Vitamin depletion/repletion studies

It is apparent that abnormally increased excretion of kynurenine metabolites after a test dose of tryptophan cannot necessarily be regarded as evidence of vitamin Bg deficiency. This means that the tryptophan load test is unreliable 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. 

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. 

More recent depletion/repletion studies, using more sensitive indices of status in which subjects were repleted with either a constant intake of vitamin Be and varying amounts of protein, or a constant amount of protein and varying amounts of vitamin Bg, have shown average requirements of 15 to 16 /xg per g of dietary protein, suggesting a reference intake of 18... 

Requirements for nongrowing adults are based on a limited number of depletion-repletion studies carried out on small numbers of subjects. These studies have been reviewed by Rodriguez and Irwin (1972). An abnormal dark adaptation was the criterion used to determine an inadequate vitamin A status in the early studies. This test provided no measure of the body pool size of vitamin A when abnormal dark adaptation occurred. Dark adaptation in adults is known to be affected by factors other than vitamin A (Fisher et al., 1970 Ripps, 1982). Thus it was difficult, even in the classic Sheffield depletion study of the British Medical Research Council (Hume and Krebs, 1949), to attribute aberrant dark adaptation specifically to the relative vitamin A status of subjects. This was particularly true because there was a poor correlation between blood levels of vitamin A and evidence of impaired dark adaptation until blood levels fell critically low and clinical signs became obvious. It is therefore most unfortunate that a carefully controlled study of adult human requirements that used labeled retinol has not been fully reported (Hodges and Kolder, 1971). From the data available (Sauberlich et al., 1974), individual variation in blood and clinical responses to... 

Estimates of riboflavin requirements are based on depletion/repletion studies to determine the minimum intake at which there is significant excretion of the vitamin. In deficiency there is virtually no excretion of the vitamin as requirements are met, so any excess is excreted in the urine. On this basis the minimum adult requirement for riboflavin is 0.5-0.8 mg/day. At intakes between 1.1 and 1.6mg/day urinary excretion rises sharply because tissue reserves are saturated. 

While the tryptophan load test is a useful index of status in controlled depletion/repletion studies to determine vitamin Bg requirements, it is not an appropriate index of status in population studies. 

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. 

As shown previously (Vorobjeva and Iordan, 1976), the content of 2 pg vitamin Bn per g of dry biomass of P. shermanii represents a threshold for the isomerization to proceed. In these studies, a number of metabolic variables were investigated in vitamin Bn-replete cells (about 1000 pg vitamin B /g biomass), Bn-deficient cells (about 10 pg vitamin B /g biomass) and Bn-depleted cells (at most 2 pg vitamin Bn/g biomass). The cellular vitamin Bn content was varied by changing the concentration of a cobalt salt in the growth medium, or by using a mutant strain that produced only traces of corrinoids. 

Analysis of the results concerning the DNA synthesis in vitamin Bn-deficient cells allowed us to conclude that a vitamin Bn-independent system may operate in these cells. This system has been investigated in a series of studies (Iordan et al., 1986 Iordan, 1992 Iordan and Petukhova, 1995). After a number of passages in a medium carefully depleted of cobalt (which simulated natural habitats) the cells of P. shermanii contained less than 2 iig of cobalamins per g biomass. These cells, adapted to the vitamin B -free medium, are referred to as vitamin B -depleted cells. The synthesis and activities of ribonucleotide reductase (RNR) were compared in vitamin B -replete (Bi2 ), Bu-deficient and Bn-depleted cells. 

Very few direct studies have been performed to determine human vitamin A requirements. In the Sheffield study (Hume and Krebs, 1949), 16 subjects were depleted of vitamin A for 2 years only three subjects showed clear signs of impaired dark adaptation. One of these subjects was repleted with 390 /rg of retinol per day, which resulted in a gradual restoration of dark adaptation the other two subjects received /3-carotene. On this basis, the minimum requirement was presumed to be 390 /rg, and the reference intake was set at 750 /rg. 

Most studies of vitamin Bg requirements have followed the development of abnormalities of tryptophan (and sometimes also methionine) metabolism during depletion and normalization during repletion with graded intakes of the vitamin. 

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