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【关键词】 methylation
Division of Clinical Pharmacology and Division of Pharmacology and Experimental Therapy, Department of Pharmacology and Toxicology, University Hospital Tübingen, Tübingen, Germany
ABSTRACT
Homocysteine is a precursor of S-adenosylmethionine (AdoMet) and a metabolite of S-adenosylhomocysteine (AdoHcy). The ratio of AdoMet to AdoHcy, defined as the methylation potential (MP), indicates the flow of methyl groups within the cells. Chronic elevations of total homocysteine (tHcy) in plasma correlate with increased AdoHcy concentrations, decreased MP, and impaired DNA methylation. However, the influence of acute hyperhomocysteinemia on MP is unknown. We induced acute hyperhomocysteinemia in 14 healthy volunteers by oral administration of L-homocysteine (65.1 μmol/kg body wt) in an open, randomized, placebo-controlled two-period crossover study. The kinetics of tHcy in blood and urine, MP in blood, and global DNA methylation in lymphocytes were studied systematically during 48 h. Plasma tHcy concentrations reached a peak at 34 ± 11 min after an oral load with L-homocysteine and decreased with a half-life of 257 ± 41 min (means ± SD). Only 2.3% of the homocysteine dose were recovered in urine. AdoHcy concentrations and MP in whole blood and erythrocytes were not affected by the oral homocysteine load. Furthermore, global DNA methylation in lymphocytes did not change under these conditions. We found no difference between the genotypes of 5,10-methylenetetrahydrofolate reductase in response to the homocysteine load. However, AdoMet content in erythrocytes was significantly higher in the C677T carriers (CT; n = 7) compared with the CC genotype (n = 7). Although chronic elevation of tHcy has been shown to affect MP and DNA methylation, acute elevation of plasma tHcy above 20 μmol/l for 8 h is not sufficient to change MP and to induce DNA hypomethylation in lymphocytes.
HOMOCYSTEINE is a sulfur-containing, nonproteinogenic amino acid biosynthesized from methionine. It plays a key role between the folate cycle and the activated methyl cycle (Fig. 1). Homocysteine can be transsulfurated to cystathionine, remethylated to methionine, synthesized to S-adenosylhomocysteine (AdoHcy), or released into the extracellular space. During remethylation, homocysteine acquires a methyl group from N-5-methyltetrahydrofolate or from betaine to form methionine. A considerable proportion of methionine is then converted to S-adenosylmethionine (AdoMet), the major methyl donor to a variety of acceptors including guanidinoacetate, nucleic acids, neurotransmitters, phospholipids, and hormones. The product of these methylation reactions, AdoHcy, inhibits most of the AdoMet-dependent methyltransferases and is therefore rapidly hydrolyzed to adenosine and homocysteine (5). The hydrolysis of AdoHcy to adenosine and homocysteine by AdoHcy hydrolase (E.C. 3.3.1.1 [EC] ; AdoHcyase) is the only metabolic pathway leading to formation of homocysteine in vertebrates. This reaction is reversible and favors synthesis of AdoHcy. However, under physiological conditions, the metabolic flux is directed toward homocysteine formation because of removal by further metabolism (17, 31).
The ratio of AdoMet to AdoHcy, defined as the methylation potential (MP), is considered to be a reliable indicator of the flow of methyl groups transferred from AdoMet to methyl acceptors within the cells (18). As recently shown, chronic elevations of plasma total homocysteine (tHcy) correlate with increased AdoHcy levels and with a decreased MP in plasma. The low MP is associated with a decreased methylation of lymphocyte DNA (36). This state may alter methylation-dependent gene regulation and may have unwanted effects on a variety of organ systems. Homocysteine is assumed to be an independent marker for increased cardiovascular risk (13, 20), and DNA hypomethylation may be one of the responsible mechanisms.
Elevated tHcy plasma concentrations can be the result of reduced enzymatic activity of many enzymes involved in homocysteine metabolism because of vitamin deficiencies or genetic mutations mainly of cystathionine -synthase or 5,10-methylenetetrahydrofolate reductase (MTHFR) (25). A well-examined polymorphism of the latter enzyme is the C677T transition (CT). If this transition occurs in both alleles, resulting in a 677TT genotype, plasma tHcy concentration is 2.6 μmol/l higher compared with 677CC carriers (CC) (3). Furthermore, hyperhomocysteinemia is regularly seen in chronic renal failure (CRF). However, the underlying mechanisms have not been fully elucidated (22).
To assess the time frame in which hyperhomocysteinemia can affect MP and thus DNA methylation, we induced acute hyperhomocysteinemia by oral administration of exogenous L-homocysteine in healthy male volunteers. With this study design, we wanted to determine whether short-time exposure to homocysteine is sufficient to induce changes in MP in erythrocytes and global DNA methylation in lymphocytes. In addition, we wanted to measure renal homocysteine excretion in relation to homocysteine plasma levels.
MATERIALS AND METHODS
Subjects
Healthy nonsmoking male volunteers (n = 14; age 2126 yr, body mass 6398 kg, body mass index 20.026.6 kg/m2) participated in the study. The study was conducted using an open, randomized, placebo-controlled two-period crossover design. The study was approved by the local Ethics Committee, and written informed consent was obtained from all participants. Individuals with a known allergic disposition or with cardiovascular risk factors including diabetes mellitus, blood pressure readings >160/95 mmHg, or clinical evidence of cardiovascular disease were excluded. The mean blood pressure values were 121 ± 7.9 (systolic) and 79 ± 7.3 (diastolic) mmHg. No volunteer had a systolic blood pressure above 130 mmHg or a diastolic blood pressure above 90 mmHg. Thus all volunteers were normotensive. All subjects were nonvegetarians and free from pharmacotherapy and intake of vitamin preparations. Their plasma concentrations of folate, pyridoxine (vitamin B6), cobalamin (vitamin B12), and tHcy were within the normal ranges. For inclusion of the volunteers into the study, we set an upper limit of plasma tHcy of 15.0 μmol/l.
Preparation of Study Medication
The homocysteine solution was prepared as described by Guttormsen et al. (10). Powdered L-homocysteine thiolactone hydrochloride (65.1 μmol/kg body wt; molecular wt 153.6 g/mol; Sigma, Munich, Germany) was dissolved in 5 ml of 5 mol/l sodium hydroxide solution and allowed to stand for 5 min to open the thiolactone ring. Then 5 ml of 5 mol/l HCl were added for neutralization, and the pH was adjusted to 45. A mixture of 90 ml water and 100 ml apple cider (to mask the unpleasant taste of homocysteine) was added to give a total volume of 200 ml. The mixture was prepared immediately before administration under the supervision of a pharmacist. The placebo control consisted of 100 ml water and 100 ml apple cider.
Study Protocol
The volunteers were required to abstain from alcohol and caffeine for the 24 h preceding each study day. They entered the study after an overnight fast in a quiet, temperature-controlled room (22 ± 2°C). They were requested to drink 300 ml/h water throughout the whole study day. After 5 min of supine rest, blood pressure and pulse rate were measured. To obtain blood samples, two 1.7-mm-diameter cannulas were inserted into an antecubital vein on either side for infusions and contralateral blood sampling. Thereafter, venous blood was withdrawn for predose determination of tHcy, AdoMet, AdoHcy, inulin, and DNA methylation in lymphocytes. A separate blood sample was withdrawn for the genotyping of MTHFR (C677T polymorphism).
Clearance protocols were started with a 5-min inulin (Inutest, Fresenius, Graz, Austria) loading infusion of 50 ml 0.9% sodium chloride solution (Delta Pharma, Pfullingen, Germany) containing 1.5 g inulin/m2 body surface (7). Directly afterward, a continuous infusion of inulin (6.25 g/m2 body surface) was administered at a rate of 50 ml/h throughout the entire duration of clearance periods. Simultaneously, an infusion of 0.45% sodium chloride and 2.5% glucose (Delta Pharma) was started at a rate of 3 ml?h1?kg body wt1. Clearance periods were performed at various time intervals, whereby blood withdrawal was made at the respective midpoint of each urine collection period. The different clearance periods were carried out according to the time course of tHcy plasma levels following the oral load. After 1 h, when urine flow and plasma inulin were stabilized, a first 1-h urine collection was carried out to obtain the basal inulin clearance value before the administration of study medication (i.e., homocysteine vs. placebo). From there on, the infusion rate of the 0.45% sodium chloride and 2.5% glucose solution was adapted at the end of each clearance period to match urinary flow rate.
The study medication (i.e., 200 ml homocysteine solution vs. 200 ml of placebo) was prepared immediately before administration and was ingested orally in less than 2 min. According to the subjects' body weights (6398 kg), the administered homocysteine doses ranged between 4.1 and 6.4 mmol (mean: 4.9 mmol). Then, blood samples were collected at +0.25 (15 min), 0.5 (30 min), 0.75 (45 min), 1, 1.5, 2, 3, 4, 6, 8, 12, 24, and 48 h after the administration of the study medication to measure tHcy plasma concentration. Additional blood samples were collected for measurements of AdoMet, AdoHcy, and tHcy in whole blood and erythrocytes at +1, 2, 4, 8, 24, and 48 h. Separate blood samples were collected for determination of DNA methylation at +1, 8, 24, and 48 h.
Urine was collected for tHcy measurement in fractions at 0, +0.5, 1, 2, 3, 4, 6, 8, and 12 h after the administration of study medication by spontaneous voiding. Urine was collected for inulin measurement in fractions 1 h before and at 0, +1, 2, 4, 6, 8, and 12 h, i.e., until the end of each clearance period. Blood samples for inulin measurement were collected at 0.5, +0.5, 1.5, 3, 5, 7, and 10 h, i.e., at the midpoint of each collection period. During clearance periods, subjects remained in a supine position.
Blood pressure and pulse rate were recorded at 0, +1, 2, 3, 4, 5, 6, 8, 10, and 12 h. Standardized meals, poor in protein and vitamins, were given at +4 and +10 h. Subjects were discharged 12 h after the administration of study medication. The blood sampling at +24 and +48 h was performed in ambulatory subjects with a single-use butterfly needle. This procedure was repeated in one of the following weeks (maximum interval of 3 wk) in a randomized crossover design.
Analytic Methods
Homocysteine measurement. Whole blood for tHcy plasma concentration was collected in 3-ml Sarstedt tubes (Sarstedt, Nümbrecht, Germany) containing EDTA, placed on ice, and centrifuged within 30 min at 3,350 g for 10 min at +4°C. Plasma was stored for a maximum of 12 wk at 25°C until analysis. For measurement of tHcy in whole blood, samples were collected with 20 μl of capillaries (Bio-Rad Laboratories, Munich, Germany) and hemolyzed with 0.2 ml hemolysis reagent (aqueous solution of EDTA and a nonionic detergent; catalogue no. 1954075, Bio-Rad Laboratories), resulting in an immediate inhibition of homocysteine-generating and -converting enzymes and a stabilization of the tHcy concentration for up to 2 days. The hemolyzed blood samples were stored at +4°C for a maximum of 2 days until analysis. Measurements of tHcy concentrations in plasma and in whole blood were performed by HPLC (Bio-Rad Laboratories) in the Department of Internal Medicine IV, Endocrinology, Metabolism, Pathobiochemistry, and Clinical Chemistry, University Hospital Tübingen.
Urinalysis. Urine samples were stored at 25°C for a maximum of 6 mo. Urinary tHcy concentrations were determined by HPLC, using a homocysteine assay by Bio-Rad Laboratories with the following modification. The internal standard of the reagent kit was replaced by mercaptopropionic acid to avoid interferences with unknown urinary compounds (Proksch B, unpublished observations). The lower limit of quantification was 1.5 μmol/l.
Clearance measurement. Clearance measurements are usually performed with the widely used polyfructosan inulin. In our study, we used the branched sinistrin (Inutest), which is easier to handle than the linear inulin due to its better water solubility. In this paper, sinistrin is referred to as inulin, which is the more common term. Inulin concentrations in plasma and urine were determined by a colorimetric method using Seliwanoff's reagent (a solution of resorcinol and HCl) (34). Clearances were calculated according to standard formulae and normalized to a body surface area of 1.73 m2 for the glomerular filtration rate.
Metabolite determination. AdoMet and AdoHcy concentrations were measured in perchloric acid extracts. Whole blood (2 ml) dropping out of the cannula was collected in tubes containing 5 ml of 0.6 N perchloric acid at 0°C. The tubes were shaken thoroughly, placed on ice, and centrifuged within 10 min at 3,350 g for 10 min at +4°C. For AdoMet and AdoHcy measurement in erythrocytes, whole blood was collected in 3-ml Sarstedt tubes containing EDTA and immediately centrifuged at 680 g for 5 min at +4°C. One milliliter of erythrocyte sediment was transferred into 4 ml of 0.6 N perchloric acid at 0°C, shaken thoroughly, placed on ice, and centrifuged at 3,350 g for 10 min at +4°C. All samples and standards were supplemented with a known amount of N-6-methyladenosine as the internal standard. The supernatant was adjusted to a pH between 5.5 and 6.0 by adding 2 M K2CO3/1 M KH2PO4. The precipitated potassium perchlorate was discarded after centrifugation at 20,000 g, and the supernatant was applied onto a solid-phase extraction column (BondElut, ICT, Bad Homburg, Germany). Elution of the compounds was performed with 0.1 M HCl, and the eluate was analyzed by reverse-phase chromatography with UV detection according to Delabar et al. (6) with the following modifications. The binary solvent system consisting of solvent A (10 mM ammonium dihydrogenphosphate, 0.6 M heptanesulfonic acid sodium salt in 3% methanol) and solvent B (like solvent A and in addition 10% acetonitrile) delivered the following gradient: immediately after sample injection, a linear gradient was started to increase solvent B to 15% with a transition rate of 1.2%/min and to 70% with a transition rate of 7.5%/min. Thereafter, solvent B was kept at 70% for 5 min. Before the next sample injection, solvent A was kept at 100% for 10 min to reequilibrate the system. Thus the chromatogram was completed within 30 min. Remote control, data acquisition, and quantification of peak areas were performed with Peak Simple Software 3.12 by SRI.
Global DNA methylation. Whole blood for assessment of DNA methylation in lymphocytes was collected in two 8-ml Sarstedt tubes containing EDTA. Preparation of lymphocytes was performed with Ficoll gradient (Bio-Chrom, Berlin, Germany). Global DNA methylation was determined as described by Pogribny et al. (23). Genomic DNA was isolated using a DNeasy Tissue Kit (Qiagen), and 2 μg were digested for 15 h with 20 units HpaII (Fermentas) according to the manufacturer's instructions. A second DNA aliquot was incubated without enzyme and served as background control. For cytosine extension assay, 0.5 μg DNA was incubated with 1x NH4 reaction buffer for PANScript Polymerase, 1 mM MgCl2, 0.25 U PANScript DNA Polymerase, and 0.1 μl [3H]dCTP (4060 μCi/mmol, New England Nuclear Life Science Products) in a total volume of 25 μl for 1 h at +56°C. Duplicate 10-μl aliquots from each reaction were filtered through Whatman DE-81 ion exchange filters, and the filters were washed three times with 3 ml 0.5 M sodium phosphate buffer (pH 7.0). Radioactivity incorporated in DNA and absorbed on the filters was determined by liquid scintillation counting.
MTHFR genotyping. For the genotyping of MTHFR, 1 blood sample/subject was withdrawn with a Sarstedt tube containing EDTA and stored at 25°C for a maximum of 6 mo. Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures with a QIAamp DNA Blood Kit (Qiagen). Two probes were prepared: a C allele-specific probe, 5'-Tet-TCT GCG GGA GcC GAT TTC ATC ATC-Tamra-3'; and a T allele-specific probe, 5'-Fam-TCT GCG GGA GtC GAT TTC ATC ATC-Tamra-3'. The primer design for PCR of the flanking region of C677T/MTHFR was as follows: forward, 5'-GGC TGA CCT GAA GCA CTT GAA-3'; and reverse, 5'-GCG GAA GAA TGT GTC ATC CT-3'. Real-time PCR was carried out with a thermal cycler (GeneAmp, PCR System 9700, Applied Biosystems). PCR was performed according to the following conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Genotyping of MTHFR C677T was performed by pyrosequencing as described by Zhang et al. (37). This procedure took place after the end of the study; thus all investigators and volunteers were blinded concerning the MTHFR C677T genotype. The analysis was carried out in the Department of Medical Genetics, University Hospital Tübingen.
Statistical Methods
Data were analyzed by ANOVA and a Student-Newman-Keuls test for multiple comparison. A difference between group data was considered to be significant when P was <0.05. Results are expressed as means ± SD.
RESULTS
Homocysteine Kinetics
tHcy plasma concentrations at predose were 8.9 ± 1.5 μmol/l (Fig. 2). The volunteers were administered a mean homocysteine dose, depending on body weight, of 754 ± 100 mg, i.e., 4,910 ± 649 μmol. Relevant tHcy kinetic parameters in plasma, whole blood, and erythrocytes were maximum concentration (Cmax; 90.4 ± 12, 72.4 ± 13, and 68.4 ± 29 μmol/l), maximum time point (tmax; 34 ± 11, 64 ± 16, and 77 ± 28 min), area under the curve (AUC; 048 h; 486 ± 69, 492 ± 125, and 470 ± 311 μmol?h1?l1), and half-life (257 ± 41, 328 ± 45, and 470 ± 313 min), respectively. Twelve hours after homocysteine administration, a mean amount of 110.6 ± 32 μmol was recovered in the urine, which corresponds to 2.3 ± 0.7% of the orally administered homocysteine dose (Fig. 3). Renal homocysteine clearance reached a maximum of 10.8 ± 4 ml?min1?(1.73 m2)1 body surface 1 h after homocysteine administration (Table 1) and fell subsequently to 1.5 ± 0.8 ml?min1?(1.73 m2)-1 body surface 12 h after homocysteine administration.
View this table:
Inulin Clearance
Inulin clearance at predose was 91.3 ± 14.1 ml?min1?(1.73 m2)1 body surface (in the homocysteine group) and 100.0 ± 27.5 ml?min1?(1.73 m2)1 body surface (in the placebo group), respectively. Homocysteine intake did not cause any significant change in the glomerular filtration rate compared with placebo.
AdoMet and AdoHcy Concentrations in Whole Blood
AdoMet and AdoHcy predose values in whole blood were 2.0 ± 0.4 μmol/l and 217.6 ± 61 nmol/l, respectively (Table 1). One hour after the administration of homocysteine, whole blood values of AdoMet and AdoHcy were not significantly changed (2.1 ± 0.5 μmol/l and 256 ± 80 nmol/l, respectively). Compared with whole blood measurements during placebo study days (AdoMet predose: 1.9 ± 0.3 μmol/l, +1 h: 1.9 ± 0.3 μmol/l; AdoHcy predose: 211 ± 53 nmol/l, +1 h: 231 ± 80 nmol/l), these changes in AdoMet and AdoHcy whole blood concentrations were significantly different in the paired t-test but not in the ANOVA. All other differences of AdoMet and AdoHcy whole blood concentrations between time points after homocysteine administration and predose measurements were not significantly different from placebo. The absolute values ranged between 2.2 ± 0.4 μmol/l (maximum; at +2 h) and 2.1 ± 0.3 μmol/l (minimum; at +4 h) for AdoMet values and between 256 ± 79 nmol/l (maximum; at +1 h) and 200.0 ± 48 nmol/l (minimum; at +4 h) for AdoHcy values. When the respective AdoMetpostdose/AdoMetpredose and AdoHcypostdose/AdoHcypredose ratios were calculated, no significant differences between verum and placebo were observed. When AdoMet and AdoHcy values or AdoMet and AdoHcypostdose/AdoHcypredose ratios were correlated with the respective tHcy plasma concentrations of each time point assessed, no significant differences between verum and placebo were observed.
MP in whole blood was 10.2 ± 4 at predose (Table 1). After homocysteine administration, MP values ranged between 8.8 ± 2.8 (minimum; at +1 h) and 10.9 ± 3.5 (maximum; at +4 h). All the differences between time points after homocysteine administration and predose measurements were not significantly different from placebo. When the respective MPpostdose/MPpredose ratios were calculated, no significant differences between verum and placebo were observed. When MP values or MPpostdose/MPpredose ratios were correlated with the respective tHcy plasma concentrations of each time point assessed, no significant differences between verum and placebo were observed.
AdoMet and AdoHcy Concentrations in Erythrocytes
AdoMet and AdoHcy predose values in erythrocytes were 4.0 ± 0.9 μmol/l and 323 ± 129 nmol/l, respectively (Table 1). After homocysteine administration, AdoMet values ranged between 4.0 ± 0.9 μmol/l (minimum; at +1 h) and 4.5 ± 1.0 μmol/l (maximum; at +24 h), and AdoHcy values ranged between 353 ± 86 nmol/l (maximum; at +1 h) and 300 ± 100 nmol/l (minimum; at +4 h). All the differences between time points after homocysteine administration and predose measurements were not significantly different from placebo. When the respective AdoMetpostdose/AdoMetpredose and AdoHcypostdose/AdoHcypredose ratios were calculated, no significant differences between verum and placebo were observed. When AdoMet and AdoHcy values or AdoMet and AdoHcy postdose/predose ratios were correlated with the respective tHcy plasma concentrations of each time point assessed, no significant differences between verum and placebo were observed.
MP in erythrocytes was 14.1 ± 6.1 at predose. After homocysteine administration, values ranged between 11.8 ± 4.4 (minimum; at +1 h) and 16.7 ± 11.1 (maximum; at +4 h). All the differences between time points after homocysteine administration and predose measurements were not significantly different from placebo. When the respective ratios MPpostdose/MPpredose were calculated, no significant differences between verum and placebo were observed. When MP values or MPpostdose/predose ratios were correlated with the respective tHcy plasma concentrations of each time point assessed, no significant differences between verum and placebo were observed.
DNA Methylation Status in Lymphocytes
In the cytosine extension assay, the extent of genome-wide methylation in lymphocytes was determined by [3H]dCTP incorporation. The values at predose were 17.5 ± 4.7 x 103 dpm/μg DNA (in the homocysteine group) and 17.2 ± 3.8 x 103 dpm/μg DNA (in the placebo group), respectively (Table 1). After homocysteine administration, a maximum of 21.9 ± 10 x 103 dpm/μg DNA was reached after 48 h. In the placebo group, a slight increase in [3H]dCTP incorporation on 19.0 ± 1.7 x 103 dpm/μg DNA (after 8 h) was noted. However, the difference in methylation status between verum and placebo was not significant. Thus global DNA methylation is not affected by acute hyperhomocysteinemia.
C677T/MTHFR Genotyping
Seven subjects (50%) were heterozygous carriers of the 677T-genotype (CT), whereas the other seven subjects (50%) were homozygous carriers of the wild-type allele (CC). There were no significant differences in the measured parameters (tHcy, AdoMet, AdoHcy, and DNA methylation) between the CC and the CT genotype of MTHFR, with one exception being a slightly but significantly elevated content of AdoMet in erythrocytes of CT carriers compared with CC. This difference was not only found at all time points but also during the placebo treatment and during homocysteine loading as well.
Adverse Events
Except for a transient headache in one of the subjects, no adverse events were reported. There were no clinically relevant changes in blood pressure or pulse rate. One volunteer experienced an anaphylactic reaction after 4 min of inulin infusion before any homocysteine was administered (9). Subsequently, he was excluded from the study and was replaced by another volunteer.
DISCUSSION
Homocysteine is assumed to be an independent marker for increased cardiovascular risk (13, 20). With a prevalence of 57% in the general population, hyperhomocysteinemia is considered to play a role in the genesis of premature atherosclerosis. However, a causal relationship between hyperhomocysteinemia and cardiovascular disease remains to be established. The recently published Vitamin Intervention for Stroke Prevention trial in 3,680 adults to study the effects of a vitamin-rich diet on recurrent cerebral infarction (primary outcome), coronary heart disease events, and death (secondary outcomes) did not change the end points during a 2-yr period, although tHcy was lowered by 2 μmol/l (28). A variety of other mechanisms of Hcy-induced atherosclerosis, including DNA hypomethylation, have been described (2, 29).
In the present study, we wanted to assess whether acute hyperhomocysteinemia can induce similar changes as found in chronic hyperhomocysteinemia (36), e.g., reduction in MP and DNA methylation. We administered the same oral dose of L-homocysteine (65.1 μmol/kg body wt) and applied the same mixing procedure as in the study by Guttormsen et al. (10). Accordingly, for Cmax, tmax, plasma AUC, and plasma half-life, similar values (means ± SD) were obtained in our investigation (90.4 ± 12, 34 ± 11 min, 486 ± 69 μmol?h1?l1, and 257 ± 41 min, respectively) compared with the results of Guttormsen et al. (10) (57.4 ± 9.9 μmol/l, 59 ± 20 min, 416 ± 41 μmol?h1?l1, and 223 ± 45 min, respectively). The reason for an earlier tmax in our study may be due to the fact that, unlike Guttormsen et al. (10), we measured tHcy plasma concentrations at 0.75 h (= 45 min) when the levels are near their Cmax values. Additionally, we have shown that Cmax of tHcy in erythrocytes was lower than in plasma and was reached at a later time point than in plasma. According to former studies (11, 30), the influence of the volunteers' protein-poor diet on postprandial changes in tHcy concentrations is negligible.
Urinary homocysteine excretion is very low as shown in studies with animals and humans (1, 22). This low excretion rate of homocysteine is the consequence of its metabolism by remethylation and transsulfuration (4, 22) and its high protein binding in humans (24). Assuming a protein binding of homocysteine of 80% in humans, 20% of tHcy enters the tubular fluid by glomerular filtration. However, in our study, only 2% of the administered homocysteine dose were recovered in urine within 12 h. This is in line with the findings of Guttormsen et al. (12), who have shown in 19 cobalamin- and folate-deficient volunteers with hyperhomocysteinemia (67.1 ± 39.5 μmol/l) and with normal renal function, that unchanged urinary excretion of homocysteine in 24 h was 6.5 ± 3.0% of an orally administered homocysteine dose. Our data show that the excretion of tHcy into the urine increases transiently up to >60% of the calculated filtered load during acute hyperhomocysteinemia. These findings indicate that the tubular uptake of homocysteine by the basic amino acid transporter is saturable.
Although animal studies have demonstrated significant renal uptake and metabolism of homocysteine (1, 15), the relevance of these findings in humans has been questioned, and renal metabolism of homocysteine appears marginal in humans (32). Furthermore, the contribution of impaired homocysteine excretion to hyperhomocysteinemia in renal failure seems to be very small. Hyperhomocysteinemia, frequently observed in CRF patients, may be explained by the loss of the homocysteine-metabolizing capacity of the entire body (26, 33). The role of the kidney in the development of hyperhomocysteinemia, however, is incompletely understood at present (19). Our finding that renal homocysteine clearance increased from the basal level of 0.5 to 10.8 ml/min (Table 1) during the first 60 min, when Hcy levels are elevated up to 80 μmol/l, indicates that the tubular uptake of filtered Hcy may be a saturable process. In patients with CRF, tubular uptake of Hcy might be impaired and could therefore contribute, at least in part, to increased Hcy plasma levels. The importance of the association between renal impairment and the risk of hyperhomocysteinemia in kidney-transplant patients has been recently emphasized (35).
During acute hyperhomocysteinemia, both AdoHcy concentration and MP did not change significantly in whole blood and erythrocytes. One reason for this observation might be due to the fast catabolism of homocysteine by remethylation (to methionine) or transsulfuration (to cystathionine) in healthy individuals (8). Therefore, a longer-lasting and stable elevation of tHcy as reported in patients with CRF or MTHFR deficiency seems to be necessary to induce changes in AdoMet and AdoHcy and thus in MP. Other previous studies (without exogenous homocysteine loading) have shown that a chronic but mild elevation in tHcy concentrations resulted in increased AdoHcy concentrations and decreased MP (16, 21, 36).
Our data clearly show that global DNA methylation in lymphocytes is not affected by transient tHcy elevation. This observation indicates that epigenetic mechanisms assessed by global DNA methylation follow a slow time course, compatible with the observation that global DNA methylation status is changed only during replication. Correspondingly, as recently shown, short-term (24 h) changes in the MP in HepG2 cells do not lead to changes in DNA methylation; however, changes in mRNA methylation were observed (14). Whether site-specific methylation at regulatory gene sequences can be induced by short-term changes in MP or tHcy concentrations awaits further studies. Finally, we would like to indicate that these results on MP and DNA methylation, which we relate to each other, stem from the two different cell compartments, MP in erythrocytes and DNA methylation in lymphocytes. However, besides methodical reasons, our approach allows a comparison with previous reports which suggested that global hypomethylation in lymphocyte DNA may be an early biomarker of abnormal methylation in other tissues (36).
With a focus on safety aspects of acute hyperhomocysteinemia achieved by a single oral homocysteine load, such a procedure may be considered not harmful, as much as MP and global DNA methylation were unchanged. In addition, we did not observe in our study any adverse effects that are likely to be caused by the intake of homocysteine. Thus the oral administration of homocysteine in the range of the used doses (65.1 μmol/kg body wt) is well tolerated and may be considered safe.
In our study, the phenotyping of the C677T mutation of the MTHFR gene showed a distribution of 50:50 between homozygous (CC) and heterozygous allele carriers (CT). Both genotypes did not differ in their response to an acute challenge of hyperhomocysteinemia. This applies to kinetics of tHcy concentrations in blood and urine, AdoMet in whole blood, AdoHcy in whole blood and erythrocytes, and to DNA methylation. However, we found one significant difference between the two genotypes, which was an elevated content of AdoMet in erythrocytes of CT carriers, independent of tHcy plasma levels. Apparently, this difference did not affect the response to Hcy loading of both genotypes.
Concerning further investigations of the influence of tHcy on MP, the question about an exogenous induction of chronic hyperhomocysteinemia may be raised. Because of the fast catabolism of homocysteine with a half-life of 4 h, it would be hardly feasible to maintain chronic and stable plasma concentrations over a longer period (e.g., several days) by administering oral homocysteine doses. Intravenous administration would be also problematic to perform due to the lack of data about administration in humans and due to the unstable nature of homocysteine in terms of storage. The induction of chronic hyperhomocysteinemia by a methionine-loading test or administration of folate antagonists for an investigation of MP, however, would induce additional changes in AdoMet and AdoHcy metabolism, masking direct effects of elevated Hcy levels in plasma. To assess the meaning of the MP in clinical studies on homocysteine metabolism, it also appears to be necessary to measure AdoMet and AdoHcy simultaneously in settings when folic acid, pyridoxine, or cobalamin are administered (27).
In summary, acute hyperhomocysteinemia did not change AdoMet, AdoHcy, and the MP in either whole blood or erythrocytes. Moreover, no induction of DNA hypomethylation in lymphocytes was observed. Although chronic elevation of tHcy has been shown to affect MP and DNA methylation, acute elevation of tHcy concentration in plasma above 20 μmol/l for 8 h is not sufficient to change MP and to induce DNA hypomethylation in lymphocytes. Only a small amount of administered homocysteine (2.3%) was recovered in urine, whereas the rest is assumed to be subject to metabolism.
GRANTS
This study was supported by the Federal Ministry of Education and Research (Grant 01EC0001) and by a "fortune project" of the Medical Faculty of the University of Tübingen (Grant 11450-0).
DISCLOSURES
The authors declare that they do not have any financial or proprietary interest in the subject matter or materials discussed in this study.
ACKNOWLEDGMENTS
We thank R. Riehle, S. Hasanovic, Dr. R. Lehmann, S. Jelesnianski, W. Beer and Dr. P. Bauer for excellent technical assistance.
FOOTNOTES
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