To investigate the sensitivity of guinea pig tissues to ascorbic acid depletion, as distinct from inanition, young male guinea pigs were maintained with either ascorbic acid restriction or total diet restriction for 8 weeks. One group (A) received no ascorbic acid for 3 weeks, then 0.5 mg/d for 5 weeks; one group (B) was weight-matched by restricted food intake to the first group; a third (marginally deficient) group (C) received 1 mg ascorbic acid/d throughout; a fourth was weight-matched to this group @); and a fifth group received the control diet adlib. (E). Both of the groups with restricted ascorbic acid intakes (A and C) developed very low tissue ascorbic acid contents, but only the first group (severely deficient group A) also exhibited a severely reduced growth rate. This group also exhibited reduced femur calcium and hydroxyproline contents and reduced skin hydroxyproline content. These changes were not seen in the corresponding weight-matched group (B). Neither plasma alkaline phosphatase (EC 3.1.3.1) activity, nor a variety of indices of vitamin D status exhibited changes which could be attributed specifically to reduced ascorbic acid intake and hence to lowered tissue ascorbic acid content. It is concluded that low tissue ascorbic acid levels in guinea pigs clearly alter the connective tissue composition of growing femur and skin, but do not necessarily produce a major, specific effect on vitamin D status. Moreover, the control of inanition is crucial to permit interpretation of the changes in metabolism that are caused by ascorbic acid deficiency.Ascorbic acid: Guinea pig: Vitamin D The sensitivity of bone connective tissue to the effect of ascorbic acid deficiency in ascorbicacid-requiring species such as the guinea pig and higher primates, including man, is well established. Earlier studies from this laboratory (Bates, 1979) showed that bone collagen synthesis, measured by the incorporation of labelled proline into collagen hydroxyproline, was second only to skin collagen synthesis in its sensitivity to ascorbic acid depletion in the guinea pig. It was also apparent that inanition, which typically accompanies ascorbic acid deficiency in guinea pigs, may exert secondary effects on metabolic processes which are not attributable to the lowered tissue ascorbic acid levels per se. It is therefore important to distinguish the non-specific effects of inanition from the specific effects of tissue ascorbic acid depletion.Recently a study was reported (Sergeev et al. 1990) suggesting that vitamin D metabolism may also be sensitive to ascorbic acid deficiency in guinea pigs, and that the consequent alterations in hormonal control of calcium may be responsible for some of the bone defects seen in ascorbic-acid-deficient animals. However, this study did not include inanition controls, and did not report growth curves; thus it was unclear whether the reported effects were specific for ascorbic acid depletion, or secondary to inanition. In addition, Weiser et

While it is clear from these studies that high concentrations or intakes of ascorbate can reduce tissue concentrations of Cu in a variety of species, there is a less clear consensus and uniformity with respect to possible functional effects of ascorbate overload on Cudependent processes in viva Several early studies on aortic rupture in chicks (Starcher et al. 1964;Carlton & Henderson, 1965;Hill & Starcher, 1965) obtained strong evidence for a gross failure of elastin cross-linking, attributed to reduced activity of the key enzyme, lysyl oxidase, in birds that were exposed simultaneously to excessive dietary ascorbate, and to low dietary Cu levels. Studies in mammalian species, however, have not reproduced such a dramatic effect. Nevertheless, in view of the potential importance of this interaction, for human subjects who choose to take daily megadoses of ascorbic acid, there is a need for further studies in relevant animal models such as the guinea-pig, which like humans has an absolute requirement for dietary ascorbate, as well as for Cu. The possibility that high levels of circulating vitamin C may increase the likelihood of Fe acting pro-oxidatively, and thereby influence outcome, has been illustrated by recent studies of human pre-term infants (Silvers et al. 1994;Powers et al. 1995). Deleterious effects of high ascorbate intakes may thus involve more than one transition metal.Another separate but related question arises from the need for better biochemical tests for suboptimum v. optimum status, with respect to those functionally-critical processes which are dependent on specific micronutrients. Collagen cross-linking is such a process. A severe lack of Cu can impair the activity of the Cu-dependent enzyme lysyl oxidase, responsible for the initiation of cross-linking (Farquharson et al. 1989;Robins, 1994). Impairment of cross-linking may then result in loss of tensile strength. Lack of ascorbic acid is well known to affect the hydroxylation of collagen lysyl residues (Kivirikko & Myllyla, 1982;Yeowell et al. 1995), and this could, in theory, alter the pattern of collagen cross-links, by altering the ratio of deoxypyridinoline (derived from lysine) to pyridinoline (derived from hydroxylysine) (Robins, 1994).The dual purpose of the present study was: first to re-examine the interactions of ascorbate and Cu, by dietary modulation in young guinea-pigs, and second, to test the hypothesis that Cu and/or ascorbate status might alter the ratio of deoxypyridinoline collagen-derived cross-links in bone and in urine. MATERIALS AND METHODS Animals and dietsThe purified guinea-pig diet contained the following components (g/kg): sucrose 331, maize starch 50, ovalbumin 300, cellulose powder 150, maize oil 73, potassium acetate 25, choline chloride 2, magnesium oxide 5 , inositol2, salt mixture 60, vitamins (see later). The salt mixture was based on that of Greenfield et al. (1969), except that the CuS04 therein was omitted. It contained (g/kg): CaC03 205, CaHP04 325, Na2HP04 185, KC1 205, MgS04 70, MnS04 4-5, Fe-citrate...

Zinc and phytate intakes of 183 rural Gambian infants were obtained from weighed records of breastmilk and food intake and measured contents in foods. Total zinc intake of 2.7 mg/d in the first month of age declined to 1.5 mg/d at 3 months, then increased to 4.3 mg/d by 17 months. Breastmilk was an important source of zinc, but the predominant cereal and groundnut-based foods had high [phytate]/[Zn] molar ratios ranging from 13 to 28, indicating potential impaired zinc bioavailability. The [phytate]/[Zn] molar ratio for the diet as a whole was low in early infancy, but increased to 13 in the second year. In contrast, this ratio was less than 6 for the diet of 48 Cambridge breastfed infants up to 18 months. A further disadvantage to the Gambian infants was indicated by their lower intake of protein of animal origin. However, calcium intake was estimated in both communities to be below the level which could give rise to zinc chelation in association with phytate. Compared to 'basal' and 'normative' requirements, total zinc intake of the Gambian infants showed the greatest shortfall between 3 and 12 months, making this the age band for maximum probable benefit from focused intervention programmes.

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Mild-to-moderate vitamin C depletion in weanling guinea-pigs affects pyridinoline:deoxypyridinoline (collagen cross-link) ratios in femur shaft and urine, attributed to impairment of hydroxylation of collagen lysine. We investigated: (1) whether the picture at two time points is compatible with progressive accumulation of abnormal collagen; (2) whether any changes are seen in skin, where little deoxypyridinoline occurs; (3) whether total food restriction has similar effects. Male weanling Dunkin-Hartley guinea-pigs were fed diets containing either 0·5 (vitamin C-restricted) or 160·0-320·0 (vitamin C-adequate) mg vitamin C/d. Two groups receiving the vitamin C-adequate diet received it ad libitum. Two other groups received the vitamin C-adequate diet in a restricted amount, limited to that which permitted nearly the same growth rate as in the vitamin C-restricted groups. Animals were fed for 4 or 8 weeks; urine was collected, and vitamin C and collagen indices were measured. In the femur shaft, the hydroxyproline content per unit weight was unaffected by vitamin C restriction or by total food restriction. Deoxypyridinoline was increased and the pyridinoline:deoxypyridinoline ratio was decreased in vitamin C-restricted groups, but not in food-restricted groups. Changes in the value of the ratio were greater after 8 than after 4 weeks. Urine indices mirrored bone indices. In skin, the main effect of vitamin C restriction was to reduce hydroxyproline. Here, the cross-link ratios changed less markedly than in bone, and there was less deoxypyridinoline. We conclude that the picture at two time points is compatible with a progressive accumulation of pyridinoline-enriched collagen in vitamin C-deprived animals, that the picture in skin differs from that of bone and urine, and that cross-link changes are not produced by total food restriction.