Kidney International, Vol. 54 (1998), pp. 1380 –1384
Serum total homocysteine concentration before and after renal transplantation MARGRET ARNADOTTIR, BJO ¨RN HULTBERG, JAN WAHLBERG, BENGT FELLSTRO ¨M, and EMO ¨KE DIM´ENY Department of Medicine, National University Hospital, Reykjavik, Iceland; Department of Clinical Chemistry, University Hospital in Lund, Lund, Departments of Transplant Surgery and Medicine, Uppsala University Hospital, Uppsala, and Department of Medicine, Umeå University Hospital, Umeå, Sweden
Serum total homocysteine concentration before and after renal transplantation. Background. Hyperhomocysteinemia is by now an established risk factor for the development of atherosclerosis. Total homocysteine concentration (tHcy) correlates inversely with glomerular filtration rate, and it is roughly three times as high in hemodialysis patients as in healthy individuals. Therefore, tHcy would be expected to fall markedly after successful renal transplantation. The aim of the present study was to assess the changes in tHcy associated with renal transplantation. Methods. tHcy was analyzed in samples collected before renal transplantation and at six months after transplantation in 55 stable patients, all of whom were treated with cyclosporine (CS). tHcy was also analyzed in samples from 55 controls characterized by markers of renal function that matched those of the posttransplant state. Results. At six months after transplantation, tHcy was significantly decreased as compared with pretransplant tHcy (27.7 6 14.8 vs. 36.9 6 21.3 mmol/liter, P , 0.001). Post-transplant tHcy was markedly higher than the tHcy of the control group (27.7 6 14.8 vs. 16.0 6 5.3 mmol/liter, P , 0.0001). The post-transplant change in tHcy ranged widely, the average change being a reduction of 14%. Sixteen patients (29%) actually manifested an increase in post-transplant tHcy. The post-transplant changes in tHcy correlated inversely with pretransplant tHcy (r 5 20.66, P , 0.0001) and directly with the changes in serum albumin concentrations (r 5 0.35, P , 0.05) and CS trough concentrations (r 5 0.29, P , 0.05). A multivariate analysis, including the posttransplant changes in serum concentrations of folate and albumin as well as creatinine clearances explained 21% of the change in tHcy (P , 0.05). After inclusion of the CS concentration, an independent predictor, the model accounted for 28% of the post-transplant change in tHcy (P , 0.01). Conclusion. The post-transplant reduction in tHcy was far smaller than expected with respect to renal function, and the post-transplant changes in the major biochemical determinants of tHcy contributed relatively little to explain the change in tHcy.
Key words: homocysteine, atherosclerosis, glomerular filtration rate, cyclosporine, endothelial toxicity, platelets and coagulation, end-stage renal disease. Received for publication December 11, 1997 and in revised form April 28, 1998 Accepted for publication April 28, 1998
© 1998 by the International Society of Nephrology
Thus, the results suggest the post-transplant introduction of one or more factors that induce an increase in tHcy. Treatment with CS appears to be such a factor.
Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis according to a few large prospective investigations [1, 2] and many small retrospective studies [3, 4]. The mechanism behind the vascular damage has not been established with certainty, but there is evidence of explanatory factors such as direct endothelial toxicity as well as functional disturbances of platelets and coagulation factors . Hyperhomocysteinemia is frequently observed in patients with renal insufficiency , which is of interest due to the greatly increased mortality in complications to atherosclerosis in this patient category . Homocysteine metabolism in renal failure has been the subject of many investigations . Plasma total homocysteine concentration (tHcy) is significantly increased early in renal insufficiency  and dialysis patients manifest about threefold increase in tHcy as compared with healthy individuals . The mechanisms behind the hyperhomocysteinemia of uremia have not been fully elucidated, but homocysteine removal may be hampered by reduced net renal uptake  and possibly enzyme inhibition . On the other hand, relatively little is known about homocysteine metabolism in renal transplant recipients. Homocysteine concentration was increased in two studies of renal transplant recipients most of which were treated with azathioprine and prednisolone [9, 10]. In one study, tHcy was higher in cyclosporine-treated renal transplant recipients than in patients on azathioprine and prednisolone only, renal function being similar in the study groups . The purpose of the present study was to assess the changes in tHcy associated with renal transplantation in patients treated with cyclosporine (CS). METHODS Patients The renal transplant recipients were consecutively recruited at the Transplant Unit of the University Hospital in
Arnadottir et al: Post-transplant homocysteine concentration Table 1. Characteristics of renal transplant recipients before (pretransplant) and at six months after transplantation (post-transplant) as well as those of renal function-matched controls
Age years Sex (M/F) Serum creatinine mmol/liter Creatinine clearance ml/min Serum albumin g/liter Serum folate nmol/liter B-cyclosporine mmol/liter Cyclosporine dose mg/kg/day Prednisolone dose mg/kg/day Azathioprine dose mg/kg/day
46 6 15 25/30 —
— — 149 6 57
51 6 16 28/27 144 6 86
51 6 21
52 6 16
41 6 5 44 6 61 —
40 6 4 12 6 12 137 6 56
38 6 4 15 6 5 —
4.2 6 1.4
0.14 6 0.04
1.25 6 0.62
Data are given as mean 6 standard deviation.
Uppsala, Sweden. Included in the study were the 55 stable patients who were available for blood sampling six months after transplantation. Demographic data are given in Table 1. Eight patients received a kidney from a living donor. Forty-three had a primary transplantation. The causes of end-stage renal disease were chronic glomerulonephritis (N 5 24), diabetic nephropathy (N 5 8), chronic interstitial nephritis (N 5 6), polycystic kidney disease (N 5 6), nephrosclerosis (N 5 5), and unspecified renal disease (N 5 6). At the time of transplantation, 49 patients were on chronic hemodialysis and 6 patients on continuous peritoneal dialysis. The dialysis patients were generally prescribed low-dose folic acid substitution (0.3 mg daily). A subgroup of 14 patients was taking folic acid in pharmacological doses explaining the high mean serum folate concentration (Table 1). The immunosuppressive protocol consisted of CS, azathioprine and prednisolone according to the Scandinavian Multicenter Trial protocol . The doses of the immunosuppressive drugs are shown in Table 1. In addition, 55 non-nephrotic patients were recruited for control purposes (Table 1). Procedure Fasting blood samples were collected immediately before transplantation and at six months after transplantation. Serum was stored at 220°C for analysis of tHcy in the same series. Before and at six months after transplantation, serum folate and albumin concentrations were analyzed. At six months after transplantation, serum creatinine concentration, endogenous creatinine clearance and trough CS concentration in whole blood were also determined. The control group was selected to match post-transplant markers of renal function. Blood samples for analysis of tHcy were collected as described above. Serum folate,
albumin and creatinine concentrations as well as creatinine clearances were also measured. Laboratory investigations tHcy was measured by high performance liquid chromatography after the reduction of disulfide-bonds by dithiothreitol . Serum creatinine concentration was analyzed by an enzymatic method and endogenous creatinine clearance was calculated after a 24-hour urine collection. Trough CS concentration in whole blood was measured by a radioimmunoassay with monoclonal antibodies (CycloTrac; Incstar Corporation, Stillwater, MN, USA). Serum albumin concentration was analyzed by the bromocresol green method and serum folate concentration by a radioimmunoassay. Statistics The data are presented as mean 6 SD. Due to the skewed distribution of tHcy values nonparametric statistical methods were used; pre- and post-transplant variables were compared by the Wilcoxon’s rank order test for the paired case, post-transplant and control variables were compared by the Mann-Whitney U-test and correlation analyses were performed with the Spearman’s rank correlation test. Multiple regression analyses were performed to identify independent predictors of tHcy. Before inclusion into the multivariate analyses, tHcy was log transformed. RESULTS Serum concentrations of folate, albumin and creatinine, trough CS concentrations in whole blood, and endogenous creatinine clearances are shown in Table 1. Mean pretransplant tHcy was significantly higher than mean post-transplant tHcy (36.9 6 21.3 vs. 27.7 6 14.8 mmol/liter, P , 0.001). The mean change was a reduction in tHcy of 14% but the changes ranged from a reduction of 85% to an increase of 142%. Mean post-transplant tHcy was significantly higher than mean tHcy of the renal function matched control group (27.7 6 14.8 vs. 16.0 6 5.3 mmol/liter, P , 0.0001). There were significant correlations between pretransplant tHcy and post-transplant tHcy (r 5 0.42, P , 0.01; Fig. 1), folate concentrations (r 5 20.38, P , 0.01), and albumin concentrations (r 5 0.43, P , 0.01). Post-transtrant tHcy correlated significantly with folate concentrations (r 5 20.28, P , 0.05), creatinine concentrations (r 5 0.45, P , 0.01) and creatinine clearances (r 5 20.42, P , 0.01) but not with albumin concentrations, CS concentrations or the doses of CS, prednisolone and azathioprine. A multiple regression analysis, including folate and albumin concentrations as well as creatinine clearances, revealed that these variables only explained 23% of posttransplant tHcy (P , 0.05) whereas the same variables
Arnadottir et al: Post-transplant homocysteine concentration
Fig. 1. A scattergram showing the relationship between pre-and posttransplant plasma total homocysteine concentrations (tHcy). The correlation coefficient was 0.42 (P , 0.05).
Fig. 2. A scattergram showing the relationship between the cyclosporine concentrations and the post-transplant changes in tHcy (DtHcy). The correlation coefficient was 0.29 (P , 0.05).
explained 65% of tHcy in the renal function-matched controls (P , 0.0001). The post-transplant changes in tHcy correlated inversely with pretransplant tHcy (r 5 20.66, P , 0.0001), that is, lower post-transplant tHcy values were associated with less post-transplant reductions. The post-transplant changes in tHcy correlated also with the changes in albumin concentrations (r 5 0.35, P , 0.05) and CS concentrations (r 5 0.29, P , 0.05; Fig. 2), but not with the changes in folate concentrations, the creatinine clearances or with the doses of CS, prednisolone and azathioprine. A multiple regression analysis showed that the posttransplant changes in folate (r 5 20.13, NS) and albumin concentrations (r 5 0.44, P , 0.01) as well as the creatinine clearances (r 5 20.27, P 5 0.06) accounted for 21% (P , 0.05) of the post-transplant changes in tHcy. The model explained 28% (P , 0.01) after the inclusion of CS concentrations (r 5 0.30, P , 0.05). tHcy increased in 16 patients (29%) after transplantation. These patients had significantly lower pretransplant tHcy than the others (24.0 6 9.0 vs. 42.0 6 22.6 mmol/liter, P , 0.001). Pre- and post-transplant folate and albumin concentrations, creatinine clearances as well as prednisolone and azathioprine doses did not differ between the groups. However, these 16 patients were receiving higher doses of CS (5.0 6 1.6 vs. 3.8 6 1.2 mg/kg, P , 0.01) and had higher CS concentrations (169 6 70 vs. 124 6 43 mmol/liter, P , 0.05) than the others.
ever, numerically, the decrease was far smaller than expected with regard to renal function. This was illustrated by the marked difference between the mean post-transplant tHcy and the mean tHcy of the renal function-matched control group. Hyperhomocysteinemia is an established cardiovascular risk factor, known to predict morbidity  as well as mortality in the general population . There is an inverse correlation between glomerular filtration rate and tHcy  and hyperhomocysteinemia is highly prevalent in dialysis patients . After successful transplantation renal function is greatly improved and, therefore, a substantial decrease in tHcy would be expected. This, in combination with the fact that renal transplant recipients manifest a markedly increased cardiovascular risk , calls for studies on posttransplant homocysteine metabolism. A few reports have shown increased tHcy in renal transplant recipients [10, 11, 15] and there is evidence that hyperhomocysteinemia is associated with increased prevalence of atherosclerotic complications in these patients [10, 11]. The purpose of the present study was to document the changes in tHcy occurring after renal transplantation. The time point of six months post-transplant was chosen for sampling because by then most patients have reached a stable phase. The mean value of pretransplant tHcy in the present study was similar to the tHcy reported by this and other laboratories, in patients with end-stage renal failure [16, 17]. After transplantation, tHcy only fell by a mean of 14%. This change was statistically significant but the post-transplant tHcy was still much higher than anticipated or almost twice the value in patients with similar function in native
DISCUSSION tHcy was significantly decreased six months after renal transplantation as compared with pretransplant tHcy. How-
Arnadottir et al: Post-transplant homocysteine concentration
kidneys. In addition, in 29% of the renal transplant recipients, tHcy actually increased after transplantation. A multiple regresssion analysis, including traditional biochemical determinants of tHcy, surprisingly showed that the improvement in renal function only had a marginal influence on the post-transplant change in tHcy and that the changes in serum folate concentrations did not have a significant predictive value. On the other hand, the CS concentrations and the changes in serum albumin concentrations were independent predictors of the post-transplant change in tHcy. These results agree with the comparison between the patients who manifested an increase in tHcy and those who showed a decrease in tHcy after transplantation; the only difference between the groups were higher CS concentrations and CS doses as well as lower initial tHcy values in the former group. The direct correlation between the pre- and post-transplant concentrations of tHcy probably reflects the genetic influence on homocysteine metabolism. Not only was the post-transplant decrease in tHcy unexpectedly small, but changes in major determinants of tHcy did not explain more than 21% of the post-transplant change in tHcy. This is in accordance with a prospective study of heart transplant recipients showing that a posttransplant increase in tHcy of 70% could not be adequately accounted for by changes in vitamin concentrations and renal function . Thus, the influence on homocysteine metabolism after transplantation must be sought outside the spectrum of the traditional determinants of tHcy. Possibly, the renal graft does not compensate for all the functions of healthy kidneys or the dialysis treatment and the associated drug treatment may play a role in the context. However, this would not account for the fact that the influence on homocysteine metabolism seems to be similar after heart transplantation and renal transplantation. Therefore, the post-transplant introduction of a factor that influences homocysteine metabolism seems more likely. Conceivably, consequences of immunological reactions or side effects of immunosuppressant(s) are involved. The present study shows an independent relationship between CS concentrations and the post-transplant changes in tHcy. Moreover, in a previous investigation we found that CS-treated renal transplant recipients had significantly higher tHcy than those treated with azathioprine and prednisolone only . In that study, there was no correlation between tHcy and folate concentrations in the CStreated patients, suggesting an effect on the folate-dependent remethylation of homocysteine. This hypothesis is supported by the present finding that the changes in serum folate concentrations did not significantly predict the posttransplant change in tHcy. Even though trough CS concentration is taken into consideration, much of the posttransplant change in tHcy is still unaccounted for. For this purpose, the area under the CS curve may be a better determinant than CS trough concentration, or the other immunosuppressants may play a role. However, in the
present study, no relationship could be found between tHcy and either prednisolone doses or azathioprine doses. Treatment with 5 to 16 mg of folic acid daily lowers tHcy in practically all hemodialysis patients, resulting in a mean decrease in tHcy of about 30 to 50% [16, 17]. In the present study, the change in tHcy varied widely after transplantation, the mean change being a reduction of 14%. Thus, it seems that folic acid administered in high dosage to dialysis patients is a more reliable tHcy-lowering treatment than renal transplantation. However, the present study was limited to one time point after transplantation, that is, six months. Future studies should cover a wider range of time in order to document the natural history of homocysteinemia in this group of patients. In this context, it should be mentioned that high-dose treatment with folic acid also lowers tHcy effectively in cyclosporine-treated renal transplant recipients . Reprint requests to Dr. Margret Arnadottir, Division of Nephrology, Department of Medicine, National University Hospital, IS-101 Reykjavik, Iceland. E-mail: [email protected]
REFERENCES 1. STAMPFER MJ, MALINOW MR, WILLETT WC, NEWCOMER LM, UPSON B, ULLMANN D, TISHLER PV, HENNEKENS CH: A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA 268:877– 881, 1992 2. ARNESEN E, REFSUM H, B¨ ONÅ KH, UELAND PM, F¨ ORDE OH, NORDREHAUG JE: Serum total homocysteine and coronary heart disease. Int J Epidemiol 24:704 –709, 1995 3. UELAND PM, REFSUM H, BRATTSTRO ¨ M L: Plasma homocysteine and cardiovascular disease, in Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function, edited by FRANCIS RB JR, New York, Marcel Dekker, Inc., 1992, pp 183–236 4. BOUSHEY CJ, BERESFORD SAA, OMENN GS, MOTULSKY AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. JAMA 274:1049 –1057, 1995 5. DENNIS VW, ROBINSON K: Homocysteinemia and vascular disease in end stage renal disease. Kidney Int 50(Suppl 57):S11–S17, 1996 6. RAINE AEG, MARGREITER R, BRUNNER FP, EHRICH JHH, GEERLINGS W, LANDAIS P, LOIRAT C, MALLICK NP, SELWOOD NH, TUFVESSON G, VALDERRABANO F: Report on management of renal failure in Europe, XXII, 1991. Nephrol Dial Transplant 7(Suppl 2):7–35, 1992 7. ARNADOTTIR M, HULTBERG B, NILSSON-EHLE P, THYSELL H: The effect of reduced glomerular filtration rate on plasma total homocysteine concentration. Scand J Clin Lab Invest 56:41– 46, 1996 8. BOSTOM A, BROSNAN JT, HALL B, NADEAU MR, SELHUB J: Net uptake of plasma homocysteine by the rat kidney in vivo. Atherosclerosis 116:59 – 62, 1995 9. WILCKEN DEL, GUPTA VJ, BETTS AK: Homocysteine in the plasma of renal transplant recipients: Effects of cofactors for methionine metabolism. Clin Sci 61:743–749, 1981 10. MASSY ZA, CHADEFAUX-VEKEMANS B, CHEVALIER A, BADER CA, DRUEKE TB, LEGENDRE C, LACOUR B, KAMOUN P, KREIS H: Hyperhomocysteinemia: A significant risk factor for cardiovascular disease in renal transplant recipients. Nephrol Dial Transplant 9:1103–1108, 1994 11. ARNADOTTIR M, HULTBERG B, VLADOV V, NILSSON-EHLE P, THYSELL H: Hyperhomocysteinemia in cyclosporine-treated renal transplant recipients. Transplantation 61:509 –512, 1996 12. LINDHOLM A, ALBRECHTSEN D, TUFVESON G, KARLBERG I, PERSSON NH, GROTH CG: A randomized trial of cyclosporine and prednisolone
14. 15. 16.
Arnadottir et al: Post-transplant homocysteine concentration
versus cyclosporine, azathioprine and prednisolone in primary cadaveric renal transplantation. Transplantation 54:624 – 631, 1992 ANDERSSON A, ISAKSSON A, BRATTSTRO ¨ M L, HULTBERG H: Determination of homocysteine and other thiols in human plasma by highperformance liquid chromatography and a thiol-specific postcolumn derivatization procedure. Clin Chem 39:1590 –1597, 1993 NYGÅRD O, NORDREHAUG JE, REFSUM H, UELAND PM, FARSTAD M, VOLLSET SE: Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 337:230 –236, 1997 FOGARTY DG, WOODSIDE J, LIGHTBODY JH, LOUGHREY CM, YARNELL J, YOUNG IS, MAXWELL AP: Plasma homocysteine in renal transplant recipients. (abstract) J Am Soc Nephrol 7:1931, 1996 ARNADOTTIR M, BRATTSTRO ¨ M L, SIMONSEN O, THYSELL H, HULTBERG B, ANDERSSON A, NILSSON-EHLE P: The effect of high dose
pyridoxine and folic acid supplementation on serum lipid and plasma homocysteine concentrations. Clin Nephrol 40:236 –240, 1993 17. BOSTOM AG, SHEMIN D, LAPANE KL, HUME AL, YOBURN D, NADEAU MR, BENDICH A, SELHUB J, ROSENBERG IH: High dose B-vitamin treatment of hyperhomocysteinemia in dialysis patients. Kidney Int 49:147–152, 1996 18. BERGER PB, JONES JD, OLSON LJ, EDWARDS BS, FRANTZ RP, RODEHEFFER RJ, KOTTKE BA, DALY RC, MCGREGOR CGA: Increase in total plasma homocysteine concentration after cardiac transplantation. Mayo Clin Proc 70:125–131, 1995 19. ARNADOTTIR M, HULTBERG B: Treatment with high-dose folic acid effectively lowers plasma homocysteine concentration in cyclosporinetreated renal transplant recipients. (letter) Transplantation 64:1087, 1997