Nephrology
Dialysis
Transplantation
Original Article
Evidence for disturbed S-adenosylmethionine : S-adenosylhomocysteine
ratio in patients with end-stage renal failure: a cause for disturbed
methylation reactions?
Franziska M. T. Loehrer1, Christian P. Angst1, Felix P. Brunner2, Walter E. Haefeli3,4 and
Brian Fowler1
1Metabolic Unit, University Children’s Hospital Basel, 2Division of Nephrology and 3Division of Clinical Pharmacology, Department of Medicine, University Hospital, Basel,4Department of Pharmacy, University of Basel, Switzerland
Abstract Key words: S-adenosylhomocysteine;
S-adenosylmeth-ionine; cardiovascular disease; end-stage renal disease;
Background. Elevated homocysteine concentrations
have been associated with premature arteriosclerosis homocysteine; methylation and with impairment of key methylation reactions
through accumulation of the homocysteine metabolite S-adenosylhomocysteine. In end-stage renal failure
Introduction
high homocysteine concentrations are commonlyfound but thus far the concentrations of related
adenosylated metabolites in plasma have not been Cardiac and cerebrovascular diseases are major causes assessed. of morbidity and mortality in chronic renal failure [1].
Methods. In this prospective study we determined However, although traditional risk factors such as plasma homocysteine and related metabolites in 25 arterial hypertension, glucose intolerance, and hyperli-patients on regular haemodialysis, and in 40 healthy pidaemia are common in uraemia [1] they are only volunteers. Blood samples from patients were drawn poor predictors of vascular events in chronic renal immediately before and in 10 patients additionally failure patients [2]. Following the finding of mild after the dialysis session. elevations of homocysteine as an independent risk
Results. Folic acid and vitamin B12 in plasma were factor for various forms of vascular disease [3], a
similar in patients (mean±SEM 25±2 nmol/l and number of studies investigated plasma total homocyst-400±41 pmol/l respectively) and controls (24±3 and eine concentrations in chronic renal failure patients 324±23 respectively). In patients plasma homo- with varying degrees of renal impairment, including cysteine, S-adenosylmethionine and S-adenosylhomo- those on maintenance dialysis, which revealed elevated cysteine were markedly elevated (36.6±3.6 mmol/l, homocysteine levels in the majority of these patients 381±32 nmol/l and 1074±55 nmol/l respectively) [4–6]. Some of these studies provided evidence that compared to the control values (6.8±0.4 mmol/l, homocysteine is an independent risk factor for vascular 60±3 nmol/l and 24.4±1.1 nmol/l respectively) events also in end-stage renal disease [6], whereas this whereas the molar ratio of plasma S-adenosyl- association was not confirmed in others [5]. Standard methionine and S-adenosylhomocysteine was signific- haemodialysis significantly lowers, but fails to normal-antly decreased (0.36±0.02 and 2.7±0.2 in patients ize homocysteine concentrations [7] despite normal and controls respectively). Haemodialysis failed to plasma concentrations of vitamin cofactors and sub-normalize the abnormal levels of these metabolites. strates (folic acid, vitamin B
12, and vitamin B6) for
Conclusion. Since the ratio of S-adenosylmethionine : homocysteine metabolism [5]. Even though some stud-S-adenosylhomocysteine is closely linked to the activity ies demonstrated homocysteine-lowering effects of folic of numerous enzymatic methylation reactions, these acid supplementation in chronic renal failure, hyperho-results suggest that methylation may be impaired in mocysteinaemia persists in most of these patients [7]. these patients. Therefore patients with end-stage renal disease remain exposed to the potential arteriosclerotic effect of long-lasting hyperhomocysteinaemia.
The sulphur-containing amino acid homocysteine is produced during methionine metabolism via the aden-Correspondence and offprint requests to: Dr Brian Fowler, University
osylated compounds S-adenosylmethionine (AdoMet) Children’s Hospital Basel, Metabolic Unit, CH-4005 Basel,
Switzerland. and S-adenosylhomocysteine (AdoHcy) [8]. Not only
On the day of the study routine clinical chemistry (Hitachi
does AdoMet play a key role in the enzymatic
regula-911 automatic analyser) and haematological parameters were
tion of homocysteine-metabolizing reactions, but it is
determined.
also the main methyl donor in numerous enzymatic
In the dialysis patient group blood samples were drawn
transmethylation reactions, which lead to formation of
immediately before haemodialysis (non-fasting) and in 10 of
AdoHcy [9]. AdoHcy acts as a competitive inhibitor
them also at the end of the haemodialysis procedure. Blood
of these AdoMet-dependent transmethylation reactions samples treated with EDTA were placed on ice immediately, and the affinity of most methyltransferases for this processed within 30 min and stored at−70°C until analysis. inhibitor has been observed to be greater than the
affinity for the substrate AdoMet [10]. It is well known
Methods
that the ratio of AdoMet to AdoHcy is crucial in the
regulation of the enzymatic transmethylation reactions Plasma samples for total homocysteine, AdoMet, and [10,11]. Alterations leading to a decrease of the AdoHcy determination were processed and measured by AdoMet : AdoHcy ratio have been shown to inhibit reversed-phase chromatography with fluorescence detection transmethylation reactions in different tissues [11], as previously described [13] with the following modification
potentially affecting biosynthesis of a wide range of of the HPLC conditions for AdoHcy and AdoMet. Etheno-derivatives of both metabolites were estimated separately on
endogenous compounds such as proteins, hormones,
a 4.0×200 mm Hypersil RP-18 (3 mm) column with a guard
phospholipids, neurotransmitters, DNA, and RNA.
column, 4.0×20 mm, filled with the same packing material.
Even though thermodynamics favour the biosynthesis
Etheno-AdoHcy was eluted at a flow rate of 0.8 ml/min with
of AdoHcy, AdoHcy normally remains low in vivo
a 0.1 mol/l sodium acetate buffer containing 4.3% acetonitrile
provided that homocysteine is removed immediately,
adjusted to pH 4.45 with acetic acid. Separation of
etheno-which is not the case in haemodialysis patients [10]. AdoMet was performed at a flow rate of 0.7 ml/min with Hence elevations of homocysteine levels may lead to the same buffer containing 10 mmol/l heptanesulphonic acid an increase in AdoHcy and therefore to alterations of and 3.95% acetonitrile and after 35 min the column was the ratio AdoMet : AdoHcy. flushed with 100% acetonitrile for 10 min followed by
equilib-Recent studies showed a decreased ratio of ration at initial conditions for 10 min.
The detection limit was 10 nmol/l in plasma for both
AdoMet : AdoHcy in erythrocytes of chronic renal
AdoHcy and AdoMet with a signal-to-noise ratio5.
failure patients [12]. However, no such data exist for
Methionine in plasma was determined by ion-exchange
plasma of these patients, which may be more important
column chromatography using ninhydrin detection as
previ-since plasma may reflect more closely metabolism in
ously described [14].
the liver, whose metabolic capabilities are different
Samples for AdoMet measurement in whole blood were
from those of red cells [12]. processed and determined by reversed-phase chromatography The aim of this study was therefore to investigate with UV detection as described previously [15]. Erythrocyte the ratio of AdoMet : AdoHcy as a metabolic con- concentrations were calculated by multiplying the difference sequence of increased homocysteine in patients with between plasma and whole-blood values by 100/haematocrit.
end-stage renal disease on maintenance haemodialysis. Total vitamin B12 and folic acid determinations were performed by dual competitive binding assay using the Dualcount Solid Phase No Boil radioassay from Diagnostic Products Corporation (Los Angeles, CA, USA).
Subjects and methods
Unpaired comparisons were tested by the Mann–Whitney U test and paired comparisons by the Wilcoxon signed rank
Subjects test. The relationship between pairs of variables was tested
by linear regression analysis. P values<0.05 were considered Twenty-five patients with end-stage renal disease, aged from significant. All tests were performed by the software package 30 to 79 years (12 males) undergoing standard bicarbonate Student Systat 1.0 for Windows (Systat Inc., Evanston, IL, haemodialysis therapy twice or three times a week (predialysis USA). Unless indicated otherwise values are expressed as mean serum creatinine 567–1320 mmol/l ), were investigated the mean±SEM.
at the Division of Nephrology of the University Hospital Basel after giving informed consent. They did not receive
any vitamin B12 or folate supplementation and their dietary
Results
intake of methionine was not restricted. They were clinically stable at the time of investigation and the causes of renal
Biochemical, haematological, and demographic
para-failure were chronic glomerulonephritis (7 patients),
poly-cystic renal disease (4), chronic pyelonephritis (2), analgesic meters of patients and control subjects are shown in
nephropathy (2), diabetic nephropathy (4) and undetermined Table 1. Homocysteine was increased above the normal
(6). Fourteen patients were treated with erythropoietin for range of 2.2–13.2 mmol/l, established in our own
anaemia and nine had previously experienced 11 vascular healthy control population [15], in all 25 patients, in events (9 coronary artery disease and 2 peripheral arterial agreement with previous studies. AdoMet and AdoHcy occlusive disease). Exclusion criteria were malnutrition
concentrations in plasma were higher than the highest
(serum albumin<30 g/1), rejection of a kidney transplant,
control value in all patients, whereas the ratio of
or vascular events within 6 months before the start of the
AdoMet : AdoHcy was lower in all of them compared
study. A group of 40 healthy randomly selected volunteers
to control values (Figure 1). The mean value of
living in the same city, aged from 34 to 82 years (21 males)
methionine was significantly decreased in patients, and
with normal renal function (serum creatinine 49–93 mmol/l )
Table 1. Patient and control subject characteristics and laboratory data
Parameter Patients (n=25) Control subjects (n=40)
Age (years) 62±2 (30–79) 62±2 (34–82)
Body mass index (kg/m2) 23±1 (17–33) 24±1 (17–35)
Serum creatinine (mmol/l ) 846±37 (567–1320)* 69±2 (49–93)
Urea (mmol/l ) 27±1 (19–41)* 5.5±0.2 (3.9–7.9)
ALAT ( U/l ) 15±1 (5–29)* 19±2 (5–63)
Haemoglobin (g/dl ) 11±0.4 (9–16)* 14±0.2 (12–17)
Haematocrit (%) 35±1 (28–50)* 43±0.5 (36–51)
AdoHcy in plasma (nmol/l ) 1074±55 (531–1587)* 24.4±1.1 (9.6–38.7)
AdoMet in plasma (nmol/l ) 381±32 (223–841)* 60±3 (35–118)
AdoMet : Adohcy 0.36±0.02 (0.17–0.69)* 2.7±0.2 (1.2–6.0)
Total homocysteine (mmol/l ) 36.6±3.6 (15.6–87.2)* 6.8±0.4 (1.7–13.3) Methionine in plasma (mmol/l ) 13.0±0.7 (7.1–20.9)* 21.6±2.6 (14.9–30.7) AdoMet in erythrocytes (mmol/l ) 2.3±0.2 (0.07–4.8) 3.6±0.1 (2.0–5.1)
Folic acid (nmol/l ) 25±2 (8–41) 24±3 (10–54)
Vitamin B12(pmol/l ) 400±41 (182–1023) 324±23 (231–463)
Values are mean±SEM (range). *P<0.001 versus control group (consistent also when gender is considered). AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine.
Table 2 summarizes the pre- and postdialysis values of the biochemical parameters in 10 patients. AdoMet and AdoHcy concentrations decreased significantly after dialysis, but remained markedly elevated com-pared to those of the control group. The ratio of AdoMet : AdoHcy increased slightly but remained significantly below the control range. Homocysteine values decreased in all patients but remained above the upper limit of our own healthy population normal range (<13.2 mmol/l, n=50 [15]). No significant changes were observed for vitamin B12 and methionine even though the latter showed a slight trend towards lower values. Folic acid decreased significantly after dialysis, reaching values significantly below those of the healthy control group (P<0.05).
Differences between gender in the patient group were observed for body mass index (25.1±1.1 kg/m2 and 21.4±0.9 for men and women respectively; P<0.05), and for homocysteine (30.9±4.7 mmol/l and 41.8±5.1 respectively; P<0.05). In the control group gender differences were found for creatinine (77.5±2.1 mmol/l and 64.2±1.6 for men and women respectively,
P<0.01); haemoglobin (15.0±0.2 g/dl and 13.7±0.2
respectively, P<0.01), and haematocrit (44.8±0.6% and 41.4±0.6 respectively, P<0.01). Linear regression analysis between each of the measured parameters
Fig. 1. Molar ratio of S-adenosylmethionine :
S-adenosylhomo-revealed a significant correlation between AdoHcy and cysteine. (AdoMet : AdoHcy) in the plasma of 25 patients and 40
healthy controls ( logarithmic scale). creatinine in men but not in women of both the patient (R=0.63, P<0.05) and the control group (R=0.44,
P<0.05). Within patients a significant correlation was
the control values. Folic acid and vitamin B
12 values observed between AdoMet and AdoHcy (R=0.71, were within the normal population range (7–39 nmol/l
P<0.01) for men, whereas for women this relationship
and 150–700 pmol/l respectively; defined by Diagnostic
was only weak and not statistically significant (R= Products Corporation (Los Angeles, CA, USA)) in all
0.51, P=0.08) (Figure 2). These results were also patients and controls, and mean values in patients did
consistent when AdoHcy was related to creatinine and not differ significantly from those of the control group.
AdoMet in a multiple regression analysis. Mean erythrocyte levels of AdoMet in patients were
significantly decreased compared to control values. No
Conclusions
differences were found between patients with andwith-out a history of vascular events or between subjects
with or without erythropoietin treatment for any of This study set out to investigate the relationship between elevated homocysteine and important closely the parameters measured.
Table 2. Effect of haemodialysis on circulating metabolites and vitamins in 10 patients
Compound Predialysis Postdialysis
AdoHcy in plasma (nmol/l ) 1324±60 (957–1587) 309±35 (99–418)*
AdoMet in plasma (nmol/l ) 484±58 (284–841) 250±71 (72–791)*
AdoMet : AdoHcy 0.36±0.03 (0.25–0.53) 0.64±0.13 (0.32–1.52)*
Total homocysteine (mmol/l ) 35.5±4.0 (23.2–62.9) 22.1±2.2 (12.9–37.2)* Methionine in plasma (mmol/l ) 14.6±0.5 (11.4–16.8) 12.7±1.0 (7.8–18.7)
Folic acid (nmol/l ) 21±1.5 (15–30) 15±1.0 (11–19)*
Vitamin B
12(pmol/l ) 275±23 (182–376) 293±29 (149–415)
Serum creatinine (mmol/l ) 916±55 (567–1169) 403±36 (251–623)*
Values are mean±SEM (range). *P<0.01 versus predialysis values. AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine. reinforces the idea of disturbed transmethylation reac-tions in end-stage renal disease. Figure 3 shows import-ant examples of the many methylated compounds synthesized in transmethylation reactions. Disturbed metabolism of monoamine neurotransmitters as well as low cerebrospinal fluid AdoMet concentrations have been reported in a variety of neurological and psychiat-ric disorders such as depression, dementia, degenera-tion of the spinal cord, and HIV-related neuropathies, as reviewed by Bottiglieri et al. 1994 [17]. Indeed, patients with end-stage renal disease have a high prevalence of for example peripheral neuropathy [18] and major depression [19], suggesting that impairment of methylation reactions may contribute to neurolog-ical syndromes in chronic renal failure. In accordance with this hypothesis clinical improvement of patients with major depression was observed after treatment with AdoMet [20], although homocysteine metabolites
Fig. 2. Correlation between S-adenosylmethionine (AdoMet) and have not been measured in that study. Whether admin-S-adenosylhomocysteine (AdoHcy) in plasma of 25 patients and 40 istration of AdoMet to dialysis patients will result in healthy controls ( logarithmic scale).
any clinical benefit requires further study. In addition to approaches aimed at lowering homocysteine and related metabolites, especially AdoMet and AdoHcy AdoHcy (e.g. folate supplementation), which have not in end-stage renal disease to provide insight into pos- achieved normalization of homocysteine in dialysis sible pathogenetic mechanisms. Substantially elevated patients [7], it may be a valuable strategy to further plasma levels of both AdoHcy and AdoMet with a
clearly reduced AdoMet : AdoHcy ratio have been shown for the first time in these patients. These findings are likely to be the consequence of markedly elevated homocysteine, since the thermodynamics of the AdoHcy hydrolase reaction favour the synthesis of AdoHcy when homocysteine is not removed immedi-ately [10].
Inhibition of transmethylation reactions by AdoHcy, a potent inhibitor of the methyltransferases [10], may lead to an accumulation of AdoMet and may explain the markedly increased AdoMet plasma concentrations in our dialysis patients. Interestingly, not only the concentration of AdoHcy but also and more import-antly the ratio of AdoMet and AdoHcy is crucial in the regulation of the transmethylation reactions [11]. Hence, transmethylation in humans may be decreased either if AdoMet concentrations are low ( limited sub-strate availability) or if AdoHcy concentrations are
high irrespective of the concentrations of the methyl Fig. 3. Examples of biologically important S-adenosylmethionine-donor AdoMet. Thus, the very low AdoMet : AdoHcy dependent transmethylation reactions (based on human and animal
studies [16 ]). ratio in plasma of our patients compared to controls
increase AdoMet because of the presence of high quality of life and morbidity of chronic renal failure patients, in addition to the prevention of vascular circulating concentrations of the inhibitor AdoHcy.
The finding of low erythrocyte AdoMet levels in disease. spite of high plasma concentrations confirms previous
Acknowledgements. These studies were supported by grants from findings that AdoMet cannot be taken up by red cells
the Swiss National Science Foundation (No. 3200–045988.95); the even in the presence of high concentrations in plasma
Treubel Foundation, Basel; F. Hoffmann–La Roche Ltd., Vitamins [10]. Furthermore, although homocysteine enters and Fine Chemicals Division Exploratory Research, Basel, erythrocytes, when extracellular levels are in the high Switzerland. We gratefully thank Marianne Zaugg for her excellent range of those measured in end-stage renal disease technical assistance. Special thanks are due to PD Dr P. Huber of the Department of Central Laboratories, University Hospital Basel, patients [12], de novo synthesis of AdoMet from
homo-Switzerland. cysteine does not occur in red cells [12].
The finding of low methionine concentrations in patients is surprising since remethylation has been
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Received for publication: 13.8.97 Accepted: 22.10.97
