Muscle Tissue Fluid Research Articles

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet – A pilot study

Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake.

Quantitative amino acid profiling and stable isotopically labeled amino acid tracer enrichment used for in vivo human systemic and tissue kinetics measurements

An important area within clinical functional metabolomics is in vivo amino acid metabolism and protein turnover measurements for which accurate amino acid concentrations and stable isotopically labeled amino acid enrichments are mandatory not the least when tissue metabolomics is determined. The present study describes a new sensitive liquid chromatography tandem mass-spectrometry method quantifying 20 amino acids and their tracer(s) ([ring-(13)C6]/D5Phenylalanine) in human plasma and skeletal muscle specimens. Before analysis amino acids were extracted and purified via deprotonization/ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-(13)C/(15)N). The method was validated according to general recommendations for chromatographic analytical methods. The calibration curve correlations for amino acids were on average; r(2)=0.998. Interday accuracy for amino acids determined in spiked plasma was on average 97.3% and the coefficient of variation (CV) was 2.6%. The ([ring-(13)C6]/D5Phenylalanine) enrichment CV's for machine reproducibility in muscle tissue fluid and plasma were 4.4 and 0.8%, and the interday variability was 3.4% and the recovery was 90.5%, respectively. In conclusion, we have developed and validated a method for quantitative amino acid profiling that meets the requirements for systemic and tissue human in vivo amino acid and protein turnover kinetics measurements. Moreover, citrulline, ornithine, π-methyl-histidine, τ-methyl-l-histidine, hydroxy-proline and carnitine were analysed but when similar precision and accuray are required an additional stable istopically labeled internal standard for these meatablites should be be added.

Salmonella Serovars and Antimicrobial Resistance Patterns on a Sample of High Seroprevalence Pig Farms in England and Wales (2003-2008)

Following the introduction of a national abattoir-based monitoring programme for Salmonella in pigs, advisory visits were made to pig farms in England and Wales with high Salmonella seroprevalence assessed by muscle tissue fluid (meat juice) ELISA. Samples (n = 15 790), including pooled pen floor faeces (n = 12 136), were collected for Salmonella culture from 296 farms, between October 2003 and February 2008. Salmonella was isolated from 4489 (28%) of all samples collected, including 3301 (27%) of pooled pen floor faecal samples, from 270 (91%) of farms visited. Salmonella Typhimurium and S. Derby were the most prevalent serovars, representing 64% and 16% of isolates serotyped, respectively. The main phage types of S. Typhimurium identified were U288 and DT193. Antimicrobial resistance (AMR) was seen in 92% of isolates tested, with the highest frequencies of resistance occurring to tetracyclines (T), sulphonamide compounds (SU), ampicillin (AM), sulphamethoxazole/trimethoprim (SXT), streptomycin (S) and chloramphenicol (C). Fifty-nine AMR patterns were observed, the most frequent of these being T, AM, SXT, C, S, SU, seen in 35% of isolates tested. Multi-drug resistance was commonly found, with 67% of isolates submitted for AMR testing showing resistance to between four and nine antimicrobials.

Influence of tracer selection on protein synthesis rates at rest and postexercise in multiple human muscles

The goal of this investigation was to assess the influence of tracer selection on mixed muscle fractional synthesis rate (FSR) at rest and postexercise during amino acid infusion in multiple human skeletal muscles. Fractional synthesis rate was measured before and 24 hours after 45 minutes of running using simultaneous infusion of [ 2H 5]-phenylalanine (Phe) and [ 2H 3]-leucine (Leu) coupled with muscle biopsies from the vastus lateralis and soleus in aerobically trained men (n = 8; age, 26 ± 2 years). Mixed muscle protein FSR was analyzed by gas chromatography–mass spectrometry combined with a standard curve using the enriched muscle tissue fluid as the precursor pool. To control for potential analytical differences between tracers, all samples and standards for both tracers were matched for m + 0 abundance. Tracer selection did not influence resting FSR for the vastus lateralis or soleus ( P > .05). Fractional synthesis rate measured 24 hours postexercise was higher ( P < .05) compared with rate at rest and was similar between tracers for the vastus lateralis (Phe, 0.110% ± 0.010%·h −1; Leu, 0.109% ± 0.005%·h −1) and soleus (Phe, 0.123% ± 0.008%·h −1; Leu, 0.122% ± 0.005%·h −1). These data demonstrate that tracer selection does not influence the assessment of resting or postexercise FSR, thereby supporting the use of both [ 2H 5]-phenylalanine and [ 2H 3]-leucine for the measurement of FSR in exercise-based studies of human skeletal muscle.

Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer

The goal of this study was to discover whether using different tracers affects the measured rate of muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions and mixed meal feeding (70 mg protein x kg fat-free mass(-1) x h(-1) x 2.5 h) by simultaneous intravenous infusions of [5,5,5-(2)H(3)]leucine and either [ring-(13)C(6)]phenylalanine or [ring-(2)H(5)]phenylalanine and analysis of muscle tissue samples by gas chromatography-mass spectrometry. Both the basal FSR and the FSR during feeding were approximately 20% greater (P < 0.001) when calculated from the leucine labeling in muscle tissue fluid and proteins (fasted: 0.063 +/- 0.005%/h; fed: 0.080 +/- 0.007%/h) than when calculated from the phenylalanine enrichment data (0.051 +/- 0.004 and 0.066 +/- 0.005%/h, respectively). The feeding-induced increase in the FSR ( approximately 20%; P = 0.011) was not different with leucine and phenylalanine tracers (P = 0.69). Furthermore, the difference between the leucine- and phenylalanine-derived FSRs was independent of the phenylalanine isotopomer used (P = 0.92). We conclude that when using stable isotope-labeled tracers and the classic precursor product model to measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly depending on the tracer amino acid used; however, the anabolic response to feeding is independent of the tracer used. Thus different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle protein synthesis, and data from studies using different tracer amino acids can be compared qualitatively but not quantitatively.

Mechanism of insulin's anabolic effect on muscle: measurements of muscle protein synthesis and breakdown using aminoacyl-tRNA and other surrogate measures

Despite being an anabolic hormone in skeletal muscle, insulin's anticatabolic mechanism in humans remains controversial, with contradictory reports showing either stimulation of protein synthesis (PS) or inhibition of protein breakdown (PB) by insulin. Earlier measurements of muscle PS and PB in humans have relied on different surrogate measures of aminoacyl-tRNA and intracellular pools. We report that insulin's effect on muscle protein turnover using aminoacyl-tRNA as the precursor of PS and PB is calculated by mass balance of tracee amino acid (AA). We compared the results calculated from various surrogate measures. To determine the physiological role of insulin on muscle protein metabolism, we infused tracers of leucine and phenylalanine into 18 healthy subjects, and after 3 h, 10 subjects received a 4-h femoral arterial infusion of insulin (0.125 mUxkg(-1)xmin(-1)), while eight subjects continued with saline. Tracer-to-tracee ratios of leucine, phenylalanine, and ketoisocaproate were measured in the arterial and venous plasma, muscle tissue fluid, and AA-tRNA to calculate muscle PB and PS. Insulin infusion, unlike saline, significantly reduced the efflux of leucine and phenylalanine from muscle bed, based on various surrogate measures which agreed with those based on leucyl-tRNA (-28%), indicating a reduction in muscle PB (P < 0.02) without any significant effect on muscle PS. In conclusion, using AA-tRNA as the precursor pool, it is demonstrated that, in healthy humans in the postabsorptive state, insulin does not stimulate muscle protein synthesis and confirmed that insulin achieves muscle protein anabolism by inhibition of muscle protein breakdown.

Interstitial muscle concentrations of rocuronium under steady-state conditions in anaesthetized dogs: actual versus predicted values

The objective of this study was to compare rocuronium effect (C(e)) and peripheral (C(2)) compartment concentrations predicted by pharmacokinetic-pharmacodynamic (PK-PD) modelling with those measured in plasma (C(p)) and in the interstitial fluid of muscle tissue (C(ISF,u)) by microdialysis in anaesthetized dogs. After approval by the Animal Care Committee, eight adult male dogs with a body weight ranging from 7 to 18 kg were anaesthetized with pentobarbital. Each dog received a 2-min rocuronium infusion of 0.15 mg kg(-1) min(-1) followed by a 118-min infusion of 60 microg kg(-1) min(-1) via the right jugular vein. Arteriovenous gradient across the hindlimb was measured at 40, 60, 100 and 120 min. Three microdialysis samples were collected at 40-min intervals. Once the infusion stopped, arterial samples were collected every 2 min for the first 10 min and every 20 min for the next 120 min. Neuromuscular function was monitored using train-of-four stimulation until full recovery. Dogs were then killed and a biopsy of muscle tissue was performed (C(m)). At steady state, the mean C(ISF,u) value was 1353 ng ml(-1). After correction for the unbound fraction in plasma, the mean C(e,corr) and C(2,corr) were 1681 and 1481 ng ml(-1), respectively. At the terminal sampling point, C(m) was 10-fold higher than C(p). Unbound concentration of rocuronium measured in the muscle interstitial fluid under steady-state conditions confirms that parametric PK-PD modelling gives reliable estimates of effect site concentrations. Rocuronium accumulates in muscle tissue, probably by non-specific protein binding in the interstitial space.

Skeletal muscle myosin heavy-chain synthesis rate in healthy humans.

Mixed muscle protein synthetic rate has been measured in humans. These measurements represent the average of synthetic rates of all muscle proteins with variable rates. We determined to what extent the synthesis rate of mixed muscle protein in humans reflects that of myosin heavy chain (MHC), the main contractile protein responsible for the conversion of ATP to mechanical energy as muscle contraction. Fractional synthetic rates of MHC and mixed muscle protein were measured from the increment of [13C]leucine in these proteins in vastus lateralis biopsy samples taken at 5 and 10 h during a primed continuous infusion of L-[1-13C]leucine in 10 young healthy subjects. Calculations were done by use of plasma [13C]ketoisocaproate (KIC) and muscle tissue fluid [13C]leucine as surrogate measures of leucyl-tRNA. Fractional synthetic rate of MHC with plasma KIC (0.0299 +/- 0.0043%/h) and tissue fluid leucine (0.0443 +/- 0.0056%/h) were only 72 +/- 3% of that of mixed muscle protein (0.0408 +/- 0.0032 and 0.0603 +/- 0.0059%/h, respectively, with KIC and tissue fluid leucine). Contribution of MHC (7 +/- 1 mg.kg-1.h-1) to synthetic rates of whole body mixed muscle protein (36 +/- 5 mg.kg-1.h-1) and whole body protein (127 +/- 4 mg.kg-1.h-1) is only 18 +/- 1 and 5 +/- 1%, respectively. This relatively low contribution of MHC to whole body and mixed muscle protein synthesis warrants direct measurement of synthesis rate of MHC in conditions involving abnormalities of muscle contractile function.

A problem in the interpretation of beta-lactam antibiotic levels in tissues.

Ceftazidime was injected iv to rabbits (25 mg/kg). Samples of the whole muscle and muscle tissue fluid were removed at 0.25, 0.5, 1, 1.5, 2, 2.5 h for assay. Muscle tissue fluid was obtained using implanted cotton threads and freshly applied paper discs. The levels of ceftazidime in muscle fluid were similar to concurrent serum levels at all times tested. Ceftazidime levels in small pieces of excised muscle were significantly lower (ten-fold) than concurrent serum or tissue fluid levels. This difference between ceftazidime levels in whole tissue and tissue fluid is explained by the fact that not all antibiotics penetrate into the cellular mass of tissue but some remain confined to the much smaller interstitial tissue fluid compartment. The assumption that antibiotics will diffuse evenly throughout a tissue is an error to be avoided in the interpretation of antibiotic levels in tissues.

Determination of extravascular concentrations of doxycycline.

Doxycycline concentration in rabbit serum and muscle fluid were studied following a single iv infusion of 10 mg/kg. Maximal levels of doxycycline in muscle tissue fluid were found within 20 min after the end of infusion. Thereafter muscle fluid levels of doxycycline were similar to concurrent serum levels and were eliminated at a similar rate.

Tissue distribution of ampicillin: assays in muscle tissue and subcutaneous tissue cage fluid from normal and nephrectomized rabbits.

Tissue distribution of ampicillin following a single intravenous injection was studied in normal and functionally nephrectomized rabbits. Serum concentrations of ampicillin were compared with those obtained in subcutaneous tissue cage fluid, muscle (measured by a direct agar diffusion method) and muscle tissue fluid (obtained from implanted cotton threads). Considerable differences were found. Penetration of ampicillin into rabbit muscle was rapid and the elimination half-life was similar to that of serum. Concentrations in tissue cage fluid were low as compared with muscle tissue fluid levels, and penetration and elimination was slower. Results from experiments in functionally nephrectomized rabbits showed that a steady state between serum and muscle seemed to be attained after 15-30 min. These results were used to estimate the degree of protein binding of ampicillin in muscle tissue fluid.

Antibiotic assays in muscle: are conventional tissue levels misleading as indicator of the antibacterial activity?

Concentrations of ampicillin and cefuroxime in serum and muscle were studied in rabbits following a single intramuscular injection of 25 mg/kg. Simultaneous measurements were performed in whole muscle tissue pieces and in muscle tissue fluid obtained by implantation of cotton threads under the muscle fascia. The concentrations in the tissue fluid showed only a short time lag to thea serum levels. However, levels in whole muscle were substantially lower than the corresponding concentrations in muscle tissue fluid, indicating that the antibiotics were not evenly distributed within the tissue. The whole muscle tissue levels seriously underestimated, by a factor of approximately 5 times, the actual antibiotic concentrations in the extracellular fluid, where the microorganisms causing soft tissue infections are most likely to be found.