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Biomedical subjects

P J Garlick

Publications and source records attributed to P J Garlick.

At least 19 recordsLinked to original sources

Non-surgical alternatives to invasive procedures in mice.

We have developed and validated catheterization protocols in mice that allow for simultaneous infusion and sampling. A sampling catheter was inserted in the lateral vein of the tail, while the animals were infused either intravenously or intragastrically through a second catheter placed in the contralateral lateral vein or via an intragastric catheter, respectively. The applicability of these methods of infusion and blood sampling were validated by conducting urea kinetics utilizing stable isotopes. These non-surgical procedures are non-invasive, inexpensive, fast to perform and animals do not require a recovery period before their use.

Animals↗

Acute alcohol administration inhibits the refeeding response after starvation in rat skeletal muscle.

This study determined whether an acute alcohol dose could inhibit the refeeding response in starved muscle. Rats starved for 24 h were pretreated with alcohol or saline before refeeding by intragastric or intravenous infusion of enteral diet (ENT), total parenteral nutrition (TPN), or saline. Refeeding by TPN or ENT stimulated increases in the fractional rate of protein synthesis (k(s)) in skeletal muscle. Alcohol prevented the increase in k(s) when refeeding occurred intragastrically (TPN or ENT) (P < 0.001) but not intravenously (TPN). Upon intragastric refeeding, alcohol inhibited the increase in both eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and p70 S6 kinase (p70(S6K)) phosphorylation in plantaris but caused only partial inhibition in soleus muscle (ENT only). When rats were refed intravenously, alcohol had no effect on the increased 4E-BP1 or p70(S6K) phosphorylation in either muscle. Plasma insulin levels were augmented by alcohol. Alcohol-related changes in plasma amino acid concentrations were similar irrespective of the route of feeding, whereas IGF-I levels showed differential changes. This is the first study to demonstrate that acute alcohol ingestion impedes the starved-to-fed response in skeletal muscle.

Amino Acids↗

Response of in vivo protein synthesis in T lymphocytes and leucocytes to an endotoxin challenge in healthy volunteers.

In vivo determination of protein synthesis in immune cells reflects metabolic activity and immunological activation. An intravenous injection of endotoxin to healthy volunteers was used as a human sepsis model, and in vivo protein synthesis of T lymphocytes and leucocytes was measured. The results were related to plasma concentrations of selected cytokines, peripheral cell counts and subpopulations of immune cells. The subjects (n = 8 + 8) were randomized to an endotoxin (4 ng/kg) or a saline group. In vivo protein synthesis was determined twice: before and 1-2.5 h after the endotoxin/saline injection. Protein synthesis decreased in isolated T lymphocytes, but increased in leucocytes. Plasma levels of TNF-alpha, IL-8, IL-6, IL-1 ra and IL-10 were elevated, whereas IL-2 and IFN-gamma, produced predominantly by T lymphocytes, did not change in response to endotoxin. Neutrophils increased, whereas lymphocytes and monocytes decreased 2.5 h after the endotoxin injection. Flow cytometry revealed a drop in total CD3+ T lymphocytes and CD56+ natural killer cells, accompanied by an increase in CD15+ granulocytes. In summary, in vivo protein synthesis decreased in T lymphocytes, while the total leucocyte population showed a concomitant increase immediately after the endotoxin challenge. The changes in protein synthesis were accompanied by alterations in immune cell subpopulations and in plasma cytokine levels.

Adult↗

Growth hormone does not affect albumin synthesis in the critically ill.

OBJECTIVE: To study the effect of growth hormone (GH) on albumin synthesis in critically ill patients. DESIGN: Prospective randomized controlled study. SETTING: Two intensive care units, university hospital and county hospital, respectively. PATIENTS: Twenty-two critically ill patients in the intensive care unit. INTERVENTIONS: Albumin synthesis was measured twice in each patient, with a 5-day interval. The patients in the control group (n = 11) received standard intensive care unit (ICU) treatment between measurements, whereas those in the GH group (n = 11) also received 0.3 U/kg daily of human recombinant GH. MEASUREMENTS AND RESULTS: Albumin synthesis was measured by labeling with L-[2H5]phenylalanine. In the control group, the fractional synthesis rate (FSR) of albumin was 16.3+/-4.1%/day (mean and SD) in the first measurement and 15.7+/-4.2%/day 5 days later (NS), whereas in the GH group the corresponding values were 17.0+/-4.7%/day and 16.7+/-5.5%/day (NS). The calculated absolute synthesis rates of albumin, based on FSR and intravascular albumin mass, also showed no effect of GH. CONCLUSION: Albumin synthesis rates were consistently higher in the two groups of critically ill patients than previously reported values in healthy subjects. However, GH treatment for 5 days neither stimulated nor inhibited albumin synthesis rates in these critically ill patients.

APACHE↗

Acute metabolic acidosis decreases muscle protein synthesis but not albumin synthesis in humans.

Chronic metabolic acidosis induces negative nitrogen balance by either increased protein breakdown or decreased protein synthesis. Few data exist regarding effects of acute metabolic acidosis on protein synthesis. We investigated fractional synthesis rates (FSRs) of muscle protein and albumin, plasma concentrations of insulin-like growth factor-I (IGF-I), thyroid-stimulating hormone (TSH), and thyroid hormones (free thyroxin [fT(4)] and triiodothyronine [fT(3)]) in seven healthy human volunteers after a stable controlled metabolic period of 5 days and again 48 hours later after inducing metabolic acidosis by oral ammonium chloride intake (4.2 mmol/kg/d divided in six daily doses). Muscle and albumin FSRs were obtained by the [(2)H(5)ring]phenylalanine flooding technique. Ammonium chloride induced a significant decrease in pH (7.43 +/- 0.02 versus 7.32 +/- 0.04; P < 0.0001) and bicarbonate concentration (24.6 +/- 1.6 versus 16.0 +/- 2.7 mmol/L; P < 0.0001) within 48 hours. Nitrogen balance decreased significantly on the second day of acidosis. The FSR of muscle protein decreased (1.94 +/- 0.25 versus 1.30 +/- 0.39; P < 0.02), whereas the FSR of albumin remained constant. TSH levels increased significantly (1.1 +/- 0.5 versus 1.9 +/- 1.1 mU/L; P = 0.03), whereas IGF-I, fT(4), and fT(3) levels showed no significant change. We conclude that acute metabolic acidosis for 48 hours in humans induces a decrease in muscle protein synthesis, which contributes substantially to a negative nitrogen balance. In contrast to prolonged metabolic acidosis of 7 days, a short period of acidosis in the present study did not downregulate albumin synthesis.

Acidosis↗

A combined stress hormone infusion decreases in vivo protein synthesis in human T lymphocytes in healthy volunteers.

In vivo protein synthesis decreases in mononuclear cells following a combined stress hormone infusion given to healthy volunteers as a human trauma model. Here, the purpose was to further investigate this finding and to measure in vivo protein synthesis in isolated T lymphocytes. Furthermore, the effects of stress hormones on the lymphocyte subpopulations and mononuclear cells, characterized by flow cytometry and phytohemagglutinin (PHA)-induced and unstimulated proliferative responses in vitro, were elucidated. Healthy volunteers (n = 16) were randomized into 2 groups to receive either a stress hormone or a saline infusion for 6 hours. In vivo protein synthesis was studied before and after the treatment by measuring the incorporation of stable isotopically-labeled phenylalanine into lymphocyte and mononuclear cell proteins. Protein synthesis decreased after stress hormone infusion in both cell populations: in T lymphocytes from 13.0% +/- 0.7%/d (mean +/- SD) to 8.6% +/- 2.1%/d (P <.01) and in mononuclear cells from 13.3% +/- 1.2%/d to 6.3 +/- 2.0%/d (P <.001). No change in proliferative responsiveness in vitro was observed. The stress hormone infusion produced a decrease in the percentage of T helper CD3/CD4 from 41% to 18% (P <.001), T cytotoxic CD3/CD8 from 27% to 15% (P <.001), as well as total T CD3 cells from 69% to 35% (P <.001). There was an increase in the percentage of natural killer (NK) cells CD16/CD56 from 17% to 55% (P <.001). Determination of phenotypes expressed on activated T lymphocytes showed that CD3/HLA-DR was unchanged and CD3/CD25 decreased from 14% to 7% (P <.01) in the stress hormone group. The study showed that the decrease of in vivo protein synthesis was 34% in T lymphocytes as compared with 53% in mononuclear cells, when determined immediately after a 6-hour stress hormone infusion. This change was associated with a pronounced decrease in all lymphocyte subpopulations, except for the NK cells, which increased substantially.

Adult↗

Stimulation of human albumin synthesis and gene expression by growth hormone treatment.

BACKGROUND AND AIMS: In this study the effects of acute (5 h) and short-term (5 days) GH treatment on albumin synthesis rates in man were investigated and related to changes in the availability of hepatic albumin mRNA. METHODS: 30 patients undergoing elective laparoscopic cholecystectomy were randomized into controls (n=10) or GH-treatment (12 U/dose) for 5 h or 5 days (n=10 in each group). Albumin mRNA levels (in liver biopsy specimens) were measured employing a quantitative polymerase chain reaction assay developed specifically for this purpose, whereas albumin synthesis was measured using [(2)H(5)]phenylalanine. RESULTS: The fractional synthesis rate of albumin was 6.0+/-0.9 %/day in the control group and 8.0+/-1.8 %/day and 8.3+/-1.7 %/day in the GH-treated groups, respectively (P<0.05 vs controls in both cases). The corresponding values for the concentration of albumin mRNA were 2.6+/-1.1 ng/microg total RNA, 2.9+/-0.8 ng/microg total RNA (NS) and 4.7+/-1.8 ng/microg total RNA in the "GH 5" group (P<0.01 vs controls). The changes in albumin synthesis were only partly explained by the differences in hepatic albumin mRNA levels (r=0.5, P<0.01). CONCLUSION: These results suggest that GH may induce a quick, gene expression-independent increase in albumin synthesis, which is sustained by a later-occurring increase in albumin gene expression.

Adult↗

Growth hormone together with glutamine-containing total parenteral nutrition maintains muscle glutamine levels and results in a less negative nitrogen balance after surgical trauma.

BACKGROUND: Muscle protein catabolism, reflected by a decrease in glutamine (GLN), a decrease in muscle protein synthesis, and a negative nitrogen balance can be reduced by either administration of GLN or growth hormone (GH). In this study, the effects of a combination of GH and GLH were studied. METHODS: Patients (n = 16) undergoing abdominal operation were given total parenteral nutrition (TPN) containing either GLN alone or GLN together with GH (GH/GLN) during 3 postoperative days. The amino acid concentration and protein synthesis in muscle tissue and the nitrogen balance were measured. RESULTS: GH/GLN reduced nitrogen losses compared with GLN alone (-5.8 +/- 1.4 g nitrogen versus -10.6 +/- 1.1 g nitrogen, P <.05). GH/GLN maintained muscle GLN at preoperative levels compared with a 47.5% +/- 6.3% decline in the GLN group. A similar decrease was seen in the fractional synthesis rate of muscle protein postoperatively in both groups. CONCLUSIONS: GH has an additive effect given together with GLN on muscle amino acid metabolism, preventing the decrease in the GLN concentration in skeletal muscle and diminishing the loss of whole body nitrogen. However, the improvements in muscle amino acid concentrations and nitrogen loss were not associated with differences between the groups in muscle protein synthesis postoperatively.

Abdomen↗

Assessment of the safety of glutamine and other amino acids.

Glutamine is used to supplement intravenous and enteral feeding. Although there have been many human studies of its efficacy, there have been very few studies with safety as a primary goal. This article analyzes the literature on the safety of glutamine and also examines the available information on high intakes of total protein and other amino acids, so that additional indicators of potentially adverse effects can be suggested. Four studies that specifically addressed glutamine safety were identified, from which it was concluded that glutamine is safe in adults and in preterm infants. However, the published studies of safety have not fully taken account of chronic consumption by healthy subjects of all age groups. To help identify potential undetected hazards of glutamine intake, the literature on adverse effects of high dietary intake of protein and other amino acids was examined. High protein is reputed to cause nausea, vomiting and ultimately death in adults, and has been shown to result in neurological damage in preterm infants. Individual amino acids cause a variety of adverse effects, some of them potentially fatal, but neurological effects were the most frequently observed. Because glutamine is metabolized to glutamate and ammonia, both of which have neurological effects, psychological and behavioral testing may be especially important.

Administration, Oral↗

Increased concentration of tracee affects estimates of muscle protein synthesis.

Muscle protein synthesis was measured by infusion of L-[2H(5)]phenylalanine in two groups of anesthetized dogs, before and during infusion of insulin with euaminoacidemia, and with differing concentrations of unlabeled phenylalanine (tracee). With the infusion of insulin, muscle protein synthesis increased 39 +/- 12% based on phenylalanyl-tRNA. Calculation with plasma phenylalanine enrichment overestimated insulin stimulation by 40% (56 +/- 12 vs. 39 +/- 12%). Raising the concentration of plasma phenylalanine twofold during infusion of insulin further increased the apparent stimulation of muscle protein synthesis based on plasma relative to phenylalanyl-tRNA by 225% (65 +/- 19 vs. 20 +/- 14%, P < 0.001). In both experiments, the stimulation of synthesis rates calculated from phenylalanine enrichment within the muscle was closer to that from phenylalanyl-tRNA (48 +/- 19%, experiment 1; 30 +/- 14%, experiment 2). Results indicate that the enrichment of a labeled amino acid within plasma and tissue amino acid pools is affected by the concentration of tracee infused. Increasing the concentration of tracee overestimates the insulin-mediated stimulation of muscle protein synthesis when amino acid pools other than aminoacyl-tRNA are used as the precursor enrichment.

Amino Acids↗

Enhanced synthesis of albumin and fibrinogen at high altitude.

The acute effects of active and passive ascent to high altitude on plasma volume (PV) and rates of synthesis of albumin and fibrinogen have been examined. Measurements were made in two groups of healthy volunteers, initially at low altitude (550 m) and again on the day after ascent to high altitude (4,559 m). One group ascended by helicopter (air group, n = 8), whereas the other group climbed (foot group, n = 9), so that the separate contribution of physical exertion to the response could be delineated. PV was measured by dilution of (125)I-labeled albumin, whereas synthesis rates of albumin and fibrinogen were determined from the incorporation of isotope into protein after injection of [ring-(2)H(5)]phenylalanine. In the air group, there was no change in PV at high altitude, whereas, in the foot group, there was a 10% increase in PV (P < 0.01). Albumin synthesis (mg. kg(-1). day(-1)) increased by 13% in the air group (P = 0.058) and by 32% in the foot group (P < 0.001). Fibrinogen synthesis (mg. kg(-1). day(-1)) increased by 40% in the air group (P = 0.068) and by 100% in the foot group (P < 0.001). Hypoxia and alkalosis at high altitude did not differ between the groups. Plasma interleukin-6 was increased modestly in both groups but C-reactive protein was not changed in either group. It is concluded that increases in PV and plasma protein synthesis at high altitude result mainly from the physical exercise associated with climbing. However, a small stimulation of albumin and fibrinogen synthesis may be attributable to hypobaric hypoxia alone.

Adult↗

Effects of smoking and abstention from smoking on fibrinogen synthesis in humans.

Cigarette smoking and hyperfibrinogenaemia are both significant risk factors for the development of cardiovascular disease. Two studies are described here which aimed to establish the metabolic mechanism responsible for the raised plasma fibrinogen concentration observed in smokers. Chronic smokers had a significantly elevated absolute rate of fibrinogen synthesis (ASR) compared with non-smokers (22.7 +/- 1.3 mg/kg per day versus 16.0 +/- 1.3 mg/kg per day; means +/- S.E.M., P < 0.01), with plasma levels of fibrinogen significantly correlated with fibrinogen synthesis (r = 0.65, P = 0.04). Unlike fibrinogen, plasma albumin concentrations were lower in smokers than in non-smokers (45 +/- 0.4 versus 47 +/- 0.7 g/l, P < 0.05), but there was no difference in rates of albumin synthesis between the two groups. Two weeks cessation from smoking by previously chronic smokers was associated with a rapid and marked fall in plasma fibrinogen concentration (from 3.06 +/- 0.11 g/l to 2.49 +/- 0.14 g/l, P < 0.001), and a significant reduction in ASR (a 33% reduction, from 24.1 +/- 1.7 to 16.1 +/- 1.0 mg/kg per day, P < 0.001). These studies suggest a primary role for increased synthesis in producing the hyperfibrinogenaemia associated with smoking. Moreover, abstention from smoking for a period of only 2 weeks induces a significant decrease in the rate of fibrinogen synthesis by the liver, with a concomitant reduction in the plasma fibrinogen concentration.

Adult↗

Lymphocyte protein synthesis is increased with the progression of HIV-associated disease to AIDS.

HIV infection has been shown to affect lymphocyte function and to reduce lymphocyte responsiveness in vitro to mitogenic stimulation, but little is known about lymphocyte metabolism in vivo and how it is affected during the course of the disease. This study investigated the metabolic activity of lymphocytes in vivo through the progression of HIV-associated disease. Lymphocyte protein synthesis was measured with L-[(2)H(5)]phenylalanine (45 mg/kg body weight) in healthy volunteers (n=7), in patients who were HIV-positive (n=7) but asymptomatic, and in patients with AIDS (n=8). The rates of lymphocyte protein synthesis [expressed as a percentage of lymphocyte protein, i.e. fractional synthesis rate (FSR)] were not altered in HIV-positive patients compared with healthy controls (7.9+/-1.28% and 9.1+/-0.53%/day respectively), but were significantly elevated in AIDS patients (14.0+/-1.16%/day; P<0.05). The serum concentration of the cytokine tumour necrosis factor-alpha (TNF-alpha) increased with the progression of the disease, and TNF-alpha levels were significantly higher in AIDS patients (6.81+/-0.88 ng/l) than in healthy controls (3.09+/-0.27 ng/l; P<0.05). Lymphocyte protein FSR was positively correlated with serum TNF-alpha concentration (r=0.55, P=0.009) and negatively correlated with CD4(+) lymphocyte count (r=-0.70, P=0.004). The elevation of lymphocyte protein synthesis in AIDS patients suggests a higher rate of turnover of lymphocytes. This may be associated with a generalized activation of the immune system, which is also reflected by the elevated serum TNF-alpha concentration in the late stages of HIV-associated disease.

Acquired Immunodeficiency Syndrome↗

Hyperventilation increases muscle protein synthesis in critically ill trauma patients.

BACKGROUND: Critically ill trauma patients are often in negative nitrogen balance and demonstrate advanced muscle protein wasting, which is in part due to a decrease in muscle protein synthesis. Previous studies have been performed on the relationship between pH and protein metabolism. Some evidence suggests that alkalosis might enhance protein synthesis. The purpose of the present study is to determine whether protein synthesis is increased in trauma patients who have a respiratory alkalosis from hyperventilation. METHODS: Trauma patients in the intensive care unit (n = 8) who were treated with hyperventilation for elevated intracranial pressures were enrolled. Muscle protein synthesis rates were determined in vivo using the flooding method with l-[(2)H(5)]phenylalanine. Measurements were performed twice on each patient within a 36-h period, first during hyperventilation and then after hyperventilation was discontinued. Hemoglobin oxygen saturation was maintained above 95% for all measurements. RESULTS: Protein synthesis in muscle was 1.38 +/- 0.11%/day during hyperventilation (pH 7.50 +/- 0.02, pCO(2) 27.3 +/- 1.0 mm Hg) and 0.93 +/- 0.15%/day after respiratory parameters were normalized (pH 7.39 +/- 0.01, pCO(2) 39.4 +/- 1.5 mm Hg). The synthesis rate was significantly higher (P < 0.01, paired t test), 0.46 +/- 0.13%/day (32.6%), at the time of hyperventilation. CONCLUSION: Muscle protein synthesis is elevated during hyperventilation in critically ill patients with traumatic brain injury. We believe this preliminary study provides data that warrant confirmation in larger clinical studies. It suggests that this ventilatory therapeutic strategy may have a role in mitigating the negative nitrogen balance and muscle protein wasting that can impair the recovery of these patients.

Adult↗

In vivo protein synthesis of circulating human T lymphocytes does not respond to a cortisol challenge within 24 h.

BACKGROUND: Although immunocompetence is often measured by assessing responsiveness of lymphocytes to mitogenic stimulation in vitro, this approach may not reflect the in vivo situation. The aim of this investigation was to determine in vivo the protein synthesis rate (FSR) in isolated T lymphocytes and to study the effect of a short-term cortisol infusion on FSR. METHODS: Healthy volunteers (n=24) were randomised into 4 groups. A continuous cortisol infusion (6 microg kg(-1) min(-1)) during 6 h was given to groups 1 and 2, whereas groups 3 and 4 served as control groups and received saline infusion. Protein synthesis was studied before and after 6 h of the cortisol/saline infusion (groups 1 and 3) or 24 h after the start of the infusion (groups 2 and 4). FSR was determined in vivo by the flooding method. The isotopic enrichment of phenylalanine in plasma and lymphocyte protein was determined with gas chromatography-mass spectrometry. RESULTS: The FSR in T lymphocytes was 13.6+/-0.9%/24 h as a mean value (+/-SD) of the first determination in 4 groups. There was no significant difference in FSR from the baseline value immediately after the cortisol infusion (group 1: 13.3+/-1.4%/24 h vs 13.5+/-2.8%/24 h) or 24 h after the start of the infusion (group 2: 13.6+/-0.7%/24 h vs 12.3+/-2.4%/24 h). CONCLUSION: The metabolic activity of circulating T lymphocytes, as reflected by a quantitative measurement of in vivo protein synthesis of human T lymphocytes, was not affected by the increased level of cortisol.

Adult↗

Albumin synthesis is diminished in men consuming a predominantly vegetarian diet.

Albumin synthesis was calculated in healthy male volunteers consuming diets differing in the relative contribution of protein from animal or vegetable sources. In one study (Study 1, n = 4) two isoenergetic and isonitrogenous diets were consumed for a period of 10 d each. One diet (diet A) was animal protein rich (74%), the other one (diet V) contained 67% of vegetable protein. Albumin synthesis rate was measured from L-[(2)H(5)]phenylalanine incorporation (43 mg/kg) at the end of each dietary period. Both albumin fractional synthesis rate (FSR) (5.7 +/- 0.6 vs. 6.7 +/- 0. 8%/d, P = 0.04) and absolute synthesis rate (ASR) (123 +/- 6 vs. 143 +/- 8 mg. kg(-1). d(-1), P = 0.05) were reduced after diet V. In a second study (Study 2, n = 8) a third dietary treatment was added (Diet VS). This was similar to diet V but supplemented with soy protein (18g/d). The results of study 2 confirmed that albumin synthesis was reduced after diet V (FSR: 5.9 +/- 0.3 vs. 6.7 +/- 0. 5%/d, P = 0.015; ASR: 126 +/- 7 vs. 146 +/- 9 mg. kg(-1). d(-1), P = 0.007), but it also showed that the drop could be prevented by adding supplemental protein to the predominantly vegetarian diet (Diet VS) (FSR: 6.4 +/- 0.3%/d, P = 0.08; ASR: 140 +/- 7 mg. kg(-1). d(-1), P = 0.03). Albumin synthesis appears to be modulated by changes in the proportion of animal vs. vegetable protein occurring in the diet. The mechanism might be related to differences in digestibility and consequently in net amino acid availability between diets.

Adult↗

Protein-sparing effect in skeletal muscle of growth hormone treatment in critically ill patients.

OBJECTIVE: To investigate the effect of growth hormone (GH) treatment on skeletal muscle protein catabolism in patients with multiple organ failure in the intensive care unit (ICU). SUMMARY BACKGROUND DATA: Skeletal muscle depletion affects the incidence of complications and the length of hospital stay. A protein-sparing effect of GH treatment in skeletal muscle of long-term ICU patients was hypothesized. METHODS: Twenty critically ill ICU patients were randomized to treatment with GH (0.3 U/kg/day) or as controls. Percutaneous muscle biopsy samples were taken before and after a 5-day treatment period starting on day 3 to 42 of the patient's ICU stay. Protein content, protein synthesis, water, nucleic acids, and free amino acids in muscle were analyzed. RESULTS: The protein content decreased by 8% +/- 11% in the control patients, with no significant change in the GH group. The fractional synthesis rate of muscle proteins increased in the GH group by 33% +/- 48%, and muscle free glutamine increased by 207% +/- 327% in the GH group. Total intramuscular water increased by 12% +/- 14% in the control group as a result of an increase in extracellular water of 67% +/- 86%; these increases were not seen in the GH group. In contrast, the intracellular water increased by 6% +/- 8% in the GH group. CONCLUSION: Treatment with GH for 5 days in patients with multiple organ failure stimulated muscle protein synthesis, increased muscle free glutamine, and increased intracellular muscle water.

APACHE↗