Homocysteine and thiol metabolites in vitamin B 12 deficiency

Clinical Science (2001) 100, 111 116 (Printed in Great Britain) 111 Homocysteine and thiol metabolites in vitamin B 12 deficiency L. R. RANGANATH*, M. BAINES and N. B. ROBERTS *Department of Chemical Pathology, Epsom General Hospital, Epsom, Surrey KT18 7EG, U.K., and Department of Clinical Chemistry, Royal Liverpool University Hospital, Liverpool L7 8XP, U.K. A B S T R A C T Homocysteine metabolism is increasingly implicated in a diverse group of clinical disorders, including atheromatous vascular disease. We studied the disposition of homocysteine via the trans-sulphuration pathway, plasma glutathione peroxidase (GPx) activity and plasma levels of the sulphated hormone dehydro-epiandrosterone sulphate (DHEAS) in six vitamin B 12 -deficient human subjects before and after 2 weeks of vitamin B 12 repletion, both in the fasting state and following an oral methionine load (0.1 g/kg body weight). Fasting plasma total homocysteine concentrations fell (P 0.03) and total cysteine concentrations rose significantly (P 0.048) after treatment for 2 weeks with vitamin B 12 injections. The magnitude of the mean fall in the fasting concentration of homocysteine (38.8 µmol/l) was similar to the mean rise in cysteine levels (36.0 µmol/l) following vitamin B 12 therapy. Circulating levels of homocysteine were increased at 4 h after a methionine load when compared with fasting levels, both before and after vitamin B 12 repletion (P 0.003 for both). Total cysteinyl-glycine was lower post-methionine than in the fasting state following vitamin B 12 therapy (P 0.007). Fasting plasma GPx fell significantly after 2 weeks of vitamin B 12 therapy (P 0.05). The change in plasma GPx between the fasting state and 4 h after methionine loading was significantly different pre- and post-vitamin B 12 therapy (P 0.05). The present study provides indirect support to the hypothesis that defects in the trans-sulphuration and remethylation of homocysteine produce hyperhomocysteinaemia in vitamin B 12 deficiency in human subjects. Elevated homocysteine levels directly or indirectly may up-regulate GPx. Sulphation status, as measured by plasma DHEAS, was unchanged. INTRODUCTION Deficiencies of folate and vitamin are not uncommon in the general population [1]. Approximately 1% of middle-aged and elderly subjects have vitamin deficiency, and folate deficiency is likewise common in states of poor nutrition [2]. These deficiencies may be subclinical and detected by plasma measurements only, or present as clinically recognizable disease states. The widely recognized clinical syndromes associated with these deficiency states are megaloblastic anaemia and neurological disease. It has long been known that vitamin and folate regulate homocysteine methionine metabolism, with vitamin acting as cofactor to the enzyme methionine synthase and folate as the precursor of tetrahydrofolate (both part of the remethylation cycle of homocysteine), and that hyperhomocysteinaemia is a feature of these deficiency states [3]. Homocysteine is increasingly recognized as a risk factor for atheromatous vascular disease [4,5], thrombophilia [6] and neural tube Key words: cysteine, dehydro-epiandrosterone, glutathione peroxidase, homocysteine, vitamin B. Abbreviations: DHEAS, dehydro-epiandrosterone sulphate; GPx, glutathione peroxidase. Correspondence: Dr L. Ranganath, Department of Clinical Chemistry, Royal Liverpool University Hospital, Prescot Street, Liverpool L7 8XP, U.K. (e-mail lrang liv.ac.uk).

112 L. R. Ranganath, M. Baines and N. B. Roberts defects [7]. Despite this potential for causing disease, classical descriptions of pernicious anaemia, known to be associated with raised homocysteine levels, do not include increased thrombogenicity or atherogenesis. The molecular mechanisms underlying the damage due to homocysteine are not fully understood, although oxidative damage generated by high homocysteine levels has been implicated [8,9]. Since homocysteine can be eliminated by remethylation to form methionine, or converted into cysteine and glutathione by trans-sulphuration mechanisms [10], we investigated the disposition of homocysteine by the latter route, which may potentially generate molecules that are protective against oxidative injury. Cellular and plasma glutathione peroxidase (GPx) enzymes form part of the antioxidant defences, including those against peroxide-induced damage [11,12]. As hyperhomocysteinaemia has been shown to injure endothelial cells via the generation of hydrogen peroxide [12] and to decrease the activity of cellular GPx [13], we wished to study the effects of the hyperhomocysteinaemia of vitamin deficiency on plasma GPx. Trans-sulphuration of homocysteine also generates cysteine, which is crucial for the eventual sulphation of endogenous and exogenous substances that is necessary for elimination from the body of endogenous as well as xenobiotic molecules [14]. The aims of the present study were: (1) to assess the disposition of homocysteine via the trans-sulphuration pathway in vitamin -deficient subjects, (2) to assess any relationship with plasma GPx, and (3) to evaluate sulphation status by measuring levels of an endogenous sulphated hormone, dehydro-epiandrosterone sulphate (DHEAS). All of these measurements were carried out both before and after 2 weeks of vitamin repletion. Homocysteine metabolism was studied in the fasting state as well as after an oral methionine load in six subjects with low plasma levels of vitamin. Oral methionine administration transiently increases circulating levels of homocysteine and other thiol compounds, reaching a peak between 4 and 8 h in healthy subjects. Methionine administration allows an assessment of the activity of the trans-sulphuration pathway [15,16]. MATERIALS AND METHODS Study protocol The programme of investigation was approved by the Epsom General Hospital Medical Research Ethics Committee. Written consent was obtained from all participants. Subjects (Table 1) Two pre-menopausal female and four male subjects with low plasma vitamin levels (but with plasma folate concentrations within the reference range) were chosen for study. All subjects had their plasma vitamin measured by macrocytosis; levels of plasma vitamin were less than the lower limit of the reference range (210 910 pg ml) in all subjects. None of the subjects was a smoker or abused alcohol or had impairment of renal function. All subjects were Caucasian, on a Western diet, and were requested to follow their usual diet and other lifestyle factors for the duration of the study. All were explicitly advised to avoid taking any extra vitamin supplementation. Experimental protocol Subjects were seen between 07.30 and 09.00 hours on two separate occasions, each following an overnight fast of 12 h. After a basal 10 ml venous blood sample had been collected, methionine (0.1 g kg body weight) flavoured with Modjul flavour system (Scientific Hospital Supplies, Liverpool, U.K.) in 400 ml of water was given orally. A second 10 ml venous blood sample was obtained 4 h later [16]. All subjects received five intramuscular injections of hydroxycobalamin (1 mg) between the first and second visits, and plasma concentrations of vitamin were greater than 2000 pg ml at the second visit. None of the subjects had any adverse reaction to oral methionine. Chemical analyses Blood samples were collected into EDTA glass collection bottles (for thiol metabolites, GPx and DHEAS) and centrifuged at 2000 g for 10 min; separated plasma was frozen within 15 min of venesection. Total homocysteine, cysteine, cysteinyl-glycine and glutathione (the terms homocysteine, cysteine, cysteinyl-glycine and glutathione in the rest of the paper refer to sum of the reduced, oxidized and protein-bound compounds, unless stated otherwise) were measured by HPLC on a Drew DS30 analyser (Drew Scientific, Barrow-in-Furness, U.K.), in an adaptation of an HPLC method [17]; the within-run coefficients of variation for homocysteine, cysteine, cysteinyl-glycine and glutathione were 2.7%, 3.3%, 6.0% and 7.7% at concentrations of 16.9, 292, 13 and 3.7 µmol l respectively (n 10). DHEAS was measured by radioimmunoassay (DPC, Llanberis, U.K.), with a within-run coefficient of variation of 10% throughout the standard range. Plasma GPx was measured by the method of Paglia and Valentine [18] (Ransel; Randox Laboratories, Antrim, U.K.) on a Falcor 300 analyser (A. Menarini, Wokingham, U.K.), with a withinrun coefficient of variation of 6.4% at a concentration of 625 units l. Statistical analyses Experimental groups were compared using the paired Student s t test. Pearson s correlation coefficients were calculated to assess relationships between groups. Values of P 0.05 were taken to be significant.

Homocysteine metabolism in vitamin B 12 deficiency 113 Table 1 Details of subjects and of fasting pre-therapy measurements M, male; F, female. Parameter Subject no. 1 2 3 4 5 6 Reference range Age (years) 45 57 68 70 86 47 Sex F M M M M F Plasma vitamin B 12 (pg/ml) 121 204 139 87 149 126 210 910 Plasma folate (ng/ml) 4.4 2.1 5.7 8.1 5.2 4.0 2.8 20.0 Red cell folate (ng/ml) 156 194 206 261 180 355 145 1100 Haemoglobin (g/dl) 13.4 14.2 15.7 14.3 14.5 13.6 F, 11.8 14.8; M, 13.3 16.7 Haematocrit 0.39 0.43 0.48 0.43 0.42 0.39 F, 36 44; M, 39 50 White blood cells ( 10 9 /l) 12 18.5 6.2 5.9 7.5 13.5 3.5 11.0 Platelets ( 10 9 /l) 287 266 278 214 219 344 150 400 Mean corpuscular volume (fl) 103 105 103 117 113 102 80 100 Homocysteine (µmol/l) 17.1 31.3 12.5 142.5 79.0 9.0 5 15* Cysteine (µmol/l) 240 129 235 194 205 167 160 260* Cysteinyl-glycine (µmol/l) 36 45 34 26 36 33 55 75* Glutathione (µmol/l) 4.3 5.3 3.2 2.4 3.2 2.7 4 20* DHEAS (µmol/l) 1.0 1.3 4.9 1.5 1.2 5.1 0.7 11.5 GPx (units/l) 436 379 380 526 380 531 300 450 * Data taken from [31]. Data provided by Ransel, Randox Laboratories, Antrim, U.K. RESULTS Homocysteine and other thiol metabolites (Tables 1 and 2) Fasting plasma homocysteine concentrations ranged from 9.0 to 142.5 µmol l, and fell significantly after treatment for 2 weeks with vitamin injections (P 0.03), with each subject showing a fall. Concentrations of homocysteine were increased at 4 h post-methionine compared with fasting levels, both before and after vitamin repletion (P 0.003). The magnitude of the rise in plasma homocysteine after administration of methionine was similar before and after the vitamin therapy. In contrast with homocysteine, fasting plasma cysteine concentrations increased following treatment for 2 weeks with vitamin injections (P 0.048), with increases in five of the six subjects. The mean increase in fasting plasma cysteine after vitamin treatment (36.0 µmol l) was similar to the mean fall in fasting homocysteine (38.8 µmol l), suggesting a reciprocal relationship. There were no other significant changes in cysteine levels, although cysteine concentrations tended to be lower post-methionine. Cysteinyl-glycine levels post-methionine tended to be lower than fasting levels both before and after vitamin treatment, although only post-vitamin levels achieved statistical significance (P 0.007). No significant differences were seen in plasma glutathione Table 2 Fasting and 4 h post-methionine plasma levels of thiol metabolites, DHEAS and GPx in six vitamin B 12 -deficient patients before and after vitamin B 12 therapy Values are means (S.E.M.). Significance of differences: *P 0.05 for after compared with before vitamin B 12 therapy; P 0.01 for after a methionine load compared with the fasting state. Before B 12 treatment After B 12 treatment Metabolite Fasting Post-methionine Fasting Post-methionine Homocysteine (µmol/l) 48.7 (21.6) 72.7 (19.2) 9.9 (0.9)* 29.2 (3.8) Cysteine (µmol/l) 195 (17.2) 188 (15.7) 231 (16.4)* 203 (16.1) Cysteinyl-glycine (µmol/l) 35.0 (2.5) 32.0 (3.6) 36.3 (2.3) 29.3 (2.2) Glutathione (µmol/l) 3.52 (0.4) 4.37 (0.9) 5.37 (1.3) 5.67 (1.5) DHEAS (µmol/l) 2.50 (0.79) 2.73 (1.04) 2.15 (0.65) 3.68 (1.26) GPx (units/l) 439 (30) 420 (35) 391 (25)* 415 (38)

114 L. R. Ranganath, M. Baines and N. B. Roberts before or after vitamin therapy, either in the fasting state or following oral methionine loading. Plasma DHEAS (Tables 1 and 2) There were no significant differences in plasma DHEAS levels measured pre-vitamin fasting (2.50 0.79 µmol l), pre-vitamin post-methionine (2.73 1.04 µmol l), post-vitamin fasting (2.15 0.65 µmol l) and post-vitamin post-methionine (3.68 1.26 µmol l). Plasma GPx (Tables 1 and 2) Fasting plasma GPx fell significantly following 2 weeks of vitamin therapy (P 0.05). The change in plasma GPx that occurred between the fasting state and 4 h after methionine loading was significantly different pre- (falling levels) and post- (rising levels) vitamin therapy (P 0.05). DISCUSSION The range of fasting homocysteine concentrations before vitamin therapy was wide, and it is noteworthy that some vitamin -deficient subjects had plasma homocysteine concentrations comparable with those found in classical homocystinuria, i.e. fasting levels of approx. 100 µmol l. It is difficult to be sure why there was such a wide range of plasma homocysteine levels in our vitamin -deficient subjects, although contributory factors could include the period of deficiency, vitamin B status and tissue vitamin status, which were not assessed in the present study. Despite this variation in fasting homocysteine levels before vitamin therapy, all subjects showed a decrease in these levels after vitamin therapy, to very low levels, with a very narrow range (7.8 12.9 µmol l; reference range 15.0 µmol l). This confirmed previous studies showing a significant fall in fasting plasma homocysteine following vitamin therapy [19]. The rise in circulating homocysteine levels and the fall in plasma cysteinyl-glycine at 4 h after a methionine load in the present study are also similar to previous findings in six healthy subjects studied by Mansoor and colleagues [15]. We did not find evidence for an increase in the plasma levels of cysteine and downstream thiol products (cysteinyl-glycineandglutathione) inhyperhomocysteinaemia of vitamin deficiency, which may potentially be protective against oxidative damage due to homocysteine. Rather, plasma cysteine levels were relatively lower before treatment and increased after vitamin repletion, in a reciprocal molar manner with homocysteine. Although deficiencies of vitamin and folic acid are known to cause hyperhomocysteinaemia due to impaired remethylation of homocysteine, the enzymes of the transsulphuration pathway are not known to be dependent on vitamin or folate as cofactors. Several possible explanations are considered here. First, the changes in homocysteine and cysteine levels noted here might suggest direct stimulation of the trans-sulphuration pathway by vitamin. Folic acid has been suggested to increase trans-sulphuration by an unknown mechanism [20]. Secondly, a change in the disposal of cysteine through alternative pathways, protein and peptide synthesis and provision of intracellular organic sulphate was a possibility [14]. However, no change in circulating DHEAS levels was seen in the present study, providing no support for an alteration in sulphation status (a surrogate marker of altered cysteine disposal). Thirdly, co-existing vitamin B deficiency could have impaired the trans-sulphuration of homocysteine through inactivation of cystathionine synthase [21,22]. However, although vitamin B was not measured in the present study, there is no reason to believe that vitamin B status differed between the two visits, and all subjects were explicitly requested not to take vitamin supplementation or change their usual diet for the duration of the study. Fourthly, cysteine and homocysteine may form mixed-disulphide bridges [20], although such an occurrence in plasma is unlikely to account for our results, since total plasma homocysteine was measured; however, intracellular homocysteine cysteine disulphide bonds may have been present during hyperhomocysteinaemia before vitamin therapy, binding more cysteine in the intracellular compartment and thus accounting for the lower plasma cysteine levels. Unlike plasma homocysteine cysteine disulphide bridges, intracellular disulphides are not measured in our assays. Support for this explanation comes from the fact that the rise in cysteine and the fall in homocysteine occuring after vitamin therapy were similar in molar terms. It is also notable that the rise in homocysteine levels after a methionine load was associated with a fall in cysteine levels. Finally, the hypothesis of Selhub and Miller [23], based on studies in rats and pigs, suggests that when remethylation of homocysteine is impaired, methionine salvage is impaired, and the S- adenosylmethionine concentration within the cells falls; a fall in intracellular S-adenosylmethionine inhibits transsulphuration [23 25]. Our studies in human vitamin deficiency, showing defects in remethylation and in transsulphuration of homocysteine, provide support for this hypothesis. Although the pathogenic mechanisms of homocysteinaemia are not fully understood, oxidative damage may be important. Homocysteine is oxidized to homocystine, and in the process hydrogen peroxide is generated. Hydrogen peroxide is a powerful oxidant and causes injury to living cells. Among the defence mechanisms against oxidative damage that are available, GPx is an important component, and is present in the plasma as well as within cells. Homocysteine has been shown to

Homocysteine metabolism in vitamin B 12 deficiency 115 inhibit intracellular GPx in vitro [12]. The present study showed that plasma GPx levels were higher before vitamin therapy, when homocysteine levels were high, and fell after vitamin therapy (in parallel with homocysteine), which was not consistent with inhibition by increased hydrogen peroxide generation. Rather, our results are consistent with an up-regulation of plasma GPx in response to high homocysteine concentrations and, possibly, increased hydrogen peroxide formation. Cellular GPx was not measured in the present study, as previous work has shown that changes in cellular activity occur over a much longer period than plasma changes, and would not be expected to change within the time scale of the present study [26]. Another potential mechanism that may account for the hyperhomocysteinaemia of vitamin deficiency is leakage from haemopoietic cells due to ineffective erythropoiesis. We do not have any direct evidence for such a mechanism, although leakage of homocysteine and GPx from the cells before vitamin therapy could explain their parallel temporal changes. Both pernicious anaemia [27] and ischaemic hypoxic damage to organs such as the heart, liver and skeletal muscle have been associated with high levels of circulating enzymes due to their release into the plasma by leakage through damaged cell membranes [28 30]. Our GPx assay recognizes both plasma and cellular GPx activity equally. In summary, the present study has shown that patients with vitamin deficiency had raised plasma homocysteine levels, which fell following vitamin therapy. Additionally, a lower plasma cysteine level pre-therapy, which increased post-therapy, was also seen in the present study; this suggests that a defect in trans-sulphuration may co-exist with a defect in the trans-methylation of homocysteine, as proposed by Miller and Selhub [23]. 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