă 2-amidinopropaneă dihydrochlorideă (AAPH),ă whichă wasă measuredăbyăerythrocyteămembraneăstabilityăassayă(0.0439ă±ă0.0069ăvsă0.1291ă±ă0.0396ăC50-ăAAPHă (mM),ă p<0.05).ăTheăinterventionă didă notăchangeăsignificantlyătheă activityă ofă erythrocyteă superoxideă dismutaseăandătheăplasmaălevelsăofăantioxidantăagents.ăăTheăresultsăindicateăthatăGeriforteăpossessesă cytoprotectiveăpropertiesăonăerythrocytesăagainstăoxidativeăchallengeăandăspecificăantioxidantăactivity,ă whichăinvolvesămainlyătheăintracellularăprotectiveăsystemsăandătheămembranes.ă Key words:ăantioxidant,ăerythrocyteămembraneăstability,ăGeriforte,ăaging.ă List of abbreviations:ă AAPH:ă azo-bisă 2-amidinopropaneă dihydrochloride;ă ABTS·+:ă 2,2’azino-bis(3-ethylbenzothiazoline-6-sulfonică acid)ă radicală cation;ă CAT:ă catalase;ă GAP:ă antioxidantă gap;ă SOD:ă superoxideă dismutase;ă TAA:ă serumă totală antioxidantă activity;ă TEAC:ă Troloxă equivalentă antioxidantăactivity.ăă Recentă dataă stronglyă leadă toă theă ideaă thată oxidativeăstressăisăaăsignificantămarkerăofăsenescence,ă beingă establishedă ină agedă humansă andă laboratoryă animals.ăThisăageărelated-oxidativeăstressăisăgeneratedă byăaăcombinationăofăincreasedăfreeăradicalsăproduction,ă decreasedă antioxidantsă levelsă andă impairedă repairă ofă oxidativeădamagesă[1].ă Ină recentă years,ă theă clinicală researchă ofă theă anti-ageingăherbalădrugsăhasăreceivedă considerableă attention,ă plantsă beingă richă sourcesă ofă compoundsă withăantioxidantăactivity.ăTheăideaăofăsuperiorityăofă multi-compoundădrugsă(e.g.ăcombinationăofădifferentă phytochemicalsă oră naturală antioxidants)ă asă beingă muchă moreă powerfulă thană single-compoundă drugsă hasămoreăandămoreăgainedătheăinterestăofăscientists.ă Hittingămultipleătargetsăhasăbeenărecentlyăproposedă toă impactă theă complexă equilibriumă ofă theă wholeă biomoleculară networkă moreă favorableă thană drugsă thată actă onă aă singleă targetă [2–4].ă Thisă correspondsă wellă withă theă mechanismă ofă actionă ofă medicinală plants,ă whichă representă complexă mixturesă ofă phytochemicalsă andă affectă hundredsă ofă differentă enzymesăandăreceptorsăsimultaneously.ă ROM.ăJ.ăINTERN.ăMED.,ă2009,ă47,ă3,ă289–295ă PreviousăstudiesăshowedăthatăGeriforte,ăaăwellă knownăAyurvedicămixtureăofăseveralăplantăingredientsă andă minerals,ă haveă someă anti-ageingă andă antioxidantă effectsăin vitroăexperimentsăorăinăanimalăstudies:ăită reducesăage-relatedăchangesăinătheăliverăandătheăbraină [5][6],ădecreasesăfreeăradicalămediatedăcytotoxicityăină lymphocytesă [7],ă hasă goodă radioprotectiveă effectsă basedă onă itsă capacityă toă scavenge·OH,ă O2·–,ă ABTS·+ă andăNOă[8][9].ăă Takingă intoă accountă thată erythrocytesă areă particularlyăsensitiveătoăoxidativeăstressădueătoăhighă O2ă tension,ă highă contentă ofă polyunsaturatedă lipids,ă andă theă presenceă ofă transitionă metalsă [10][11],ă theyă areăconsideredătoăbeăanăexcellentămodelăforătheăstudyă ofătheăbiomembraneăoxidativeădamage.ăNevertheless,ă theă effectsă ofă Geriforteă onă erythrocyteă membraneă stabilityă haveă notă beenă previouslyă beenă addressed,ă despiteă widespreadă interestă ină theă relevanceă ofă antioxidantăpropertiesăofăGeriforte.ăă Keepingă theă aboveă factsă ină view,ă theă presentă studyă wasă designedă toă evaluateă theă cytoprotectiveă activitiesă ofă Geriforteă againstă freeă radicală damageă usingăerythrocytesăasămodelăsystem,ăandătoăestablishă MarilenaăGîlc ăet al.ă 290ă ă theăpreferentialătargetăofăGeriforteăantioxidantăaction:ă cellulară oră plasmaă antioxidantă systems.ă Weă studiedă erythrocytesă andă plasmaă antioxidantsă ină healthyă subjectsă beforeă andă afteră Geriforteă supplementationăă (2ă tabletsă twiceă dailyă foră 8ă weeks).ă Thisă isă theă firstă clinicalăstudy,ăwhichăevaluatedătheăeffectăofăGeriforteă onăantioxidantăsystemsăinăhumans.ă MATERIAL AND METHODS Theă studyă wasă approvedă byă theă locală ethicală committeeă andă conductedă accordingă toă theă ethicală obligationsă ofă theă Declarationă ofă Helsinki.ă Theă contentă ofă theă studyă wasă explainedă toă theă volunteers.ăWrittenăinformedăconsentăwasăobtainedă fromăsubjectsăpriorătoăinclusionăinătheăstudy.ăă SUBJECTSă Subjectsă wereă recruitedă fromă twoă Asylumsă forăAgeingăPeople,ăadministrativeăandămedicalăstaffă ofă “Carolă Davila”ă Universityă andă Universityă Hospital,ă Bucharestă andă healthyă volunteersă whoă haveăhelpedăinăotherăstudiesăwithinătheăDepartment.ă Screeningă includedă aă medicală history,ă physicală examinationă andă bloodă tests.ă Subjectsă withă anyă acuteă oră chronică diseaseă (diabetesă mellitus,ă cardiovasculară diseases,ă gastrointestinală diseases,ă etc.),ă anyă medication,ă ingestionă ofă vitamin,ă herbală oră minerală supplementsă oră withă haemoglobin,ă albumină levelă belowă normală rangesă quotedă ină oură laboratoryăwereăexcluded.ăă 94ă subjectsă wereă screenedă foră thisă study.ăă 40ăsubjectsăwereăexcludedăonătheăgroundsăofăseriousă diseaseăorăabnormalăhaemoglobinăorăalbuminălevels,ă whileă 7ă subjectsă wereă takingă vitamină oră herbală supplements.ăAfterăscreening,ăaătotalăofă37ăsubjectsă wereăincludedăinătheăstudy.ăă STUDYăăDESIGNă Thisăwasăanăopenăclinicalăstudy.ăTheădurationă ofă theă trială foră eachă participantă wasă 8ă weeks.ă Theă subjectsăwereăsupplementedăwithă2ătabletsăofăGeriforteă twiceădailyăforă8ăweeks.ăTheădataăobtainedăafterătheă supplementationă periodă wereă comparedă withă theă meană valuesă obtainedă beforeă theă supplementation.ă Allă subjectsă wereă freeă livingă andă consumedă selfselectedă dietsă throughoută theă study.ă Beforeă andă ată theăendăofă8ăweekăsupplementationăperiodă10ămlăofă ă 2 bloodăwasăcollectedăintoăheparinizedăvacutainerătubesă andă5ămlăofăbloodăinăEDTA-coatedăvacuumătubesăfromă overnightăfastingăandă10ăminăofărestingăindividuals.ăă 0.5ă mlă ofă EDTA-mixedă bloodă haveă beenă immediatelyă usedă foră theă erythrocyteă membraneă stabilityă assayă andă theă restăhasă beenă centrifugedă andă processedă foră theă measurementă ofă theă otheră parameters.ă Afteră centrifugationă theă plasmaă wasă removedăandăretainedăonăiceăforătheăassayăofătotalăfreeă thiolsă groupsă andă Troloxă equivalentă antioxidantă activityă (TEAC).ă Theă remainingă erythrocytesă wereă washedă threeă timesă withă 0.9%ă NaClă andă lysedă ină ultrapureă Chelexă treatedă water.ă Theă redă cellă stromaă wasă removedă byă centrifugationă (10ă minutesă ată 4ºC)ă andăonăclarifiedăsupernatantă(keptăonăice)ăsuperoxideă dismutaseăactivity,ăcatalaseăactivityăandătotalăandăfreeă thiolsă levelsă wereă assayed.ă Followingă centrifugationă theăplasmaăandăerythrocytesăaliquotsăwereăstoredăată– 80°Că untilă use.ă Reagentsă andă ultraă pureă wateră wereă treatedăwithăChelexă100ă(Merck,ăDarmstadt,ăGermany)ă toăbindătransitionalămetals.ăAllăreagentsăwereăofăpureă analyticală qualityă andă wereă purchasedă fromă SigmaAldrichăChemieă(Steinheim,ăGermany),ăifănotăotherwiseă indicated.ă Allă assaysă wereă performedă byă spectrophotometryă onă aă Perkin-Elmeră Lambdaă EZă 210ă (Perkin-ElmerăInc,ăBoston,ăUSA),ăexceptingăerythrocyteă membraneăstabilityăassay,ăwhichăwasăperformedăonă aă Microplateă Readeră BIO-RADă (USA),ă andă wereă carriedă oută onă triplicateă samples.ă Theă followingă parametersăwereămeasured:ă ERYTHROCYTESăăăMEMBRANEăăăSTABILITYă Theă assayă isă basedă onă theă sensitivityă toă haemolysisă ofă aă 0.6–0.8%ă erythrocyteă suspensionă (obtainedăfromăbloodăsamples)ăonătheăintroductionăofă AAPHă (azo-bisă 2-amidinopropaneă dihydrochloride),ă aăradicalăinitiatoră[12].ă Theăerythrocytesăwereăisolatedăbyăcentrifugationă ată300×găforă15ăminutesăată4ºCăandăplasmaăremoved.ă Phosphate-bufferedă salineă (PBS:ă 10mMă sodiumă phosphate,ă Phă7.4,ă150ă mMăNaCl)ă wasăaddedăbackă toă theă originală wholeă bloodă volume,ă andă 0.5ă mlă ofă theă resuspendedă cellsă dilutedă intoă 25ă mlă PBSă toă obtainăaă0.6–0.8%ăerythrocyteăsuspension.ă100ăµlăofă thisă suspensionă wasă mixedă withă 100ă µlă solutionă ofă differentăAAPHăconcentrationsăină96-wellămicroplatesă andă theseă mixturesă wereă incubatedă ată 37ºCă underă gentleăstirringăată1000ărpmăforă20ăhours.ăTheăproperă concentrationă ofă AAPHă andă incubationă timeă wereă determinedă byă preliminaryă experiments.ă Theă sameă 3ă ProtectionăofăerythrocyteăandăGeriforte amountă ofă AAPHă wasă addedă toă controlă wellsă containingă erythrocytesă lysedă byă additionă ofă Tritonăă X-100ă toă aă finală concentrationă ofă 1%.ă Thisă controlă allowedăcorrectionăforătheăamountăofăerythrocytesăină theăbloodăsample,ăasăwellăasăforătheăeffectăofăAAPHă onă absorbanceă ofă hemoglobin.ă Afteră 20ă hours,ă theă microplatesăwereăcentrifugedăată900×găforă3ăminutesă andă theă supernatantă containingă theă releasedă hemoglobină(Hb)ăfromătheălysedăcellsăwasăevaluatedă byămeasuringătheăabsorbanceăată405ănmă(Soretăbandă absorptionă ofă hemoglobin).ă Theă absorbanceă ofă eachă wellăwasăreadăată405ănmăusingăaăMicroplateăReaderă (BIO-RAD,ăUSA).ăTheăpercentageăofăhaemolysisăwasă determined.ă Erythrocyteă stabilityă wasă expressedă asă AAPHăconcentrationănecessaryătoăobtaină50%ăofătotală haemolysisă afteră 20ă hoursă (C50-AAPH).ă C50-AAPHă wasă calculatedă accordingă toă theă methodă ofă Stagstedă [12]ă fromă aă fită ofă dataă toă aă sigmoidă curve,ă usingă GraphPadă Prism4ă (Sană Diego,ă CA,ă USA).ă Theă experimentăwasăperformedăinătriplicate.ă Erythrocyte catalase (CAT)ăactivityăwasămeasuredă usingătheămethodădescribedăbyăMannervikă[13].ăTheă erythrocyteă lysateă wasă dilutedă ină 0.05Mă potassiumă phosphateă bufferă (pH=7)ă andă theă reactionă wasă startedă byă addingă 10mMă hydrogenă peroxide.ă Theă decreaseăinăabsorbanceăată240nmăwasămeasuredăforă 30ăseconds.ăTheăenzymeăactivityăwasăcalculatedăasăaă functionă ofă theă rateă constantă ofă theă firstă orderă reactionă(k)ăandăwasăexpressedăasăk/găHb.ă ERYTHROCYTEăăSUPEROXIDEăăDISMUTASEă(SOD)ă SODă activityă wasă determinedă accordingă toă Marklundămethodă[14].ăTheămethodăisăbasedăonătheă abilityă ofă SODă toă inhibită pyrogallolă autoxidation.ă CuZnSODăfromăerythrocytesăwasăextractedăwithăcoldă ethanol/chloroformă 62.7/37.5ă (v/v).ă Theă supernatantă fluidă wasă mixedă withă0.2ă mMăpyrogallolăandăTRISă bufferă(tris-hydroxymethyl-aminomethane-ăhydrochloride)ă containingă cacodylică acidă andă ethyleneă diaminopentaaceticăacidă(pH=8.2),ăinătheăpresenceăofăoxygen.ă Theă auto-oxidationă ofă pyrogallolă wasă monitoredă ată 420nmă foră 3ă minutesă againstă theă blankă sample.ă SODă activityăwasăexpressedăasăU/găHb,ăoneăunitărepresentingă theăamountăofăenzymeărequiredătoăinhibitătheărateăofă pyrogallolăautoxidationăbyă50%ăată25°C.ă Serum total antioxidant activity (TAA)ăefficientlyă reflectsă theă reală totală antioxidantă effect,ă takingă intoă accountă theă synergismă betweenă antioxidantsă andă alsoă exogenousăantioxidantsă(e.g.ăherbalăflavonoids,ăetc.).ăAă summationă ofă individuală measurementsă mayă notă 291 accuratelyăreflectătheătotalăantioxidantăeffect.ăTherefore,ă TAAă isă moreă adequate,ă cheaperă andă lessă timeconsumingătoămeasureătheăserumăantioxidantăstatusăofă subjectsăthanăindividualămeasurementăofăantioxidants.ă TAAăwasădeterminedăbasedăonătheă6-hydroxy2,5,7,8-tetramethylchroman-2ăcarboxylicăacidă(Trolox)ă equivalentăantioxidantăcapacityă(TEAC)ăassayădevelopedă byăMillerăet al.ă[15],ăwithăminorămodificationsă[16].ă Theă TEACă assayă measuresă theă relativeă abilitiesă ofă antioxidantsă ină plasmaă oră otheră biologicală fluidsă toă scavengeătheă2,2’-azino-bisă(3-ethylbenzothiazoline-6sulfonică acid)ă (ABTS)ă radicală cationă (ABTS+)ă ină comparisonăwithătheăantioxidantăpotencyăofăstandardă amountsă ofă Trolox,ă theă wateră solubleă vitamină Eă analog.ă Theă ABTS+ă radicală wasă generatedă fromă theă interactionăbetweenăABTSăandăpotassiumăpersulphate.ă Solutionă containingă ABTS+ă wasă addedă toă theă serumă samplesăandătheăabsorbanceăwasăreadăafteră1ăminăată 734ă nmăagainstăaăreagentăblankă(preparedăwithă5ămMă phosphateăbufferătreatedăasătheăsample).ăTheăpercentageă inhibitionă ofă theă absorbance,ă whichă isă directlyă proportională toă theă antioxidantă activityă ofă theă sample,ă wasă calculated.ă Theă assayă wasă calibratedă againstă aă calibrationă curveă withă Troloxă asă standard,ă andă theă resultsăwereăexpressedăasămmol/LăTrolox.ă SERUMăăRESIDUALăăANTIOXIDANTăăACTIVITYă (“ANTIOXIDANTăăGAP”;ăăGAP)ă Theă principală antioxidantsă (byă massă andă activity)ă ofă humană plasmaă areă albumină andă urică acid,ă whichă accountă foră 51–57%ă ofă theă totală antioxidantă activityă [15,17].ă Antioxidantă gapă reflectsă theă combinedă activityă ofă otheră extracellulară antioxidantsă andă wasă calculatedă byă subtractingă theă antioxidantă activityă ascribableă toă albumină andă urică acidă fromă theă TAAă valueă foră eachă sampleă accordingătoătheăformula:ăă GAPă =ă TAAă –ă [(Albumineă ×ă 0.69)ă +ă urică acid]ă where:ă 0.69ă isă theă TEACă valueă foră humană serumă albumină whileă 1.0ă isă theă TEACă valueă foră serumăuricăacidă(GAPă=ăserumăresidualăantioxidantă activity;ă TAAă =ă serumă totală antioxidantă activity;ă Albumină =ă serumă albumină concentrationă (expressedă asămmol/L);ăUricăacidă=ăserumăuricăacidăconcentrationă (expressedă asă mmol/L)).ă Theă resultsă wereă expressedă asămmol/LăTroloxăactivity.ă TOTALăăPLASMAăăFREEăăTHIOLSăăLEVELă Foră theă estimationă ofă totală –SHă groupsă contentă aliquotsă ofă plasmaă wereă mixedă withă MarilenaăGîlc ăet al.ă 292ă ă phosphateă bufferă (pHă =ă 8)ă andă 10%ă sodiumă dodecylă sulphate.ă Thenă Ellmană reagentă wasă addedă andă samplesă wereă incubatedă ată 37Că foră oneă hour.ăă Theă absorbanceă wasă readă ată 412nmă againstă aă reagentă blank.ă Resultsă wereă calculatedă usingă aă calibrationă curveă withă GSHă asă standardă andă wereă expressedăasă…mol/găproteină[18].ă Erythrocyte cell free thiols (glutathione)ăwereă determinedă onă erythrocyteă lysateă afteră precipitationă ofăproteinsăusingăaăprecipitatingăsolutionăcontainingă metaphosphorică acid,ă disodiumă ethylenediamineă tetraacetică acidă andă sodiumă chloride.ă Theă mixtureă wasăallowedătoăstandăforă5ăminutesăandăthenăfiltered.ă 0.3MăsodiumăphosphateăsolutionăandăEllmanăreagentă wereă thenă addedă toă theă filtrateă andă theă absorbanceă wasă readă ată 412ă nm.ă Resultsă areă calculatedă usingă aă calibrationă curveă withă GSHă asă standardă andă areă expressedărelativeătoăhemoglobinăcontentă[19].ă Plasma uric acid, plasma total proteins, plasma albumină wereă measuredă usingă standardă laboratoryă techniquesă (kită Humană Gesellschaftă füră Biochemicaă undăDiagnosticaămbH,ăWiesbadenăGermany).ă ă 4 STATISTICS Theăresultsăwereăexpressedăasămeană±ăstandardă erroră ofă theă meană (SEM).ă Theă dataă wereă analyzedă usingă GraphPadă Instată andă GraphPadă Prism4ă (Sană Diego,ăCA,ăUSA)ăstatisticalăanalysisăprogram.ăAllătheă valuesă wereă normallyă distributed.ă Theă statisticală significanceă ofă differencesă betweenă individuală valuesă wasă assessedă byă Students’să pairedă t-test.ă Differencesă wereăconsideredăstatisticallyăsignificantăatăpă<ă0.05.ăă RESULTS Theă statisticală analysisă foră theă comparisonsă betweenă theă twoă momentsă isă shownă ină Tableă I.ă Significantă differencesă observedă wereă asă follows.ă Erythrocytesă membraneă stabilityă measuredă asă C50AAPHă(mM)ăandăcatalasaăactivitytăwereăsignificantlyă increasedă (pă <ă 0.01,ă respectivelyă pă <ă 0.05)ă afteră Geriforteăsupplementation.ăThereăwereănoăsignificantă differencesă ină serumă levelsă ofă theă individuală antioxidantsăbeforeăandăafterăGeriforteăadministration.ăăă Table I MedicinalăplantsăcontainedăinăGeriforteătabletsă Extracts mg/tb mg/tb 100mgă 13.8mgă Ashvagandhaă(Withania somnifera)ă Shatavariă(Asparagus racemosus)ăă 30mgă 20mgă Kasaniă(Cichorium intybus)ă Daruharidraă(Berberis aristata)ă 13.8mgă 10mgă Mandukaparniă(Centella asiatica)ă Haritakiă(Terminalia chebula)ă 20mgă 15mgă Vasakaă(Adhatoda vasica)ăă Arjunaă(Terminalia arjuna)ăă Biranjasiphaă(Achillea millefolium)ă Kasamardaă(Cassia occidentalis)ăă 10mgă 6.4mgă 3.2mgă 3.2mgă Vriddadaruă(Argyreia speciosa)ă Musaliă(Asparagus adscendens)ă Udakiryakaă(Caesalpinia digyna)ăă Bhringarajaă(Eclipta alba)ă Jatiphalamă(Myristica fragrans)ă Kapikachchuă(Mucuna pruriens)ă Jaatipatreeă[Myristica fragransă(Mace)]ă Pippaliă(Piper longum)ă Kumkumaă(Crocus sativus)ă Yawaniă(Carum copticum)ă 10mgă 10mgă 10mgă 10mgă 10mgă 10mgă 10mgă 10mgă 7mgă 5mgă Jyothishmatiă(Celastrus paniculatus)ă 5mgă Haridraă(Curcuma longa)ă 5mgă DISCUSSION Theăpresentăstudyăindicatedăstrongăcytoprotectiveă activityăofăGeriforteăinăerythrocytesăagainstăoxidativeă challengeă inducedă byă AAPH.ă Theă hydrophilică azoă compoundă (AAPH)ă usedă ină thisă studyă isă knownă toă decomposeă spontaneouslyă ină theă aqueousă regionă ată 37ºCă intoă carbon-centeredă radicalsă that,ă ină turn,ă ă Powders Chyavanprashăconcentrateă Himsraă(Capparis spinosa)ă produceă peroxylă radicalsă ină theă cellă membranesă leadingătoătheăhemolysisă[20].ăAfterăsupplementationă withă Geriforte,ă theă C50-AAPHă valueă significantlyă increased,ă indicatingă aă higheră resistanceă ofă erythrocytesăagainstăAAPH-inducedăoxidation.ăă Theăerythrocyteăhasăseveralărobustăsystemsătoă protectă itselfă againstă oxidativeă damageă andă hemolysis,ă includingă CATă andă SOD,ă whichă mightă beăoverwhelmedăbyăaălargeăoxidantăinsultălikeăthată 5ă 293 ProtectionăofăerythrocyteăandăGeriforte otheră naturală antioxidantsă thată haveă beenă revealedă toă protectă biologicală membranesă againstă AAPHă inducedă oxidativeă damageă [21,22],ă oură resultsă mightă beă correlatedă withă thisă abilityă ofă Geriforteă ingredientsătoăinteractăwithătheăerythrocyteămembraneă oră differentă cytoplasmică components,ă otheră thană SODăorăevenăCAT.ăTheălackăofăcorrelationăbetweenă C50-AAPHă andă CATă activityă isă supportedăă alsoă byă theă factă thată AAPHă doesă notă passă theă membrane,ă bută generatesă reactiveă radicalsă outsideă theă cells,ă whichă reactă withă moleculesă ină proximityă toă theă siteă ofă generationă (e.g.ă membraneă lipidsă oră proteins)ă[12].ă inducedă byă AAPH.ă Althoughă erythrocyteă CATă valuesă wereă significantlyă increasedă afteră Geriforteă supplementation,ă noă significantă changesă wereă detectedă ină theă erythrocyteă SODă values,ă whenă comparedă withă initială values.ă Moreover,ă noă correlationă wasă foundă betweenă C50-AAPHă valuesă andăCATăactivity.ăTherefore,ăneitherătheăincreaseăofă CATă activityă oră SODă activityă cană explaină theă Geriforteă protectionă againstă AAPHă inducedă hemolysis.ăTakingăintoăaccountăthatăGeriforteăingredientsă (e.g.ăPhyllantus emblica, Withania somnifera, Centella asiatica, Tinospora cordifolia,ă Curcuma longa,ă zincă oxide,ăetc.)ăareăpotentialăsourcesăofăflavonoidsăandă ă Table II InfluenceăofăGeriforteăonăerythrocyteămembraneăstabilityăandăantioxidantăagentsăinăhealthyăsubjectsă Parameter Before the intervention with Geriforte (n= 37) Average ± SEM After the intervention with Geriforte (n =37) Average ± SEM Student’s test P Erythrocyteămembraneăstabilityăă C50-AAPHă(mM)ă Erythrocyteăcatalaseăă K/găHb/mlă Totalăplasmaăfreeăthiolsă µmol/mgăproteine/mlă Erythrocyteăfreeăthiolsă (glutathione)ăµmol/găHb/mlăă Serumătotalăantioxidantăactivityă (TAA)ămMăTroloxă Serumăresidualăantioxidantăactivityă (GAP)ămMăTroloxă ErythrocyteăSODăactivityăU/găHbă 0.0439ă±ă0.0069ă 0.1291ă±ă0.0396ă P<0.05ă 265.745ă±ă15.768ă 352.329ă±ă18.480ă P<0.01ă 7.135ă±ă0.450ă 7.318ă±ă0.276ă n.s.ă 221.268ă±ă10.248ă 206.751ă±ă8.951ă n.s.ă 1.440ă±ă0.019ă 1.432ă±ă0.026ă n.s.ă 0.652ă±ă0.031ă 0.629ă±ă0.052ă n.s.ă 403.243ă±ă37.74ă 368.436ă±ă16.601ă n.s.ă SEMă–ăstandardăerrorăofătheămean;ăn.s.ă–ănon-significantă Ană increaseă ină serumă TAAă andă GAPă wereă expectedădueătoătheăfollowingăfacts:ă 1) Antioxidantă activityă ofă variousă Geriforteă ingredients:ă vită Că ină Phyllantus emblica [23],ă curcuminăinăCurcuma longa [22],ă sitoindosidesă VII–XăandăwithaferinăAăinăWithania somniferaă [24,25],ăetc.ă 2) AAPH,ă whichă isă hydrophilic,ă generatesă radicalsă inătheăaqueousăregionă(e.g.ăserum,ăplasma),ăwhileă hydrophilică antioxidants,ă suchă asă vitamină Că andă uricăacid,ăwhichăareăwellăknownăasăscavengersăofă radicalsă ină thisă aqueousă phase,ă suppressă theă oxidationăinitiatedăwithăAAPHă[26].ă Paradoxically,ă allă theă antioxidantsă evaluatedă ină theă plasma,ă includingă TAA,ă GAP,ă totală plasmaă freeă thiolsă showedă noă significantă changesă withă Geriforteăsupplementation.ă Aă newă reviewă ofă theă literatureă concerningă Geriforteăpropertiesăsuggestedăusăaăpossibleăexplanation.ă ă Ită hasă beenă alreadyă showedă theă selectiveă influenceă ofăGeriforteăonădifferentătissueăorămolecularătargets.ă Foră instance,ă Geriforteă producesă lesseră changeă ină extrahepatică(e.g.ălungs)ăthenăinăhepaticăantioxidantă defense,ă maybeă dueă toă theă factă thată herbală constituentsă haveă firstă undergoă moreă oră lessă metabolismă byă liveră ină theă caseă ofă orală routeă ofă administrationă beforeă findingă theiră wayă intoă theă systemicăcirculationă[27].ăEvenăinătheăsameăorgan,ă Geriforteă hasă specifică effectsă onă theă differentă antioxidantă agentsă (e.g.ă increasesă theă activityă ofă hepaticăcatalase,ăGPXăandăSOD,ăbutăhasănoăchangeă inătheăhepaticăactivityăofăglutathioneăreductaseăandă hepaticălevelăofăreducedăglutathione).ă Oură results,ă suggestingă ană increaseă ină erythrocyteă bută notă plasmaă antioxidantă defense,ă correlatedă withă literatureă data,ă suggestă aă possibleă differentialăaffinityăofăGeriforteăphytochemicalsăforă certaină cellulară compartmentsă andă biomoleculesă 294ă ă MarilenaăGîlc ăet al.ă (e.g.ă membrane,ă CAT).ă Ită wasă proposedă thată AAPHă enhancedă lipidă peroxidationă ină cellulară systemsă andă oxidationă ofă methionine,ă tyrosine,ă andă tryptofană residuesă thată leadsă toă theă alterationă ofă surfaceă hydrophobicityă andă precipitationă ofă proteinsă [28,29].ă Therefore,ă hydrophobică structuresă (e.g.ă membranes)ă representăpotentialătargetsăofăAAPH-inducedăoxidation.ă Geriforte,ă whichă reducesă theă susceptibilityă towardsă AAPH-ă inducedă damage,ă bută doesă notă modifyă theă majoră plasmaă hydrophilică antioxidantsă (glutathione,ă uricăacid,ăalbumin),ămightăhaveăaăspecialăaffinityăforă certainăhydrophobicăstructures.ăă Oură studyă supportsă theă conceptă thată theă consumptionă ofă naturală antioxidantă supplementsă cană beă associatedă withă improvementă ină antioxidantă defenseă mechanisms.ă Weă suggestă thată theă aboveă 6 resultsăcontributeătoătheăreportedăpositiveăneurologicală healthăbenefitsăofăGeriforteăadministration.ă Nevertheless,ătheămainăquestionăisăstillăwithoută answer:ăDoesăGeriforteăprolongătheălifeăspan?ăă Althoughă Geriforteă cană selectivelyă enhanceă certainăantioxidantădefenseămechanismsăandăreduceă theă erythrocyteă biomembraneă oxidativeă damage,ă ită isă notă cleară yetă ifă thisă herbominerală geriatrică tonică managesătoăextendălifeăspan.ăă Furtheră workă involvingă theă characterizationă ofă theă membraneă bindingă andă membraneă actionăă ofă Geriforteă ingredientsă isă clearlyă warranted.ăă Also,ă additională studiesă areă requiredă toă findă theă effectiveă doseă andă durationă foră whichă Geriforteă mayă beă takenă orallyă toă bringă clinicală anti-ageingă effectsăinăhumans.ă Studiul prezent a investigat influenţa Geriforte, un remediu natural indian, asupra sistemelor antioxidante eritrocitare şi plasmatice la subiecţi umani sănătoşi. A fost evaluată deasemenea capacitatea Geriforte de a inhiba liza eritrocitelor indusă de dihidroclorura de azo-bis 2-amidinopropan (AAPH). Suplimentarea cu Geriforte creşte activitatea catalazei eritrocitare (265.745 ± 15.768 vs 352,329 ± 18.480 K/g Hb/ml, p<0.01) şi reduce citotoxicitatea mediată de radicali liberi indusă via AAPH, care a fost estimată prin intermediul stabilităţii membranare eritrocitare (0.0439 ± 0.0069 vs 0.1291 ± 0.0396 C50- AAPH (mM), p<0.05). Intervenţia nu a modificat semnificativ activitatea superoxide dismutazei eritrocitare şi concentraţiile plasmatice ale agenţilor antioxidanţi. Rezultatele indică faptul că Geriforte posedă activitate citoprotectoare în cazul eritrocitelor stresate oxidative şi deasemenea o activitate antioxidantă specifică ce implică în special sistemele protectoare intracelulare şi membranele. ă ă ă Corresponding author: Assist.ăProf.ăMarilenaăGîlc ,ăMD,ăPhD,ăă BiochemistryăDepartment,ăFacultyăofăMedicineă“CarolăDavila”ăUniversity,ă 8,ăBd.ăEroilorăSanitari,ăBucharest,ă050471,ăRomaniaă E-mail:ă[email protected]ă REFERENCES 1. 2. 3. 4. 5. 6. 7. ă BECKMANăK.B.,ăAMESăB.N.,ăThe free radical theory of aging matures.ăPhysiologicalăReviews,ă1998,ă78ă(2):ă547–81.ă AGOSTONă V.,ă CSERMELYă P.,ă PONGORă S.,ă Multiple weak hits confuse complex systems: a transcriptional regulatory network as an example.ăPhys.ăRev.ăE.ăStat.ăNonlin.ăSoftăMatterăPhys.,ă2005,ă71:ă1–8.ă CSERMELYă P.,ă AGOSTONă V.,ă PONGORă S.,ă The efficiency of multi-target drugs: the network approach might help drug design.ăTrendsăPharmacol.ăSci.,ă2005,ă26:178–82.ă KOEHLERăK.F.,ăHELGUEROăL.A.,ăHALDOSENăL.A.,ăWARNERăM.,ăGUSTAFSSONăJ.A.,ăReflections on the discovery and significance of estrogen receptor β.ăEndocrineăReviews,ă2005,ă26(3):ă465–78.ă BARDHANăP.,ăSHARMAăS.K.,ăGARGăN.K.,ăEffect of Geriforte in vivo and in vitro on age related enzyme change in liver and brain of rats.ăInd.ăJ.ăExp.ăBiol.,ă1985,ă23:ă323–26.ă SINGHă B.,ă SHARMAă S.P.,ă GOYALă R.,ă Evaluation of Geriforte, a herbal geriatric tonic, on antioxidant defense system in Wistar rats.ăAnn.ăNYăAcad.ăSci.,ă1994,ă717:ă170–3.ă BANSALă A.,ă SAIRAMă M.,ă PRASADă D.,ă SHARMAă S.K.,ă ILLAVAZHAGANă G.,ă KUMARă D.,ă SELVAMURTHYă W.,ă Cytoprotective and immunomodulatory properties of Gerifote, a herbomineral preparation, in lymphocytes.ă Phytomedicine,ă 2001,ă6(86):ă438–44.ă 7ă 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 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Inhibition of oxidation of soybean phosphatidylcholine and methyl linoleate in aqueous dispersions by uric acid.ăBull.ăChem.ăSoc.ăJpn.,ă1986,ă59:471–77.ă PATHANIAăV.,ăSYALăN.,ăHUNDALăM.K.,ăKHANDUJAăK.L.,ăGeriforte stimulates antioxidant defense system.ăInd.ăJ.ăExp.ă Biol.,ă1998,ăAprilă(36):ă414–17.ă MIKIăM.,ăTAMAIăH.,ăMINOăM.,ăYAMAMOTOăY.,ăNIKIăE.,ăFree-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by α-tocopherol.ăArch.ăBiochem.ăBiophys.,ă1987,ă258:ă373–80.ă CHIEN-CHUNGă C.,ă YAN-SHANă M.A.,ă EARLă R.ă STADTMAN,ă Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems.ăProc.ăNatl.ăAcad.ăSci.ăUSA,ă1997,ă94:ă2969–74.ă ReceivedăMayă25,ă2009ă ă ă ă ă ă ă ă ă 295 ă 296ă ă ă ă ă ă ă MarilenaăGîlc ăet al.ă 8 CASE REPORTS Young Woman with Polyserositis, Ovarian Cystic Mass and Increased Level of CA-125. Case Report of Peritoneal and Pleural Tuberculosis C. JURCU 1, CRINA FILIŞAN1, CLAUDIA POPOVICI2, LIANA TOMA3, B. PANAITE4, O. NICODIN4, I. COPACI1 1 3rd Internal Medicine Department, 2 Pneumology Department, 3 Pathology Department, 4 Gynecology Department, “Dr. Carol Davila” Central Clinical Emergency Military Hospital, Bucharest, Romania A 21-year old woman was addressed to our department for progressive abdominal swelling, fatigue and fever. The clinical examination, the ultrasound examination and the computed tomography showed the presence of polyserositis (ascites and pleural effusion) and revealed a cystic mass at the level of right ovary. The laboratory work-up showed an increased level of CA-125, suggesting a malignancy. The thoracoscopy with visualization of the pleura revealed disseminated small white spots. The laparoscopic exploration of the pelvis and of the peritoneum also showed the same disseminated lesions and a cystic-like mass at the level of the right ovary which was excised and diagnosed as a benign cyst. At the analysis of the frozen and paraffin sections, the diagnostic of pleural and peritoneal tuberculosis was made and the specific quadruple treatment was started with a good evolution at two months and with the normalization of the CA-125 level. This case report underlines the importance of tuberculosis in the differential diagnosis of patients with polyserositis and increased levels of CA-125. Key words: peritoneal tuberculosis, pleural tuberculosis, ascites, ovarian cystic mass, CA-125. CASE REPORT A 21-year old woman was addressed to our department for progressive abdominal swelling, fatigue, fever and dry cough beginning in the last 2 weeks before admissions. She was treated with a short-course of empiric antibiotic therapy, but with the persistence of fever. Subsequently, an ultrasound examination performed upon her GP indication revealed the presence of ascites. At presentation she was very anxious. The appetite was normal and there was no history of weight loss. She had no past history of any diseases. There was no family history of tuberculosis or other infectious, inflammatory or genetic diseases or any cancers. The physical examination revealed a distended abdomen due to ascites, discretely tender at the level of right iliac fossa. Dullness and the diminution of the pulmonary sounds were noted at the lower zones of both lungs. She had fever (up to 38.8°C). There were no palpable lymph nodes, no hepatomegaly or splenomegaly and the bowel sounds were audible. ROM. J. INTERN. MED., 2009, 47, 3, 297–299 The standard thoracic X-ray revealed the presence of the bilateral pleural fluid without other lesions. The ultrasound examination showed the moderate ascites and a cystic-like mass at the level of right ovary. The genital examination and the intravaginal ultrasound confirmed the cystic-like lesion of the right ovary, the ascites and the thickening of the pelvic peritoneum. The computed tomography confirmed also the bilateral pleural effusion and the presence of moderate ascites, as well as the cystic lesion of the right ovary, without abdominal or pelvic adenopathies. The laboratory work-up revealed a mild anemia (Hbţ10.2 g/dL), a mild thrombocytosis (602 000/mm3), monocytosis and lymphopenia, an increased C-reactive protein (96 mg/L) and fibrinogen (937 mg/dL), negative rheumatoid factor, negative antinuclear factor. The serology for hepatitis B and C virus and for HIV were negative. An increased level of CA-125 was noted (286.1 UI/mL; normal values Ţ 35 U/mL). The analysis of the ascites and pleural fluid showed exudate features, with an increased number C. Jurcu et al. 298 of the lymphocytes without malignant cells and without acid fast bacilli at the Ziehl-Nielsen stain. The bronchoscopy examination was unremarkable, with no acid fast bacilli at the Ziehl-Nielsen stain of the aspirate. The thoracoscopy with visualization of the pleura revealed disseminated small white deposits suggestive of carcinomatosis or miliary. A pleural biopsy was performed. The laparoscopic exploration of the pelvis and of the peritoneum revealed disseminated miliary lesions, a normal uterus and the cystic-like 2 mass at the level of right ovary which was excised and diagnosed as being a benign cyst. At the analysis of the frozen and paraffin sections, the diagnostic of pleural and peritoneal tuberculosis was made (Fig. 1) and the specific quadruple treatment was started (Isoniazid, Rifampicine, Pyrazinamide and Ethambutol). The evolution at two months was excellent with the resolution of the ascites and of the pleural fluid and with the normalization of CA-125 level (23.1 UI/mL). She will continue the same treatment, according to standard recommendations. Fig. 1. – Peritoneal tuberculous granuloma with lymphocytes, epithelioid cells, Langhans giant cell surrounding the central caseous necrosis (Hematoxylin and eosin stain, original magnification 20×). DISCUSSION The cancer antigen 125 (CA-125) is a “clasical” marker used for the diagnosis and the follow-up of the patients with ovarian carcinoma. However, increased values of the CA-125 were reported in other malign or benign conditions [1]. There are some reported isolated cases and small series of patients with tuberculous peritonitis and concomitent increased levels of CA-125 [2][3]. In the study of Mas et al, the levels of CA-125 were increased in 10 patients with proven tuberculous peritonitis, and these levels were significantly lower after 4-month of antituberculous treatment [4]. The median value of CA-125 in patients with peritoneal tuberculosis was 331 U/ml in the cohort of 10 patients with peritoneal tuberculosis of Bilgin et al. [5], but values higher than 1000 U/ml were also cited [6]. In our patient, the presence of a benign ovarian cyst was coincidental and created differential diagnosis difficulties. Some authors [2][5][6] reported cases of peritoneal tuberculosis mimicking ovarian cancer, underlying the importance of the correct differential diagnosis. Thus, the diagnosis of tuberculosis was established in 1.2% of women suspected to have ovarian cancer [6]. Therefore, even if the diagnosis of peritoneal tuberculosis is rare among suspected women, it must be keept always in mind especially in developing countries with an increased incidence of tuberculosis, as well as in imunocompromised patients. The diagnosis is usually established at pathological examination after taking biopsies during laparoscopy or laparotomy performed in the gynecology 3 Peritoneal and pleural tuberculosis clinics. However, the place of the internist might be important, some of the patients being initially referred to internal medicine clinics because of systemic features, like fever or polyserositis. CONCLUSIONS In patients with peritoneal tuberculosis the values of CA-125 were reported to be increased and to normalize after the specific treatment. The diagnostic 299 of peritoneal tuberculosis must be kept in mind in women with ascites and increased levels of CA-125. The diagnostic needs a multidisciplinary approach, but is establish at the pathological examination of the biopsies specimens obtained by laparoscopy or laparotomy. Some of the patients are initially reffered to internal medicine clinics because the systemic symptoms. Therefore, the internists should be aware of the diagnostic of peritoneal tuberculosis and of the conditions that may increase the CA-125 levels. Pacienta, în vârstǎ de 21 de ani, a fost îndrumatǎ cǎtre serviciul nostru pentru creşterea progresivǎ în volum a abdomenului, fatigabilitate şi febrǎ. Examenul clinic, explorarea ultrasonograficǎ şi tomografia computerizatǎ pun în evidenţǎ poliserozita (lichid de ascitǎ şi la nivel pleural) şi prezenţa unei formaţiuni chistice la nivelul ovarului drept. Un nivel crescut al CA-125 este evidenţiat la bilanţul de laborator, sugerând o posibilǎ etiologie neoplazicǎ. Toracoscopia cu vizualizarea pleurei relevǎ multiple leziuni albicioase de mici dimensiuni diseminate la nivel pleural. Explorarea laparoscopicǎ a pelvisului şi peritoneului evidenţiazǎ leziuni similare. Formaţiunea chisticǎ ovarianǎ a fost excizatǎ şi diagnosticatǎ ca fiind de origine benignǎ. Examenul histopatologic al fragmentelor pleurale şi peritoneale stabileşte diagnosticul de leziuni tuberculoase. Evoluţia clinicǎ a pacientei a fost favorabilǎ la douǎ luni de cvadruplǎ terapie tuberculostaticǎ, cu normalizarea valorilor CA-125. Cazul subliniazǎ faptul cǎ tuberculoza este un element important în diagnosticul diferenţial al pacienţilor cu poliserozitǎ şi valori crescute ale CA-125. There are no conflicts of interest to be disclosed. Corresponding author: C. Jurcu , MD, PhD Student 3rd Internal Medicine Department, “Dr. Carol Davila” Central Clinical Emergency Military Hospital, Str. Mircea Vulcanescu 88, Bucharest, Romania Tel: +40213193051, Fax: +40213193079 E-mail: [email protected] REFERENCES 1. 2. 3. 4. 5. 6. SEVINC A., ADLI M., KALENDER M.E., CAMCI C., Benign causes of increased serum CA-125 concentration. Lancet Oncol 2007; 8:1054–1055. NISTAL DE PAZ F., HERRERO FERNÁNDEZ B., PÉREZ SIMÓN R., FERNÁNDEZ PÉREZ E., NISTAL DE PAZ C., ORTOLL BATTLE P. et al., Pelvic-peritoneal tuberculosis simulating ovarian carcinoma: report of three cases with elevation of the CA 125. Am. J. Gastroenterol., 1996; 91:1660–1661. UZUNKOY A., HARMA M., HARMA M., Diagnosis of abdominal tuberculosis: experience from 11 cases and review of the literature. World J. Gastroenterol., 2004; 10:3647–3649. MAS M.R., CÖMERT B., SAĞLAMKAYA U., YAMANEL L., KUZHAN O., ATEŞKAN U. et al., CA-125; a new marker for diagnosis and follow-up of patients with tuberculous peritonitis. Digest Liver Dis., 2000; 32:595–597. BILGIN T., KARABAY A., DOLAR E., DEVELIOĞLU O.H., Peritoneal tuberculosis with pelvic abdominal mass, ascites and elevated CA 125 mimicking advanced ovarian carcinoma: a series of 10 cases. Int. J. Gynecol. Cancer, 2001; 11:290–294. KOC S., BEYDILLI G., TULUNAY G., OCALAN R., BORAN N., OZGUL N. et al., Peritoneal tuberculosis mimicking advanced ovarian cancer: a retrospective review of 22 cases. Gynecologic Oncology, 2006; 103:565–569. Received June 18, 2009 298 C. Jurcu et al. 2 Incidental Findings During ENT Routine Examination for Head and Neck Trauma DIANA ZANFIR1, SABINA ZURAC2, FLORICA STĂNICEANU2, B. ANDREESCU3, A. REBOŞAPCĂ3 1 Department of ENT, “Grigore Alexandrescu” Hospital, Bucharest, Romania Department of Pathology, “Colentina” University Hospital, Bucharest, Romania 3 Department of Plastic Surgery, “Colentina” University Hospital, Bucharest, Romania 2 Several patients with head and neck disorders are incidentally diagnosed during clinical examination for traumatic events. Our study refers to 76 patients with head and neck trauma evaluated in the emergency ward of Phonoaudiology and ENT Functional Surgery dr Dorin Hociota Institute in 2007. During routine ENT consultation, 13 patients were identified with trauma unrelated lesions: nasal septum deviations (5 cases), basal cell carcinomas (3 cases), lentiginous malignant melanoma, keratoacantoma, branchial cyst, nasal lobular capillary hemangioma, squamous cell carcinoma of the tongue (one case each). We strongly recommend ENT examination in patients with head and neck trauma not only for establishing the gravity and the extend of the traumatic lesions in the forensic approach but also for revealing unknown underlying disease in some cases with incredible results for the well-being of the patient. Key words:head and neck trauma, incidental findings, nasal septum deviation, skin tumor. Head and neck trauma represents one of the most frequent location of a traumatic event and a major cause of death or debilitating disease due to mechanic injuries. However, even in major trauma, most of the lesions of the cephalic extremity are minor injuries, most often not requiring any ENT or oral surgical treatment [1]. In these circumstances, especially in severe trauma, ENT exam is postponed until life-threatening lesions are stabilized. Due to minor symptoms, frequently spontaneously resolved during recovery for other lesions, the patient will skip the head and neck examination. Our study underlies the opportunity of an ENT complete examination that may disclose unknown lesions, some of them with heavy impact onto the prognosis of the patient and the quality of its life. MATERIAL AND METHODS Our study refers to 76 patients reported in 2007 at the emergency ward of Phonoaudiology and ENT Functional Surgery dr. “Dorin Hociota” Institute for head and neck trauma. We analyzed several parameters: age, sex, cause of trauma, complexity of the trauma, location of the head and neck injuries, severity of the traumatic lesions, trauma unrelated lesions. We ROM. J. INTERN. MED., 2009, 47, 3, 301–305 studied also the association between the location of the traumatic injuries, severity of the symptoms and the location of the trauma unrelated lesions. Statistical analysis was performed – Chi test (χ test) – with a level of statistical significance of P < 0.05. Whenever it was necessary Fisher distribution was used to ensure an extrapolation to a more homogeneous distribution of the cases. The statistical calculations were performed using EXCEL and EPIINFO programs. RESULTS Most of patients were injured through heteroaggression, few of them during car crashes and one patient in a work accident (Table I). There was a female predilection (male to female ratio = 1:2) (Table II). The female predilection may be related to the main cause of the trauma, i.e. heteroaggression some of them in family environment, as previously reported [2]. Most of the patients were young (63.16% of the patients being 20–39 years old), also in conjecture with cause of the trauma (Table III). Two thirds of the patients presented head and neck limited trauma (67.11% of the cases, Table IV). There are two possible explanations for this finding: either the heteroaggression as main cause of the Diana Zanfir et al. 302 trauma associates a head and neck likelihood of the injury location or the pre-presentation selection of the patients considering the specific of the institute and the gravity of the associated lesions (a severe injured patient with multiple traumatic injuries with head and neck, chest and abdominal location will be referred to an emergency department with multi disciplinary possibilities of diagnosis and treatment and not to an ENT hospital). Table I – – – – – 2 3 patients with basal cell carcinoma of the left temporal area, right nasal wing, right inferior eyelid (3.95%) one patient with lentiginous malignant melanoma of the left malar area, Breslow index of 0,42 mm, Clark score II (1.32%) one patient with a branchial cyst (1.32%) one patient with a lobular capillary hemangioma (pyogenic granuloma) of the nasal mucosa (1.32%) one patient with a squamous cell carcinoma of the tongue (1.32%) Cause of trauma Cause of trauma Heteroaggression Number of cases 63 Car crash 12 Work accident Total 1 76 Table V % 15.79% 1.32% – Table II Sex distribution Cause of trauma Number of cases % Male 25 Female Total 51 76 32.89% 67.11% – Anatomic area Facial area Temporal bone, petrous part Number of cases 62 11 Cervical region 2 Two areas associated 1 Total 76 Age (years) Number of cases % 20–29 30–39 40–49 50–59 60–69 Over 70 Total 26 22 10 7 8 3 76 34.21% 28.95% 13.16% 9.21% 10.53% 3.95% – Moderate (controlled with nonsteroid anti inflammatory drugs) Severe (controlled with opioid drugs) Total Trauma complexity Polytraumatisms 25 Total 76 14.47% 2.63% 1.32% – Number of cases % 45 59.21% 28 3 76 36.84% 3.95% – Soft tissue lesions Table IV Number of cases 51 81.58% Severity of the lesions Pain Mild (analgesics not required) Age distribution % Table VI Signs and symptoms Table III Complexity of the case ENT area limited lesions Location of the injury 82.89% Swellings 76 Bruises Erosions Hematoms 59 16 5 Deep cuts (fatty tissue) 5 6.58% 6.58% 59.21% Bone lesions % 67.11% Present 45 32.89% – Absent 31 Total 76 Most of the patients presented facial lesions (81.58%, Table V) with mild symptoms and lesions (Table VI). 13 patients presented trauma unrelated lesions: – 5 patients with nasal septum deviation (6.58%) – one patient with keratoacantoma of the left temporal area (1.32%) 100.00 % 77.63% 21.05% 40.79% – The presence of nasal septum deviation was far more frequent in our group of study. 56 patients were diagnosed with this lesion, but in most of the cases (40 patients) the posttraumatic nature of the illness was easily traceable (severe edema and fracture of the nasal bones). At the opposite part of the spectrum there were 5 patients with nasal septum 3 303 ENT incidental findings in trauma patients deviation without any lesion, no matter how mild, in the nasal area; also, they denied the nasal location of the trauma (ocular region, frontal area, temporal area and lateral mandibular area in two patients were acknowledged as site of trauma). However, there were quite a significant number of patients (11 cases, 19.64% of the patients with nasal septum deviation) presenting both nasal septum deviation and mild trauma of the nose. In these cases it is very difficult to establish the pretraumatic or post-traumatic nature of the septum deviation and we preferred to exclude them from our study. The five patients with skin tumors were referred to plastic surgeons. The lesions were resected, the final diagnosis being established after histopathologic diagnosis. Four of the cases were tumor lesions with low impact on the prognosis of the patients, at least in the moment of the diagnosis (3 cases of basal cell carcinomas and one keratoacantoma). However, one patient presented an irregular pigmented lesion on left cheek clinically suspicious for a dysplastic nevus; after surgical resection, the tumor was histopathologically diagnosed as lentiginous malignant melanoma with minute areas of vertical invasion within the papillary dermis (Clark score II) (Fig. 1); the overall prognosis of the patient is favorable, considering the Breslow index of 0.42 mm – thin melanoma; the patient is well, without any recurrences or metastases, 23 months after resection. The patient with the branchial cyst presented abnormal slightly fluctuent tumefaction in the lateral cervical area. He was a victim of a street altercation and he received one direct blow in the face with subsequent fracture of the nasal bones, but no trauma of the neck was acknowledged. An open excision biopsy of the lesion was performed, the histopathological diagnosis being that of a branchial cyst (Fig. 2). The patient with the pyogenic granuloma of the nasal mucosa was also the victim of a heteroaggression in domestic environment with multiple lesions in the facial area (bruises of the left eye, left cheek and nose and several erosions of the left cheek). The trauma was less than 24 h old. During complete ENT examination a small cherrylooking nodule (1.2 cm in the biggest diameter) was identified adherent of the nasal mucosa covering the vestibular part of the septum; the lesion looked ulcerated in the surface and bled easily when touched. It was unclear if the nodule was secondary to trauma but the size and presence of ulceration were arguments in favor of a preexisting lesion. The nodule was surgically removed; the histopathologic examination revealed a pyogenic granuloma (lobular capillary hemangioma) (Fig. 3) that was obvious preexisting to the traumatic event. No supplementary burden from the legal point of view was added to the situation of this patient. The most impressive case was that of a 42 years old male, victim of a car crash. His head injuries were minor (mild pain and tumefaction of the face) but the examination of the oral cavity reveal a white area adherent to the underlying mucosa (leucoplakia) on the left lateral margin of the tongue. The lesion was slightly elevated with irregular surface. Based on his history of heavy smoking (30 cigarettes a day for the last 19 years) and moderate alcohol consumption, a biopsy was performed revealing microinvasive squamous carcinoma (Fig. 4). An MRI of the neck was perfomed without revealing enlarged nodules. A tongue partial resection with tumor invasion up to 1.1 cm from the nearest lateral resection margin and 0.4 cm from the deepest resection margin was performed. Clean surgical margins were present in respect of dysplastic lesions. The patient was referred to a Radiotherapy Department for further treatment. He is free of disease 2 years and 9 month after first diagnosis. Most of the trauma unrelated lesions were present in the same location as the traumatic ones (Table VII); however, 3 cases (almost one quarter of the cases) had lesions incidentally discovered in nontraumatized areas: the patient with keratoacanthoma, the patient with branchial cyst and the patient with squamous cell carcinoma of the tongue who presented with cervical, facial and ear trauma, respectively. Table VII Location of the trauma unrelated lesions versus the location of the trauma Location of the trauma unrelated lesions in the area of the trauma outside the area of the trauma Total Number of cases 10 3 13 % 76.92% 23.08% – We compared the severity of the traumatic lesions in patients with trauma unrelated lesions with severity of the traumatic lesions in the whole group (Table VIII). No association was present Diana Zanfir et al. 304 between the severity of the pain and presence of trauma unrelated lesions (P chi test 0.07), the severity of the soft tissue lesion and presence of trauma unrelated lesions (P chi test 0.76) or the presence of fractures and presence of trauma unrelated lesions (P chi test 0.14). Table VIII Severity of the traumatic lesions in patients with trauma unrelated lesions Signs and symptoms Number of cases % Mild (painkillers not required) 12 Moderate (controlled with nonsteroid anti inflammatory drugs) Severe (controlled with opioid drugs) Total 1 92.31% Pain 0 13 7.69% 0.00% – Swellings 13 Bruises 6 100.00% Erosions 2 Hematoms 1 Deep cuts (fatty tissue) 0 Soft tissue lesions Bone lesions Present 0 Absent 13 Total 13 46.15% 15.38% 7.69% 0.00% 0.00% 100.00% – DISCUSSION Several patients with head and neck disorders are incidentally diagnosed during clinical examination for different complaints. One of these situations is the ENT examination in forensic medicine onset. Some patients are diagnosed with severe diseases previously unsuspected or unreported by the patient [3][4]. In our group, five patients presented skin tumors; diagnosing these lesions was mere consequence of patient presentation in a medical institution and not ENT examination related. Eight of our patients benefit from the ENT consultation because their previously unknown illnesses were diagnosed during specific maneuvers 4 performed by ENT doctor. We included in this group 5 patients with nasal septum deviation. This lesion is usually difficult to analyze because there are no clear cut criteria to link it to the current trauma or to establish its occurrence before the traumatic event. For this reason we decide to include in patients with trauma unrelated lesions group the patients with nasal septum deviation without any evidence of trauma in the nasal area, no matter how mild. It is obvious that a patient with severe swelling and bruises of the nose and maybe fracture of the nasal bones has a trauma related nasal septum deviation while a patient with mild edema of the nose has a nontraumatic nasal septum deviation. Based on these principles, it is more likely that the real incidence of nasal septum deviation not related to the traumatic event is actually higher than we considered [5–7]. These cases underline the importance of a complete ENT examination in trauma. There is not any correlation between the location or the signs or symptoms of the traumatic and nontraumatic lesions, but there were several cases where the nontraumatic lesions were present in patients with mild symptoms and different location to that of the trauma. Their discovery was possible only due to a complete ENT screening not required by the current clinical onset. The type of the lesion incidentally discovered during ENT exam for head and neck trauma revealed 6 malignancies or tumors with uncertain behaviour, two of them with extreme impact on the prognosis of the patient (malignant melanoma and squamous cell carcinoma of the tongue). The other 4 tumors (keratoacanthoma and basal cell carcinomas) grow slowly; basal cell carcinomas represent a source of aesthetic impairment and invasive potential in the deep structures, imposing disfiguration surgical excisions to accomplish complete excision [8–10]. We strongly recommend ENT examination in patients with head and neck trauma, not only for establishing the gravity and the extend of the traumatic lesions in the forensic approach, but also for revealing unknown underlying disease in some cases with incredible results for the well-being of the patient. Mai mulţi pacienţi cu afecţiuni ale capului şi gâtului sunt diagnosticaţi întâmplător în cursul examinării clinice pentru evenimente traumatice. Studiul nostru se referă la 76 de pacienţi cu traumatisme ale capului şi gâtului evaluaţi la camera de gardă a Institului de Fonoaudiologie şi Chirurgie Funcţională ORL 5 ENT incidental findings in trauma patients 305 dr. Dorin Hociotă în 2007. În cursul examinării ORL de rutină au fost identificaţi 13 pacienţi cu leziuni netraumatice: 5 cazuri de deviaţii de sept nazal, 3 carcinoame bazocelulare şi câte un pacient cu melanom malign lentiginos, keratoacantom, hemangiom capilar lobular nazal şi carcinom scuamocelular de limbă. Recomandăm examinarea ORL la pacienţii cu traumatisme de cap şi gât, nu numai pentru stabilirea gravităţii şi extinderii leziunilor traumatice din punct de vedere medicolegal, dar şi pentru identificarea unor afecţiuni anterior ignorate de către pacient, în unele cazuri cu rezultate incredibile pentru starea de sănătate a acestuia. Corresponding author: Diana Zanfir Department of ENT, “Grigore Alexandrescu” Hospital 30, Iancu de Hunedoara Street, Bucharest, Romania E-mail: [email protected] REFERENCES GASSNER R., TULI T., HÄCHL O., RUDISCH A., ULMER H., Cranio-maxillofacial trauma: a 10 year review of 9,543 cases with 21,067 injuries J. Craniomaxillofac. Surg., 2003; 31:51–61. 2. 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