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A sensitive and reliable biomarker has yet to be identified for marginal copper deficiency in humans. The need for such a biomarker is critical, because increased cases of human copper deficiency evolve following bariatric surgery and other secondary factors besides diet. Four experiments were devised to induce marginal copper deficiency through copperdeficient (CuD) diets (5 wk for mice and 4 wk for rats). In Expt. 1 and 2, male postweanling mice were raised in either solidbottom plastic cages (Expt. 1) or stainless steel hanging cages (Expt. 2) and compared. Postweanling rats (Expt. 3) and adult mice (Expt. 4) were also studied using stainless steel cages. Copper-adequate controls were fed a semipurified diet containing 9 mg Cu/kg. CuD rats exhibited the most severe changes in biomarkers due to copper limitation, including major reductions in plasma ceruloplasmin (Cp) and erythrocyte superoxide dismutase (Sod1) and augmentation in copper chaperone for Sod1 (CCS). The CuD mice in Expt. 2 were more deficient than the CuD mice in Expt. 1, likely due to coprophagia differences. In fact, the CuD mice in Expt. 1 had unaltered Sod1 or Cp levels. Importantly though, these marginally deficient mice and CuD adult mice that had no changes in Cp activity or liver copper level had robust augmentation of CCS. Erythrocyte CCS was the only consistent biomarker to change in copper deficiency for all dietary groups, suggesting that CCS may be an excellent biomarker for human confirmation of marginal copper deficiency. J. Nutr. doi: 10.3945/jn.111.150755.

In previous work identification of urinary metabolites of 4'-deoxypyridoxine which had been oxidized in the 5'-position and long-term dilution of labeled urinary metabolites with unlabeled molecules suggested possible microbial contributions. In the current studies germfree guinea pigs were able to convert 4'-deoxypyridoxine to 4'-deoxy-5-pyridoxic acid demonstrating that the ability to oxidize the 5'-position is not restricted to microorganisms. Labelling curves for urinary pyridoxic acid in rats continuously fed [14C]pyridoxine since weaning were similar in conventional and germfree animals indicating that any vitamin B-6 synthesized in the intestinal tract was not readily absorbed and metabolized. Therefore, coprophagy did not make a detectable contribution to vitamin B-6 metabolism in rats receiving a nutritionally complete diet. The difficulty in achieving comparable labeling in adult animals is probably due to very slow turnover of portions of the vitamin B-6 pool and not to microbial production of vitamin B-6. The total pool calculated from the radioactivity in the germ-free rats averaged 16.2 +/- 0.8 nmol vitamin B-6 compounds/g body wt. Only 10% of the ingested label was recovered in the feces. In addition, only about 50% of the label excreted in the urine appeared as 4-pyridoxic acid in rats. These observations suggest that it may be difficult to quantitate the total urinary and fecal excretion of ingested vitamin B-6 without using tracers.

The rate of passage of digesta and the digestibility of a nonpurified diet were studied in adult female rats prevented from coprophagy on alternate weeks by confinement to their normal feeding tunnels in metabolism cages. In food-restricted rats a decrease in the time for the first appearance in the feces of a digesta marker was noted when prevention of coprophagy was followed by permitting rats to feed on their feces while being maintained on a restricted food intake, as compared to control rats permitted coprophagy throughout. The prevention of coprophagy had no effect on the rate of passage of digesta along the small intestine. The prevention of coprophagy had no effect on the apparent digestibility of dry matter, organic matter, energy or protein when the rats were fed ad libitum. However, when rats had lost 20% or more of their body weight by restricted feeding, digestibility of the measured constituents of the food offered in restricted amounts increased, but this effect was abolished when coprophagy was prevented.

The effect of coprophagy prevention and antibiotics on acetyl- choline levels of kidney, brain and small intestine in weanling rats fed choline- deficient diets was studied. It was found that 5 to 6 days of dietary choline deficiency resulted in decreased concentration of acetylcholine in kidney (64% ), brain (28% ) and small intestine (30% ). Coprophagy prevention or the inclu sion of antibiotics in the diet maintained the tissues' acetylcholine at normal or near-normal levels in the choline deficient rats. These results are similar to those which have been obtained by others in germfree rats. J. Nutr. 101: 25â€"28, 1971.

When a diet containing raw soybean was fed to rats, there was an increase in the synthesis of pancreatic protein, presumably exocrine protein, as evidenced by an increased uptake of [35S]cystine. There was also an increased transsulfuration of methionine sulfur as indicated by labeled sulfur transformation from methionine to cystine. This same pattern of events was produced in rats receiving a casein-containing diet when 50 mg of crystalline trypsin inhibitor was administered by gavage. However, if coprophagy was prevented, the increased uptake of [35S]cystine and [35S]methionine transsulfuration under both dietary conditions was blocked. It was found that prevention of coprophagy was without effect upon these two processes if supplementary dietary cystine was provided or if a dietary protein source with adequate cystine, i.e., heat-treated soybean, was provided. It was concluded that by practicing coprophagy, sufficient fecal cystine was being returned to the upper intestinal tract to permit some synthesis of pancreatic exocrine protein and with this stimulation of synthesis, transulfuration could proceed. This assumes that the biosynthesis of cystine is dependent upon the availability of sufficient cystine to permit active protein synthesis. In another situation where cystine requirement is high, namely, the rapidly growing rat, a limited amount of cystine was fed by providing a 12% casein diet. Either supplementary cystine or methionine was provided and it was found that both amino acids gave optimal growth in conventional rats, but when coprophagy was prevented, optimal growth was achieved only with the cystine-supplemented diet.

Effects of ascorbic acid ingestion on the severity of vitamin B-12 deficiency were investigated by feeding weanling rats experimental diets containing 0-100 micrograms of vitamin B-12 activity per kg of diet, with or without 6.0 mg of ascorbic acid per ml supplied in drinking water for 15 wk. This daily consumption of up to 150 mg of ascorbic acid did not impair growth, but did result in significantly higher concentrations of ascorbic acid in plasma, liver, adrenal glands and feces. When rats were fed diets deficient or marginally deficient in vitamin B-12, liver concentrations of vitamin B-12 were markedly lower than in liver of rats fed adequate vitamin B-12. Ascorbic acid ingestion raised values significantly in the vitamin B-12-deficient diet group. Urinary methylmalonic acid was significantly elevated in the deficient rats. However, it was significantly reduced to more normal values by ascorbic acid in rats with both low and marginal vitamin B-12 status, as defined by dietary and liver concentrations of vitamin B-12 activity. Although coprophagy was not prevented, rats showed no increased consumption of feces with the higher ascorbic acid content. Thus, the results of this research indicate that vitamin C ingestion partially protects rats from vitamin B-12 deficiency.

The prevention of coprophagy decreased the apparent digestibility of dry matter, organic matter, crude protein, ether extract and acid detergent fiber when guinea pigs were fed alfalfa meal or a semipurified diet. The feces of guinea pigs fed the semipurified diet were analyzed for eight minerals by mass spectroscopy, and the prevention of coprophagy increased the amount of minerals appearing in the feces. The addition of 250 ppm molybdenum to the semipurified diet did not increase the amount of copper in the feces. It was concluded that coprophagy is important in the utilization of nutrients by the guinea pig and that coprophagy must be considered in digestion trials with any rodents. The importance of coprophagy in the nutrition of the rat and rabbit has received considerable study but little work has been conducted with guinea pigs. For example, coprophagy is not mentioned by Reid (1) in her review of the nutrition of the guinea pig, nor is the guinea pig discussed in a recent review dealing with coprophagy (2). Therefore, the following trials were initiated to study the effect of coprophagy on digestion and mineral excretion in the guinea pig. EXPERIMENTAL