The Methionine Load Test

As discussed in Section 10.3.4.2, the metabolic fate of homocysteine arising from methionine is determined not only by the activity of cystathionine synthetase and cystathionase, hut also the rate at which it is remethylated to methionine (which is dependent on vitamin B12 and folate status) and the requirement for cysteine. 

It is apparent that abnormally increased excretion of homocysteine and cystathionine metabolites after a test dose of methionine cannot necessarily be regarded as evidence of vitamin Be deficiency. This means that, like the tryptophan load test, the methionine load test is unreUahle as an index of status in epidemiological studies, although it is (probably) reliable in depletion/ repletion studies to determine requirements. 

Vitamin Be requirements have been estimated both by isotopic tracer studies to determine turnover of the body pool (Section 9.6.1) and also by depletion/ repletion studies using a variety of indices of status (Section 9.6.2). These studies have generally been conducted on young adults, and there is inadequate information to determine the requirements of elderly people, because apparent status assessed by a variety of indices declines with increasing age, despite intake as great as in younger people (Bates et al., 1999a). As discussed in Section 9.6.3, there is also inadequate information to estimate the requirements of infants. 

1 Vitamin Be Requirements Estimated from Metabolic Turnover 

There is a variety of estimates of the body pool of vitamin Be. Short-term studies with isotopic tracers suggest a total body content of between 160 to 600 ixmo (40 to 150 mg), with a half-life of 33 days, suggesting a minimum requirement for replacement in the wide range between 0.6 to 2.27 mg per day. 

About 80% of the toted body vitamin Be is in skeleted muscle glycogen phos-phorylase, with a relatively slow turnover. Based on longer term tr acer studies, Coburn (1990, 1996) has suggested a toted body pool of 250 mg, or 15 nmol (3.7 fig) per g of body weight, with a loss of about 0.13% per day, hence a 

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Impairment of remethylation is strongly implicated as the cause of hyper-homocysteinemia in patients with CKD, which has been demonstrated in a radioisotope study in patients with CKD (Van Guldener et al. 1999), although reduced clearance of homocysteine has been suggested as the possible cause in some reports. We reported that supplementation with folic add and methyl-cobalamin normalized the remethylation pathway (Koyama et al. 2002). Compared with the decreases in homocysteine of 17.3 8.4% after supplementation with folic add alone and 18.7 7.5% after that with methylcoba-lamin alone, a combination of foUc acid and methyleobalamin decreased homocysteine by approximately 60% and normalized the findings of the methionine loading test Table 47.1. This result suggest that both coenzymes, folic acid and methyleobalamin, were insufficient due to reduced availability of these coenzymes in patients with CKD. There is also a report that increased MMA in dialysis patients was reduced by the administration of methylcoba-lamin (Nakamura et al. 2002). 

Table 47.3 Methionine loading on patients with CKD Stage V. Group A was treated with folic acid 15 mg/day orally, methylcobamain 500 pg intravenously after each hemodialysis session and vitamin Bg 60 mg/day orally. Group B was treated with folic acid and methyl-cobalamin (without vitamin Bg). All patients were treated for three weeks. A methionine-loading test was conducted before and after supplementation. Amino acid level was measured at fasting and two hours and four hours after methionine load (0.05 g/kg orally). Both groups showed normal findings of homocysteine profile during the methionine loading test after treatment whether with vitamin Bg or not. However, profiles of methionine and cysteine were not normalized. Reproduced with permission from Koyama (2011).

The metabolism of methionine, shown in Figure 11.22, includes two pyridoxal phosphate-dependent steps cystathionine synthetase and cystathionase. Cystathionase activity falls markedly in vitamin deficiency, and as a result there is an increase in the urinary excretion of homocysteine and cystathionine, both after a loading dose of methionine and under basal conditions. However, as discussed below, homocysteine metabolism is affected more by folate status than by vitamin status, and, like the tryptophan load test, the methionine load test is probably not reliable as an index of... 

Figure 4 Methionine metabolism, the basis of the methionine load test for vitamin Be status.

Some 10-25% of the population have a genetic predisposition to hyperhomocysteinemia, which is a risk factor for atherosclerosis and coronary heart disease, as a result of polymorphisms in the gene for methylenetetrahydrofolate reductase. There is no evidence that supplements of vitamin Bg reduce fasting plasma homocysteine in these subjects, and like the tryptophan load test, the methionine load test may be an appropriate index of status in controlled depletion/repletion studies to determine vitamin Bg requirements, but not in population studies. 

Early studies of vitamin Be requirements used the development of abnormalities of tryptophan or methionine metabolism during depletion, and normalization during repletion with graded intakes of the vitamin. Although tryptophan and methionine load tests are unreliable as indices of vitamin Be status in epidemiological studies (Section 9.5.4 and Section 9.5.5), under the controlled conditions of depletion/repletion studies they do give a useful indication of the state of vitamin Be nutrition. More recent studies have used more sensitive indices of status, including the plasma concentration of pyridoxal phosphate, urinary excretion of 4-pyridoxic acid, and erythrocyte transaminase activation coefficient. 

Her very low-protein diet was continued, but in order to ensure an adequate supply of essential amino acids for growth she was fed a mixture of the ketoacids of threonine, methionine, leucine, isoleucine and valine. After each feed she again became abnormally drowsy and markedly ketotic, with significant acidosis. Her plasma ammonium ion concentration was within the normal range, and a glucose tolerance test was normal, with a normal increase in insulin secretion after glucose load. 

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