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

Patients
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).

Results

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).

Discussion

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.

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