Is D-methionine bioavailable to the dairy cow?

Rumen-protected forms of Met contain an equimolar mixture of the D- and L-isomers. Only L-Met can be directly used for protein synthesis, but it is unclear how much of the D-isomer can be transformed into L-Met in ruminants. Four lactating dairy cows, with an average milk yield of 32.4 kg/d, received a basal diet (12.5% crude protein, supplying 1,718 g/d of metabolizable protein) in 12 equal meals per day plus an abomasal infusion of amino acids (590 g/d, casein profile without Met). They were used in 3 consecutive studies to determine utilization of D-Met. First, the cows each received portal vein infusions for d of 5, 10, or 15 g/d of DL-Met in a Youden square. On the last day of each period, 6 arterial samples were collected at 45-min intervals. Concentrations of L- and D-Met were determined on a chiral column by gas chromatography-mass spectrometry. Portal infusion of 5, 10, and 15 g/d of DL-Met increased plasma total Met concentrations (19.7, 25.0, and 34.4±0.6 µM) and the proportion of Met as D (19.4, 30.5, and 37.3±0.7%). The fractional removal of D-Met was 6 to 7 times lower than the fractional removal of L-Met, with mean half-lives of 52 versus 8 min, respectively. Second, the same cows were infused for 8 h with L[methyl-(2)H(3)]Met at 1.3 mmol/h; at 2 h, cows received a bolus injection i.v. of D-[1-(13)C]Met (6.8 mmol), and arterial samples were collected after 10, 20, 30, 40, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min. Expressed relative to L-[(12)C]Met; that is, as tracer:tracee ratios, enrichments of plasma D-[1-(13)C]Met and L-[1-(13)C]Met averaged 1.77±0.14 and 0.144±0.026, respectively, 10 min after the bolus injection and declined exponentially thereafter. A minimum of 75±3% of the D-[1-(13)C]Met was transformed into L-[1-(13)C]Met. Third, the cows received, in a crossover design, an abomasal infusion for D of either DL-Met or L-Met (1g/d) and, on the last day of each experimental period, blood samples were collected simultaneously from arterial, portal, hepatic, and mammary vessels. Arterial total Met concentrations were higher with DL- versus L-Met infusions (37.4 vs. 25.4±0.5 µM), with 37.1±5.0% as D-Met. The mammary gland did not extract any D-Met. Hepatic removal of D-Met was observed, but was numerically lower than the fractional extraction of L-Met. In conclusion, much of the D-Met is transformed into L-Met by the dairy cow but at a slow rate. No uptake of D-Met occurs across the mammary gland but L-Met synthesized from the D-isomer elsewhere in the body can be utilized for milk protein synthesis.

Hepatic metabolism of 2-hydroxy-4-methylthiobutyrate in growing lambs.

This study was undertaken to determine how, and where, 2-hydroxy-4-methylthiobutyrate (HMTBA) can augment Met metabolism in lambs. Four lambs (initial body weight of 50 kg, SE = 2, and 6 mo of age) prepared with catheters in the mesenteric, portal, hepatic, and jugular veins plus the aorta, were fed at 1.5x maintenance on a grass hay, barley, fish meal, molasses/pre-mix (5:3:1:1, as fed) diet, supplied as hourly meals. Lambs were infused for 10 h with [methyl-2H3]Met (0.11 mmol/h) in a jugular vein and p-aminohippurate into the mesenteric vein. From 1 h onwards, successive 3-h infusions of saline (control), 0.55 mg/min (3.67 micromol/min), and 4.44 mg/min (29.6 micromol/min) of HMTBA were also infused into the mesenteric vein. Plasma, sampled continuously, was collected every 20 min during the last 60 min of each infusion. All infused HMTBA was recovered at the portal vein with 25% extracted subsequently by the liver. Portal appearance of total Cys and Met was unaltered by HMTBA infusion, but net splanchnic appearance of Cys increased (0.04, 0.08, 0.23 mmol/h, SEM = 0.05), whereas Met decreased (0.14, -0.01, -0.21 mmol/h, SED = 0.05). Despite this, arterial Met increased (27.0, 30.7, 51.5 microM, SEM = 2.1) as did Met irreversible loss rate (27.6, 28.7, 40.1 micromol/h, SEM = 0.51), equivalent to 40% of the HMTBA reentering the plasma after conversion to Met. These data indicate that, in ruminants, HMTBA is probably converted to Met within peripheral tissues; that is, where the metabolic need for Met exists.

Absorption of 2-hydroxy-4-methylthiobutyrate and conversion to methionine in lambs.

Absorption and metabolism of the Met hydroxy analog 2-hydroxy-4-methylthiobutyrate (HMTBA) was examined using stable isotopes. In the first trial, Dl[1-13C]HMTBA was infused for 6 h (7.4 micromol/min) into the abomasum, and [2H3]Met was infused into the mesenteric vein, of 4 lambs prepared with vascular catheters across the splanchnic bed. Daily, lambs were offered 35 g of a mixed forage-concentrate feed/kg. Recovery of HMTBA at the portal vein was 87%, and of this, 63% bypassed the liver. In contrast, hepatic extraction of Met equaled or exceeded net absorption. Only small quantities of Met synthesized from HMTBA were exported from either the digestive tract or liver, but there was substantial and significant input from posthepatic tissues. In a second experiment, 3 of the lambs were killed following 4-h infusions of DL[1-13C]HMTBA and [2H3]Met with enrichments monitored in 15 tissues. Only kidney showed [1-13C]Met enrichment higher than plasma, which suggests that it must be a primary source of plasma Met derived from HMTBA. Based on comparison of plasma and intracellular [1-13C]:[2H3]Met enrichments, all tissues synthesized Met from HMTBA but to significantly different extents. The lowest values were for muscle, skin, brain, and lung; intermediate conversions occurred in rumen, omasum, abomasum, duodenum, jejunum, ileum, and cecum; and the greatest synthesis, equivalent to 22 to 24% of Met entry into cells, was observed for liver and kidney. Therefore, although liver and kidney both converted HMTBA to Met, it was retained by the former and exported by the latter. Under these experimental conditions, synthesis of Met from HMTBA completely eliminated use of dietary Met.

Increased protein turnover despite normal energy metabolism and responses to feeding in patients with lung cancer.

We have examined the responses of energy and protein metabolism to nutrient intake in nine patients with lung carcinoma, of whom none were cachexic and only one had distant metastases, compared with nine control patients for elective aneurysm surgery, who were comparable in terms of age, body mass index, and smoking habits. Whole-body protein turnover and leucine oxidation were assessed by primed continuous infusion of L-[13C]leucine. Indirect calorimetry was used to determine energy expenditure and rates of carbohydrate and fat utilization. Lean body mass (LBM) was estimated from dilution of deuterium oxide. Measurements were made over an 8-h period, including 4 h postabsorptive followed by 4 h of feeding, during which small hourly meals were consumed. In the post-absorptive state, the rate of incorporation of leucine into protein was higher in the cancer group (mean +/- SD, cancer versus control: 102 +/- 21 versus 86 +/- 8 mumol/kg LBM/h, P less than 0.05), as was the release of leucine by protein degradation (126 +/- 19 versus 110 +/- 10 mumol/kg LBM/h, P less than 0.01), but there was no difference in rates of leucine oxidation (27 +/- 6 versus 27 +/- 5 mumol/kg LBM/h) or leucine balance (-25 +/- 7 versus -24 +/- 4 mumol/kg LBM/h). There were no differences between the cancer and control groups with respect to either resting energy expenditure (37.3 +/- 3.5 versus 35.2 +/- 3.8 kcal LBM/day) or the postabsorptive pattern of nutrient utilization (61 +/- 13% fat, 26 +/- 10% carbohydrate, and 13 +/- 2% protein versus 65 +/- 7%, 21 +/- 7%, and 14 +/- 2%, respectively). During feeding, leucine oxidation rose relative to the postabsorptive state, incorporation into protein remained the same, and release by protein degradation fell. Incorporation (106 +/- 20 versus 89 +/- 7 mumol/kg LBM/h, P less than 0.05) and release (59 +/- 12 versus 42 +/- 14 mumol/kg LBM/h, P less than 0.02) remained higher in the cancer group than in controls, but leucine oxidation (43 +/- 15 versus 43 +/- 12 mumol/kg LBM/h) and leucine balance (+48 +/- 10 versus +47 +/- 12 mumol/kg LBM/h) were the same. Energy expenditure during feeding increased to 43.8 +/- 5.1 versus 43.2 +/- 4.2 kcal/kg LBM/day, derived from 32 +/- 11% fat, 52 +/- 9% carbohydrate, and 16 +/- 5% protein in cancer patients and 36 +/- 7%, 48 +/- 8%, and 16 +/- 4%, respectively, in controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Short-term growth hormone administration at the time of opportunistic infections in HIV-positive patients.

OBJECTIVES: A 12-week course of recombinant human growth hormone is an effective but expensive therapy for established HIV-related wasting. Wasting in HIV disease is often episodic, coinciding with bouts of acute opportunistic infection. We hypothesized that a short course of growth hormone, targeted at the time of opportunistic infection, might improve protein metabolism thereby reducing lean tissue loss. METHODS: HIV-infected men with acute opportunistic infections, who received standard antimicrobial treatment for their infection as well as intensive nutritional counselling and oral energy supplements, were randomized to receive growth hormone or placebo for 14 days. Principal assessments were protein metabolism (measured by 13C-leucine infusion), body composition (measured by DEXA) and safety. RESULTS: There were no significant changes in outcome parameters in the placebo group (n = 11). In the growth hormone group (n = 9), protein catabolic rate decreased by 60% in the fasted state (P = 0.02 versus placebo), lean body mass increased by 2.2 kg (P = 0.03 versus baseline) and fat mass decreased by 0.7 kg (P = 0.002 versus baseline). There was no increase in adverse or serious adverse events in the growth hormone as compared with the placebo group. CONCLUSIONS: A two-week course of growth hormone at the time of acute opportunistic infection in HIV-infected patients improves protein metabolism and body composition during therapy and appears to be safe. This may represent a rational and economical approach to the use of growth hormone therapy.

Identification of the NH2-terminal blocking group of calcineurin B as myristic acid.

The NH2-terminal blocking group of the Ca2+-binding B-subunit of calcineurin (protein phosphatase-2B) has been identified as myristic acid by fast atom bombardment mass spectrometry and gas chromatography. The sequence, myristyl-Gly-Asn-Glu-Ala-, is very similar to that of the catalytic subunit of cyclic AMP-dependent protein kinase, the only other protein known to contain this fatty acid. This finding, and the elution of all myristyl peptides at 57% acetonitrile on reverse phase HPLC, may facilitate the identification of other proteins with this blocking group.

Whole-body protein turnover from leucine kinetics and the response to nutrition in human immunodeficiency virus infection.

Whole-body protein metabolism was investigated in human immunodeficiency virus (HIV) infection by primed constant infusion of L-[1-13C]leucine in 8 control and 22 HIV-infected subjects (8 stage II; 14 stage IV disease), in postabsorptive and fed states. Postabsorptive leucine flux was increased 25% in subjects with stage IV HiV infection vs that in control subjects (130 +/- 13 vs 103 +/- 10 mumol leucine.kg-1.h-1, P < 0.001); both leucine disposal by protein synthesis (111.6 +/- 12.1 vs 82.3 +/- 9.2, P < 0.001) and release by protein degradation (129.7 +/- 13.1 vs 103.4 +/- 10.2, P < 0.001) were increased. No difference in leucine balance or oxidation was found but fat oxidation was greater in subjects with HIV infection (61.1 +/- 13.0% of energy) than in control subjects (47.6 +/- 13.7% of energy, P < 0.025). Stage II subjects had intermediate values of leucine flux, not significantly different from those of control subjects. Provision of parenteral nutrition for 4 h increased leucine flux with a switch in leucine balance from net loss to net gain; this response was quantitatively similar in all groups. HIV infection increases whole-body protein turnover but does not quantitatively impair the acute anabolic response to intravenous nutrition.

The role of degradation in the acute control of protein balance in adult man: failure of feeding to stimulate protein synthesis as assessed by L-[1-13C]leucin infusion.

The effect of feeding on whole-body protein turnover was measured in six healthy volunteers using the essential amino acid, L-[1-13C]leucine, as a tracer for protein metabolism. Varied lengths of periods of feeding and isotope infusion produced different apparent responses to feeding. When parameters of protein turnover were estimated from 8-hour infusions, the change from post-absorptive in the first four hours to mixed feeding during the final four hours was found to produce positive leucine balance by decreasing degradation from 89.5 +/- 5.0 to 31.7 +/- 7.3 mumol leucine/kg/h (P less than .001), with no apparent change in synthesis. By contrast, when tracer was infused for 24 hours with 12 hours of feeding followed by 12 hours of fasting, the estimate of protein synthesis during feeding was 35% higher than during fasting (P less than .01). However, when tracer infusion during the 12-hour feeding/12-hour fasting protocol was limited to the last four hours of each nutritional period, the estimates of fed and fasted protein synthesis showed no significant difference, 71.3 +/- 6.5 and 66.2 +/- 5.6 respectively, while the calculated rate of protein degradation was 43% lower during feeding (P less than .002). As relatively higher levels of enrichment in plasma leucine were detected in comparable nutritional states following longer infusions, the possibility of significant recycling of label was investigated. Residual tracer was still detectable in both breath and plasma 12 hours after cessation of a 12-hour tracer infusion, supporting the conclusion that significant errors in estimates of protein turnover due to recycling of label arise with prolonged infusions.(ABSTRACT TRUNCATED AT 250 WORDS)

Method for the determination of 15NH3 enrichment in biological samples by gas chromatography/electron impact ionization mass spectrometry.

An alternative method for the determination of [15N]ammonia enrichment in biological fluids was developed. It is based on the use of glutamate dehydrogenase of bovine liver (EC 1.4.1.2.) with 2-oxopentanoic acid as substrate, to convert the ammonia present in the sample into norvaline, the enrichment of which can be measured by gas chromatography/mass spectrometry as its tertiary butyldimethylsilyl (TBDMS) derivative under electron impact selective ion recording (SIR) conditions. The principal advantage of the present approach is that it is simpler and quicker than the previously described methods, because the synthetic product, norvaline, is not present in biological fluids and pre-processing of the sample is unnecessary. The procedure includes a pre-incubation stage which allows removal of contaminant ammonia present in the reagents used for the enzyme reaction. The contributions of other sources of nitrogen to norvaline production have been checked and quantified: these may provide limitations of the technique when samples for analysis are low in ammonia (e.g. arterial or hepatic venous blood). To reduce these contributions, short times of incubation are proposed. The results from two experiments in vivo in which two sheep were infused with [15N]ammonium chloride in the mesenteric vein are presented and the biological implications which arise from the results are discussed. The validity of the procedures was demonstrated by the quantitative recovery from the mesenteric and portal veins of [15N]ammonia infused.

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.

The effect of insulin suppression on postprandial nutrient metabolism: studies with infusion of somatostatin and insulin.

Utilization of fat, carbohydrate and protein before and after feeding was studied in six healthy subjects using simultaneous respiratory gas exchange measurement and [1-13C] leucine infusion. The role of insulin was investigated by repeating a control study with the addition of an infusion of somatostatin, a hormone which can suppress insulin release. Where near-complete insulin suppression was effected, subjects were studied on a third occasion with the further addition of exogenous insulin infusion. The normal switch on feeding from fat to carbohydrate as principal energy source was reproduced at insulin levels of only 17%-33% of control values, which were inadequate to prevent hyperglycaemia. At fed levels below 10%, a fat-predominant pattern persisted unless insulin was infused. Protein degradation was reduced and synthesis unaffected by feeding, regardless of insulin concentration. Leucine oxidation was dependent on its plasma concentration in the presence of circulating insulin. Thus insulin appears to be necessary for the normal switch to carbohydrate oxidation on feeding but not for postprandial changes in protein metabolism.

Identification of methyl-branched fatty acids from the triacylglycerols of subcutaneous adipose tissue of lambs.

A concentrate of branched chain fatty acids (as methyl esters) was prepared from the triacylglycerols of subcutaneous adipose tissue lipids of lambs receiving a carbohydrate-rich (cereal diet). This was accomplished by procedures which allowed the removal of unsaturated components by peroxidation and straight chain saturated components by urea-adduct formation. The concentrate was analyzed by high resolution gas chromatography in combination with mass spectrometry and was shown to consist of a complex mixture of saturated methyl-substituted fatty acids. Methyl substitution occurred on even-numbered carbon atoms (relative to the carboxyl group) and the chain lengths of the acids ranged from 10 to 18 carbon atoms. Acids with one methyl substituent in the fatty acyl chain were most abundant; di-, tri- and tetramethyl-substituted acids were also present. The biosynthesis of these methyl-substituted acids is discussed briefly.

Subclinical infection with the nematode Trichostrongylus colubriformis increases gastrointestinal tract leucine metabolism and reduces availability of leucine for other tissues.

Gastrointestinal (GI) tract leucine metabolism was measured in 6- to 9-mo-old lambs subjected to trickle infection with Trichostrongylus colubriformis larvae and in separate animals that were not infected. Animals prepared with a jejunal catheter and with indwelling catheters into the aorta and the portal- (PDV) and mesenteric- (MDV) drained viscera were infused simultaneously with [1-13C] and [5,5,5-2H3] leucine to determine GI tract sequestration of leucine from arterial and luminal amino acid pools by tracer and tracee arteriovenous concentration differences. Leucine oxidative losses and net fluxes were also determined across the GI tract. Infection had no detectable effect on whole-body leucine flux, but it increased total GI tract leucine sequestration by 24% (P<.05) and GI tract oxidative losses of leucine by 22 to 41% (P<.01). Net PDV fluxes of leucine were decreased by 20 to 32% during the infection. The infection did not alter either the proportion of precursor leucine used by GI tract metabolism that was derived from the arterial leucine pool (.84 to .88) or the proportional sequestration of digesta-derived leucine during "first pass" absorptive metabolism (.12 to .18). These findings help to elucidate the metabolic basis for the reduced growth rates and nitrogen retention observed when animals are subjected to subclinical nematode infection.

Characterization of ethyl substituted fatty acids from lamb subcutaneous triacylglycerols by combined gas chromatography mass spectrometry.

Mid-chain ethyl substituted fatty acids with chain lengths ranging from 10 to 16 carbon atoms were identified in a concentrate of branched chain fatty acids isolated from the triacylglycerol fatty acids of the subcutaneous adipose tissue of barley-fed lambs. The acids were identified by gas chromatography in combination with mass spectrometry and their mass spectral features are discussed.

Measurement of 13C enrichment of plasma lactate by gas chromatography/isotope ratio mass spectrometry.

An application of a gas chromatography/isotope ratio mass spectrometry (GC/IRMS) method for stable carbon isotope analysis of blood plasma lactic acid is presented. The method involves a simple extraction procedure followed by derivatization with diazomethane. It is shown that derivatization is by single methylation, thus minimizing the dilution of the derivative's 13C content, yet still ensuring good chromatographic behaviour on a polar capillary column. This ensures a high sensitivity of the isotopic analysis. Repeatability, expressed by the coefficient of variation, varied from 0.3% to 19%, depending on sample enrichment. Reproducibility was 2.3% over a 10 day period. The detection limit, defined as 2SD, was about 0.0004 atom % excess (APE), equivalent to 0.001 mol % excess, when based on a measured precision of about 0.2/1000 in delta notation. A comparison is made between enrichments obtained using a calibration curve and those obtained using a correction for the added methyl carbon. The two methods agreed well, with a relative difference (delta APE/APE x 100%) of less than 0.5% for samples enriched with between 0.004 and 1.28 APE. It is concluded that the method provides simple and precise isotope analysis of picomole quantities of blood lactate.

Protein turnover rates in sick, premature neonates during the first few days of life.

Rates of protein turnover were measured in 19 infants during the first few days of life while they were receiving i.v. glucose. The technique consisted of a continuous i.v. infusion of L-[1-13C]leucine to measure whole body leucine flux and determination of total urinary nitrogen excretion to assess leucine oxidation rates. Subsequent to each of the studies, the decision to start total parenteral nutrition (TPN) was made by the clinician concerned, with the result that seven infants did not start TPN and 12 did. There were significantly greater urinary nitrogen excretion (p less than 0.001) and lower rates of whole body protein synthesis (p = 0.024) and breakdown (p = 0.015) in those who did start TPN compared with those who did not. The marked difference in nitrogen excretion between the two groups suggests that this could be a useful determinant for deciding which neonate should start TPN.

Muscle and whole body metabolism after norepinephrine.

The effect of an infusion of norepinephrine (0.42 nmol.kg-1.min-1) on energy metabolism in the whole body (using indirect calorimetry and the arteriovenous forearm catheterization techniques in eight healthy young male adults. The activity of the triglyceride-fatty acid cycle, which mainly operates in nonmuscular tissues, was also assessed by measuring glycerol turnover using [2H5]glycerol (to indicate lipolysis) and indirect calorimetry (to indicate net fat oxidation). Norepinephrine increased whole body oxygen consumption by almost 10% (P < 0.01), but the estimated oxygen consumption of muscles tended to decrease. Muscle blood flow (measured by 133Xe) and forearm blood flow (measured by strain-gauge plethysmography) were not significantly affected by norepinephrine, but the rate of uptake of nonesterified fatty acids and beta-hydroxybutyrate increased severalfold (P < 0.05), whereas that of glucose did not. The activity of the triglyceride-fatty acid cycle increased fourfold after norepinephrine administration, having a marginal effect on resting energy expenditure (approximately 1.5%) but accounting for approximately 15% of the increase in whole body energy expenditure. This study provides no evidence that skeletal muscle is an important site for norepinephrine-induced thermogenesis and suggests that an increase in the activity of the triglyceride-fatty acid cycle contributes to the norepinephrine-induced increase in energy expenditure of nonmuscular tissues.

Hepatic protein synthesis in the sheep: effect of intake as monitored 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.

Conversion of [15N]ammonia into urea and amino acids in humans and the effect of nutritional status.

Hepatic NH3 detoxification by ureagenesis requires an input of aspartate-N, originating either from amino acid-N or NH3-N. The relative importance of these two routes may depend on the nutritional state. To test this, four volunteers were given a liquid diet for 2 d and then on day 3 were either fed every 20 min or fasted. Doses of 15NH4Cl were taken orally every 20 min for 6 h (total 1.5 g) and blood was sampled hourly. Urea-N elimination under fasted conditions was only 0.75 of that for the fed state. Considering the increase in body urea pool during feeding, ureagenesis during fasting was probably closer to 0.6 of that during feeding. Since the [14N15N]urea enrichment was not different between the fed and fasted states, the proportion of the 15NH3 dose converted to urea during fasting was also 0.6 of that during the fed condition. No change in [14N15N]urea and [amide-15N]glutamine enrichment suggested that NH3 enrichment was also not affected by nutritional state. Enrichment of [15N15N]urea was approximately 0.05 that of [14N15N]urea which indicates that 15NH3 can also enter the aspartate route, the importance of which is yet unknown. Both [15N15N]urea and [amino-15N]glutamine enrichment in the fasted state were approximately 1.7 times that in the fed state, indicating increased labelling of precursors and/or increased NH3 flux through the aspartate route. Glutamate, valine, leucine and isoleucine showed comparable increases in enrichment during fasting. Arginine enrichment was unaltered by nutritional state, but was lower than [14N15N]urea, indicating incomplete equilibration with the arginine pool in periportal hepatocytes. The present study indicates that hepatic NH3 detoxification may use the aspartate route, gaining importance in the fasted state. The majority of urea was supplied with only one N atom from NH3, thus provision of the other may have consequences for alternative substrates, in particular amino acids.

Amino acid use by the gastrointestinal tract of sheep given lucerne forage.

Essential amino acid (EAA) utilization by gastrointestinal tract (GIT) tissues has been investigated in sheep given 800 and 1,200 g/day lucerne pellets. Animals prepared with indwelling catheters into the aorta and the portal drained viscera plus cannulas into the small intestine were infused with mixed U-13C-labeled amino acid or (1-13C]leucine tracers into the jugular vein or directly into the small intestine. GIT sequestration of EAA from arterial and luminal AA pools was determined from tracer and tracee arterioportal concentration differences at both levels of intake. Proportional tracer 13C-labeled EAA extraction of the arterial supply, on first pass across the GIT during jugular infusion, ranged from 0.063 for histidine to 0.126 for leucine. Recovery of intestinally infused tracer 13C-EAA at the portal vein ranged from 0.61 for histidine to 0.83 for valine. These data were independent of intake. Calculated rates of tracee sequestration by GIT tissues represented 0.45-0.65 of whole body EAA flux, except for histidine, for which the values were much lower (0.25-0.32). With the exception of phenylalanine, more than 0.8 of the EAA used by the GIT was extracted from circulating blood, thus calling into question the theory that GIT tissues make preferential use of EAA during absorptive metabolism, restricting supply to peripheral tissues such as skeletal muscle (growth) or mammary glands (lactation). Instead the GIT seems to compete very successfully with these tissues for circulating blood EAA.