Ramazan ÇETINKAYA, Ali Rıza ODABAŞ, Yılmaz SELÇUK, Esin AKTAŞ, Fatih AKÇAY

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1 17 KRONİK HEMODİYALİZ HASTALARINDA PRİMER RENAL HASTALIKLARIN PLAZMA HOMOSİSTEİN DÜZEYLERİNE ETKİLERİ EFFECTS OF PRIMARY RENAL DISEASE ON PLASMA HOMOCYSTEINE CONCENTRATION IN CHRONIC HEMODIALYSIS PATIENTS Ramazan ÇETINKAYA, Ali Rıza ODABAŞ, Yılmaz SELÇUK, Esin AKTAŞ, Fatih AKÇAY Departments of Nephrology, Microbiology and Biochemistry, Ataturk University, School of Medicine, Erzurum, Turkey Özet Amaç: Ateroskleroz için bağımsız ve potansiyel bir risk faktörü olan hiperhomosisteinemi (hhom), son dönem böbrek yetmezlikli (SDBY) hastalarda sıktır. SDBY inde hhom nin temel nedeni homosistein metabolizmasındaki bozukluktur. Çalışmamızda primer böbrek hastalıklarının kronik hemodiyaliz hastalarında hhom de etkilerinin olup olmadığını araştırmayı amaçladık. Metod: Çalışmaya 103 kronik hemodiyaliz hastası alındı. Hastalar primer böbrek hastalıklarına göre 8 gruba ayrıldı. İki aylık folik asid ve B 12 tedavisi öncesi ve sonrası total plazma Hcy (tphcy), serum vitamin B 12 ve serum folik asid (FA) düzeyleri ölçüldü. Gruplara göre tphcy düzeyleri ve tedavi etkinlikleri açısından fark olup olmadığı incelendi. Bulgular: Tedavi öncesi tphcy 24.4±6.7 µmol/l bulundu. Gruplar arasında istatistiksel anlamlı farklılık tesbit edilmedi (p= 0,497). Tedavi sonunda tphcy düzeyleri istatistiksel olarak anlamlı bir şekilde azaldı. (16.2±4.5 µmol/l, p<0.0001). Tedavi etkinliklerinde farklılık izlenmedi (p=0.669). Sonuç: Kronik hemodiyaliz hastalarında homosistein düzeyleri ve hiperhomosisteinemi tedavisinin etkinliği primer etyolojilere bağlı olarak değişmemektedir. Bütün kronik hemodiyaliz hastaları primer böbrek hastalıklarına bakılmaksızın tedavi edilmelidir. Anahtar kelimeler: Hemodiyaliz, Hiperhomosisteinemi, Primer böbrek hastalığı Summary Background: Hyperhomocysteinemia (hhcy) is prevalent in more than 85% of patients with end-stage renal disease (ESRD) and is an independent and potential risk factor for atherosclerotic cardiovascular disease. The main cause of hhcy in ESRD is abnormalities of homocysteine (Hcy) metabolism. The aim of this study was to verify the effects of primary renal disease (PRD) on plasma total homocysteine (pthcy). Methods: The study included 103 chronic hemodialysis patients. The patients were divided in to 8 groups according to PRD. pthcy, serum folic acid (FA) and serum vitamin B 12 concentrations were measured before therapy and after 2 months FA (6 mg/day) and B 12 (2 mg/week) therapy. The levels of pthcy were examined according to the groups and whether there was a difference with regard to treatment effectivity. Results: pthcy before treatment was found as 24.4±6.7 µmol/l. A statistically significant difference with regard to pthcy between the groups was not found (p= 0,497). At the end of the treatment, tphcy decreased to statistically significant levels (16.2±4.5 µmol/l, p<0.0001). A significant difference among treatment activities was not observed (p=0.669). Conclusions: pthcy and the effectiveness of the treatment of hhcy in chronic hemodialysis patients have not changed depending on PRD. All chronic hemodialysis patients should be treated without considering PRD. Key words: Hemodialysis, Hyperhomocysteinemia, Primary renal disease AÜTD 2003; MJAU 2003;

2 18 AÜTD 2003 Table 1. Demographic and Clinical Characteristics of Patients groups N sex (f/m) age (years) duration of HD (months) CPN 25 (%24.3) 19/ ±14.4 (24-74) 51.60±41.19(11-149) CGN 24 (%23.4) 10/ ±13.33 (15-65) 46.87±46.44(4-200) DNP 15 (%14.6) 9/ ±12.85 (21-72) 22.86±22.20 (8-80) HT 10 (%9.8) 5/ ±8.53 (41-65) 39.80±33.75 (13-108) amyl 8 (%7.8) 3/ ±12.85(22-65) 21.62±15.75 (8-46) OUP 7 (%6.7) 1/ ±15.2 (27-71) 16.85±5.11 (10-24) PCKD 7 (%6.7) 3/ ±8.86 (44-70) 47.71±41.10 (6-124) other 7 (%6.7) 5/ ±8.29 (41-65) 28.71±18.67 (9-54 total 103 (%100) 55/ ±13.91 (15-74) 38.85±36.66 (6-200) CPN: chronic pyelonephritis, CGN: chronic glomerulonephritis, DNP: diabetic nephropathy, HT: hypertension, Amyl: amyloidosis, OUP: obstructive uropathy, PCKD: polycystic kidney disease Introduction Cardiovascular complications are the leading cause of death in patients with end stage renal disease (ESRD). There are several risk factors for accelerated atherosclerosis in ESRD. These factors include lipid abnormalities, haemostatic disorders and hyperhomocysteinemia (hhcy). Various studies show that hhcy is an independent and putative risk factor for atherosclerotic coronary heart disease, stroke and peripheral vascular disease (1-3). In ESRD the frequency of hhcy is much greater than in normal population. hhcy occurs in nearly 85% of patients undergoing maintenance hemodialysis (HD) and is associated with increased morbidity and mortality. hhcy is present in approximately 5% of the normal population and 40% of the patients with atherosclerosis who are not in renal failure. An increase of 1 µmol/l of total Hcy propagates the risk of atherosclerosis by 1% in ESRD patients (4,5). Major determinants of elevated pthcy in chronic renal failure (CRF) patients include deficiencies in folate and vitamin B 12, methylenetetrahydrofolate reductase (MTHFR) genotype and renal function. Consequently, it is suggested that administration of folic acid (FA), vitamin B 6 and vitamin B 12 may decrease homocysteine. There is a powerfully negative correlation between Hcy and glomerular filtration rate. In ESRD patients, the protein-bound fraction of Hcy generally elevates but Hcy sulfhydryl may not be elevated (6,7). Diabetic glomerulosclerosis, hypertensive nephrosclerosis, primary glomerular, vascular, systemic and cystic diseases may cause CRF. Nevertheless, it is not know that these diseases influence Hcy and Hcy lowering therapy (8,9). In this study our aim was to verify the effects of PRD on pthcy concentration before and after treatment of hhcy. Materials and Methods One hundred and three chronic HD patients (55 females, 48 males, mean age: ±13.91 years and HD durations: 38.85±36.66 months) undergoing HD at Ataturk University Hospital and Erzurum State Hospital HD Centers were included in the study. The patients were dialyzed for 4 hours three times weekly, using a low-flux dialyser of cellulosynthetic membrane (Hemophane, Kavasumi Laboratories, Japan), glucose-containing dialysate with bicarbonate, blood flow between ml/min, and a dialysate flow of 500 ml/min. The patients were divided into 8 groups according to PRD. The renal diagnoses were chronic pyelonephritis (n=25), chronic glomerulonephritis (n=24), diabetic nephropathy (n=15), hypertensive nephrosclerosis (n=10), amiloidosys (n=8), reflux nephropathy (n=7), polycystic disease (n=7) and others (n=7). Table 1 shows characteristics of patients. No patient was on FA, vitamin B 12 and vitamin B 6 supplementation during the 4-week period before starting the study. The nature, purpose and potential risks of the study were explained to all patients before study and local Ethics Committee approved the study protocol. In all patients, pthcy, serum FA and serum vitamin B 12 were determined before therapy and 2 months after FA (6 mg/day) and B 12 (2 mg/week) therapy. Serum albumin (SA), and Kt/V values were obtained in the onset of therapy. Before therapy period, the levels of pthcy according to the groups were examined. Then, in all groups, the levels of pthcy and treatment effectivity were compared 2 months after therapy. The relationship between the levels of pthcy and SA, FA, vitamin B 12, and Kt/V values was examined. Venous blood samples were collected at a morning clinic after an overnight fast. All samples were taken from non-fistula arm and on the same day of the week, which, in the HD patient, was prior to the first dialysis of the week. The sample intended for Hcy analysis was immediately put on ice and centrifuged within 15 min, and the plasma stored at 20 0 C until analysis. pthcy was measured by enzyme immunoassay method (Axis Homocysteine EIA, Axis- Shield AS). The normal values of pthcy ranged from 5

3 19 Table 2. The Effects of FA and Vitamin B12 on pthcy Levels According to Groups groups before treatment tphcy (µmol/l) after treatment tphcy (µmol/l) p CPN 24.5±7.4 ( ) 16.8±4.2 ( ) < CGN 25.4±6.2 ( ) 16.5±4.7 ( ) < DNP 23.6±6.1 ( ) 15.8±4.6 ( ) =0.001 HT 23.5±3.4 ( ) 16.3±2.3 ( ) =0.005 amyl 19.7±5.2 ( ) 12.3±2.6 ( ) =0.01 PCKD 26.6±8.4 ( ) 15.7±2.6 ( ) =0.01 OUP 23.7±5.9 ( ) 17.3±5.2 ( ) =0.02 other 27.0±10.1 ( ) 17.6±8.0 ( ) =0.01 total 24.4±6.7 ( ) 16.2±4.5 ( ) < to 15 µmol/l. FA and B 12 concentrations in plasma were measured by chemiluminometric method (ACS 180). The normal range for plasma folate concentrations was nmol/l, and for vitamin B pmol/l. Blood urea nitrogen (BUN) was measured on fresh blood using enzymatic methods. SA was measured by colorimetric method (ADVİA 1650 Bayer). Single-pool Kt/V was estimated according to Daugirdas second-generation formula (10): Kt/V of treatment = Ln (R x t) + (4 3.5 x R) x UF/W ([R= post-dialysis BUN / pre-dialysis BUN], t= the time of duration dialysis session (hours), UF= total ultrafiltration on dialysis session (L), W= post-dialysis patient body weigh). Statistics Statistical analyses were performed using the statistical package SPSS 10.0 for Windows. Key subject characteristics and parameters are presented as mean, standard deviation (SD) and 95% confidence interval. Difference in the mean value of the groups was evaluated with the Student s t-test, Kruskall-Wallis H test, Wilcoxon rank sum test and Mann-Whitney U test. Spearman rank correlation coefficients were determined to study the association between variables. Multiple linear regression analysis test was used to study the association between pthcy and possible determinants. Values of P<0.05 were considered to be statistically significant. Results tphcy Concentrations pthcy before treatment was found as 24.4±6.7 µmol/l. A statistical significant difference with regard to pthcy before treatment between the groups was not found (p= 0,497). After treatment, pthcy levels reduced to 16.2±4.5 µmol/l by a decrease of % It was also possible to indicate that pthcy levels after treatment were significantly lower than the values before treatment (p<0.0001). In all groups, pthcy levels after treatment were lower than the values before treatment significantly (Table2). No difference was observed among the groups with respect to treatment activity (p=0.669). Serum Folic Acid and Vitamin B 12 Levels Serum FA level was measured as 7.23±3.0 ng/ml before treatment and as 19.4±5.2 ng/ml after treatment (p<0.0001). There was no significant difference between the levels of FA in groups before and after the treatment (p=0.5, p=0.720, respectively). The levels of vitamin B 12 were found 340.3±132.4 pmol/l before treatment and 561.2±293 pmol/l after treatment (p<0.0001). There was no significant difference between the levels of B 12 in groups during both before and after treatment (p=0.603, p=0.960, respectively). Effective Parameters on tphcy Levels A strong negative correlation was found between SA levels [3.78±0.41 ( ) g/dl] and tphcy levels [24.4±6.7 ( ) µmol/l] (r=-0.250,p=0.01). There was also another expressive negative correlation between Kt/V 1.33±0.17 value and tphcy levels 24.4±6.7 µmol/l (r=-0.536, p<0.0001). A meaningful negative correlation was found between Serum FA levels and pthcy levels (r= , p<0.0001). In addition, a significant negative correlation was also identified between serum vitamin B 12 and tphcy (r= , p<0.0001). FA, vitamin B12 and Kt/V values were found as the predictive factors on tphcy levels in multiple linear regressions analyze (p<0.000,p=0.001, p<0.0001, respectively). Discussion In the ESRD population, multiple metabolic and hemodynamic abnormalities adversely affect endothelial function. In addition, irreversible structural

4 20 AÜTD 2003 vascular disease already may be present. hhcy is one of the significant factors, which spoils the endothelial functions. Current models favor direct angiotoxicity involving endothelial and vascular smooth muscle cells, and impaired thrombolysis. The treatment of hhcy should probably be an integral part of an endothelial protective regimen, consisting of lipidlowering agents, antihypertensives and antioxidant vitamins (11-13). FA, vitamin B 12 and other B vitamins, which are used in treatment of hhcy, are acting as the co-factors of the enzymes in Hcy metabolism. Remethylation and transsulphuration are the most important two metabolic pathways in Hcy metabolism. In ESRD patients, both pathways have been affected (7,11). It was pointed out that a decrease of approximately 30-40% in Hcy levels was obtained when FA is used solely or in any combination in various experiments. These drugs, mainly FA, have reduced such disorders in endothelial functions (14-16). In our study, we applied a treatment of oral 6 mg/day FA and intra-muscular 2-mg/week vitamin B 12 to chronic HD patients following a wash out period of 4 weeks. After treatment, it was noticed that a decrease of approximately 33.6% was obtained in pthcy levels with respect to basic values. Such reduction was expressive in statistical mean and an effective treatment was achieved. Although the treatment was effective, the completely normal pthcy levels with respect to the reference values could not be obtained accordingly. In various studies, normohomosisteinemia was notable to be obtained despite an effective treatment (14,17). ESRD may occur depending upon various reasons. Is it possible to say that the PRD causing CRF may affect Hcy metabolism except the improved kidney failure? The numbers of prospective studies, which were executed under this question, are so limited. The studies carried out in patients with diabetic nephropathy have shown that the increase in Hcy quantities has a direct relation with GFR (18,19). A significant relation between pthcy levels and GFR was also proved in patients with nephrotic syndrome depending upon various primary reasons (20). In another study carried out in patients with idiopathic membranous glomerulonephritis, a similar result was obtained (21). However, such studies do not provide sufficient and definite information on the effectiveness of primary diseases. In addition, no study, which was carried out to prove if there was any difference in treatment effectiveness compared with the primary disease, was noted in literature. In our study, it was researched whether the PRD have any contribution on pthcy levels as an independent factor from renal insufficiency and if there was any difference in achievement of hhcy treatment with regard to PRD. No significant difference was noted between both in basic pthcy levels and in pthcy levels after treatment in 8 different ESRD patient groups whom we have arranged in accordance with the PRD. In each group, pthcy levels after treatment were found lower significantly than basic pthcy levels. On the other hand, there was no difference between the groups with respect to the treatment effectiveness. Consequently, the fundamental effective factor in hhcy improvement could be the metabolic and hemodynamic differences occurred depending upon directly kidney insufficiency. In a similar way, it could be also possible to identify a result that the PRD do not have significant roles on hhcy treatment effectiveness. A significant negative correlation was found between serum FA, vitamin B 12 and pthcy levels in our study. At the same time, serum FA and vitamin B 12 levels were identified as the factors having predictive values for Hcy levels. There was no significant difference between serum FA and vitamin B 12 levels in each group in periods after and before treatment. pthcy levels increase gradually depending upon respective GFR in patients with kidney disorders in pre-dialysis period. In such term, when the effect of HD and peritoneal dialysis on pthcy levels was searched, it was found that the patients in peritoneal dialysis had lower pthcy levels compared with the ones, who were under treatment to reduce the Hcy levels. (22-24). It was identified that a significant negative correlation was present between Kt/V, which is an indicator of HD adequate, and having a predictive value on pthcy. We have also found that an expressive negative correlation was present between SA levels and pthcy levels but SA levels did not have any predictive value on pthcy. Finally, the treatment of hhcy in ESRD patients could be achieved effectively by supplying FA and vitamin B 12. PRD was not found as an effective factor on both pthcy levels and treatment of hhcy. On the other hand, it was found that there was a negative and meaningful relation between Kt/V, serum FA, serum vitamin B 12, SA, and tphcy concentrations. In addition, Kt/V, serum FA and serum vitamin B 12 were found as the predictive factors for tphcy. References 1. Oishi K, Nagake Y, Yamasaki H, Fukuda S, Ichikawa H, Ota K, Makino H. The significance of atherogenic indices in patients on hemodialysis. Am J Nephrol 2000;20: Suliman ME, Lindholm B, Barany P, Bergstrom J. HHcy in chronic renal failure patients: relation to nutritional status and cardiovascular disease. Clin Chem Lab Med 2001;39:

5 21 3. Alfthan G, Aro A, Gey KF. Plasma homocysteine and cardiovascular disease mortality [Letter]. Lancet. 1997;349: Jungers P, Joly D, Massy ZA, et al. Sustained reduction of hyperhomocysteinaemia with folic acid supplementation in predialysis patients. Nephrol Dial Transplant 1999;4: Sunder-Plassmann G, Fodinger M, Buchmayer H, et al. Effect of high dose folic acid therapy on hhcy in hemodialysis patients: Results of the Vienna Multicenter Study. J Am Soc Nephrol 2000;11: Miner SES, Evrovski J, Cole DEC. Clinical chemistry and molecular biology of homocysteine metabolism: an update. Clinical Biochemistry 1997;30: Henning BF, Riezler R, Tepel M, et al. Evidence of altered homocysteine metabolism in chronic renal failure. Nephron 1999;83: Lazarus JM, Brenner BM. Chronic renal failure. In: Fauci AS, Braunwald E, Isselbacher KJ et al. (eds); Harrison s Principles of Internal Medicine 14 th edit. The Mc Graw Hill Companies, 1998; 2: Luke GR. Chronic renal failure.in: Goldman L, Bennett JC (eds); Cecil Textbook of Medicine 21 th edit. Philadelphia, W.B. Saunders Company, 2000: Daugirdas JT. Second generation logarithmic estimates of singlepool variable volume Kt-V: An analysis of error. J Am Soc Nephrol 1993; 4: Mallamaci F, Zoccali C, Tripepi G, et al. Hyperhomocysteinemia predicts cardiovascular outcomes in hemodialysis patients. Kidney Int 2002; 61: Wrone EM, Zehnder JL, Homberger JM, Mc Cann LM, Coplon NS, Fortmann SP. An MTHFR variant, homocysteine, and cardiovascular comorbidity in renal disease. Kidney Int 2001; 60: Mezzano D, Pais EO, Aranda E, et al. Inflammation, not hyperhomocysteinemia, is related to oxidative stress and hemostatic and endothelial dysfunction in uremia. Kidney Int 2001; 60: Ashfield-Wat PAL, Moat SJ, Doshi SN, McDowell IFW. Folate, homocysteine, endothelial function and cardiovascular disease. What is the link? Biomed Pharmacother 2001; 55: Mattson MP, Kruman II; Duan W. Folic acid and homocysteine in age related disease. Ageing Research Reviews 2002; 1: De Vriese AS, Verbeke F, Schrijvers BF, Lameire NH. Is folate a promising agent in the prevention and treatment of cardiovascular disease in patients with renal failure. Kidney Int 2002; 61: Moat SJ, Doshi SN, McDowell IFW. Folate, homocysteine, endothelial function and cardiovascular disease. What is the link? Biomed Pharmacother 2001;55: Oishi K, Nagake Y, Yamasaki H, et al. The significance of serum homocysteine levels in diabetic patients on haemodialysis. Nephrol Dial Transplant 2000;15: Gonzales-Clemente JM, Deulofeu R, Mitjavila J, Gloria Galdon. Plasma homocysteine is not increased in microalbuminuric patients with type 2 diabetes without cardiovascular disease. Diabetes Care 2002;25: Joven C, Arcelus R, Camps J et al. Determinants of plasma homocysteine in patients with nephrotic syndrome. J Mol Med 2000;78: Arnodottir M, Hultberg B, Berg AL. Plasma total homocysteine concentration in nephrotic patients with idiopatic membranous nephropathy. Nephrol Dial Transplant 2001;16: Ducloux D, Heuze-Lecornu L, Gibey R, Bresson-Vautrin C, Vautrin P, Chalopin Jm. Dialysis adequacy and homocysteine concentrations in peritoneal dialysis patients. Nephrol Dial transplant 1999; 14: Moustapha A, Gupta A, Robinson K et al. Prevelance and determinants of hyperhomocysteinemia in hemodialysis and peritoneal dialysis. Kidney Int 1999; 55: Arnodottir M, Berg AL, Hegbrant J, Hultberg B. Influence of haemodialysis on total homocysteine concentration. Nephrol Dial Transplant 1999; 14: Correspondence: Ramazan ÇETİNKAYA Atatürk Üniversitesi Postanesi PK: , Erzurum, Türkiye ramazancetinkaya@yahoo.com Phone: /1093 Fax: