After bolus and continuous enteral feeding of the same protein, different digestion and absorption kinetics and anabolic responses are observed. Establishing which mode of feeding has the highest anabolic potential in patients with chronic obstructive pulmonary disease (COPD) may aid in the prevention of muscle wasting, but an important confounding factor is the duration of assessments after bolus feeding. We hypothesized that the anabolic response to bolus and continuous feeding in COPD patients is comparable when methodological issues are addressed. Twenty-one older adults (12 patients with stage II–IV COPD and 9 healthy controls) were studied after intake of a fast-absorbing hydrolyzed casein protein–carbohydrate mixture either as a single bolus or as small sips (crossover design). Whole body protein synthesis (PS), breakdown (PB), net PS (PS − PB) protein efficiency (netPSPE), net protein balance (phenylalanine (PHE) intake – PHE hydroxylation) protein efficiency (netBalPE), and splanchnic PHE extraction (SPE PHE ) were assessed using stable isotope tracer methodology. Bolus feeding assessments were done at 90, 95, and 99% of the calculated duration of the anabolic response. At 99%, netBalPE was higher for sip feeding than bolus feeding in both groups ( P <0.0001). Nevertheless, bolus feeding was associated with a lower SPE PHE ( P <0.0001) and higher netPSPE ( P <0.0001). At 90% compared with 99%, PS and netBalPE after bolus feeding was significantly overestimated. In conclusion, several factors complicate a comparison of the anabolic capacity of bolus and continuous feeding in acute studies, including the critical role of SPE calculation and assumptions, and the duration of postprandial assessments after bolus feeding.

Leucine modulates muscle protein synthesis (MPS), with potential to facilitate accrual/maintenance of muscle mass. Animal models suggest that leucine boluses shortly after meals may prolong MPS and delay onset of a “muscle-full” state. However, the effects of nutrient “top-ups” in humans, and particularly older adults where deficits exist, have not been explored. We determined the effects of a leucine top-up after essential amino acid (EAA) feeding on anabolic signaling, MPS, and muscle energy metabolism in older men. During 13 C 6 -phenylalanine infusion, 16 men (∼70 years) consumed 15 g of EAA with ( n =8, FED + LEU) or without ( n =8, FED) 3 g of leucine top-up 90 min later. Repeated blood and muscle sampling permitted measurement of fasting and postprandial plasma EAA, insulin, anabolic signaling including mTOR complex 1 (mTORC1) substrates, cellular ATP and phosphorylocreatine, and MPS. Oral EAA achieved rapid insulinemia (12.5 iU·ml −1 25 min post-feed), essential aminoacidemia (3000 μM, 45–65 min post-feed), and activation of mTORC1 signaling. Leucine top-up prolonged plasma EAA (2800 μM, 135 min) and leucine availability (1050 μM, 135 min post-feed). Fasting FSRs of 0.046 and 0.056%·h -1 (FED and FED + LEU respectively) increased to 0.085 and 0.085%·h -1 90–180 min post-feed and returned to basal rates after 180 min in both groups. Phosphorylation of mTORC1 substrates returned to fasting levels 240 min post-feed in both groups. Feeding had limited effect on muscle high-energy phosphates, but did induce eukaryotic elongation factor 2 (eEF2) phosphorylation. We demonstrate the refractoriness of muscle to nutrient-led anabolic stimulation in the postprandial period; thus, leucine supplements should be taken outside of meals, or with meals containing suboptimal protein in terms of either amount or EAA composition.

Background: Exercise and citrulline (CIT) are both regulators of muscle protein metabolism. However, the combination of both has been under-studied yet may have synergistic effects on muscle metabolism and performance. Methods: Three-month-old healthy male rats were randomly assigned to be fed ad libitum for 4 weeks with either a citrulline-enriched diet (1 g·kg −1 ·day −1 ) ( CIT ) or an isonitrogenous standard diet (by addition of nonessential amino acid) ( Ctrl ) and trained (running on treadmill 5 days·week −1 ) ( ex ) or not. Maximal endurance activity and body composition were assessed, and muscle protein metabolism (protein synthesis, proteomic approach) and energy metabolism [energy expenditure, mitochondrial metabolism] were explored. Results: Body composition was affected by exercise but not by CIT supplementation. Endurance training was associated with a higher maximal endurance capacity than sedentary groups ( P <0.001), and running time was 14% higher in the CITex group than the Ctrlex group (139±4 min versus 122±6 min, P <0.05). Both endurance training and CIT supplementation alone increased muscle protein synthesis (by +27% and +33%, respectively, versus Ctrl , P <0.05) with an additive effect (+48% versus Ctrl , P <0.05). Mitochondrial metabolism was modulated by exercise but not directly by CIT supplementation. However, the proteomic approach demonstrated that CIT supplementation was able to affect energy metabolism, probably due to activation of pathways generating acetyl-CoA. Conclusion: CIT supplementation and endurance training in healthy male rats modulates both muscle protein and energy metabolisms, with synergic effects on an array of parameters, including performance and protein synthesis.

Previous studies have provided conflicting conclusions concerning the efficacy of improving protein balance in patients by standard intravenous nutrition [TPN (total parenteral nutrition)], which is either explained by suboptimal nutritional regimens or insensitive clinical methods. The aim of the present study was therefore to evaluate the effects on the initiation of translation of skeletal muscle proteins by standard overnight TPN. A total of 12 patients who underwent standard surgery were included. TPN was provided as an all-in-one treatment by constant infusion [0.16 gN·kg −1 of body weight·day −1 (30 kcal·kg −1 of body weight·day −1 )]. Saline-infused patients served as controls. Rectus abdominis muscle biopsies were taken at the time of the operation. The phosphorylation state of the proteins for initiation of translation was quantified. Plasma glucose, and serum insulin, glycerol, triacylglycerols (triglycerides) and NEFAs (non-esterified fatty acids; ‘free fatty acids’) were not significantly altered during TPN infusion, whereas total plasma amino acids increased, as shown by increases in methionine, phenylalanine, threonine, alanine, arginine, aspartic acid, glycine and histidine ( P <0.05). Overnight TPN increased the formation of active eIF4G–eIF4E (where eIF is eukaryotic-initiation factor) complexes ( P <0.05), whereas the inhibitory complex 4E-BP1 (eIF4E-binding protein)–eIF4E was moderately decreased ( P <0.06). TPN increased the amount of the most phosphorylated form of 4E-BP1 ( P <0.05), and increased the amount ( P <0.04) and phosphorylation ( P <0.01) of p70 S6K (70 kDa ribosomal protein S6 kinase). In conclusion, an overnight pre-operative constant infusion of standard TPN altered initiation factor complexes, indicating activation of the initiation of protein translation in rectus abdominis muscle in the presence of increased plasma amino acid levels, but without a concomitant increase in energy substrates and insulin. In contrast with our results from previous studies, the methodology used in the present study appears to be more sensitive in reflecting directional changes in human muscle protein synthesis compared with traditional methods, particularly based on measurements of amino acid flux.

The palatine tonsils are constantly exposed to ingested or inhaled antigens which, in turn, lead to a permanent activation of tonsillar immune cells, even in a basic physiological state. The aim of the present study was to investigate if the immunological activation of the human palatine tonsil is reflected by a high metabolic activity, as determined by in vivo measurement of protein synthesis. The protein synthesis rate of the tonsil was also compared with that of the circulating T-lymphocytes, the total blood mononuclear cells and the whole population of blood leucocytes. Phenotypic characterization of immune-competent cells in tonsil tissue and blood was performed by flow cytometry. Pinch tonsil biopsies were taken after induction of anaesthesia in healthy adult patients ( n =12) scheduled for ear surgery, uvulopalatopharyngoplasty or nose surgery. Protein synthesis was quantitatively determined during a 90-min period by a flooding-dose technique. The in vivo protein synthesis rate in the palatine tonsils was 22.8±5.7%/24 h (mean±S.D.), whereas protein synthesis in the circulating T-lymphocytes was 10.7±3.4%/24 h, in mononuclear cells was 10.8±2.8%/24 h and in leucocytes was 3.2±1.2%/24 h. CD3 + lymphocytes were the most abundant cell population in the tonsil. The in vivo protein synthesis rate in human tonsils was higher compared with the circulating immune cells. This high metabolic rate may reflect the permanent immunological activity present in human tonsils, although cell phenotypes and activity markers do not explain the differences.

Muscle protein catabolism is a considerable clinical problem following surgery. However, the impact of surgical trauma on muscle protein synthesis is not well characterized. In this pilot study, we therefore investigated whether the severity of surgical trauma is related to a decrease in muscle protein synthesis rate in humans. Metabolically healthy patients ( n =28) were included in the study. Eight of the patients were day-care patients undergoing minor breast surgery (defined as minor surgery). The other 20 patients were subjected to major abdominal surgery and were therefore scheduled to stay overnight in the recovery room during the first postoperative night (defined as major surgery). Protein FSRs (fractional synthesis rates) in skeletal muscle were determined during a measurement period of 90 min before surgery and immediately after termination of surgery. FSR in skeletal muscle of the minor surgery patients was 1.72±0.25%/24 h before surgery and 1.67±0.29%/24 h after surgery ( P =0.68). In the major surgery group, FSR was 1.62±0.30%/24 h before surgery and 1.57±0.40%/24 h ( P =0.59) immediately following surgery. The observations made in this pilot study could not confirm a size-related decrease in muscle protein synthesis immediately following minor and major surgery. This finding is discussed in relation to confounders, postoperative course and to muscle protein degradation. The shortage of knowledge in this field is emphasized.

The amino acid arginine has been shown to affect the growth of several tumours, although the mechanisms of its action are not clear. In the present study, using a human breast tumour cell line (MCF-7), we investigated the arginine requirements of tumour cells for optimal protein synthesis and growth, and the metabolic pathway responsible for the arginine-dependent growth. The results showed that MCF-7 cells are highly dependent on arginine for growth and that the requirement for arginine is much higher than for an indispensable amino acid, leucine, indicating that arginine is needed for pathways other than protein synthesis. In arginine-free cultures, growth could be completely restored by the urea cycle intermediate citrulline. However, arginine could not be replaced by the urea cycle intermediate and the direct precursor for polyamine synthesis, ornithine, or by the polyamine putrescine, suggesting that the high dependence on arginine is not due to a requirement for polyamine synthesis. Moreover, inhibition of NOS [NO (nitric oxide) synthase] did not affect cell protein synthesis and growth, and the arginine analogue and substrate for NOS, homoarginine, could not replace arginine, implying that the conversion of arginine into NO is not involved in the growth-promoting effects of arginine. The major determinant for the high dependence of MCF-7 cells for arginine was found to be the irreversible conversion of this amino acid into ornithine by the intracellular enzyme arginase. The conversion into ornithine caused a progressive depletion of arginine from the culture medium, which ultimately inhibited cell protein synthesis and halted growth. Intracellular arginase activity may be the major factor determining the requirement for arginine of all cells in culture.

The effect of sepsis on liver synthesis of albumin remains controversial, with studies in man suggesting that synthesis increases, whereas in animals increased, decreased and unaltered synthesis have been reported. To reconcile these conflicting data, total and relative albumin synthesis was measured in rats 24 h after caecal ligation and puncture (CLP) by immunoprecipitation of albumin following a flooding dose of L -[4- 3 H]phenylalanine. Following CLP, animals were starved for 18 h and then received intravenous infusions of saline or parenteral nutrition (PN) with or without glutamine for 6 h. In animals receiving PN, parenteral injections of growth hormone (GH) or saline vehicle were also administered. Fractional rate of liver total protein synthesis was elevated and total albumin synthesis rate was reduced in all CLP groups when compared with non-operated animals. Total albumin synthesis was also lower in all animals receiving PN than those receiving saline alone, although these differences did not attain statistical significance, except for the group receiving PN+GH. Relative albumin synthesis was also reduced after CLP, and was significantly lower in animals receiving PN than in those receiving saline alone. These findings suggest that in sepsis hepatic protein synthesis is reprioritized away from the production of albumin towards the production of acute-phase proteins and that this change is not influenced by the provision of nutritional support, glutamine or the administration of GH.

Chronic alcohol muscle disease is characterized by reduced skeletal muscle mass precipitated by acute reduction in protein synthesis. The pathogenic mechanisms remain obscure, but several lines of evidence suggest that increased oxidative stress occurs in muscle in response to alcohol and this may be associated with impaired α-tocopherol status. Potentially, this implies a therapeutic role for α-tocopherol, especially as we have shown that supplemental α-tocopherol may increase the rate of protein synthesis in normal rats [Reilly, Patel, Peters and Preedy (2000) J. Nutr. 130 , 3045–3049]. We investigated the therapeutic effect of α-tocopherol on plantaris muscle protein synthesis in rats treated either acutely, chronically or chronically+acutely with ethanol. Protein synthesis rates were measured with a flooding dose of L -[4- 3 H]phenylalanine. Protein, RNA and DNA contents were determined by standard laboratory methods. Ethanol caused defined metabolic changes in muscle, including decreased protein, RNA and DNA contents in chronically treated rats. In acute or chronic+acute studies, ethanol suppressed fractional rates of protein synthesis. α-Tocopherol supplementation did not ameliorate the effects of either acute, chronic or chronic+acute alcohol on plantaris muscle protein content or rates of protein synthesis. In control animals (not treated with alcohol), α-tocopherol supplementation decreased muscle protein content owing to increases in protein turnover (both synthesis and degradation). α-Tocopherol supplementation is not protective against the deleterious effects of alcohol on protein metabolism in skeletal muscle.

We have investigated sequential changes in skeletal muscle and hepatic protein synthesis following sepsis, and their relationship to changes in circulating and tissue glutamine concentrations. Male Wistar rats underwent caecal ligation and puncture (CLP) or sham operation, with starvation, and were killed 24, 72 or 96 h later. A group of non-operated animals were killed at the time of surgery. Protein synthesis was determined using a flooding dose of l -[4- 3 H] phenylalanine, and glutamine concentrations were measured by an enzymic fluorimetric assay. Protein synthesis in gastrocnemius muscle fell in all groups. Gastrocnemius total protein content was reduced after CLP and at 72 and 96 h after sham operation. After CLP, protein synthesis was lower at 24 h, and total protein content was lower at 72 and 96 h, than in sham-operated animals. CLP was associated with increased liver protein synthesis at all time points, whereas there was no change after sham operation. Liver protein content did not change after CLP, but was lower at 72 and 96 h after sham operation than in non-operated animals. Plasma glutamine concentrations were reduced at 24 h after sham operation, and at 72 and 96 h after CLP. Muscle glutamine concentrations were reduced in all groups, with the decrease being greater following CLP than after sham operation. In the liver, glutamine concentrations were unchanged after CLP, but increased after sham operation. In rats with sepsis, decreases in muscle protein synthesis and content are associated with markedly reduced muscle glutamine concentrations. Plasma glutamine concentrations are initially maintained, but fall later. In liver, protein synthesis is increased, while glutamine concentrations are preserved. These results support a peripheral-to-splanchnic glutamine flux in sepsis.

Epidemiological evidence shows that small size at birth is associated with an increased risk of developing cardiovascular and metabolic disease in adult life. We have examined the relationships between size at birth and maternal body composition and protein turnover in normal pregnant women. A group of 27 multiparous Caucasian women with singleton pregnancies were studied at around 18 and 28 weeks' gestation. Body composition was determined by anthropometry, and whole-body protein turnover was estimated by using a single oral dose of [ 15 N]glycine and the end-product method. The baby's weight and length were measured within 48 h of birth. Mothers with a greater lean body mass had higher rates of protein turnover at 18 weeks' gestation. This association was largely accounted for by differences in the mother's visceral, rather than muscle, mass. Mothers who had higher protein turnover at 18 weeks' gestation had babies that were longer at birth. After adjustment for the duration of gestation and the baby's sex, 26% of the variation in length at birth was accounted for by maternal protein synthesis at 18 weeks' gestation. Maternal protein intake was not associated with the baby's birth length. Thus the mother's ability to nourish her fetus is influenced by her body composition and her rate of protein turnover. Dietary intake does not adequately characterize this ability.

1. The variability between normal individuals in the efficiency of postprandial protein utilization (PPU), a determinant of the apparent protein requirement, was examined in relation to the relative responses of protein synthesis and proteolysis to protein feeding by means of [1– 13 C]leucine turnover and balance studies. 2. Twenty-five healthy adults were infused intravenously with l -[1- 13 C]leucine continuously for 9 h. This was started in the postabsorptive state (PA, 3 h) and followed by low-protein feeding (LP, 3 h), and then by isoenergetic high-protein feeding (HP, 3 h). This allowed protein intake to be varied against a constant postprandial insulin level so that the extent of any amino-acid-mediated responses which were additional to those exerted by insulin could be investigated. Leucine oxidation, O , and balance (intake-oxidation), protein synthesis, S , and degradation, D , were calculated from plasma [1- 13 C]α-ketoisocaproic acid enrichment and 13 CO 2 excretion. 3. PPU protein , calculated as change in leucine balance/change in intake (HP-LP), varied from 0.58 to 0.99 (mean = 0.81±0.10), independently of age or sex. PPU protein varied directly with the inhibition of D and inversely with the increase in leucine concentration and stimulation of O and S . 4. Efficient PPU, as demonstrated by the top quintile of individuals categorized in terms of PPU protein , involves maximal inhibition of D by protein feeding with minimal increases in free amino acid concentrations, O and S . Lesser inhibition of D and greater stimulation of S and O characterized the lower, less efficient quintile. This indicates that the efficiency of protein utilization in individuals, and a component of their apparent protein requirement, is determined by the sensitivity of the insulin-mediated inhibition of proteolysis to amino acid supply.

1. Sepsis was induced in rats by an intravenous injection of live bacteria. Infected and pair-fed animals were studied before the infection, in an acute septic phase (day 2 post-infection), in a chronic septic phase (day 6) and in a late septic phase (day 10). Protein synthesis rates were measured in vivo after administration of a flooding dose of l[1- 13 C]valine. 2. During the acute phase, muscle protein loss associated with infection resulted from both a decrease in protein synthesis and an increase in proteolysis. During the chronic phase and the late phase, the increase of proteolysis in infected rats as compared with pair-fed animals persisted, worsening muscle atrophy. Skin protein synthesis rates were not significantly modified by infection. However, skin protein content decreased 6 and 10 days after infection, suggesting an increased proteolysis in response to sepsis. 3. Protein synthesis in liver of infected rats was twice that of pair-fed animals. Liver protein synthesis remained elevated in infected rats compared with pair-fed animals until day 10. Hypoalbuminaemia and high plasma concentrations of fibrinogen were evident at all periods studied. α 2 -Macroglobulin and α 1 -acid glycoprotein reached peak concentrations during the acute phase (concentrations increased 50 times in infected rats). On day 10, the levels of these proteins were still about 12-fold higher. 4. Protein synthesis rates were significantly increased in the digestive tract and lung of infected rats compared with pair-fed groups on days 2 and 6, but were similar in the two groups on day 10 postinfection. The fractional protein synthesis rate was increased 3-fold over the entire experimental period in the spleen. 5. The results show that sepsis stimulates protein synthesis in various tissues over a long time, and that skin, like muscle, can provide amino acids to the rest of the body.

1. The present study sought to determine the possible existence of a pool of proteins which turn over with life-time kinetics. The pattern of enrichment of ammonia and urea in hourly samples of urine was determined in normal adults to whom oral doses of [ 15 N]glycine were given hourly for 36 h. The subjects received hourly meals throughout, and in six the study commenced at 06.00 hours, in five at 12.00 hours and in two at 18.00 h. 2. A plateau level of enrichment was achieved in urinary ammonia within 4–6 h. Regardless of the time at which the study started this plateau was held until about midnight, at which time there was an increase in enrichment, with a second higher plateau 5–6 h later. The second plateau was held to the end of the study. For urinary urea the rate of rise in enrichment was slower and smoother, because of the slow turnover of the urea pool. 3. Protein synthesis, derived from the first ammonia plateau, 179 mg h −1 kg −1 , was significantly higher than that derived from the second plateau, 118 mg h −1 kg −1 . Using the plateau in urea towards the end of the 36 h, the estimate of protein synthesis was 153 mg h −1 kg −1 . 4. The results are considered to provide evidence of a pool of proteins for which degradation takes place in harmony with a circadian rhythm.

1. The stimulation and depression of peripheral blood lymphocytes has previously been studied in vitro , showing an immune depression postoperatively; however, it is difficult to interpret these in vitro findings. Therefore, an in vivo technique has been established for determination of the fractional protein synthesis rate, as an index of metabolic activity in human peripheral blood lymphocytes, by using a stable isotope technique. 2. The rate of protein synthesis was calculated from the increase in enrichment of l-[ 2 H 5 ]phenylalanine in protein of a mixed population of mononuclear leucocytes, isolated by density gradient, after an intravenous flooding dose of l-[ 2 H 5 ]phenylalanine. A linear time course of isotopic incorporation into the cells was demonstrated. 3. The fractional rate of protein synthesis of a mixed population of mononuclear leucocytes was studied in relation to surgical interventions and to potential modifiers of the response. The fractional synthesis rate increased 24 h after open and laparoscopic cholecystectomy (49 ± 19% and 40 ± 14% respectively, P > 0.02), irrespective of postoperative total parenteral nutrition or preoperative glucose infusion. In contrast to surgery, insulin did not stimulate protein synthesis in peripheral mononuclear leucocytes.

1. The healing of an abdominal muscle wound after surgery is associated with a considerable increase in the rate of protein synthesis. We have investigated whether this increase in protein synthesis is affected by chronic undernutrition, and whether this causes a delay in wound healing. 2. A group of rats was fed 58% of the voluntary food intake of a matched control group. After 7 days half the rats in each group underwent abdominal surgery. Forty-eight hours later all the rats were killed and muscle protein synthesis rate was measured by the flooding dose technique. 3. In a second experiment using the same dietary regimen rats were placed in metabolic cages after surgery and killed 7 days later. In addition to measurements of muscle protein synthesis, wound breaking strength was measured with a tensiometer and collagen content was also measured at the wound site. 4. Dietary restriction caused a loss of body weight, a decrease in nitrogen balance and a deficit in muscle protein mass. It also caused a decrease in protein synthesis rate in gastrocnemius muscle and in parts of the abdominal muscle distant from the site of the wound. However, it had no effect on the rate of muscle protein synthesis at the site of the wound either 2 or 7 days after surgery. The tensile strength and the collagen content of the wound were also unaffected by food restriction. 5. It is concluded that the wound healing process is uniquely protected from the effects of moderate undernutrition such as might be experienced by a chronically ill patient.

1. We studied the effect of sepsis and the regulation by glutamine of protein synthesis in enterocytes isolated from the small intestine of rats. 2. Sepsis was induced by caecal ligation and puncture; control rats were sham operated. Enterocytes were isolated from the jejunum and incubated in a medium containing [ 3 H]phenylalanine. 3. Sixteen hours after caecal ligation and puncture, protein synthesis, measured as incorporation of radioactivity into protein, was increased by 65%, 89% and 137% respectively in enterocytes from the tips and mid-portions of the villi and from the crypts. 4. Addition of glutamine to incubated enterocytes stimulated protein synthesis in a dose-dependent manner, and this effect was most pronounced in crypt cells from septic rats. The effect of glutamine on protein synthesis was duplicated by equimolar concentrations of acetoacetate or 3-hydroxybutyrate, both of which may serve as fuel for enterocytes, and was blocked by the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. 5. The results suggest that sepsis stimulates protein synthesis in enterocytes and that glutamine regulates protein synthesis in the same cells, probably by energy provision.

1. n -3 polyunsaturated fatty acids decrease responses to cytokines and inflammatory agents. The present study examines how different intakes of n -6 and n -9 fatty acids influence the metabolic response to endotoxin in Wistar rats. 2. Weanling male rats were, for 4 weeks, fed diets containing 50, 100 or 200 g/kg fat in the form of maize oil (rich in linoleic acid), butter (poor in linoleic acid, rich in oleic acid) or olive oil (adequate in linoleic acid, rich in oleic acid) or standard laboratory chow. All butter and olive oil diets included 10 g/kg maize oil, in total fat, to avoid essential fatty acid deficiency. 3. Rats subsequently received 800 μg/kg Escherichia coli endotoxin or sterile saline subcutaneously. Twenty-four hours after injection, the rate of tissue protein synthesis was measured in liver, lung, kidney, tibialis muscle and spleen by the ‘flooding dose’ method. Protein and zinc concentrations were assayed in all tissues and serum albumin and caeruloplasmin measured. 4. In animals fed chow, protein synthetic rate increased by 18%, 29% and 27% in liver, lung and kidney respectively. Tissue zinc concentrations increased by 33% in kidney, and tissue protein increased by 17%, 23% and 17% in liver, lung and kidney respectively. Serum caeruloplasmin increased by 60% and albumin concentration fell by 14%. 5. In animals consuming the 50 g/kg maize oil diet, protein synthetic rate increased by 56%, 36% and 34% in liver, lung and kidney respectively. Tissue zinc concentration increased by 14%, 15% and 17% in the three tissues respectively, and tissue protein concentration increased by 7%, 9% and 51% respectively. Serum caeruloplasmin increased by 172% and albumin concentrations fell by 22%. 6. No change in any parameter occurred in response to endotoxin in rats given diets containing fat predominantly as butter (50 and 100 g/kg), or olive oil (50, 100 and 200 g/kg). 7. In animals fed maize oil diets, responses increased in magnitude in parallel with dietary intake. 8. Responses in rats fed fat (200 g/kg) predominantly as butter were similar to those seen when diets contained 50 g/kg fat as maize oil. 9. The data suggest that the metabolic response to endotoxin is enhanced by n -6 and suppressed by n -9 unsaturated fatty acids. The modulatory influence of fats on responses to inflammatory agents may depend on the relative proportions of these substances.

1. Sepsis is associated with marked changes in cardiac muscle protein synthesis. Such changes may be the result of altered transcription of specific myofibrillar protein mRNAs. 2. In order to investigate myofibrillar protein gene expression, a rat model of sepsis was used. Adult rats were given a single sub-lethal dose of lipopolysaccharide by the intraperitoneal route. At various times thereafter, rats were killed and ventricular muscle was removed. RNA was extracted and transferred to nylon membranes. Changes in expression of mRNA for α- and β-myosin heavy chain, α-actin, cardiac troponin C and carbonic anhydrase III were detected by Northern hybridization. 3. After treatment with lipopolysaccharide, mRNA for β-myosin heavy chain increased to 260% of control values at 24 h and reached a maximum of 310% at 48 h. α-Myosin heavy chain mRNA levels fell to 72% of control values at 24 h. mRNA levels for α-actin, cardiac troponin C and carbonic anhydrase III remained unchanged. 4. In order to investigate the role of tumour necrosis factor-α in this process, some rats were pretreated with monoclonal antibody against tumour necrosis factor-α before receiving lipopolysaccharide. Such animals showed an absence of tumour necrosis factor-α bioactivity in plasma, but changes in myocardial protein mRNA levels were no different from those seen in animals receiving lipopolysaccharide alone. 5. The reduction in protein synthesis in cardiac muscle in sepsis does not appear to be the result of reduced expression of genes for structural or soluble muscle protein. Rather there is a paradoxical increase in β-myosin heavy chain expression, which may represent a protective mechanism. Tumour necrosis factor-α does not appear to be involved in the mediation of these changes.

1. We report here the extent to which changes in protein turnover contribute to the previously described inhibition of growth of rat tibial length and skeletal muscle mass in response to protein deficiency [1], energy restriction and corticosterone treatment [2]. Measurements of 35 S uptake in vivo also enabled the qualitative pattern of changes in proteoglycan synthesis in bone and muscle to be established. 2. Protein deficiency was examined by ad libitum feeding of 20%, 7%, 3.5% and 0.5% protein diets with measurements at 1, 3 and 7 days (all diets), and 14 and 21 days (0.5% protein). In bone this induced delayed inhibition of tibial growth with parallel inhibition of protein synthesis, as measured by the phenylalanine flooding dose method. This was mediated by reductions in both ribosomal capacity (RNA/protein ratio) and activity (protein synthesis/RNA) in the 0.5% protein group. The pattern of inhibition of proteoglycan sulphation, measured as 35 S uptake 60 min after injection of a tracer dose of labelled sulphate, was similar to that of protein synthesis. 3. In muscle there was an intermediate graded inhibition of protein synthesis by protein deficiency, mediated by reductions in both ribosomal capacity and activity in the 0.5% protein group, which preceded growth inhibition in the 7% and 3.5% groups, and which was progressive with time. Transient increases in proteolysis contributed to the growth inhibition is some groups, but the rate fell eventually in the 0.5% group. The pattern of response of proteoglycan sulphation differed from protein synthesis with a delayed inhibition, but with subsequent marked reduction. 4. Energy restriction was induced by diets fed for 4 or 8 days at 75%, 50% and 25% ad libitum intakes with protein intakes held constant, and corticosterone treatment involved a dose of 10 mg day −1 100 −1 g (subcutaneous) with ad libitum feeding. In bone this induced a pattern of length growth inhibition which was dissociated from inhibition of protein synthesis in the moderately restricted (75% and 50%) groups. Only in the 25% group and in the 8 day corticosterone group was protein synthesis inhibited, through reductions in ribosomal capacity and activity. 35 S uptake was also dissociated from growth inhibition, with reduced 35 S uptake observed only after corticosterone treatment or 8 days of the 50% or 25% diets. 5. In muscle the energy restriction and corticosterone treatment induced parallel inhibitions of growth and protein synthesis, mediated by similar graded reductions in the RNA/protein ratios and in the 25% group in the K RNA . Proteolysis was unchanged in all except the 4-day corticosterone group (elevated by 25%) and the day 8 25% group (elevated by 40%) and corticosterone group (elevated by 60%). 35 S uptake was inhibited in parallel to muscle growth and protein synthesis. 6. These data show that inhibition of protein synthesis and 35 S uptake is an invariable element of muscle growth inhibition, and a usual but not invariable element of bone growth inhibition. Partial correlation analysis of the interactions between dietary protein, bone growth and muscle protein and proteoglycan synthesis shows that bone growth (as indicated by epiphyseal cartilage width) is significantly correlated with muscle protein synthesis and especially 35 S uptake, suggesting that the regulation of muscle growth by passive stretch consequent on bone lengthening includes muscle connective tissue growth as an important target.

1. In a previous study we found that the protein synthesis rate was increased by 50–60% in the mucosa of the jejunum and ileum during sepsis in rats. It is not known if sepsis affects protein turnover in other parts of the gastrointestinal tract as well. 2. In the present study, the influence of sepsis on mucosal protein synthesis in different parts of the gastrointestinal tract, from the stomach to the rectum, was determined in rats. 3. Sepsis was induced by caecal ligation and puncture; control rats underwent sham-operation. Protein synthesis rate was measured in vivo after administration of a flooding dose of [ 14 C]leucine. 4. Basal mucosal protein synthesis rates were lower in the colon than in the rest of the gastrointestinal tract. Sixteen hours after caecal ligation and puncture, the protein synthesis rates were increased by 40–85% in the mucosa of the small and large intestine and the rectum, whereas in the gastric mucosa, the protein synthesis rate was reduced by approximately 40%. 5. The results suggest that mucosal protein synthesis rates differ in the various regions of the gastrointestinal tract, and that the metabolic response to sepsis is different in the stomach than in the rest of the gastrointestinal tract. The finding of a reduced protein synthesis rate in the gastric mucosa may partly explain the tendency to gastric stress ulcers and bleeding seen clinically in sepsis.

1. The ‘flooding’ dose technique was used to measure rates of lymphocyte protein synthesis after infusion of [1- 13 C]leucine (20 atoms% enrichment, 4 g/70 kg body weight). Lymphocyte protein synthesis was measured in healthy subjects and in patients with metastatic colorectal cancer before and during infusion of recombinant interleukin-2. Rates of protein synthesis were compared with thymidine uptake in vitro and phenotypic analysis of lymphocytes. 2. The median rate of lymphocyte protein synthesis in four healthy subjects was 9% (range 7.2–11.4%/day) and in seven patients with colorectal cancer was 6.4% (range 4.2–8.2%/day). After recombinant interleukin-2 treatment the median rate of lymphocyte protein synthesis was 27.8% (range 25.2–33.7%/day). 3. The increased rates of lymphocyte protein synthesis in vivo , after recombinant interleukin-2 infusion, corresponded with increased rates of thymidine uptake and changes in the phenotypic expression of lymphocytes, but these were less consistent than the measured rates of protein synthesis. 4. It is concluded that lymphocyte activation is accompanied by a marked increase in lymphocytic protein synthesis which may have important implications for whole body protein metabolism. Furthermore, measurement of lymphocyte protein synthesis may provide a determination of lymphocyte activation in vivo .

1. The influence of an acute-phase reaction on the ability of protein synthesis rates in liver and three different muscles (gastrocnemius, soleus and heart) to respond to a short intravenous infusion of nutrients (glucose plus amino acids) was investigated during experimental inflammation induced by injection of human recombinant interleukin-1β or turpentine in young male rats. 2. Interleukin-1β induced a consistent increase of 3°C in body temperature between 3 and 5 h after injection, whereas turpentine induced a delayed fever, peaking by 13 h. 3. Interleukin-1β and turpentine stimulated fractional rates of protein synthesis in liver. The synthesis rate was inhibited by interleukin-1β in gastrocnemius and soleus muscle, but an elevation was seen in heart muscle. In this study there was no significant response of muscle to turpentine injection. 4. Two hours of parenteral nutrition increased fractional synthesis rates in all tissues when compared with Ringer's lactate. Somewhat larger responses to feeding were observed as a result of either interleukin-1β or turpentine injection in all tissues, but these improvements were not significant. 5. We conclude that the response of protein synthesis rates in liver and skeletal muscle to parenteral nutrition is not inhibited, and may be somewhat enhanced, during acute inflammatory conditions in the growing rat.

1. The fractional synthesis rate of protein is commonly measured by either the constant infusion method or the flooding dose method. The two methods often give different results. 2. An underlying assumption of the traditional flooding dose formula is that the protein synthesis rate is not stimulated by the flooding dose. A new formula for calculation of the fractional synthesis rate is derived with the alternative assumption that the protein synthesis rate is stimulated by an amount proportional to the change in the intracellular concentration of the infused amino acid. The alternative formula is: where E B and E F are the enrichments of bound and free amino acid, respectively (atom per cent excess), and C=1-( E F / E I ), where E I is the enrichment of the infusate. This approach defines the lowest possible value for the fractional synthesis rate. The traditional equation gives a maximal value for the fractional synthesis rate. 3. When data from the literature are considered, the fractional synthesis rate of muscle protein as calculated by the constant infusion technique falls between the values of fractional synthesis rate calculated by the two flooding dose formulae when leucine is the tracer, suggesting that a flooding dose of leucine exerts a stimulatory effect on the rate of protein synthesis, but that the increase is not as great as the increase in the intracellular concentration of leucine. 4. The precision of the formula for the calculation of fractional synthesis rate is limited by the accuracy of the underlying assumptions regarding the effect of the flooding dose on the fractional synthesis rate. At present, the best approach would appear to be the use of both equations to calculate the upper and lower bounds of the true fractional synthesis rate.

1. The amino acid L-arginine has been shown to enhance immune mechanisms and inhibit tumour growth in experimental animals, but although many of the immunological effects of arginine have been reproduced in man there have been few studies of its effects on human tumours. In this study the effects of arginine on human breast cancers were determined by measuring tumour protein synthesis and comparing this with immunohistochemical assessments of cell proliferation. 2. Patients with breast cancer were randomized to receive either a standard diet or arginine supplementation. At the time of surgery, the rate of tumour protein synthesis was measured by the incorporation of the stable isotope [1- 13 C]leucine into tumour protein. Tumours were also assessed histologically and by staining for the presence of the activation antigen Ki67. 3. The median rate of tumour protein synthesis was 10%/ day (range 5.5–15.8%/day) in the control patients and 25.6%/day (range 9-37%/day) in the patients receiving arginine supplements ( P < 0.005, Wilcoxon rank sum test). The rates of protein synthesis correlated with Ki67 expression within these tumours ( r =0.78, P < 0.001). A double-staining technique confirmed that tumour cells, rather than tumour-infiltrating lymphoreticular cells, expressed Ki67. 4. This study demonstrates that, in contrast to animal studies, L-arginine stimulates human tumours in vivo. This represents the first direct evidence that a single amino acid can modulate the behaviour of a human cancer.

1. The rate of protein synthesis in quadriceps muscle of healthy subjects estimated from the incorporation of l -[1- 13 C]leucine given by continuous infusion was 1.1%/day. The estimate of protein synthesis from the incorporation of a flooding amount of labelled leucine was 1.8%/day ( sd 0.65). The possibility that the higher rate obtained with the flooding technique arose from stimulation of protein synthesis by the large amount of leucine is unlikely. 2. The same rate of protein synthesis (1.7%/day, sd 0.3) was obtained with a flooding amount (0.05 g/kg) of a different amino acid, l -[1- 13 C]phenylalanine, as was obtained with leucine. 3. Incorporation of l -[1- 13 C]phenylalanine was not affected by simultaneous injection of leucine (1.7%/day, sd 0.7) or valine (1.6%/day, sd 0.4). 4. Protein synthesis, assessed in a completely different way from the proportion of polyribosomes isolated from the skeletal muscle, was unaltered by the injection of 0.05 g of l -leucine/kg (44.6%, sd 8.5 versus 43.8%, sd 7.7). 5. Good agreement in estimates of protein synthesis was observed in subjects in whom both legs were measured with both l -[1- 13 C]leucine (mean difference 0.16%/day) and l -[1- 13 C]phenylalanine (mean difference 0.2%/day).

1. A method is described for measuring the rates of protein synthesis in vivo in human colorectal and breast tumours by the intravenous injection of l -[1- 13 C]leucine as a ‘flooding dose'. 2. The incorporation of isotope into colorectal tumour protein was measured in six patients, whose tumours were biopsied after the injection. Fractional rates of protein synthesis were calculated from the enrichment of leucine in protein and the average free leucine enrichment in plasma. The range of rates obtained was 17.2–33.9%/day, with a mean rate (± sem ) of 22.5 ± 2.6%/day. 3. Tumour protein synthesis rates were also measured in 15 patients with breast cancer. The range of rates obtained was 5.3–15.9%/day, with a mean rate (± sem ) of 10.3 ± 0.8%/day. These rates are significantly lower than those obtained with colorectal tumours ( P < 0.001). 4. In 9 of the breast cancer patients, protein synthesis was measured in multiple random biopsies taken from the same tumour. The mean (± sem ) difference between the highest and lowest rates in biopsies from the same tumour was only 1.1 ± 0.3%/day. Only 13% of the variation in protein synthesis between separate tumours could be explained by sampling error because of variation within the tumour itself, the remainder being genuine variation between individual tumours.

1. The effect of uraemia on the rates of protein synthesis and protein degradation in liver, heart and vastus lateralis muscle were examined in the rat. Uraemia was induced by a five-sixths nephrectomy and the rates of protein turnover were compared with pair-fed sham-operated littermate controls. 2. The procedure produced plasma concentrations (means ±sem) of urea of 7.3 ±0.4 mmol/l in control and 39 ± 2 mmol/l in uraemic rats, and of creatinine of 41 ± 1 μmol/l in control and 133 ± 7 μmol/l in uraemic rats. 3. Uraemia reduced the rates of protein synthesis in liver, heart and muscle by 35 ±5, 4.0 ±1.2 and 4.0 ± 0.6%/day, respectively, compared with control rats ( P < 0.01). Since the reductions in tissue growth rate were too small to be accounted for by the reduction seen in protein synthesis alone, this implied that protein degradation was also reduced. Uraemia also caused an 18% reduction in the rate of growth as measured by the increase in tail length ( P < 0.01). 4. Uraemia reduced both protein synthesis and protein degradation. Protein synthesis exceeded protein degradation in all tissues. This would cause a reduced rate of protein accumulation in the uraemic compared with the control rats, and hence a reduced rate of growth.

1. The influence of elevated concentrations of stress hormones on the concentration of ribosomes and the relative proportion of polyribosomes, reflecting protein synthesis in vivo , in human skeletal muscle was investigated. Healthy volunteers were given a 6 h infusion of adrenaline ( n = 8), Cortisol ( n = 8), a triple-hormone combination of adrenaline, Cortisol and glucagon ( n = 8), or saline ( n = 8) 2. The total ribosome concentration declined by 30.4 ± 7.2% in the triple-hormone group ( P <0.01), by 26.9 ± 8.6% in the Cortisol group ( P <0.05) and by 24.8 ± 11.2% in the adrenaline group ( P <0.05). The proportion of polyribosomes to total ribosomes decreased by 8.5 ± 2.2% in the triple-hormone group ( P <0.05) 3. During hormone infusion the serum glucose levels were enhanced. The insulin concentrations in serum were elevated in the adrenaline group and the triple-hormone group, but not in the Cortisol group. Serum insulin decreased in the control group 4. The results indicate an effect of the combined stress hormone infusion on the total ribosome concentration as well as on the relative abundance of polyribosomes. The single hormones influenced the total ribosome concentration only. The results suggest a critical role for stress hormones in producing the decline in muscle protein synthesis seen after trauma.

1. Rates of protein synthesis were measured, in vivo , in lung, liver, heart and skeletal muscle of young male rats. Groups of rats were exposed for 1 h duration to one of the following anaesthetic regimens: 1.4% halothane, 2.2% halothane, 1.4% halothane in 66% nitrous oxide, intravenous pentobarbitone (20 mg/kg) and intravenous midazolam (18 mg/kg) combined with fentanyl (2 μg/kg). Fractional rates of protein synthesis were determined by injecting [ 3 H]phenylalanine (150 μmol/100 g body weight) 2. Liver protein synthesis was depressed significantly by all regimens, except midazolam/fentanyl, by up to 37.7% of control values. Lung protein synthesis was significantly reduced by all the anaesthetic agents by up to 30% of control rates 3. The effects of the anaesthetic agents on skeletal muscle and heart were small and not statistically significant 4. There was no evidence of ventilatory depression as manifested by changes in arterial blood gas partial pressures of CO 2 and O 2 , except in the group treated with 2.2% halothane.

1. The ‘flooding dose’ technique for measuring the rate of protein synthesis in tissues in vivo involves the injection of a large amount of unlabelled amino acid together with the tracer to minimize differences in isotopic enrichment of the free amino acid in plasma and tissue compartments. This approach has been investigated in human muscle by taking biopsies from postabsorptive male volunteers given [1- 13 C]leucine. 2. Intravenous injection of 4 g of unlabelled leucine resulted in a rapid rise in free leucine concentration of seven- to eleven-fold in plasma and five-fold in muscle. Values were still elevated by two-fold after 2 h. 3. Five minutes after injection of [1- 13 C]leucine (0.05 g/kg) the isotopic enrichment of plasma leucine was 82% that of the injected material, falling to 44% at 120 min. The enrichment of free leucine in sequential muscle biopsies was close to that in plasma and almost identical to that for plasma α-ketoisocaproate. 4. The rate of protein synthesis was determined from the increase in leucine enrichment in protein of muscle biopsies taken before and 90 min after injection of [1- 13 C]leucine (0.05 g/kg; 19 or 39 atom% excess) and the average plasma α-ketoisocaproate enrichment over this period (taken to represent muscle free leucine). The mean rate of muscle protein synthesis in 10 subjects was 1.95 (sem 0.12)%/day. Rates of protein synthesis calculated from plasma leucine as precursor enrichment were only 5% lower than those calculated from plasma α-ketoisocaproate. 5. It is concluded that a ‘flooding dose’ of 13 C-labelled amino acid is a useful and convenient technique for determining the rate of protein synthesis in tissues of human volunteers and patients.

1. The effect of fenbufen (γ-oxo-[1,1′-biphenyl]-4-butanoic acid), a known cyclo-oxygenase inhibitor, on the changes in muscle and liver protein metabolism in response to Escherichia coli endotoxin has been investigated in the rat. 2. Young male rats were fed a purified diet [18% (w/w) casein], with or without fenbufen (1.2 g/kg of diet). Groups of animals were injected with either endotoxin (LPS; Escherichia coli lipopolysaccharide 0.127 B8; 3 mg/kg body weight) or saline. Rectal temperature and food intake were measured over the following 24 h period, after which time measurements were made of muscle and liver protein content and synthesis in vivo , muscle protein degradation as the difference between protein synthesis and growth rates, muscle glutamine concentration and plasma insulin. 3. Fenbufen treatment alone tended to lower rectal temperature. It also reduced plasma insulin, slightly reduced food intake and slowed growth and muscle protein turnover, although muscle glutamine concentrations were unchanged. The slower protein synthesis mainly reflected reduced translational activity, which was consistent with the reduced insulin concentration. 4. LPS treatment increased rectal temperature by 1.6°C, and this was abolished by fenbufen, indicating that the dose of the drug was sufficient to block prostaglandin production in the hypothalamus. 5. LPS treatment induced similar losses in body weight and muscle protein in both control and fenbufen groups, despite a 50% lower food intake in the LPS plus fenbufen group compared with the LPS-only group. The loss of muscle protein in both groups reflected reduced protein synthesis and increased protein degradation. LPS treatment alone induced elevated plasma insulin, but fenbufen blocked this response and the insulin levels remained depressed. Muscle glutamine concentration fell in both LPS-treated groups, suggesting that the depression of protein synthesis and the development of the insulin resistance might be linked to the loss of intracellular glutamine. 6. LPS induced a relative increase in hepatic protein content, and total protein synthesis (by approximately 40%); fenbufen had no influence on these responses. 7. It is concluded that whereas treatment with fenbufen has marked effects on plasma insulin concentration, it has no influence on muscle and liver protein metabolism during endotoxaemia. Alternative mechanisms to those involving prostaglandins as mediators of the catabolic response of muscle are discussed, including those involving the glutamine transporter.

1. The rate of paraspinal (multifidus) muscle protein synthesis was measured bilaterally at the top, apex and bottom of the thoracic curve in nine children with an idiopathic scoliosis, using the stable-isotope-labelled amino acid l -[1- 13 C]leucine. 2. No significant difference was observed in rates of muscle protein synthesis between the two sides of the spine, at the levels of the first vertebrae in neutral alignment at the top and bottom of the curve. However, in every patient, at the apex of the spinal curve, synthesis was higher on the convexity than on the concavity (0.077 ±0.04 %/h convex, 0.052 ±0.02 %/h concave, means ± sd , P < 0.01). 3. Muscle RNA activity (μg of protein synthesized h −1 μg −1 of RNA) was lower at the curve apices on the concave than the convex side (0.019 ± 0.09 μg h −1 μg −1 convex apex, 0.016 ±0.06 μg h −1 μg −1 concave apex, P < 0.05). Activities were similar on the two sides at the top and bottom of the curve. 4. Differences in muscle histology between the two sides were also observed only at the apex, with a lower type I fibre diameter (50.9 ±8.5 μm convex, 38.3 ±2.4 μm concave, P < 0.05) and a lesser proportion of type I fibres (63 ± 12% convex, 49 ±9% concave, P < 0.05) on the concavity. 5. The results are consistent with effects on muscle protein turnover secondary to an increased muscle contractile activity on the curve convexity and functional immobilization of the muscle on the curve concavity.

1. The effects of a single dose of ethanol (75 mmol/kg body weight) on rates of muscle protein synthesis were examined in young rats. Fractional rates of protein synthesis were measured in the soleus, plantaris, gastrocnemius, diaphragm and stomach by the large ‘flooding-dose’ technique. 2. After 150 min, the fractional synthesis rates of all muscles were reduced by 15–35%. Skeletal muscles containing a predominance of anaerobic (fast-twitch, type II) fibres showed greater changes when compared with skeletal muscles with a predominance of aerobic (slow-twitch, type I) fibres. 3. Gastrocnemius muscles were separated into sarcoplasmic, stromal and myofibrillar protein fractions. Protein synthesis was reduced similarly in all fractions by ethanol treatment, by approximately 30%. 4. As skeletal muscle mass comprises 40% of body weight, the responses have important physiological implications and may also be responsible for the muscle atrophy observed in alcoholic patients.

1. Protein synthesis was measured in muscle of fed and fasted rats. Synthesis rates were 29% higher with feeding. Treatment with indomethacin before feeding completely blocked the stimulation of protein synthesis. 2. Whole-body protein synthesis was measured in six healthy volunteers with [ 15 N]glycine in fasted and fed states. Feeding increased synthesis rates by 50%. An oral dose of indomethacin 2 h before the measurement of protein synthesis had no effect in either the fasted or fed state.

1. Quadriceps muscle protein turnover was assessed in the post-absorptive state in six men immediately after the end of unilateral leg immobilization (37 ± 4 days) in a plaster cast after tibial fracture. A primed-constant intravenous infusion of l -[1- 13 C]leucine was administered over 7 h. Quadriceps needle biopsies, taken bilaterally at the end of the infusion, were analysed for muscle protein leucine enrichment with 13 C. 2. Quadriceps muscle protein synthetic rate, calculated from the fractional incorporation of [ 13 C]leucine into protein compared with the average enrichment of blood α-ketoisocaproate, was 0.046 ±0.012%/h in the uninjured leg, but was only 0.034 ±0.007%/h in the quadriceps of the previously fractured leg ( P > 0.05, means ± sd ). 3. Muscle RNA activity (i.e. protein synthetic rate per RNA) fell from 0.27 ±0.08 μg of protein synthesized h −1 μg −1 of RNA in the control leg to 0.14 ±0.03 μg of protein synthesized h −1 μg −1 of RNA in the immobilized leg ( P > 0.02). 4. Immobilization was associated with a significant atrophy of type I muscle fibres (mean diameter 69.5 ±21 μm immobilized, 81.1 ±18 μm control, P > 0.05), but no significant change occurred in type II fibre diameter. Mean quadriceps fibre volume calculated from the values for fibre diameter and percentage of each fibre type, was smaller in the injured leg by 10.6%; this value was near to the calculated difference in muscle thigh volume (calculated from thigh circumference and skin-fold thickness) which was less by 8.3%. 5. From estimated mean daily values for quadriceps protein synthetic rate (1.65 ±0.44%/day in the control legs and 1.22±0.28%/day in the injured legs) and change in fibre volume, mean daily muscle protein breakdown rates were calculated as 1.65%/ day and 1.53%/day respectively, suggesting that muscle protein breakdown was not enhanced and may have fallen. 6. The results suggest a decrease in muscle protein turnover during limb immobilization in man, with the decrement in muscle mass being due mainly to a substantial (25%) depression of muscle protein synthesis.

1. The rate of protein synthesis in the whole body was measured in one fed subject with seven 15 N-labelled amino acids (intravenous and oral doses) and two 15 N protein mixtures (oral doses only). The rates were determined individually from the urinary excretion of ammonia and total urea over a 12 h experimental period. 2. Except with oral glycine and alanine, the synthesis rates given by ammonia and urea were appreciably different within each study when calculated on the assumption of a single pool of metabolic nitrogen in the body. In general, intravenous administration of the tracers gave higher rates with urea and the oral route gave higher rates with ammonia. 3. The differences between intravenous and oral doses of 15 N could be reduced significantly by calculating synthesis rates from either the arithmetic or harmonic average of flux rates given by ammonia and urea. The averages correspond to estimates of the total flux in a two-pool model of metabolic nitrogen when it is assumed either that both pools receive an equal amount of tracer (arithmetic) or that both have the same rate of nitrogen turnover (harmonic). 4. By so reducing the effect of physical separation of nitrogen in the body, the metabolic aspects of compartmentation of the tracer could be examined. The results show that the absolute value obtained for protein synthesis depends on the source of labelled nitrogen. The data are discussed in this empirical context.

1. Whole body nitrogen turnover and protein synthesis were calculated by the method of D. Picou & T. Taylor-Roberts [ Clinical Science and Molecular Medicine (1969) 36, 283–296] except that plateau plasma enrichment of [ guanidino - 15 N]arginine was used in place of the [ 15 N]urea enrichment after a constant infusion of [ 15 N]-glycine. With this approach metabolic pool turnover and protein synthesis were 637.2 ± 73.0 mg of N day −1 kg −1 and 2964.0 ± 409.5 mg of protein day −1 kg −1 respectively. 2. Virtually identical isotopic enrichment in [ guanidino - 15 N]arginine and [ 15 N]urea were observed in a healthy young adult who took repeated oral doses of [ 15 N]glycine for a period of 60 h: 0.47 (arginine) and 0.48 (urea) atom% excess. 3. The turnover of glycine nitrogen and of urea, determined from the constant infusion of [ 15 N]glycine and [ 13 C]urea, was 66.2 ± 3.3 mg of N day −1 kg −1 and 156.2 ± 4.3 mg of urea day −1 kg −1 respectively. The ratio of steady-state enrichment in arginine to that in glycine, reflecting the fraction of arginine derived from glycine, was 10.5%. By using the [ guanidino - 15 N]arginine enrichment as representative of the expected enrichment in [ 15 N]urea at plateau, it was calculated that approximately 25% of glycine N flux is directed toward the synthesis of urea, with the remainder directed to protein and quantitatively minor products like haem and creatinine. 4. Unlike steady-state [ 15 N]urea labelling, which is achieved only after infusion of [ 15 N]-glycine for several days, plateau isotopic abundance in [ guanidino - 15 N]arginine was attained after only 1–2 h of [ 15 N]glycine infusions, thereby allowing estimation of whole body nitrogen kinetics and the rate of transfer of glycine nitrogen to urea in a relatively brief experiment.

1. Measurements have been made of whole-body and skeletal muscle protein synthesis in fed and fasted adults with l -[1- 13 C]leucine. 2. The marked increase in whole-body synthesis on feeding largely reflects the changes in protein synthesis in muscle, which doubles on feeding, compared with a 40% increase in that of the rest of the body. 3. Skeletal muscle in fed man contributes more than half to total protein synthesis occurring in the whole body.

1. The combination of a wire-mesh support with the roller-tube technique is described as a procedure for the culture of human jejunal mucosa in vitro. 2. The technique has been applied to fragments (approximately 10 mg) of jejunal biopsies from both normal subjects and patients with coeliac disease. 3. The cultured tissue has been shown by radioautography to incorporate [ 3 H]leucine into proteins of the villus epithelial cells and [ 3 H]thymidine into nucleic acid, predominantly by the enterobiasis. 4. Although the tissue protein and DNA contents fall during culture, it was found that the combined tissue and medium DNA content remained constant during culture and may be used as a reference for enzyme and biochemical studies on cultured intestinal biopsies.

1. Membrane-bound and free polyribosomes were isolated from parathyroid glands of normal dogs by a discontinuous density-gradient technique. 2. The conditions necessary for optimum incorporation by bound and free ribosomes of [ 3 H]-phenylalanine into protein were determined for asays in vitro directed by both endogenous messenger ribonucleic acid (mRNA) and polyuridylic acid [poly(U)]. 3. When the specific cofactors were available in optimum amounts, the rate of incorporation of amino acids into protein was directly proportional to the number of ribosomes present. This applied to assays directed by endogenous mRNA and poly-(U). 4. The results indicate that it is possible to isolate and directly study the protein synthetic activity of membrane-bound and free parathyroid ribosomes.

1. The release of 57 Co-labelled vitamin B 12 ([ 57 Co]B 12 ) and synthesis of transcobalamin II (TCII) by the isolated perfused rat liver were studied 10–42 days after the parenteral administration of a trace dose of 15 pmol (approximately 20 ng) of radioactive cyanocobalamin. 2. The rate of release of [ 57 Co]B 12 into plasma and bile was linear and constituted approximately 0.9% and 0.3% respectively of the initial hepatic radioactivity per hour of perfusion. 3. [ 57 Co]B 12 released into plasma was bound to TCII. Saturation of the total TCII by the addition of cyanocobalamin before perfusion resulted in the appearance of the hepatic [ 57 Co]B 12 in the free form. 4. These data were found to be compatible with the following observations in vivo : (i) rates of [ 57 Co]B 12 release as measured by urinary [ 57 Co]B 12 excretion after saturation of plasma binders with non-labelled cyanocobalamin; (ii) rates of biliary excretion of [ 57 Co]B 12 . 5. Liver damage produced by hypoxaemia was associated with a fall in the rate of release of [ 57 Co]B 12 . 6. TCII release occurred at a linear rate of almost twenty times that required for the binding of newly released hepatic vitamin B 12 . 7. Cycloheximide at a dose sufficient to inhibit release of TCII did not prevent the release of [ 57 Co]B 12 from the liver into plasma or bile. 8. Alteration of perfusate composition to contain either high plasma concentrations of vitamin B 12 and low concentrations of unsaturated TCII or high plasma concentrations of vitamin B 12 and high concentrations of unsaturated TCII had no effect on the rate of [ 57 Co]B 12 release into plasma or bile. 9. It is concluded that the fluxes of hepatic vitamin B 12 and TCII are very rapid and that the release of vitamin B 12 by the rat liver is controlled in the short term by factors other than the synthesis of TCII and the concentration of vitamin B 12 or unsaturated transcobalamin in the plasma.

1. Monolayer cultures of human parathyroid cells were established for a study of the nature of secreted human parathyroid hormone (HPTH). 2. [ 14 C]Leucine and 75 Se-labelled methionine were used as amino acid precursors for the biosynthesis of HPTH. 3. Immunoreactive labelled HPTH in the medium was extracted by affinity chromatography. 4. In cultures from glands, six adenomatous and one hyperplastic, radioactive HPTH in the media was indistinguishable in size and charge from highly purified bovine parathyroid hormone (PTH). 5. In two cultures from adenomata, media contained an immunoreactive basic peptide resembling ‘pro-PTH’ in its electrophoretic behaviour. The freeze-dried cells contained the same basic peptide.