Clinical Science (1996) 91. 79-86, FMT Loehrer et al.
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Franziska M. T. Loehrer, Walter E. Haefeli, Christian P. Angst, Garry Browne, Greta Frick and Brian Fowler
Metabolic Unit, University Children's Hospitol Basel, and Division of Clinical Pharmacology, University Hospitol Basel, Basel, Switzerland
The essential protein amino acid methionine is converted to S-adenosylmethionine by methionine adenosyltransferase (EC 220.127.116.11). S-adenosylmethionine is the methyl donor in many important transmethylation reactions that lead to the formation of the very short-lived S-adenosylhomocysteine . Enzymic hydrolysis yields homocysteine, which can be catabolized via the irreversible transsulphuration pathway, the first step in which is catalysed by the pyridoxal phosphate-dependent enzyme cystathionine b-synthase (CS; EC 18.104.22.168). Alternatively, it can be remethylated to methionine by 5-methytetrahydrofolate--homocysteine methyltransferase (methionine synthase, MS; EC 22.214.171.124), which requires vitamin B12, or by betaine-homocysteine methyl-transferase (EC 126.96.36.199). Regulation of these pathways depends on many factors [2-5], but a vital role of S-adenosylmethionine in the coordinate control of remethylation and trans--sulphuration seems evident from studies in vitro [6,7]. S-Adenosylmethionine acts as an allosteric inhibitor of 5,10-methylenetetrahydrofolate reductase (MTHFR; EC 188.8.131.52), which is crucial for 5-methyltetrahydrofolate synthesis  and as an activator of CS  at micromolar concentrations.
Disturbances of methionine metabolism are associated with a variety of disease states. For example, inborn errors due to CS, MTHFR and MS deficiencies lead to hyperhomocysteinaemia and severe disease, including ocular, skeletal, neurological and vascular pathology [1,8,9]. Furthermore, elevated plasma homocysteine levels, either in the fasting state or after methionine loading, have been found in a significant proportion of patients with coronary artery [10-13], peripheral arterial occlusive [14,15] or cerebral vascular disease [15-17], pointing to mild hyperhomocysteinaemia as an indep-endent risk factor for arteriosclerotic disease. The exact cause of this is so far unknown, but moderate deficiencies of MTHFR [18-20] and CS  and nutritional deficiencies of vitamin B12, and folate [22,23] have been implicated.
Administration of methionine followed by measurement of sulphur amino acids in blood and urine has been used to study homozygous and heterozygous CS deficiency [24-27]. Furthermore, Beers et al.  reported post-methionine load increases in homo-cysteine similar to those in obligate heterozygotes for CS deficiency in 36% of 25 patients with peripheral and 28% of 25 patients with cerebrovascular disease. Several studies have confirmed this abnormality in patients with different forms of vascular disease [21,28-30].
It is thought that methionine loading mainly stresses catabolism through homocysteine transsulphuration . However, Clarke et al.  showed an inverse relation between vitamin B12, or folate and post-load homocysteine in patients with different forms of vascular disease. On the other hand, in previous studies by Brattstrom et al.  and Andersson et al. , no correlation between post-load homocysteine and vitamin B12, folate or pyridoxal phosphate was found in patients with vascular disease. Previous studies on the relationship between folate and homocysteine have all used measurement of the total blood concentrations of the vitamin rather than the methyl form, which specifically participates in the remethylation reaction.
Until now methionine loading studies have concentrated on changes in sulphur amino acid levels [33,34], and little is known about the influence of methionine on levels of the numerous other metabolites and cofactors in humans.
In this study the effect of oral methionine on plasma levels of key compounds involved in the transmethylation pathway were studied over 24 h. In particular, and in addition to the often measured homocysteine, we determined the levels of 5-methyltetrahydrofolate, the form of folate active in remethylation of homocysteine, and the intermediate methionine metabolites S-adenosylmethionine and S-adenosylhomocysteine. Additionally, the heat stable activity of MTHFR in lymphocytes was assessed.
|Fig. 2. Concentrations of total homocysteine (a), S-adenosyl-methionine (b), S-adenosylhomocysteine (c) and 5-methyltetra-hydrofolate (d) in plasma after methionine loading in 12 healthy subjects (closed symbols) and without methionine administration in three control subjects (open symbols) over a period of 24 hours. Results are expressed as mean " SEM.|
This study set out to investigate the effects of methionine loading on S-adenosylhomocysteine, S-adenosylmethionine and 5-methyltetrahydrofolate and their relation to homocysteine in normal subjects.
That the 12 subjects handled methionine normally was suggested by the finding of normal fasting homocysteine values, which increased to a mean of 32.6" 10.3Fmol/l, which compares with the mean of 35"6Fmol/l found by Mansoor et al. . However, this does not completely exclude the heterozygous state for CS deficiency in all individuals as some overlap of post-methionine homo-cysteine levels between control subjects and obligate heterozygotes has been reported . The variable time to peak values in different subjects in our study emphasizes individual variation in such metabolic studies in humans. It helps to explain the better discrimination of obligate heterozygotes from control subjects observed when several post-load samples were analysed than in simplified tests using a single post-load measurement. Such variable peak level times have important implications both for the studies in vascular disease patients and for investigations of metabolic inter-relationships as in this study.
The major conclusions from this study are that methionine loading in normal human subjects leads to simultaneous increases in homocysteine and S-adenosylmethionine, without significant changes in S-adenosylhomocysteine, and to a later fall of 5-methyltetrahydrofolate. These changes must be consequent to methionine loading and do not simply reflect circadian variations, as demonstrated by the lack of such changes in three control subjects who received no methionine. The increase in S-adenosylmethionine probably reflects liver metabolism of methionine and increases in S-adenosylmethionine concentration as seen in rats injected with a single dose of methionine . This is supported by studies in mammals, which showed that adaptation to an excess of methionine occurs in liver as only the liver-specific isoenzyme of methionine adenosyltransferase can adapt immediately to changes in methionine concentrations . The absence of a change in S-adenosylhomocysteine concentration after methionine loading despite the large increase in homocysteine is surprising. It was shown by Hoffman et al.  that in isolated rat liver the administration of homocysteine results in an accumulation of S-adenosylhomocysteine if homocystene is not removed immediately. However, Guttormsen et al.  reported no change in S-adenosyl-homocysteine concentration after homocysteine loading, which resulted in similar concentrations of homocysteine to those found after meth ionine loading. If the lack of increase in S-adenosylhomocysteine in plasma does indeed reflect tissue levels, this could be explained simply by a transient increase in homocysteine to concentrations below those required to inhibit S-adenosylhomocysteine hydrolase  and by a fully active trans-sulphuration pathway owing to normal enzyme activities in normal subjects.
The decrease in 5-methyltetrahydrofolate found in this study could result from an increase in homocysteine levels, which may result in an enhanced turnover of the remethylation reaction, thereby depleting the co-substrate 5-methyltetrahydrofolate. Alternatively, elevated S-adenosylmethionine could lead to allosteric inhibition of MTHFR, as shown previously in vitro , at the concentrations of S-adenosylmethionine obtained in rat liver by injection of similar doses of methionine . It is possible that the two effects may operate in combination. The significant positive correlation between fasting level and both the maximum change and the AUC(t0-24) in 5-methyltetrahydrofolate indicates smaller decreases in 5-methyltetrahydrofolate in subjects with lower baseline values. This suggests that the extent of remethylation in the presence of homocysteine excess may also depend on the availability of 5-methyltetrahydrofolate. The one exceptional subject (VIII) who showed no decrease in 5-methyltetrahydrofolate but the highest post-load level of S-adenosylhomocysteine may reflect the extreme of normal variation. However, these findings could also result from a disturbed remethylation pathway, preventing normal conversion of 5-methyltetra-hydrofolate to methionine, and subsequent stress of the trans-sulphuration pathway reflected by increased S-adenosylhomocysteine. However, this subject exhibited 36% heat-stable activity of MTHFR, which is well above the range of 2.5-4.5 SDs below the mean value, which was defined by Kang et al.  as indicating the thermolabile MTHFR mutation.
An additional finding in this study was that the S-adenosylmethionine/S-adenosylhomocyste ratio in fasting human plasma, at 1.2, is lower than that reported for erythrocytes (3.3)  and cerebrospinal fluid (6.9) , indicating variation of this ratio between different tissues and compartments. Previous workers have shown that this ratio and not S-adenosylmethionine alone is a critical factor in the influence of methyltransferases .
The finding that methionine loading leads to decreases in 5-methyltetrahydrofolate subsequent to increases in homocysteine and S-adenosylmethionine indicates that a change in either homocysteine or S-adenosylmethionine may cause reductions in 5-methyltetra-hydrofolate. This must be considered in evaluating the relationship between folate and homocysteine in vascular disease. The metabolic relationships illustrated in this study should be evaluated in the search for pathogenetic mechanisms of mild hyperhomocysteinaemia and vascular disease.