A. G. Calder

Hepatic detoxification of ammonia in the ovine liver: possible consequences for amino acid catabolism

The effects of either low (25 mumol/min) or high (235 mumol/min) infusion of NH4Cl into the mesenteric vein for 5 d were determined on O2 consumption plus urea and amino acid transfers across the portal-drained viscera (PDV) and liver of young sheep. Kinetic transfers were followed by use of 15NH4Cl for 10 h on the fifth day with simultaneous infusion of [1-13C]leucine to monitor amino acid oxidation. Neither PDV nor liver blood flow were affected by the additional NH3 loading, although at the higher rate there was a trend for increased liver O2 consumption. NH3-N extraction by the liver accounted for 64-70% of urea-N synthesis and at the lower infusion rate the additional N required could be more than accounted for by hepatic removal of free amino acids. At the higher rate of NH3 administration additional sources of N were apparently required to account fully for urea synthesis. Protein synthesis rates in the PDV and liver were unaffected by NH3 infusion but both whole-body (P < 0.05) and splanchnic tissue leucine oxidation were elevated at the higher rate of administration. Substantial synthesis of [15N]glutamine occurred across the liver, particularly with the greater NH3 supply, and enrichments exceeded considerably those of glutamate. The [15N]urea synthesized was predominantly as the single labelled, i.e. [14N15N], species. These various kinetic data are compatible with the action of ovine hepatic glutamate dehydrogenase (EC 1.4.1.2) in periportal hepatocytes in the direction favouring glutamate deamination. Glutamate synthesis and uptake is probably confined to the perivenous cells which do not synthesize urea.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of blood and plasma amino acids using isotope dilution electron impact gas chromatography/mass spectrometry with U‐13C amino acids as internal standards

A method to quantitate blood and plasma amino acids by isotope dilution gas chromatography/mass spectrometry (GC/mS) is described. Samples were spiked with U-(13)C amino acids as internal standards and the tert-butyldimethylsilyl derivatives (tBDMS) separated by capillary column gas chromatography. Linear regression curves, generated for individual amino acids, gave correlation coefficients of 0.9999. The reproducibility of the method was assessed from the analysis of 10 replicate blood and plasma samples. For most amino acids a coefficient of variance (CV) of </=1% was obtained with the exception of aspartate which gave a value of 1.8%. This was probably due to the low concentration of this amino acid in the samples analysed. Recovery of amino acids added to plasma was between 96 and 103%. The use of electron impact ionization (EI) allows the method to be used in laboratories where only the more basic GC/mS is available and reduces the time spent on instrument maintenance. The method should prove useful in areas of work where accurate and precise amino acid concentrations are required.

Effect of food intake on hind-limb and whole-body protein metabolism in young growing sheep: chronic studies based on arterio-venous techniques

Whole-body protein synthesis, estimated by the irreversible loss rate procedure, and hind-leg protein metabolism determined by arterio-venous techniques were monitored in response to three nutritional conditions (approximately 0.6, 1.2 and 1.8 x energy maintenance (M)) in ten wether lambs (33 kg average live weight). In all lambs and treatments measurements were based on radiolabelled phenylalanine, but the terminal procedures (five at 0.6 x M and five at 1.8 x M) also included infusion of [1-13C]leucine; this permitted comparison of amino acids catabolized (leucine) and non-metabolized (phenylalanine) by the hind-limb tissues. Whole-body protein synthesis increased with intake and the relationship with energy expenditure was slightly lower than that reported previously for pigs and cattle. The efficiency of protein retention:protein synthesis did not exceed 0.25 between the two intake extremes. Effects of intake on amino acid oxidation were similar to those observed for cattle. Hind-limb protein synthesis also increased significantly (P < 0.001) in response to intake. Estimates of protein gain, from net uptake values, indicated that the tissues made a greater proportional contribution to total protein retention above M and to protein loss below M, emphasizing the role played by muscle tissue in providing mobile protein stores. The rates of protein synthesis calculated depended on the selection of precursor (blood) metabolite, but rates based on leucine always exceeded those based on phenylalanine when precursor from the same pool was selected. The incremental efficiency of protein retained:protein synthesis was apparently unity between 0.6 and 1.2 x M but 0.3 from 1.2 to 1.8 x M. Blood flow through the iliac artery was also proportional to intake. Leucine and oxo-acid catabolism to carbon dioxide increased with intake such that the metabolic fate of the amino acid was distributed in the proportion 2:1 between protein gain and oxidation. The rates of oxidation were only 1-3% the reported capacity of the rate-limiting dehydrogenase enzyme in muscle, but sufficient enzyme activity resides in the hind-limb adipose tissue to account for such catabolism.

Splanchnic-bed transfers of amino acids in sheep blood and plasma, as monitored through use of a multiple U-13C-labelled amino acid mixture

The response in whole-body and splanchnic tissue mass and isotope amino acid transfers in both plasma and blood has been studied in sheep offered 800 g lucerne (Medicago sativa) pellets/d. Amino acid mass transfers were quantified over a 4 h period, by arterio-venous procedures, across the portal-drained viscera (PDV) and liver on day 5 of an intravenous infusion of either vehicle or the methylated products, choline (0.5 g/d) plus creatine (1.0 g/d). Isotopic movements were monitored over the same period during a 10 h infusion of a mixture of U-13C-labelled amino acids obtained from hydrolysis of labelled algal cells. Sixteen amino acids were monitored by gas chromatography-mass spectrometry, with thirteen of these analysed within a single chromatographic analysis. Except for methionine, which is discussed in a previous paper, no significant effects of choline plus creatine infusion were observed on any of the variables reported. Whole-body protein irreversible-loss rates ranged from 158 to 245 g/d for the essential amino acids, based on the relative enrichments (dilution of the U-13C molecules by those unlabelled) of free amino acids in arterial plasma, and 206-519 g/d, when blood free amino acid relative enrichments were used for the calculations. Closer agreement was obtained between lysine, threonine, phenylalanine and the branched-chain amino acids. Plasma relative enrichments always exceeded those in blood (P < 0.001), possibly due to hydrolysis of peptides or degradation of protein within the erythrocyte or slow equilibration between plasma and the erythrocyte. Net absorbed amino acids across the PDV were carried predominantly in the plasma. Little evidence was obtained of any major and general involvement of the erythrocytes in the transport of free amino acids from the liver. Net isotope movements also supported these findings. Estimates of protein synthesis rates across the PDV tissues from [U-13C] leucine kinetics showed good agreement with previous values obtained with single-labelled leucine. Variable rates were obtained between the essential amino acids, probably due to different intracellular dilutions. Isotope dilution across the liver was small and could be attributed predominantly to uni-directional transfer from extracellular sources into the hepatocytes and this probably dominates the turnover of the intracellular hepatic amino acid pools.

Hepatic protein synthesis in the sheep: effect of intake as by use of stable-isotope-labelled glycine, leucine and phenylalanine

Rates of protein synthesis for the liver, plasma albumin and total plasma protein were quantified in sheep either offered a supra-maintenance intake or fasted for 3 d. The technique of continuous infusion over a 12 h period was employed with the simultaneous infusion of [1-13C]glycine, [1-13C]leucine and [2H5]phenylalanine. Blood and plasma samples were removed at timed intervals from the hepatic portal and hepatic veins plus the aorta. Enrichments of the free amino acids (AA) were determined in all blood and plasma samples as was the protein-bound AA in an apolipoprotein B100 extract. Protein-bound phenylalanine enrichments were also measured in albumin and total protein from plasma plus samples from liver biopsies. The apolipoprotein B100 enrichments agreed well with those of the free AA in hepatic (and hepatic portal) plasma but were lower than for arterial free AA and greater than liver homogenate free AA. This adds support to the concept that export proteins may preferentially use AA directly from extracellular sources. Intake had no significant effect on constitutive liver protein synthesis and the values agreed well with those obtained by other isotopic approaches. There were, however, significant declines, based on hepatic venous free phenylalanine enrichment, at the lower intake in both the fractional (3.4 v. 4.7% per d; P = 0.024) and absolute (2.4 v. 4.2 g/d; P = 0.011) synthesis rates of albumin, which matched the estimated decrease in total plasma albumin content (52 v. 67 g, P < 0.01). In contrast, there was a smaller reduction in total plasma protein mass (145 v. 151 g, P = 0.035) with no observed significant difference in kinetic parameters. Albumin synthesis was calculated to account for a maximum of 17% of total liver protein synthesis in the fed condition and this may fall to 8% during moderate fasts.

Responses in tissue protein synthesis to sub- and supra-maintenance intake in young growing sheep : comparison of large-dose and continuous-infusion techniques

In ten lambs (average live weight 33 kg), five offered 300 g/d (approximately 0.6 x maintenance; L) and five 900 g/d (1.8 x maintenance; H), tissue protein synthesis was measured by three procedures simultaneously. The techniques involved continuous infusion of [U-14C]phenylalanine and [1-13C]leucine over 7-8 h followed by a terminal large dose of [15N]phenylalanine during the last 30 or 60 min. Rates of protein synthesis were then calculated based on the free amino acid or oxo-acid isotopic activity in either arterial, iliac venous blood or tissue homogenate for the continuous-infusion studies, or on plasma or tissue homogenate for the large-dose procedure. For muscle (> 99%), and to a lesser extent skin (85-93%), effective flood conditions were achieved with the [15N]phenylalanine but were either not established or maintained for liver and tissues of the gastrointestinal tract (< 50%). The large dose of phenylalanine also caused changes in the concentration and isotopic activity of blood leucine and 4-methyl-2-oxo-pentanoate. Based on the assumption that the large-dose procedure yields the closest value for the true rate of protein synthesis (L 1.97%/d, H 2.85%/d) then, for muscle, only values based on the homogenate as precursor gave comparable results for both leucine (L 1.83%/d, H 3.01%/d) and phenylalanine (L 1.67%/d, H 2.71%/d) continuous infusion. The values based on the arterial or venous amino or oxo-acid were significantly less, more so at the lower intake. In contrast, for skin, a tissue dominated by export protein synthesis, values from the large-dose procedure (L 6.37%/d, H 10.98%/d) were similar to those derived with arterial or venous metabolites as precursor (L 5.23 and 6.93%/d, H 9.98 and 11.71%/d for leucine), but much less than those based on homogenate data. Based on the large-dose technique, protein synthesis increased with intake in muscle (P < 0.001), skin (P = 0.009) and liver (26.7 v. 30.5%/d; P = 0.029). The contributions of muscle and skin to total protein synthesis were approximately equal. The incremental efficiency of conversion for muscle of synthesized protein into deposition appeared to be similar to values reported for rodents.

The fate of absorbed and exogenous ammonia as influenced by forage or forage–concentrate diets in growing sheep

Changes in splanchnic energy and N metabolism were studied in sheep, prepared with vascular catheters across the portal-drained viscera (PDV) and the liver, and maintained on supramaintenance intakes of either gross or grass + barley pellets. The animals were challenged, on both diets, with 4 d intramesenteric vein infusions of NH4Cl (25 mumol/min) plus NH4HCO3 (at either 0 or 125 mumol/min). On the final day of each treatment the natural abundance NH4Cl was replaced with 15NH4Cl over a 10 h infusion while over the same period [1-13C]leucine was infused via a jugular vein. Measurements were made of blood flow plus mass transfers of NH3, urea, free amino acids and O2 across the PDV and liver. Enrichments of [14N15N]urea and [15N15N]urea plus [15N]glutamine, aspartate and glutamate were also monitored. Whole-body urea flux was determined by infusion of [14C]urea. At the end of the study the animals were infused for 3 h with 15nH4Cl, killed and liver samples assayed for intracellular free amino acid enrichments and concentrations. Blood flows across the splanchnic region were unaffected by either diet or level of ammonium salt infusion. At the lower ammonium salt infusion there was a trend for greater absorption of NH3 across the PDV (P < 0.10) with grass + barley than with the grass diet, while removal of urea was unaltered. At the higher ammonium salt infusions there was a significantly greater appearance of NH3 across the PDV and this exceeded the extra infused. Urea-N removal, however, was also elevated and by more than that required to account for the additional NH3. The PDV contributed 19-28% to whole-body O2 consumption and the liver 23-32%. Hepatic extraction of absorbed NH3 was complete on all treatments and systemic pH remained constant. The fractions of urea-N apparently derived from NH3 were similar on the grass (0.50-0.64) and grass + barley (0.64-0.67) diets. Hepatic production of urea agreed well with urea flux measurements. Between the two levels of ammonium salt infusion and within diets the additional NH3 removed across the PDV was accounted for by the increased urea-N production. The [14N15N]:[15N15N] ratio of the urea produced was 97:3, while the enrichment of hepatic intracellular free aspartate was lower than that of [14N15N]urea. Glutamine enrichments were 0.23-0.37 those of [14N15N]urea, indicating a minor role for those hepatocytes (probably perivenous) which contain glutamine synthetase (EC 6.3.1.2). Leucine kinetics, either for the whole body or splanchnic tissues, were not different between diets or level of ammonium salt infusion, except for oxidation which was less on the grass + barley ration. Amino acid concentrations were lower on the grass + barley diet but net PDV absorptions were similar. The pattern of essential amino acids absorbed into the PDV showed good agreement with the published composition of mixed rumen microbial protein. Fractional disappearances of absorbed free essential amino acids across the liver varied from 0.4 (branched chains) to near unity (histidine, phenylalanine).

Determination of 15N isotopic enrichment and concentrations of allantoin and uric acid in urine by gas chromatography/mass spectrometry.

A method for the determination of 15N enrichment and concentration of allantoin and uric acid simultaneously in urine using gas chromatography/mass spectrometry (GC/MS) is described. The urine samples contained [1,3-15N2] uric acid and its oxidation product allantoin. The uric acid and allantoin were isolated using an AG1-X8 (Cl-form) anion-exchange column and heated with a mixture containing 1:1 dimethylformamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). The tert-butyldimethylsilyl (TBDMS) derivatives of allantoin and uric acid formed were injected into a gas chromatograph interfaced with a mass spectrometer operated under electron impact ionization conditions. Isotope ratio measurements were made from the abundance of the M-57 ions at m/z 398, 399 and 400 for allantoin and at m/z 567 and 569 for uric acid. 15N2 allantoin (99 at.%) was produced from [1,3-15N2] uric acid by treatment with uricase and used as a standard. Quantitation of allantoin and uric acid was based on isotopic dilution by spiking the urine sample with known quantities of 99 at.% [15N] uric acid and allantoin internal standards. The observed isotope ratio measurements from the prepared standards matched the theoretical values. Coefficients of variation in measurements of isotope ratio and concentration were 0.2 and 0.5%, respectively. The method was applied in a study to measure the urinary recovery of [1,3-15N2] uric acid continuously infused for 8-10 h into the blood of four sheep each on two occasions. Within 24 h, 65.9 +/- 9.1% of the tracer was excreted in the urine unchanged. Little was converted into allantoin (approximately 7% of the dose). The total recovery (5 days) of the infused tracer averaged 69.5 +/- 7.6% as uric acid and 76.8 +/- 9.3% as the sum of uric acid and allantoin. Uricase activities in plasma, liver and kidney of sheep were also measured using [1,3-15N2] uric acid as a substrate. Uricase activity was estimated to be 0.6 mU g-1 wet tissue in the liver and there appeared to be none in plasma and kidney. The low uricase activities in sheep tissues appeared to explain the limited conversion of the intravenously administered [15N] uric acid to allantoin but did not explain the large quantities of allantoin excreted in urine (8.96 +/- 0.86 and 1.36 +/- 0.25 mmol d-1 for allantoin and uric acid, respectively). The GC/MS method for the determination of 15N enrichment and concentration of allantoin and uric acid in urine is accurate and precise and provides a useful tool for studies on uric acid and allantoin metabolism.

KINETICS OF BLOOD FREE AND MILK CASEIN-AMINO ACID LABELLING IN THE DAIRY GOAT AT TWO STAGES OF LACTATION

The kinetics of blood free amino acids (AA) transfer into milk casein were compared in goats (n 4) at 61 (SE 5) d (Expt 1; post-peak, 4.51 (SE 0.26) kg milk/d) and at 180 (SE 6) d (Expt 2; late, 2.36 (SE 0.16) kg milk/d) of lactation during non-primed, continuous (Expt 1, 12 h; Expt 2, 16 h) intravenous infusions of mixtures of L-[1-13C]leucine and L-[1-13C]phenylalanine with either L-[1-13C]valine (Expt 1) or L-[5-13C]methionine (Expt 2). The 13C enrichments of blood free and casein-bound AA were fitted to a single exponential model to estimate isotopic plateaux and the fractional rate constant for milk casein labelling. Milk protein output and its contribution to whole-body flux was higher in Expt 1 (post-peak) than in Expt 2 (late lactation), but the kinetics of 13C labelling of the casein-bound AA were similar for all AA tracers in both experiments. At both stages of lactation the delay (6-8 h) between the attainment of isotopic plateau for the blood free AA and the corresponding attainment of plateau for the casein-bound AA indicated that the blood free pool was not the immediate precursor pool for milk casein biosynthesis. Plateau enrichments of casein-bound AA were generally higher than those for the corresponding blood free AA in both experiments. These results indicate that the relative contributions of different AA sources to the immediate precursor pool for milk casein biosynthesis are similar at different stages of lactation despite major changes in the partitioning of whole-body flux towards milk protein output. Non-milk protein fluxes were also similar in post-peak and late lactation.

The effects of breed and level of nutrition on whole-body and muscle protein metabolism in pure-bred Aberdeen Angus and Charolais beef steers

Eighteen pure-bred steers (live weight 350 kg) from each of two breeds, Aberdeen Angus (AA) and Charolais (CH), were split into three equal groups (six animals each) and offered three planes of nutrition during a 20-week period. The same ration formulation was offered to all animals with amounts adjusted at 3-week intervals to give predicted average weight gains of either 1.0 kg/d (M/M group) or 1.4 kg/d (H/H group). The remaining group (M/H) were offered the same amount of ration as the M/M group until 10 weeks before slaughter when the ration was increased to H. Data on animal performance, carcass characteristics and fibre-type composition in skeletal muscle are presented elsewhere (Maltin et al. 2000; Sinclair et al. 2000). On three occasions (17, 10 and 2 weeks before slaughter) the animals were transferred to metabolism stalls for 1 week, during which total urine collection for quantification of Ntau-methylhistidine (Ntau-MeH) elimination was performed for 4 d. On the last day, animals were infused for 11 h with [2H5]phenylalanine with frequent blood sampling (to allow determination of whole-body phenylalanine flux) followed by biopsies from m. longissimus lumborum and m. vastus lateralis to determine the fractional synthesis rate of mixed muscle protein. For both breeds, the absolute amount of Ntau-MeH eliminated increased with animal age or weight (P < 0.001) and was significantly greater for CH steers, at all intake comparisons, than for AA (P < 0.001). Estimates of fractional muscle breakdown rate (FBR; calculated from Ntau-MeH elimination and based on skeletal muscle as a fixed fraction of live weight) showed an age (or weight) decline for M/M and H/H groups of both breeds (P < 0.001). FBR was greater for the H/H group (P = 0.044). The M/H group also showed a lower FBR for the first two measurement periods (both at M intake) but increased when intake was raised to H. When allowance was made for differences in lean content (calculated from fat scores and eye muscle area in carcasses at the end of period 3), there were significant differences in muscle FBR with intake (P = 0.012) but not between breed. Whole-body protein flux (WBPF; g/d) based on plasma phenylalanine kinetics increased with age or weight (P < 0.001) and was similar between breeds. The WBPF was lower for M/M compared with H/H (P < 0.001) based on either total or per kg live weight0.75. Muscle protein fractional synthesis rate (FSR) declined with age for both breeds and tended to be higher at H/H compared with M intakes (intake x period effects, P < 0.05). Changing intake from M to H caused a significant increase (P < 0.001) in FSR. The FSR values for AA were significantly greater than for CH at comparable ages (P = 0. 044). Although FSR and FBR responded to nutrition, these changes in protein metabolism were not reflected in differences in meat eating quality (Sinclair et al. 2000).