Sulphate recycling and metabolism in sheep and cattle.

Merino wethers and Brahman x Shorthorn steers, offered lucerne or spear grass hay, were used to study the movements of sulphate through pools in plasma and ruminal liquor. The irreversible loss of sulphate from ruminal liquor was 60 and 76% of sulphur ingested for both species fed lucerne and spear grass respectively. The irreversible loss of sulphate from the plasma averaged 67 and 56% of sulphur ingested for animals fed lucerne and spear grass respectively. Daily recycling of sulphate to the rumen of sheep was 98 mg sulphur on the lucerne diet and 3.9 mg sulphur on the spear grass diet. Sulphate recycling in cattle fed lucerne was 533 mg sulphur; in cattle fed spear grass the value was 234 mg sulphur. Over 6 days following an intravenous injection of [35S]sulphate into sheep and cattle fed lucerne, 5-10% of the dose was excreted in the faeces and c. 10% was retained. Corresponding values for animals fed spear grass were 23-31% in faeces and 40-51% of the dose retained. After intraruminal injections of [35S]sulphate, animals fed lucerne excreted 15-18% of the dose in the faeces and retained 25-30% of the dose over 6 days. Values for animals fed spear grass were 22-26% in faeces and 62-70% retained. It was concluded that sulphate recycling to the rumen is a limiting factor in microbial synthesis for sheep fed low-quality roughage, and that secretion of endogenous sulphur into the postruminal tract of ruminants is of importance in the metabolism of sulphate.     

The availability of inorganic sulphate in blood for sulphate conjugation of drugs in rat liver in vivo. (35S)Sulphate incorporation into harmol sulphate.

1. When Na235SO4 is injected intravenously in rats, it is immediately available for sulphate conjugation of the phenolic drug harmol (7-hydroxyl-1-methyl-9H-pyrido[3,4-b]indole) in the liver. This was established by following the time course of the biliary excretion of the sulphate conjugate of harmol, and the incorporation of [35S]sulphate into harmol sulphate. 2. During the 10min immediately after injection of Na235SO4 re-distribution of [35S]sulphate took place, which resulted in a rapid initial decrease in the plasma concentration of [35S]sulphate; a concomitant decrease in the amount of [35S]sulphate incorporated into harmol sulphate was observed, indicating that the co-substrate of sulphation, adenosine 3'-phosphate 5'-sulphatophosphate, equilibrates rapidly with [35S]sulphate in plasma. 3. The results suggest that the pool size of adenosine 3'-phosphate 5'-sulphatophosphate is very small; therefore the specific radioactivity of [35S]sulphate in plasma determines the specific radioactivity incorporated into sulphate esters at any time.     

The low availability of dietary choline for the nutrition of the sheep.

1. Choline, which is present in the diet of the sheep either in the non-esterified form or combined in phospholipids, is rapidly degraded in the rumen. The ultimate product formed from the N-methyl groups is methane. 2. Analysis of the non-esterified choline and the phosphatidylcholine in ruminal and abomasal digesta indicate that the phospholipid is the main vehicle for the passage of choline to the lower digestive tract. 3. The concentration of phosphatidylcholine in abomasal digesta is lower than that of ruminal digesta, which is in line with a selective retention of protozoa in the rumen as observed by others. 4. On defaunation of the rumen to remove ciliated protozoa the concentration of phosphatidylcholine in ruminal digesta falls markedly and becomes lower than that in abomasal digesta. 5. Calculation shows that the adult sheep obtains at most only about 20--25 mg of effective choline per day from its diet (0.002--0.0025% of dietary total dry-weight intake). This is some fifty times less than the minimum required to avoid pathological lesions and death in other species investigated (0.1%+ of dietary dry-weight intake). 6. Sheep liver can synthesize choline from [14C]ethanolamine both in vitro and in vivo, but the synthesis of choline per kg body weight is many times less than it is in the rat. 7. The intact sheep oxidizes an injected dose of [1,2-14C]choline to CO2 at a rate that is several times less than that observed for the rat. This could help to explain the apparent minimal requirement of sheep for dietary choline.     

Absorption, serum levels and urinary excretion of inorganic sulfate after oral administration of sodium sulfate in the conscious rat.

The absorption of inorganic sulfate after ingestion was investigated in rats. After oral administration of Na235SO4, 35S radioactivity was measurable in plasma already after 15 min and its plasma concentration reached a peak after about 1.5--2 h. The 35S-radioactivity excreted in urine during 24 h after ingestion of Na235SO4 together with varying amounts of unlabelled Na2SO4 (0.25--5.0 mmol Na2SO4 per rat) indicated an almost complete absorption of inorganic sulfate from the gastrointestinal tract. Determination of the inorganic sulfate concentration in rat serum 2 h after oral administration of 5.0 mmol Na2SO4 revealed a three-fold increase in serum sulfate concentration. The data suggest a rapid and almost complete absorption of inorganic sulfate after oral administration in the rat. Its importance in relation to the sulfate availability for sulfate conjugation of drugs is discussed.     

The influence of dietary sulphur loading on metabolism and health in young sheep fed low fibre and high starch diet

The purpose of the experiment was to evaluate the long-term effect of a low roughage diet (7-8% CF) with or without sulphur (S) supplementation (elemental and sodium sulphate 1:1) on basal dietary components, Zn, Cu and S availability, rumen metabolism and health in growing sheep. The control diet contained 0.2% and the supplemented diet 0.8% of S on a DM basis. The experiment lasted 12 weeks. The intake of the diet with 0.8% of S resulted in an increase in rumen acetic acid concentration. Rumen lactic acid concentrations in S-supplemented versus control sheep were higher in the first and lower in the third month, and decreased at the end of the third month of the experiment in both groups. Blood plasma pyruvate was lower at the end of the experiment in S-supplemented sheep than in control sheep. In the 5th week of the experiment, the high S diet depressed basal nutritional components as well as Zn and Cu availability. After 12 weeks of feeding of this diet, polioencephalomalacia had developed in all sheep.     

Gastro-intestinal tract function in sheep infused with somatostatin

The effect of a 5-day continuous intravenous infusion of somatostatin (4.6 ng min-1 kg-1) was studied, using anoestrous ewes given 791 g dry matter per day of a 60:40 lucerne hay:oat grain pelleted diet from a continuously moving belt. 51Cr-EDTA, 103Ru-phenanthroline and lignin were used as markers to determine digesta mean retention times (MRT) by a continuous infusion-total sampling procedure. The somatostatin infusion increased the concentration of somatostatin in venous plasma within the physiological range from 10 to 76 ng/l, decreased plasma concentrations of prolactin and thyroxine, but had no effect upon plasma concentrations of insulin and glucagon. It had no effect upon digesta-free weight of the rumen and omasum but consistently decreased the weight of all post-ruminal segments of the gastrointestinal (GI) tract. The infusion increased the accumulation of digesta in the abomasum and caecum. Total MRT of all three markers in the entire GI tract was unaffected by somatostatin infusion, but the proportion of total MRT spent in the abomasum + small intestine + caecum increased and the proportion spent in the large intestine and rumen decreased. Somatostatin infusion decreased apparent endogenous abomasal secretion, increased water flow from the rumen and into the abomasum and decreased voluntary water consumption. It is proposed that the prime site of somatostatin action was in the abomasal to caecal region, where somatostatin-secreting D cells are found in greatest concentration, that effects observed in the large intestine and rumen may represent secondary compensatory mechanisms and that the effects observed were due to direct action of somatostatin and were not mediated by other GI hormones.     

[35S]Thiosulphate oxidation by rat liver mitochondria in the presence of glutathione

1. Rat liver mitochondria incubated in oxygen with glutathione and [(35)S]-thiosulphate produced labelled sulphate. 2. Inner-labelled thiosulphate (S.(35)SO(3))(2-) was converted into [(35)S]sulphate more rapidly than outer-labelled thiosulphate ((35)S.SO(3))(2-). 3. Thiosulphate labelled in both sulphur atoms was formed during ((35)S.SO(3))(2-) oxidation; the outer sulphur atom before oxidation to sulphate was incorporated into the inner position. 4. A thiosulphate cycle in the metabolic pathway of sulphate formation in animal tissues is discussed.     

Inhibition of sulphur redistribution into new leaves of vegetative soybean by excision of the maturing leaf

When soybean plants are pulsed with [³⁵S]sulphate, label is subsequently redistributed from the roots to the leaves. This confounds studies to measure the redistribution of label from leaves. Accordingly, soybean plants ( Glycine max [L.] Merr. cv. Stephens) were grown in 20 μ M sulphate and a small portion of the root system (donor root) was pulsed with [³⁵S]sulphate for 24 h. After removing the donor root, the plants were transferred into unlabelled solution, either without sulphate (S20→SO) or with 20 μ M sulphate (S20→20) (intact plants). Also at this time, the expanding leaf (L3) was excised from half of the plants in each treatment (excised plants). Immediately after the pulse, only ca 15% of the label occurred in the roots and ca 40% in the expanding leaf, L3, mostly in the soluble fraction. In intact S20→20 plants, ³⁵S-label was exported from the soluble fraction of L3, mostly as sulphate, whilst L4 and L5 imported label. Similar responses occurred in S20→SO plants except that export of label from L3 was more rapid. Excision of L3 from S20→S20 plants inhibited labelling of leaves L4-L6 but not total sulphur, whereas in S20→SO plants, excision of L3 inhibited the import of both total sulphur and ³⁵S-label in leaves L4, L5 and L6. The data suggest that the soluble fraction of almost fully expanded leaves is an important reserve of sulphur for redistribution to growing leaves. The ³⁵S-label in the root system exhibited fluctuations consistent with its proposed role in the recycling of soluble sulphur from the leaves.     

Sulfur and methionine metabolism in sheep. I. First approximations of sulfur pools in and sulfur flows from the reticulo-rumen.

Sulfur pools in the rumen and sulfur flows from the rumen were investigated in two experiments with sheep on a diet containing equal parts of oaten and lucerne chaffs. The diet was fed at two levels, either chopped or pelleted, and with intraruminal DL-methionine supplements. Ruminal fluid volumes and fluid flows to the omasum were measured. None of the treatments influenced ruminal fluid volume. Fluid flow to the omasum, however, was increased by increasing dry matter intake (DMI), and was further enhanced by feeding chaffed hay rather than the same materials ground and pelleted; the DL-methionine supplement had no effect. First approximation of the ruminal sulfur pools and of sulfur flows to the omasum were derived from the concentration of sulfur in true digesta and the ruminal fluid volume or fluid flow. Increasing DMI from 500 to 1000 g/day resulted in larger ruminal pools of total (1096 v. 792 mg), neutral (1016 v. 731 mg) and protein (479 v. 419 mg) sulfur, but the reducible sulfur pools were not affected by the level of DMI. Infusions of DL-methionine increased the ruminal sulfide sulfur pool irrespective of level of DMI. The first approximation of total sulfur flow was increased by 1660 mg/day at the higher level of DMI, due mainly to increases of 710 mg S/day as protein sulfur and 859 mg S/day as non-protein neutral sulfur. Flows of inorganic sulfate and ester sulfate sulfur, although small in comparison with organic sulfur flows, increased with level of DMI. Sulfide sulfur flows were also increased at the higher level of DMI, and were almost doubled by intraruminal infusions of DL-methionine.     

Passage of the intravenously administered 15N urea into the digestive tract and its excretion in the sheep.

The experiments performed on two wethers provided with simple rumen cannulas and reentrant cannulas, inserted into the proximal duodenum and ileum, showed a passage of 15N from labelled urea, injected intravenously, from the blood to the digestive tract. The amount of the 15N in the digesta was the highest in duodenum, slightly lower in the rumen and slightly lower in ileum. Approximately 50% of the injected 15N was excreted in urine. The amount of the 15N eliminated with feces was very small; 0.6 to 2.8% of the dose injected per day. About 73--84% of the 15N which passed the duodenum was absorbed in further parts of the digestive tract. It can be concluded that all parts of the digestive tract take part in utilization of the endogenous urea.     

[35S]Thiosulphate oxidation by Thiobacillus strain C

1. Thiobacillus strain C oxidized [(35)S]thiosulphate completely to sulphate. 2. During thiosulphate oxidation [(35)S]sulphate was formed more rapidly from (S.(35)SO(3))(2-) than from ((35)S.SO(3))(2-). (35)S disappeared less rapidly from thiosulphate with ((35)S.SO(3))(2-) as substrate than with (S.(35)SO(3))(2-). 3. Thiosulphate labelled in both atoms was produced during ((35)S.SO(3))(2-) oxidation, but not during (S.(35)SO(3))(2-) oxidation. 4. No (35)S was precipitated as elementary sulphur either in the presence or absence of exogenous unlabelled sulphur. 5. During [(35)S]thiosulphate oxidation, appreciable quantities of [(35)S]trithionate accumulated and later disappeared. Other polythionates did not accumulate consistently. 6. [(35)S]Trithionate was formed initially at a greater rate from (S.(35)SO(3))(2-) than from ((35)S.SO(3))(2-), but subsequently at a similar rate from each. 7. Trithionate formed from (S.(35)SO(3))(2-) was labelled only in the oxidized sulphur atoms, but that formed from ((35)S.SO(3))(2-) was labelled in both oxidized and reduced atoms. The proportion of (35)S in the oxidized atoms increased as more trithionate accumulated. 8. The results eliminate some mechanisms of trithionate formation but are consistent both with a mechanism of thiosulphate oxidation based on an initial reductive cleavage of the molecule and with a mechanism in which thiosulphate undergoes an initial oxidative reaction.     

Enzyme activities and pH variations in the digestive tract of gilthead sea bream

Two investigations were carried out with 150 g gilthead sea bream Sparus aurata to determine the relative activity of six digestive enzymes (pepsin, trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B and amylase) and the pH variation in the lumen of different parts of the gut of fish fed one or two meals per day. Pepsin activity was found exclusively in the stomach, whereas activities of the other enzymes studied were found in all regions of the gut, including the stomach. The lack of localization of enzyme production in the digestive tract of S. aurata is similar to many other species as reported in the literature. The pH variations found in the different regions of the gut could be explained by general digestive physiology following the flow of digesta along the digestive tract. The range of pHs recorded in the various regions of the gut were generally outside the cited optima for many digestive proteases in this species.     

Dietary magnesium increases calcium absorption of ovine small intestine in vivo and in vitro

The purpose of this study was to investigate whether or not an increase in dietary Mg intake increases Ca absorption in the ovine gastrointestinal tract. In an in vivo experiment, an increase in the infused MgCl2 level (0.0, 25.0 and 75.0 mg Mg x kg BW(-1) x day(-1) with 75.0 mg Ca x kg BW(-1) x day(-1) as CaCl2) into the rumen for ten days significantly decreased fecal excretion but increased urinary excretion (P < 0.05) of Ca in five castrated male sheep. Apparent Ca absorption tended to increase (P = 0.067) whilst the retention and plasma concentration of Ca were not changed. In an in vitro experiment with isolated segments from the rumen, upper jejunum, cecum and upper colon under the presence of an electrochemical gradient, the mucosal to serosal Ca flux rate was significantly greater in the presence of 60.0 mM as compared with 1.2 mM MgCl2 (P < 0.05). From these results, we conclude that the mucosal Mg has the ability to increase the Ca absorption in the gastrointestinal tract in sheep when the dietary Mg level is raised.     

Disappearance of immunoreactive glycinin and beta-conglycinin in the digestive tract of piglets

Soybean allergy represents a health threat to human and animals. Glycinin and beta-conglycinin, the main storage proteins in soybean, have been identified as major food/ feed allergens. The present study was conducted to investigate the disappearance of immunoreactive glycinin and beta-conglycinin in the digestive processes of piglets. Twelve crossbred piglets, weaned at 21 days of age, were allocated to three dietary treatments in a complete block design, each treatment with four replicates (female/male = 1:1). From day 22-28, the control group was fed diets without leguminous products, while the two treatment groups received diets containing 2.2% purified glycinin or beta-conglycinin. All piglets were slaughtered at 29 days of age and digesta was sampled from stomach, middle jejunum, caecum and colon. Results indicated that immunoreactive glycinin and beta-conglycinin decreased as the digesta descended down the digestive tract to 0.12% and 0.47%, respectively. Little immunoreactive glycinin was found in the digesta of caecum and colon, while immunoreactive beta-conglycinin was detected in the colon. Along the whole digestive tract the disappearance of immunoreactive glycinin was significantly higher than beta-conglycinin (p < 0.05).     

Induction of primary alkylsulphatases and metabolism of sodium hexan-1-yl sulphate by Pseudomonas C12B

Sodium hexan-1-yl sulphate and certain related alkyl sulphate esters have been shown to serve as inducers of the formation of primary alkylsulphatases (designated as P1 and P2) in Pseudomonas C12B. When the organism is grown on sodium hexan-1-yl [(35)S]sulphate as the sole source of sulphur or as the sole source of carbon and sulphur only the P2 alkylsulphatase is formed and inorganic (35)SO(4) (2-) is liberated into the media. Cell extracts contain this anion as the major (35)S-labelled metabolite although two unidentified labelled metabolites as well as choline O-[(35)S]sulphate occur in trace quantities in some extracts. Dialysed cell extracts are capable of liberating inorganic (35)SO(4) (2-) from sodium hexan-1-yl [(35)S]sulphate without the need to include cofactors known to be required for the bacterial degradation of n-alkanes. The collective results suggest that sodium hexan-1-yl sulphate can act as an inducer of P1 alkylsulphatase formation without the need for prior metabolic modification of the carbon moiety of the ester.     

Sulphate uptake and metabolism in the chrysomonad, monochrysis lutheri.

The intracellular concentration of inorganic 35SO4 in Monochrysis lutheri cells exposed to 0.513 mM Na235SO4 for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this 35SO4 with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 mu moles/g cells/hr). The flux of free 35SO4 to organic 35S was 0.029 mu moles/g cells/hr. Assuming an internal electrical potential in the cells of -70 mV, this intracellular concentration of inorganic 35SO4 was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a Km of 3.2 X 10(-5) M and Vmax of 7.9 X 10(-5) mu moles sulphate/hr/10(5) cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the protein(s) associated with sulphate transport.     

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.     

Heparan sulfate at the surface of HeLa cells.

HeLa cells, labeled with Na235SO4, release into the culture medium 35SO4 bound to plasma membrane vesicles next to 35SO4-glycoproteins and free 35SO4. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain 35SO4 and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.     

Kinetics of particulate and solute marker passage in sheep supplemented with cationomycin and lasalocid antibiotics. Comparisons among methods for calculating mean retention time.

Passage of particulate and solute markers in the digestive tract was studied in sheep fed a roughage-pelleted diet supplemented or not with ionophore antibiotics. Methods of marker administration (continuous infusion vs pulse-dose) and mathematical treatment of data were investigated. Antibiotic supplementation did not affect markers' mean retention time (MRT) significantly, regardless of sampling site, technique of marker administration or MRT calculation methods used. The pulse dose technique led to 20-30% higher estimate of particle markers MRT in the entire gut and stomachs of sheep than did the continuous infusion method. Similar results were obtained between these methods for solute markers MRT at both sampling sites. Total MRT in the digestive tract could be partitioned among compartments using a deterministic model when applied to pulse-dose kinetics. Such a partitioning appeared unsatisfactory with continuous-infusion data. Estimations of particle or solute markers MRT in the rumen from duodenal and faecal marker kinetics were significantly different and rarely correlated.     

Carbon and sulphur utilization during growth of Pseudomonas fluorescens on potassium d-glucose 6-O-sulphate as the sole sulphur source

Pseudomonas fluorescens N.C.I.B. 8248 was adapted to grow on potassium d-glucose 6-O-sulphate as the sole carbon and sulphur source. Adapted bacteria grew optimally at 37 degrees C on 1.6% (w/v) sulphate ester and growth coincided with the disappearance of the ester from the culture medium at a rate of 2.4mg/h per ml. Three sulphated compounds were detected in the culture fluid at the termination of growth. One of these was present in traces only and has not been identified. The second was present in somewhat greater amounts and was identified as the 6-O-sulphate ester of d-gluconate, and the major metabolite was identified as d-glycerate 3-O-sulphate. Sulphur utilization by the organism was not associated with the appearance of a glycosulphatase enzyme in the cells. However, a novel enzyme system (or systems) was present that liberated inorganic (35)SO(4) (2-) ions from dipotassium d-gluconate 6[(35)S]-O-sulphate and from dipotassium dl-glycerate 3[(35)S]-O-sulphate. Activity towards the latter substrate could not be detected when the adapted or parent Pseudomonas strain was cultured on d-glucose and potassium sulphate as respective carbon and sulphur sources. Some properties of the enzyme acting on the glycerate ester are recorded.