Effect of light-load resistance exercise on postprandial amino acid transporter expression in elderly men.

An impaired amino acid sensing is associated with age-related loss of skeletal muscle mass. We tested whether light-load resistance exercise (LL-RE) affects postprandial amino acid transporter (AAT) expression in aging skeletal muscle. Untrained, healthy men (age: +65 years) were subjected to 13 h of supine rest. After 2 1/2 h of rest, unilateral LL-RE was conducted (leg extensions, 10 sets of 36 repetitions) at 16% 1RM Thereafter, the subjects were randomized into groups that orally ingested 40 g of whey protein either as hourly drinks (4 g per drink) (PULSE, N = 10) or two boluses (28 g at 0 h and 12 g at 7 h) (BOLUS, N = 10), or hourly isocaloric maltodextrin drinks (placebo, N = 10). Quadriceps muscle biopsies were taken at 0, 3, 7, and 10 h postexercise from both the resting and exercised leg, from which the membrane protein and mRNA expression of select AATs were analyzed by Western Blot and RT-PCR, respectively. LAT1 and PAT1 protein expression increased in response to LL-RE in the PULSE group, and SNAT2 and PAT1 protein expression increased in the BOLUS group when plasma BCAA concentration was low. In all three groups, LL-RE increased LAT1 mRNA expression, whereas a time course decrease in SNAT2 mRNA expression was observed. LL-RE increased membrane-associated AAT protein expression and mRNA expression. Altered AAT protein expression was only seen in groups that ingested whey protein, with the greatest effect observed after hourly feeding. This points toward an importance of AATs in the anabolic response following LL-RE and protein intake.

Existence of life-time stable proteins in mature rats-Dating of proteins' age by repeated short-term exposure to labeled amino acids throughout age.

In vivo turnover rates of proteins covering the processes of protein synthesis and breakdown rates have been measured in many tissues and protein pools using various techniques. Connective tissue and collagen protein turnover is of specific interest since existing results are rather diverging. The aim of this study is to investigate whether we can verify the presence of protein pools within the same tissue with very distinct turnover rates over the life-span of rats with special focus on connective tissue. Male and female Lewis rats (n = 35) were injected with five different isotopically labeled amino acids tracers. The tracers were injected during fetal development (Day -10 to -2), after birth (Day 5-9), at weaning (Day 25-32) at puberty (Day 54-58) and at adulthood (Day 447-445). Subgroups of rats were euthanized three days after every injection period, at different time point between injection periods and lastly at day 472. Tissue (liver, muscle, eye lens and patellar tendon) and blood samples were collected after euthanization. The enrichment of the labeled amino acids in the tissue or blood samples was measured using GC-MS-MS. In muscle and liver we demonstrated a rapid decrease of tracer enrichments throughout the rat's life, indicating that myofibrillar and cytoskeleton proteins have a high turnover. In contrast, the connective tissue protein in the eye lens and patellar tendon of the mature rat showed detainment of tracer enrichment injected during fetal development and first living days, indicating very slow turnover. The data support the hypothesis that some proteins synthesized during the early development and growth still exist much later in life of animals and hence has a very slow turnover rate.

Glucose and glycerol concentrations and their tracer enrichment measurements using liquid chromatography tandem mass spectrometry.

The present study describes a new liquid chromatography tandem mass spectrometry method for high-throughput quantification of glucose and glycerol in human plasma using stable isotopically labeled internal standards and is suitable for simultaneous measurements of glucose and glycerol enrichments in connection to in vivo metabolic studies investigating glucose turnover and lipolytic rate. Moreover, in order to keep up with this new fast analysis, simple derivatization procedures have been developed. Prior to analysis, glucose and glycerol were derivatized using benzoyl chloride in order to form benzoylated derivatives via new simplified fast procedures. For glucose, two internal standards were evaluated, [U-(13) C(6)]glucose and [U-(13) C(6), D(7)]glucose, and for glycerol, [U-(13) C(3), D(8)]glycerol was used. The method was validated by means of calibration curves, quality control samples, and plasma samples spiked with [6,6-D(2)]glucose, [U-(13) C(6)]glucose, and [1,1,2,3,3-D(5)]glycerol in order to test accuracy, precision, and recovery of the method. Moreover, post preparative and freeze-thaw sample stability were tested. The correlation of calibration curves for the glucose concentration were r(2) = 0.9998 for [U-(13) C(6)]glucose and r(2) = 0.9996 for [U-(13) C(6), D(7)]glucose, and r(2) = 0.9995 for the glycerol concentration. Interday accuracy for glucose using [U-(13) C(6)]glucose and glycerol determined in spiked plasma were respectively 103.5% and 106.0%, and the coefficients of variation were 2.0% and 9.7%, respectively. After derivatization, plasma samples were stable for at least 14 days. In conclusion, we have developed and validated a novel, accurate, and sensitive high-throughput liquid chromatography tandem mass spectrometry method for simultaneous determination of glucose and glycerol concentrations and enrichment of infused tracers most commonly used in human metabolic kinetic studies.

Determination of human muscle protein fractional synthesis rate: an evaluation of different mass spectrometry techniques and considerations for tracer choice.

In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically labeled amino acid will be incorporated within the few hours of a typical laboratory experiment. GC combustion isotope ratio MS (GC-C-IRMS) has thus far been considered the 'gold' standard for the precise measurements of these low enrichment levels. However, advances in liquid chromatography-tandem MS (LC-MS/MS) and GC-tandem MS (GC-MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N-acetyl-n-propyl, phenylisothiocyanate, or N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) for GC-C-IRMS, LC-MS/MS, and GC-MS/MS analysis, respectively. A second derivative, heptafluorobutyric acid (HFBA), was also used for GC-MS/MS analysis as an alternative for MTBSTFA. The machine reproducibility or the coefficients of variation for delta tracer-tracee-ratio measurements (delta tracer-tracee-ratio values around 0.0002) were 2.6%, 4.1%, and 10.9% for GC-C-IRMS, LC-MS/MS, and GC-MS/MS (MTBSTFA), respectively. FSR determined with LC-MS/MS compared well with GC-C-IRMS and so did the GC-MS/MS when using the HFBA derivative (linear fit Y = 1.08 ± 0.10, X + 0.0049 ± 0.0061, r = 0.89 ± 0.01, P < 0.0001). In conclusion, (1) IRMS still offers the most precise measurement of human muscle FSR, (2) LC-MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC-C-IRMS, and (3) If GC-MS/MS is to be used, then the HFBA derivative should be used instead of MTBSTFA, which gave unacceptably high variability.

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.

Erythropoietin down-regulates proximal renal tubular reabsorption and causes a fall in glomerular filtration rate in humans.

Recombinant human erythropoietin (rHuEPO) elevates haemoglobin concentration both by increasing red blood cell volume and by a decrease in plasma volume. This study delineates the association of rHuEPO-induced changes in blood volumes with changes in the renin­aldosterone system and renal function. Sixteen healthy males were given rHuEPO for 28 days in doses raising the haematocrit to 48.3±4.1%.Renal clearance studieswith urine collections (N = 8) were done at baseline and at days 4, 11, 29 and 42. Glomerular filtration rate (GFR) was measured by 51Cr-EDTA.Renal clearance of lithium (CLi)was used as an index of proximal tubular outflow and to assess segmental renal tubular handling of sodium and water. rHuEPO-induced increases in haematocrit occurred from day 10 onwards and was caused by both an increase in red cell volume and a fall in plasma volume. Well before that (from day 2 and throughout the treatment time), rHuEPO decreased plasma levels of renin and aldosterone (N = 8) by 21­33% (P < 0.05) and 15­36% (P < 0.05), respectively. After cessation of rHuEPO, values returned to baseline. On days 11 and 29, CLi increased (P < 0.02) indicating a significant 10­16% decrease in absolute proximal reabsorption of sodium and water (APR = GFR − CLi, P < 0.05). GFR decreased slightly, albeit significantly, on day 4 (P < 0.05). In conclusion, rHuEPO promptly, and before any changes in blood volumes and haematocrit can be detected, causes a down-regulation of the renin­aldosterone system. The results are compatible with a rHuEPO-induced reduction in proximal reabsorption rate leading to activation of the tubuloglomerular feedback mechanism and a fall in GFR. Therefore, treatment with rHuEPO may result in suppression of endogenous EPO synthesis secondary to a decrease in intrarenal oxygen consumption.

Screening for recombinant human erythropoietin using [Hb], reticulocytes, the OFF(hr score), OFF(z score) and Hb(z score): status of the Blood Passport.

Haemoglobin concentration ([Hb]), reticulocyte percentage (retic%) and OFF(hr score) are well-implemented screening tools to determine potential recombinant human erythropoietin (rHuEpo) abuse in athletes. Recently, the International Cycling Union implemented the OFF(z score) and the Hb(z score) in their anti-doping testing programme. The aim of this study is to evaluate the sensitivity of these indirect screening methods. Twenty-four human subjects divided into three groups with eight subjects each (G1; G2 and G3) were injected with rHuEpo. G1 and G2 received rHuEpo for a 4-week period with 2 weeks of "boosting" followed by 2 weeks of "maintenance" and a wash-out period of 3 weeks. G3 received rHuEpo for a 10-week period (boost = 3 weeks; maintenance = 7 weeks; wash out = 1 week). Three, seven and eight of the 24 volunteers exceeded the cut-off limits for OFF(hr score), [Hb] and retic%, respectively. One subject from G1, nobody from G2, and seven subjects from G3 exceeded the cut-off limit for Hb(z score.) In total, ten subjects exceeded the cut-off limit for the OFF(z score); two subjects from G1, two subjects from G2 and six subjects from G3. In total, indirect screening methods were able to indicate rHuEpo injections in 58% of subjects. However, 42% of our rHuEpo-injected subjects were not detected. It should be emphasised that the test frequency in real world anti-doping is far less than the present study, and hence the detection rate will be lower.