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Volume: 4 Issue: 1 June 2006


Determinants of Fasting Total Serum Homocysteine Levels in Liver Transplant Recipients

Objectives: Homocysteine (HCY) is a sulfur-containing amino acid considered to be a marker for a relative folate deficiency. Hyperhomocysteinemia is a known risk factor for development of cardiovascular disease, vascular dementia, depression, and possibly some carcinogeneses. Liver transplant recipients have an increased risk for cardiovascular disease because of a high incidence of obesity, arterial hypertension, diabetes mellitus, and hyperlipidemia. The aim of this study is to elucidate the determinants for hyperhomocysteinemia as an additional risk factor in these patients.

Materials and Methods: Seventy stable liver transplant recipients, 48 men (mean age, 50 ± 11 years) and 22 women (mean age, 52 ± 13 years) had their serum homocysteine levels tested after orthotopic liver transplantation. For mainstay immunosuppression, 53 patients were treated with tacrolimus, 10 with cyclosporine, 3 with mycophenolate mofetil, and 4 with sirolimus. Fasting blood samples were obtained and analyzed immediately (within 1 hour) for total serum homocysteine by high-performance liquid chromatography.

Results: In all patients, mean homocysteine levels were 22.7 ± 14 µmol/L (normal range, 9-15 µmol/L). Forty-six patients were found to have homocysteine levels > 15 µmol/L, and all 70 recipients had homocysteine levels > 9 µmol/mL. In our patients, increased homocysteine levels correlated well with body mass index and renal function. Homocysteine levels in patients receiving cyclosporine were higher than those in patients receiving tacrolimus (22.3 ± 6 vs 17.9 ± 12 µmol/L, P < .05).

Conclusions: Overall, homocysteine levels are significantly increased in liver transplant recipients. Homocysteine levels correlate well with obesity, renal function, and the particular immunosuppressant protocol. Therefore, a specific treatment for patients after liver transplantation (eg, one with folates) might reduce the risk of complications resulting from hyperhomocysteinemia.

Key words : Liver transplantation, Immunosuppressive drugs, Homocysteine, Folate, Obesity

Hyperhomocysteinemia is frequently associated with folate deficiency. Homocysteine is derived from cellular methionine, which is an essential amino acid. Intracellular homocysteine is normally secreted extracellularly at rapid rates. Elevated concentrations of homocysteine in plasma are related to cardiovascular diseases as well as to such cerebrovascular diseases as vascular dementia and mental cognitive dysfunction [1-4]. High homocysteine levels recently have been implicated as a risk factor for cancer and might be considered as a new tumor marker [5] and a proliferation-inducing metabolite in vitro [6].

Homocysteine metabolism is closely associated with folate metabolism. The folate metabolic pathway has been well elucidated [7]. At a specific point in its metabolic cascade, folic acid is essential for the synthesis of S-adenosylmethionine (SAM) and thereby is involved in DNA methylation. Furthermore, folate is metabolized (via dihydrofolate reductase) to dihydrofolate and tetrahydrofolate and reduced to 5-methyltetrahydrofolate (5-MTHF) via 5,10-methylenetetrahydrofolate through 5,10-methylenetetrahydrofolate reductase (MTHFR) [7]. 5-Methyltetrahydrofolate serves as a methyl donor in the remethylation of homocysteine to methionine, which in turn is converted to S-adenosylmethionine. SAM methylates specific cytosines in DNA and therefore regulates gene transcription. Folate (as 5-MTHF) and vitamin B12 are cofactors of methylation and require 2 enzymes, methionine synthase and methyltetrahydrofolate reductase [8]. Renal function seems to play a key role in homocysteine clearance. Impaired renal function leads to elevated homocysteine levels [9].

Liver transplant recipients have an increased risk for cardiovascular disease because of a high incidence of obesity, arterial hypertension, diabetes mellitus, and hyperlipidemia [10]. The atherogenic mechanism of homocysteine has not, as yet, been well defined, but it might be that homocysteine induces oxidative stress leading to consequent injury of the vascular wall and other tissues [11].

The aim of this study was to evaluate homocysteine levels in liver transplant recipients as an interdisciplinary risk factor and to find determinants for elevated fasting homocysteine serum levels.

Materials and Methods

Seventy stable liver transplant recipients, 48 men (mean age, 50 ± 11 years) and 22 women (mean age, 52 ± 13 years) seen regularly at our outpatient clinic were included in this study. Patient demographics are presented in Table 1. Patients were tested for serum homocysteine, on average, 8 ± 6 years after transplantation. Participants provided written informed consent before entering the study, and the protocol was approved by the local ethics committee. The protocol of the study conformed with the ethical guidelines of the 1975 Helsinki Declaration. Patients were treated with different immunosuppressant protocols: For mainstay immunosuppression, 53 patients received tacrolimus, 10 received cyclosporine, 3 received mycophenolate mofetil, and 4 received sirolimus (Table 2). Indications for liver transplantation are summarized in Table 3.

Clinical chemistry
Blood samples were obtained after overnight fasting and immediately (within 1 hour) analyzed for total serum homocysteine according to the manufacturer’s instructions (Immundiagnostik, Bensheim, Germany). In brief, samples were centrifuged at 2000 x g for 10 minutes. After protein denaturation, 20 µL of the probe was injected into a high-performance liquid chromatograph (Merck-Hitachi, Darmstadt, Germany) and subsequently analyzed via Chromeleon version 6.11 (Dionex, Idstein, Germany) [12].

Statistical analyses
Results are expressed as means ± SD. Differences in continuous variables between the 2 groups were analyzed using unpaired t tests. Relationships between serum homocysteine level (the dependent variable) and independent continuous variables were assessed using simple linear regression analyses. Statistical analyses were performed using the computer software Excel (Microsoft Corporation, Redmond, Wash, USA), Winstat (Fitch Software Inc, Staufen, Germany), and SPSS software (Statistical Package for the Social Sciences, version 13.0, SSPS Inc, Chicago, Ill, USA).


Serum homocysteine levels were elevated in most of our liver transplant recipients. All patients tested had homocysteine levels greater than 9 µmol/L, and 66% of these recipients had homocysteine levels greater than 15 µmol/L. There was no correlation between vitamin B12 and/or folate levels when compared with elevated homocysteine levels (data not shown), whereas serum homocysteine levels correlated strongly with serum creatinine concentrations (Table 4). However, homocysteine levels were significantly higher in recipients with a body mass index (BMI) greater than 25 (overweight) when compared with patients whose BMI was less than 25 (Table 5). Recipients with a BMI greater than 25 had homocysteine levels that were twice as high as recipients with a BMI less than 25. Furthermore, homocysteine levels were significantly elevated in recipients receiving cyclosporine and rapamycin compared with those receiving mainstay immunosuppression with tacrolimus (Table 6).


Elevated homocysteine levels were found in all of our patients after liver transplantation. Homocysteine, a sulfur-containing amino acid, is a reliable marker for impaired folate metabolism. There was no correlation found between vitamin B12 and/or folate levels and elevated homocysteine levels in our patients. Elevated homocysteine concentrations are a well-described risk factor for vascular diseases and are an additional important predictor of mortality in patients with coronary heart disease [13]. In transplant recipients, the main causes for late secondary organ loss are vascular diseases and their complications, and the undesirable effects of chronic rejection [14].

Kidney function plays an important role in homocysteine metabolism. Elevated homocysteine levels are frequently observed in patients with impaired kidney function and chronic renal failure [15,16]. Serum creatinine, a commonly used marker, is consistently one of the most reliable determinants of homocysteine in the renal disease population [17-19]. In a previous study of renal transplant recipients, elevated homocysteine levels correlated well with impaired kidney function and increased creatinine levels. It is known that minimal amounts of serum homocysteine are actively excreted through this organ independently of kidney function. Under normal conditions, however, the kidney actively clears and metabolizes plasma homocysteine [20]. Supportive evidence includes the existence of homocysteine-uptake mechanisms and metabolizing enzymes in the proximal renal tubulus, the region in which other amino acids are metabolized and filtered. Creatinine is also linked to homocysteine through the production of creatine, its precursor molecule, by an S-adenosylmethionine-dependent methyl transfer reaction [21]. Impaired renal function might be one cause for homocysteine elevation in our patients, even though an increased BMI (> 25) in liver transplant recipients and other organ recipients has never been suspected of influencing homocysteine serum levels. In a previous study by Jacques and colleagues, performed in nonrecipients, the relation between BMI and homocysteine concentrations suggests that persons with the largest weight-to-height ratio (BMI >= 30.7) had slightly greater serum homocysteine concentrations than did those with a BMI less than 30.7 [22]. Koehler and coworkers [23] also reported a weak positive relation between BMI and homocysteine concentrations, but Lussier-Cacan and colleagues [24] observed no particular association. The Hordaland Homocysteine Study investigators reported a U-shaped association between BMI and homocysteine concentrations that disappeared after adjusting for other determinants of homocysteine concentrations [25]. In a recent publication by Hirsch and colleagues [26], serum total homocysteine was not elevated in patients with a severe nonalcoholic liver disease (NAFLD). This might lead to the conclusion that hyperhomocysteinemia is specific in obese patients after liver transplantation. Prednisone intake, which is a major factor for obesity in liver transplant recipients, did not correlate with homocysteine levels in our patients (data not shown).

Liver transplant recipients treated with cyclosporine had higher plasma homocysteine concentrations than did patients treated with tacrolimus. Independent of cyclosporine levels and individual dosage, it is not known how cyclosporine interferes with homocysteine metabolism. Cyclosporine, which has well-elucidated tubular effects [27], might therefore interfere with the homocysteine clearance and/or catabolism at this level. However, the interference in the transsulfuration pathway might be another cause for increased homocysteine levels in these patients. Overall, rapamycin-treated recipients showed the highest levels of serum homocysteine. In turn, patients given tacrolimus as mainstay immunosuppression showed significantly lower levels of serum homocysteine compared with patients treated with cyclosporine, mycophenolate mofetil, or rapamycin. In the present study, the numbers of patients in the cyclosporine and rapamycin groups were rather low compared with the number of patients in the tacrolimus group. Quiroga and colleagues showed higher homocysteine levels in cyclosporine-treated renal transplant recipients than in patients treated with tacrolimus [28]. Conversely, Fernandez-Miranda and coauthors could not confirm these findings in renal recipients, but could in patients with liver transplants [29, 30].

In our patients, elevated homocysteine levels are a frequent phenomenon obviously resulting from different causes. However, renal function, obesity, and immunosuppressant protocol were determinants for elevated homocysteine levels. Improving renal function, reducing weight, and modifying immunosuppressant protocols might therefore be possible ways to influence increased homocysteine levels in liver transplant recipients, although they are often difficult to achieve.

The importance of homocysteine in developing vascular complications remains unclear. Further studies are needed to determine whether patients with elevated homocysteine levels have different rates of survival compared with recipients with lower levels. Furthermore, it is unknown, if treatments with substances like folic acid are able to change the morbidity and mortality after liver transplantation by lowering total serum homocysteine.


  1. Miller JW, Nadeau MR, Smith J, Smith D, Selhub J. Folate-deficiency-induced homocystinemia in rats: disruption of S-adenosylmethionine’s co-ordinate regulation of homocysteine metabolism. Biochem J 1994; 298 (Pt 2): 415-419
  2. Duthie SJ, Whalley LJ, Collins AR, Leaper S, Berger K, Deary IJ. Homocysteine, B vitamin status, and cognitive function in the elderly. Am J Clin Nutr 2002; 75: 908-913
  3. Fassbender K, Mielke O, Bertsch T, Nafe B, Froschen S, Hennerici M. Homocysteine in cerebral macroangiography and microangiopathy. Lancet 1999; 353: 1586-1587
  4. Schachinger V, Britten MB, Elsner M, Walter DH, Scharrer I, Zeiher AM. A positive family history of premature coronary artery disease is associated with impaired endothelium-dependent coronary blood flow regulation. Circulation 1999; 100: 1502-1508
  5. Wu LL, Wu JT. Hyperhomocysteinemia is a risk factor for cancer and a new potential tumor marker. Clin Chim Acta 2002; 322: 21-28
  6. Akoglu B, Milovic V, Caspary WF, Faust D. Hyperproliferation of homocysteine-treated colon cancer cells is reversed by folate and 5-methyltetrahydrofolate. Eur J Nutr 2004; 43(2): 93-99
  7. Mason JB. Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism. J Nutr 2003; 133: 941S-947S
  8. Cravo M, Fidalgo P, Pereira AD, Gouveia-Oliveira A, Chaves P, Selhub J, et al. DNA methylation as an intermediate biomarker in colorectal cancer: modulation by folic acid supplementation. Eur J Cancer Prev 1994; 3: 473-479
  9. Friedman AN, Rosenberg IH, Selhub J, Levey AS, Bostom AG. Hyperhomocysteinemia in renal transplant recipients. Am J Transplant 2002; 2: 308-313
  10. Johnston SD, Morris JK, Cramb R, Gunson BK, Neuberger J. Cardiovascular morbidity and mortality after orthotopic liver transplantation. Transplantation 2002; 73: 901-906
  11. Stanger O, Weger M, Renner W, Konetschny R. Vascular dysfunction in hyperhomocyst(e)inemia. Implications for atherothrombotic disease. Clin Chem Lab Med 2001; 39: 725-733
  12. Minniti G, Piana A, Armani U, Cerone R. Determination of plasma and serum homocysteine by high-performance liquid chromatography with fluorescence detection. J Chromatogr A 1998; 828: 401-405
  13. Ueland PM, Refsum H, Beresford SA, Vollset SE. The controversy over homocysteine and cardiovascular risk. Am J Clin Nutr 2000; 72: 324-332
  14. Mahony JF. Long term results and complications of transplantation: the kidney. Transplant Proc 1989; 21: 1433-1434
  15. Shemin D, Bostom AG, Selhub J. Treatment of hyperhomocysteinemia in end-stage renal disease. Am J Kidney Dis 2001; 38: S91-S64
  16. Hannedouche TP, Kunz K, Muller S, Chantrel F. Homocysteine and chronic renal failure. Adv Nephrol Necker Hosp 1998; 28: 287-310
  17. Bostom AG, Gohh RY, Beaulieu AJ, Han H, Jacques PF, Selhub J, et al. Determinants of fasting plasma total homocysteine levels among chronic stable renal transplant recipients. Transplantation 1999; 68: 257-261
  18. Ducloux D, Motte G, Challier B, Gibey R, Chalopin JM. Serum total homocysteine and cardiovascular disease occurrence in chronic, stable renal transplant recipients: a prospective study. J Am Soc Nephrol 2000; 11: 134-137
  19. Brattstrom L, Lindgren A, Israelsson B, Andersson A, Hultberg B. Homocysteine and cysteine: determinants of plasma levels in middle-aged and elderly subjects. J Intern Med 1994; 236: 633-641
  20. Friedman AN, Bostom AG, Selhub J, Levey AS, Rosenberg IH. The kidney and homocysteine metabolism. J Am Soc Nephrol 2001; 12: 2181-2189
  21. Brosnan JT, Hall B, Selhub J, Nadeau MR, Bostom AG. Renal metabolism of homocysteine in vivo. Biochem Soc Trans 1995; 23: 470S
  22. Jacques PF, Bostom AG, Wilson PW, Rich S, Rosenberg IH, Selhub J. Determinants of plasma total homocysteine concentration in the Framingham Offspring cohort. Am J Clin Nutr 2001; 73: 613-621
  23. Koehler KM, Romero LJ, Stauber PM, Pareo-Tubbeh SL, Liang HC, Baumgartner RN, et al. Vitamin supplementation and other variables affecting serum homocysteine and methylmalonic acid concentrations in elderly men and women. J Am Coll Nutr 1996; 15: 364-376
  24. Lussier-Cacan S, Xhignesse M, Piolot A, Selhub J, Davignon J, Genest J Jr. Plasma total homocysteine in healthy subjects: sex-specific relation with biological traits. Am J Clin Nutr 1996; 64: 587-593
  25. Nygard O, Vollset SE, Refsum H, Stensvold I, Tverdal A, Nordrehaug JE, et al. Total plasma homocysteine and cardiovascular risk profile. The Hordaland Homocysteine Study. JAMA 1995; 274: 1526-1533
  26. Hirsch S, Poniachick J, Avendano M, Csendes A, Burdiles P, Smok G, et al. Serum folate and homocysteine levels in obese females with non-alcoholic fatty liver. Nutrition. 2005; 1(2): 137-141
  27. Shihab FS. Cyclosporine nephropathy: pathophysiology and clinical impact. Semin Nephrol 1996; 16: 536-537
  28. Quiroga I, Morris-Stiff G, Baboo R, Darby CR, Lord RH, Jurewicz WA. Differential homocysteine levels in renal transplant patients receiving neoral versus tacrolimus. Transplant Proc 2001; 33: 1209-1210
  29. Fernandez-Miranda C, Gomez P, Diaz-Rubio P, Estenoz J, Carillo JL, Andres A, Morales JM. Plasma homocysteine levels in renal transplanted patients on cyclosporine or tacrolimus therapy: effect of treatment with folic acid. Clin Transplant 2000; 14: 110-114
  30. Fernandez-Miranda C, Sanz M, de La Calle A, Loinaz C, Gomez P, Diaz-Rubio P, et al. Determinants of increased plasma homocysteine in 221 stable liver transplant patients. Clin Chem 2001; 47: 2037-2040

Volume : 4
Issue : 1
Pages : 462 - 466

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First Department of Internal Medicine and Gastroenterology, Johann Wolfgang Goethe-University, Theodor Stern Kai 7, D-60596 Frankfurt, Germany
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