Vitamin D deficiency (plasma 25-hydroxycholecalciferol (25(OH)D)<50 nmol/l) is highly prevalent, increases risk of non-communicable diseases (NCD) and associates with increased oxidative stress in obese subjects, the elderly and patients suffering from NCD. If confirmed as an independent driver of oxidative stress, nutritional and other public health strategies to improve vitamin D status would be strongly supported. We investigated vitamin D/oxidative stress links without the confounding effects of advanced age, obesity, smoking or pre-existing disease. Plasma 25(OH)D and biomarkers of oxidative stress and antioxidant status (plasma allantoin, oxidised LDL, ferric reducing antioxidant power (FRAP), ascorbic acid, urine 8-oxo-7,8-dihydro-2'-deoxyguanosine) were measured in fasting samples from 196 consenting, healthy adults aged 18–26 years. Correlation between 25(OH)D and each biomarker as well as biomarker differences across 25(OH)D quartiles and groups (<25/25–49/≥50 nmol/l) were investigated. Median 25(OH)D was 40 nmol/l; >70 % of participants were vitamin D deficient. No significant correlations and no biomarker differences across 25(OH)D quartiles or groups were seen except for total antioxidant status. A weak direct association (r 0·252, P<0·05) was observed between 25(OH)D and FRAP, and those in the lowest 25(OH)D quartile and group had significantly lower FRAP values. Results did not reveal a clear link between vitamin D status and oxidative stress biomarkers in the absence of advanced age, obesity and disease, though some evidence of depleted antioxidant status in those with vitamin D deficiency was seen. Poor antioxidant status may pre-date increased oxidative stress. Study of effects of correction of deficiency on antioxidant status and oxidative stress in vitamin D-deficient but otherwise healthy subjects is needed.

The objectives of the present study were to investigate the recovery of [14C]allantoin in urine of sheep dosed intravenously and degradation of allantoin by rumen micro-organisms. The recovery of [14C]allantoin in the urine of eight sheep was measured during three periods in two experiments. Individual values of [14C]allantoin recovery varied from 66 to 95 % (mean value 83 (SE 1·6) %). The recovery of [14C]allantoin showed no relation to the level of feed intake. There was some evidence that glomerular filtration rate was an important factor affecting the amount of urinary allantoin recovered in one experiment. Incomplete recovery of plasma [14C]allantoin in the urine indicated losses of plasma [14C]allantoin via non-renal routes. This is supported by the disappearance of 14C from rumen contents incubated in vitro with [14C]allantoin for 48 h (88 %) and the presence of 14C in saliva in vivo from sheep sampled after dosing with [14C]allantoin. However, the amount of 14C activity in the saliva was very low (equivalent to only 1·5 % of the total dose in sheep producing saliva at a rate of 15 litres/d). The proportion of renal and non-renal excretion of purine derivatives was found to be unpredictable both between and within individual animals. The factors responsible for this variability need to be identified, and existing models of excretion of purine derivatives may need to be modified accordingly to improve their accuracy of prediction. A single intravenous injection of [4,5-14C]allantoin provides a simple alternative to infusion methods used to measure the proportion of plasma allantoin excreted in the urine of sheep. Using this method it may be feasible to validate PD excretion models in other ruminant livestock.

Allantoin net flux through the plasma allantoin compartment was determined in sheep given a roughage diet by means of a continuous infusion of [4,5-14C]allantoin for 17 h. Unlabelled allantoin was infused intravenously during the last 7 h of the tracer infusion to increase the allantoin flux by approximately 75 %. When unlabelled allantoin was infused, the specific radioactivity of allantoin in plasma and urine declined exponentially to approach a lower plateau some 2–3 h later. The estimate of net flux during the infusion of unlabelled allantoin, estimated from blood plasma and urine, was on average 79 and 90 % of expected values. Expected values of allantoin net flux during infusion of unlabelled allantoin were calculated as the sum of allantoin net flux pre-loading plus the known rate of infusion of the allantoin load. It is probable that endogenous allantoin synthesis was decreased by the infusion of allantoin: allosteric inhibition of uricase appears a plausible explanation for this observation, and for lower estimates of net flux. Appearance of labelled allantoin-C in ruminal or blood bicarbonate was negligible. Our results indicate that net flux of allantoin through blood plasma is a good predictor of the entry rate of allantoin into the primary compartment and should be a better predictor of rumen microbial outflow than urinary allantoin excretion. However, measurements of allantoin-specific radioactivity, during continuous infusion, should be taken after a period of 24 h, at which time the true plateau specific radioactivity value of allantoin in plasma would be attained.

The kinetics of allantoin metabolism were studied in rumen-cannulated sheep by means of a single intravenous injection of [4,5-14C]allantoin. The decline in the specific radioactivity of allantoin in plasma following the injection of tracer was best described by a double exponential function, indicating that allantoin moves in and out of two or more kinetically distinct compartments. Sequestering of tracer in secondary or tertiary compartments in the body water appears likely to have resulted in overestimation of net flux of allantoin through the blood in the present study. In future studies, sampling of blood for several days after administration of tracer should alleviate this problem. About 80 % of the [14C]allantoin injected was recovered as [14C]allantoin in urine during the 12 h after tracer injection, increasing to 94 % after 4 d. Allantoin-C also passed through the blood bicarbonate pool, suggesting that allantoin is degraded in the gastrointestinal tract. A small amount of allantoin-C (4 % of the net flux of allantoin through the blood pool) was apparently degraded to form bicarbonate-C in the rumen and postruminally, and subsequently appeared in blood bicarbonate-C. Transfer of allantoin-C into the rumen via saliva was insignificant. In view of these findings, the net flux of allantoin through the blood should be a better predictor of rumen microbial outflow than urinary allantoin excretion, because urinary excretion of purine derivatives must be adjusted for conversion of allantoin-C to blood bicarbonate when used to predict the flow of microbial protein from the rumen.

The potential of milk allantoin as an index ofmicrobial protein supply was evaluated in two experiments conducted with 12 multiparous Holstein-Friesian dairy cows that examined the effects of altering the supply of metabolizable energy (ME) and fermentable metabolizable energy (FME). In the first experiment, late lactation cows received a 14·3 kg dry matter (DM) per day basal diet consisting of (g/kg DM) chopped barley straw (415), soya-bean meal (322), molassed sugar-beet pulp (197), molasses (33), urea (17) and a vitamin and mineral supplement (17) for a 21-day co-variance period. During four 16-day periods, six treatments were allocated randomly to cows, consisting of potato starch (1, 2 or 3 kg DM per day) or fat supplements (0·64, 1·27 or 1·91 kg DM per day). In the second experiment, early lactation cows received 40 kg/day (fresh weight, (FW)) of silage (307 g/kg toluene-corrected dry matter, 149 g/kg DM crude protein and 11·6 MJ/kg ME). During three 21-day periods, four treatments were evaluated consisting of supplements of either 4·1 (LI) or 8·1 (12) kg fresh weight per day of a low fat concentrate (acid hydrolysis ether extract (AHEE) 45 g/kg DM) or 3·8 (HI) or 7·5 (H2) kg fresh weight per day of a high fat concentrate (AHEE 110 g/kg DM). Both experiments showed individual cow milk allantoin concentration or excretion to be poorly correlated with urinary purine derivative excretion or calculated microbial protein supply. Use of treatment mean (TM) values dramatically improved these relationships. For pooled TM (no. = 10) values from both experiments, close relationships existed between milk allantoin excretion and concentration with milk yield (r values 0·991 and 0·883, respectively). Auto-correlation with milk yield appeared to account for milk allantoin excretion and concentration being highly correlated with urinary purine derivative excretion (r values 0·908 and 0·934, respectively) and calculated microbial protein supply (r values 0·938 and 0·945, respectively). Current experimental data indicates that measurement of milk allantoin is not a reliable indicator of microbial protein supply for individual cows.

The green alga Chlamydomonas reinhardtii can use the ureides allantoin and allantoate as sole nitrogen sources. Once the uptake systems for allantoin and allantoate were induced, the uptake and growth rates were identical for the two ureides. However, the enzymatic activities involved in the degradation of the two ureides (allantoinase and allantoicase) were regulated differently. Allantoinase seems to be constitutive, since it was detected in all the nitrogen sources studied, while allantoicase behaved as an inducible enzyme, since it was present only in cells cultured in ureides or any metabolic precursor of these compounds. Neither allantoinase nor allantoicase activities were repressed by ammonium in the presence of ureides. Allantoicase activity was not induced under nitrogen starvation conditions, while it was induced in cells that had been cultured with allantoin or allantoate in the dark. Allantoin uptake showed a pattern similar to that of allantoate under all nutritional and environmental conditions tested. Inhibition of allantoin and allantoate uptake by N-ethylmaleimide suggests that thiol (SH–) groups are involved in both uptake systems. The use of both allantoin and allantoate was similarly inhibited by the metabolic poisons tested (cyanide, azide, 2,4-dinitrophenol and 3′-(3,4-dichlorophenyl)-1′,1′-dimethyl urea) but only at very high concentrations. The possibility that uptake of allantoin and allantoate might take place through two independent systems is discussed.

To investigate the effects of energy source and protein level of diets on milk protein content, 12 multiparous Holstein-Friesian cows were used in a 4 × 4 Latin square change-over experiment with 4-week periods. Four diets were offered, with ad libitum silage as proportionately 0·40 of the diet, and the remaining 0·60 as one of four concentrates, two based on barley and two on molassed sugar-beet pulp. Two protein levels were achieved by altering the amounts of digestible undegraded protein in the concentrates, with all diets formulated to supply equal quantities of rumen degradable protein. There was no effect of diet on dry-matter intakes. Both starch and high dietary protein levels significantly increased milk protein concentration (P < 0·05), but had no effects on milk fat and lactose concentrations. Mean milk yields were significantly higher (P < 0·05) with increased dietary protein. Dietary protein significantly affected the yields of milk protein (P < 0·01) and lactose (P < 0·05) but not that of fat. Urinary allantoin excretion was significantly greater with both high protein (P < 0·05) and starch-based diets (P < 0·05). No significant interaction effects were found. It is concluded that dietary effects were due largely to differences in supply of rumen degradable protein; increases in milk protein concentration were therefore brought about by increasing the protein supply to the animal.

Estimations of purine derivatives excretion and urinary-nitrogen loss were used to test the response of rumen fermentation to supplementation of straw with different sources of carbohydrate. Two groups of Rasa Aragonesa ewes (44 (s.e. 0·75) kg live weight were given ad libitum basal diets of either ammonia-treated (ATS) or urea-supplemented (USS) barley straw, with 12 animals per basal diet group. Three supplements, barley grain, sugar-beet pulp or grass hay, respectively, were given to each basal diet group, giving a total of six dietary treatments with four animals per treatment group. Four levels of supplementation were studied (150, 300, 450 and 600 g air dry matter per day), one in each of four experimental periods. Each 45-day experimental period comprised 38 days of adjustment followed by a 7-day measurement period. Digestible organic matter (DOM) intake was higher in animals receiving ATS than in animals receiving USS (504 v. 474 (s.e. 21·1) g/day, P < 0·005) and higher in animals receiving barley grain and sugar-beet pulp than in those receiving grass hay (512 and 496 v. 370 (s.e. 25·9) g/day, P < 0·005). DOM intake also increased with the level of supplementation and this increase was greater with barley grain (504 to 634 and 314 to 554 g/day for ATS and USS) and sugar-beet pulp (440 to 582 and 315 to 522 g/day) than with grass hay (430 to 407 and 267 to 370 for ATS and USS). Urinary excretions of hypoxanthine, xanthine and uric acid were not affected by the experimental treatment whereas allantoin excretion (y, mmol) increased in response to DOM intake (x, kg) (y = 13·72 × − 0·26; r = 0·79; P < 0·001; no. = 96). The response in allantoin excretion was mainly explained by the increase in DOM intake. However when data were expressed per unit of DOM intake significant differences were still evident. Allantoin/DOM intake (mmol·kg) ratio and calculated microbial nitrogen (g·kg DOM intake) supply were lower with USS diets and sugar-beet pulp supplemented diets (P< 0·05) and increased significantly with level of supplementation (P < 0·001).

During a series of routine experiments to evaluate 16 foods offered to wether sheep, conventional measurements of digestibility were augmented by measurement of fractional outflow rates (FOR) of solids (ks) and liquid (k1) from the rumen and urinary allantoin nitrogen excretion (UAN) as an index of microbial yield. Fixed (1 kg) amounts of ‘dry’ foods were offered to sheep ranging in weight from 42 to 89 kg which permitted a retrospective analysis of plane of nutrition effects on diet digestibility, ks, k1 and UAN. Each diet was simultaneously offered to three sheep (one each of large, medium and small size). Smaller sheep had significantly higher FOR, significantly lower apparent digestibilities of dry matter and significantly higher UAN (g/kg digestible organic matter (DOM)). Higher UAN (g/kg DOM) indicated higher microbial energetic efficiencies with the smaller sheep.

The saliva of sheep was shown to contain significant concentrations of uric acid (16 (sd) 4.5) μmol/l) and allantoin (120 (sd 16.4) μmol/l), sufficient to recycle purine derivatives equivalent to about 0.10 of the normal urinary excretion. When allantoin was incubated in vitro in rumen fluid, it was degraded at a rate sufficient to ensure complete destruction of recycled allantoin. In a series of experiments in which allantoin was infused into the rumen of sheep fed normally, or into the rumen or abomasum of sheep and the rumen of cattle completely nourished by intragastric infusion of volatile fatty acids and casein, no additional allantoin was recovered in the urine. These losses were probably due to the degradation of allantoin by micro-organisms associated with the digestive tract. It is concluded that all allantoin and uric acid recycled to the rumen via saliva will be similarly degraded. Therefore, the use of urinary excretion of purine derivatives as an estimator of the rumen microbial biomass available to ruminants will need to be corrected for such losses.