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1Department of Sports Studies, University of Stirling, Stirling, United Kingdom; Departments of 2Human Health and Nutritional Sciences and 3Animal and Poultry Science, University of Guelph, Guelph, Ontario; and 4Department of Medicine, McMaster University, Hamilton, Ontario, Canada
Submitted 15 April 2008 ; accepted in final form 20 June 2008
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ABSTRACT |
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60% peak oxygen consumption (
O2peak). In the first part of the study, after an overnight fast, 7 volunteers (28 ± 3 yr, 184 ± 2 cm, 88.0 ± 6.6 kg) ingested 1.66 g oral taurine doses with breakfast (8 AM) and lunch (12 noon), and blood samples were taken throughout the day. In the second part of the study, eight men (22 ± 1 yr, 181 ± 1 cm, 80.9 ± 3.8 kg, 4.21 ± 0.16 l/min O2peak) cycled for 2 h after 7 days of placebo (P) ingestion (6 g glucose/day) and again following 7 days of T (5 g/day). In the first part of the study, plasma taurine was 64 ± 4 µM before T and rose rapidly to 778 ± 139 µM by 10 AM and remained elevated at noon (359 ± 56 µM). Plasma taurine reached 973 ± 181 µM at 1 PM and was 161 ± 31 µM at 4 PM. In the second part of the study, seven days of T had no effect on muscle taurine content (mmol/kg dry muscle) at rest (P, 44 ± 15 vs. T, 42 ± 15) or after exercise (P, 43 ± 12 vs. T, 43 ± 11). There was no difference in muscle glycogen or other muscle metabolites between conditions, but there were notable interaction effects for muscle valine, isoleucine, leucine, cystine, glutamate, alanine, and arginine amino acid content following exercise after T. These data indicate that 1) acute T produces a 13-fold increase in plasma taurine concentration; 2) despite the ability to significantly elevate plasma taurine for extended periods throughout the day, 7 days of T does not alter skeletal muscle taurine content or carbohydrate and fat oxidation during exercise; and 3) T appears to have some impact on muscle amino acid response to exercise. amino acids; cycling; muscle glycogen
50–60 mmol/kg dry muscle) in relation to plasma (30–60 µmol/l in plasma) and an absence of incorporation of taurine into protein within skeletal muscle (16, 25). The reported metabolic actions of taurine have been largely related to glucose tolerance, insulin sensitivity, and substrate uptake, storage, and oxidation in skeletal and cardiac muscle. Taurine has been observed to improve insulin sensitivity in the fructose-fed rat (2), to stimulate glycolysis and glycogenesis in isolated perfused rat hearts (30), to reduce postprandial glucose oxidation assessed by respiratory gas exchange, and to increase skeletal muscle glycogen storage in Type 2 diabetic rats (21). Furthermore, Collivichi et al. (8) have demonstrated that taurine maintains hormone-stimulated glucose uptake in streptozotocin-induced diabetic rats. In rodents, taurine supplementation appears to increase muscle taurine content (43); however, it is unknown if supplementation alters human muscle taurine content.
Prolonged endurance exercise to exhaustion (100 min) has been observed to cause a decline in rodent skeletal muscle taurine content from 1 to 0.8 µmol/g wet weight, with a greater drop in taurine content (0.5 µmol/g wet weight) in fast-twitch fiber types (34). Other data have demonstrated a greater urinary loss of taurine following endurance running in humans (9) and a decline in plasma and tissue taurine content with ageing in rodents (10). However, a fall in muscle taurine content with exercise has not been observed in human studies (13, 33), and no difference was noted in femoral arterial-venous exchange of taurine during 60 min of exercise (5). Furthermore, it has been noted that endurance-trained human subjects have a higher resting and postexercise muscle taurine content than untrained subjects (16). This differential response of muscle taurine content to prolonged exercise may be related to the intensity and duration of effort (>100 min may be necessary to observe a change; Ref. 27); the initial glycogen content of the muscle; and/or the methionine and cyst(e)ine concentrations, which serve as the metabolic precursors to taurine.
Given that rodent studies have demonstrated changes in muscle contractile ability following taurine depletion (11, 15) and severe skeletal muscle exercise capacity impairment in taurine transporter knockout mice (42), it is evident that taurine depletion causes marked effects in some animal models. However, in human skeletal muscle the lack of a decline in muscle taurine with exercise suggests that supplemental taurine is unnecessary, yet taurine is added to energy drinks in the belief that it will aid athletic performance (1). We therefore hypothesized that acute oral taurine supplementation would increase plasma taurine concentration but that chronic oral taurine supplementation (7 days) would not increase the taurine content, or content of other amino acids in human skeletal muscle, nor alter the metabolic responses to prolonged (120 min) moderate-intensity [60% peak oxygen consumption (O2peak)] cycling exercise in healthy active men.
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METHODOLOGY |
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To determine the magnitude of the plasma taurine response to oral taurine supplementation during the course of 1 day, seven healthy active volunteers (28 ± 3 yr, 184 ± 2 cm, 88.0 ± 6.6 kg; 4 men, 3 women) volunteered to attend the laboratory on a single occasion following an overnight fast. On arrival at the laboratory, subjects lay down and a venous cannula was inserted into an antecubital vein. Following a period of 15 min of seated rest, a baseline fasted blood sample was drawn before subjects ate breakfast (8 AM). Two gelatin capsules totaling 1.66 g of taurine (NOW, Bloomingdale, IL) were ingested with the meal. Further blood samples were drawn each hour following ingestion for a period of 4 h (until 12 noon) when lunch was eaten, again with two gelatin capsules containing taurine (1.66 g). Blood samples were then drawn every hour for a further 4 h (until 4 PM). No other food or fluid (other than water) was allowed to be consumed during the day, and no food or fluid containing supplemental taurine was ingested at each meal time. Meal selection was independent, but foods high in taurine content were excluded from the trial.
Chronic Taurine Supplementation
For the second part of the study, eight healthy recreationally active men [22 ± 0 yr; 80.9 ± 3.8 kg; 181.3 ± 0.9 cm; O2peak 4.21 ± 0.16 l/min] volunteered to undertake two 7-day periods of oral supplementation of their diet with placebo and taurine in a single-blind ordered fashion. Subjects were instructed to maintain their regular activity habits during the study period and to refrain from ingesting any products/beverages containing supplemental taurine. Subjects were provided with written and verbal information about the study and procedures involved and gave their written informed consent for participation.
This study was approved by the Ethics Committee of McMaster University and conformed to the Declaration of Helsinki for studies on human volunteers.
Preliminary Testing
Before beginning any supplementation, subjects reported to the laboratory on two occasions. On the first visit, subjects performed an incremental cycling (Lode Excalibur, Quinton Instrument) test to exhaustion to determine O2peak. Respiratory gases were collected and analyzed using a metabolic cart (Sensormedic, Vmax229, Yorba Linda, CA). On the second visit 1 wk later, subjects attended the laboratory in the morning and cycled for 2 h at 60% O2peak to establish the correct power output for the main trials and to ensure that they could exercise for the required duration.
Supplementation
Immediately following the second visit subjects were provided with a 7-day supply of placebo capsules (2 capsules taken 3 times daily with meals, each containing 1 g of glucose). Supplementation commenced that day and finished on the morning of the 7th day on which the first main 2-h exercise trial occurred. Following a 1-wk recovery period, subjects embarked on a second 7-day supplementation period with taurine (2 capsules taken 3 times daily with meals, each containing 0.83 g of taurine, total dose per day 4.98 g). A final exercise trial was conducted at the end of the 7-day period with the final supplements ingested on the morning of the exercise trial.
Due to the lack of information that currently exists on the potential duration of washout of chronic taurine supplementation from skeletal muscle, supplements were administered in a single-blind ordered fashion with placebo ingested first. To ensure that the initial 2-h exercise trial had no effect on muscle taurine content, we included a resting skeletal muscle biopsy midway through the study before supplementing with taurine (Fig. 1).
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60% O2peak
Subjects performed a 2-h cycling trial at a moderate intensity (60% O2peak) following each period of supplementation. For each of these trials, subjects arrived at the laboratory 2–4 h postprandial. They abstained from strenuous exercise and recorded their diet in the 24 h before the trial and repeated this routine for the second trial. A Teflon catheter was inserted into the antecubital vein for blood sampling, and the catheter was kept patent by flushing with 0.9% saline. One leg was prepared for percutaneous needle biopsy sampling of the vastus lateralis muscle. Three incisions were made in the skin under local anesthesia (2% xylocaine without epinephrine) for three separate biopsies. Immediately before exercise, venous blood (6 ml) and one muscle biopsy (150 mg wet mass) were obtained while the subject rested on a bed. All muscle samples were immediately frozen in liquid nitrogen for subsequent analysis. Subjects then cycled for 2 h at 60% O2peak at a constant cadence (70–90 rpm) on the Lode ergometer. Respiratory gases were collected during 13–17, 28–32, 58–62, 88–92, and 115–119 min of exercise for the measurements of O2 and carbon dioxide production (CO2) and the calculation of the respiratory exchange ratio (RER). These parameters were used to calculate whole body fat oxidation (fat ox) and carbohydrate oxidation (CHO ox) using the nonprotein RER table (14) and according to the following equations: CHO ox = 4.585CO2 – 3.226O2; fat ox = 1.695O2 – 1.701CO2 (35). Venous blood samples were obtained at 15, 30, 60, 90, and 120 min of exercise. Immediately following exercise, two muscle biopsies were taken on the ergometer. Throughout exercise heart rate was recorded, and water ingestion was prescribed (200 ml every 20 min) in an attempt to maintain euhydration throughout the exercise period.
The same procedures were repeated on the second main trial with muscle biopsies taken from the other leg. An additional biopsy was taken at rest 1 wk after the first trial and before the start of taurine supplementation. This midstudy biopsy was obtained from the same leg as used in the first trial.
Blood Analyses
Venous blood was collected and dispensed into a K-EDTA tube. A portion (200 µl) was removed from the potassium-EDTA tube and added to 1 ml of ice-cold 0.6 N perchloric acid, shaken vigorously, and centrifuged, and the supernatant was analyzed for blood glucose and lactate using fluorometric techniques (3). A second portion (1.5 ml) was centrifuged, and the plasma was analyzed for free fatty acids (FFA) using an enzymatic colorimetric technique (Wako NEFA C test kit, Wako Chemicals, Richmond, VA).
Plasma and Muscle Amino Acids
One milliliter of whole blood from the K-EDTA tube was removed and centrifuged with 300 µl of plasma, then removed and stored at –80°C for subsequent analysis of plasma amino acids. Amino acids in plasma were obtained using HPLC and a prederivatization with phenylisothiocyanate method adapted from Bidlingmeyer et al. (4) and Heinrikson and Meredith (26). For muscle amino acid analysis, a 3- to 4-mg sample of freeze-dried muscle, powdered and free from visible connective tissue, fat, and blood, was homogenized in an ice-cold Eppendorf tube for 1 min with 100 µl of distilled deionized (MilliQ) water. The sample was then centrifuged for 3 min at 12,500 g, and 75 µl of the supernatant was subsequently used in the derivatization step using the method adapted from Bidlingmeyer et al. (4) and Heinrikson and Meredith (26).
Muscle Metabolites
A second portion of the resting and postexercise muscle biopsy was freeze-dried, powdered, and dissected free of visible connective tissue, fat, and blood. One aliquot of freeze-dried powdered muscle (10 mg) was extracted in 0.5 M HClO4-1 mM EDTA and neutralized with 2.2 M KHCO3. The supernatant was used to measure creatine, phosphocreatine (PCr), ATP, and lactate by enzymatic spectrophotometric assays (3, 22). Acetyl CoA and acetylcarnitine were assessed using a radiometric method (7). Free ADP (ADPf) and AMP (AMPf) concentrations were calculated as previously described (36). Duplicate aliquots (2–4 mg each) were extracted in 0.1 M NaOH and neutralized with 0.1 M HCl-0.2 M citric acid-0.2 M Na2PO4, and amyloglucosidase was added to breakdown glycogen to glucose, which was measured spectrophotometrically to assess glycogen content. The total creatine content of freeze-dried muscle samples was similar in both trials, and therefore all freeze-dried measurements were normalized to the highest total Cr measured among all biopsies from each subject. Furthermore, muscle water content was assessed from creatine corrected wet-to-dry ratio determined from mass pre- to postfreeze drying.
Statistics
All data are presented as means ± SE. Muscle and plasma data were analyzed by two-way repeated-measures ANOVA (time x trial) to determine significant differences between trials, rest, and end of exercise, and interactions between trial and exercise time. Following observation of a main effect, specific differences were identified using a post hoc Student's t-test. Statistical significance was accepted at a level of P < 0.05.
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RESULTS |
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Plasma taurine response to an acute oral taurine load.
There was a dramatic and significant (P < 0.05) elevation in plasma taurine in response to an acute oral dose administered with breakfast at 8 AM in the morning (Fig. 2). An 13-fold elevation in plasma taurine concentration was observed 2 h postingestion, and an 6-fold elevation was maintained at 4 h following supplementation. Plasma taurine concentration was then further elevated by the second taurine dose with lunch at 12 noon, with the concentration rising to 16-fold above baseline at 5 h (1 h following the 2nd ingestion time point). The plasma taurine concentration then fell over the following 3 h but remained significantly elevated above baseline at the 8-h time point.
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In the second part of the study in which subjects undertook two 7-day supplementation periods (placebo and taurine), all subjects complied with the supplementation protocol and only one experienced any side effects (slight muscle cramping), which occurred during the taurine ingestion period.
Muscle taurine and amino acids. Oral taurine supplementation (4.98 g/day) did not significantly affect resting skeletal muscle taurine content and did not affect the muscle taurine response to prolonged moderate-intensity exercise. Two hours of prolonged exercise itself did not result in any change in skeletal muscle taurine content after oral supplementation with either placebo or taurine (Fig. 3).
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Creatine-corrected muscle wet-to-dry mass change was not different between resting biopsies following placebo or taurine ingestion (mass change of 78.2 ± 1.7% vs. 75.8 ± 0.50% for placebo and taurine, respectively). Similarly, the mass loss from wet to dry for immediately postexercise biopsies was not different between trials (mass loss of 77.5 ± 0.1% vs. 79.5 ± 1.5% for placebo and taurine, respectively), with no main interaction effect (trial x time) observed in the statistical analysis.
Blood metabolites. Blood glucose and plasma FFA at rest and in response to exercise were not different between trials (Table 3). Blood lactate at rest was not different between trials; however, there was a significant treatment effect (P < 0.05), a significant time effect (P < 0.01), but no interaction effect.
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O2, and RER responses to exercise were not different between placebo and taurine-supplemented trials (Table 3). Subsequently, the estimated rates of carbohydrate and fat oxidation and estimated total carbohydrate and fat oxidation during exercise were also not different between trials (Fig. 7).
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DISCUSSION |
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60 µM to 750–1,000 µM. Despite these sustained elevations in plasma taurine with acute oral ingestion, a 7-day period of supplementation with three 1.66-g doses/day (breakfast, lunch, and dinner) did not alter resting skeletal muscle taurine content. Taurine supplementation also had no effect on the whole body and muscle metabolic responses to 120 min of exercise at 60% O2peak but did induce a postexercise increase in muscle content of indispensable amino acids. Plasma and Muscle Taurine Levels
Previous work with rodents reported an increase in the taurine content of skeletal muscle following the administration of a 20- to 30-ml dose of a taurine solution (0.5 g/kg) once per day over a 14-day period (43). In the present study this dose would equate to an average intake of 40–44 g/day in our two groups of human subjects. Although large doses of taurine (up to 15 g/day) appear to be well tolerated in human studies (39), doses larger than 15–20 g/day have been reported to result in gastrointestinal problems (37).
The magnitude of elevation in plasma taurine concentration in the present study was similar to that which has been observed following large acute doses of creatine monohydrate. The difference between the two dietary supplements is that a portion of the plasma creatine enters the skeletal muscle pool in the first 1–3 days of loading producing 15–25% increases in total muscle creatine content (23), whereas we have now shown that this is not the case for taurine. It is believed that the large increase in plasma creatine concentration, following oral creatine ingestion, acutely stimulates the activity of the plasma membrane creatine transporter (40), but this effect may be short lived. Taurine is transported into cells through a sodium-dependent taurine transporter (TauT). Taurine transporter activity appears to be enhanced by hyperosmotic conditions within cells, thus increasing taurine transport into cells to assist in cell volume regulation (19), but regulation of taurine transport appears to be also affected by cytokines, which act to increase taurine efflux (31). However, high circulating taurine concentration, and β-alanine both result in reduced cellular taurine uptake (20, 29). Thus taurine transport into tissue appears to be tightly regulated without even a short-lived period of activation in response to high circulating extracellular taurine concentrations. Thus the absence of a change in resting skeletal muscle taurine content may reflect downregulation of TauT activity with supplementation, which should be determined in a future study by examining TauT total protein changes in response to oral taurine supplementation.
It is also worth noting that the taurine content of skeletal muscle is partly determined by fiber type distribution, and type I fibers have been shown to contain the majority of the muscle taurine pool (24, 34). The reported proportion of type I fiber in the human vastus lateralis muscle is in the region of 44–57% but shows considerable interindividual variation (18, 32). Although we determined the taurine content in whole muscle homogenate from the vastus lateralis muscle, any meaningful change in type I fiber taurine content in response to supplementation should still have been detectable in whole muscle homogenate. The coefficient of variation (%) of our analytical method for taurine would allow us to detect a physiologically significant change in the region of 5–6%. The absence of any effect of oral supplementation over 7 days, or any effect of 120 min of exercise on human skeletal muscle taurine content, suggests that the muscle taurine pool is tightly regulated in humans, but further analysis is required to confirm that individual fiber types in humans are not influenced differently by supplemental taurine.
Muscle Glycogen and Muscle/Blood Metabolites
We did not observe any difference in resting muscle glycogen content following taurine supplementation. This contradicts the findings of others who have reported that supplemental taurine potentiated the action of insulin in animal models, and increased glycogen storage (30, 21). However, a study involving 8 wk of low-dose taurine supplementation (1.5 g/day) in overweight adult humans did not result in an exaggerated insulin response to a glucose load in an intravenous glucose tolerance test and did not impact on glucose disposal during a hyperinsulinemic euglycemic clamp (6). Furthermore, we did not observe any changes in other measured metabolites within skeletal muscle following taurine supplementation either at rest or immediately postexercise. These findings indicate that in humans the inability to alter the skeletal muscle taurine pool probably explains the lack of support for the actions of taurine noted in some rodent models where muscle taurine content has been elevated by supplementation.
Muscle Amino Acid Responses to Supplementation/Exercise
An increase in the free pool of skeletal muscle amino acids at rest or following prolonged exercise is dependent on many possible mechanisms, including 1) increased protein breakdown, 2) decreased protein synthesis, 3) increased synthesis of the dispensable amino acids, 4) increased transport into muscle, 5) reduced release from muscle, or 6) reduced amino acid oxidation. The increased intracellular threonine concentration at rest is of interest as it is a dietary indispensable amino acid (12) and cannot be synthesized in vivo. Reduced oxidation, reduced incorporation of threonine into protein, increased transport into muscle, and/or increased breakdown all seem unlikely as most of the indispensable amino acids would presumably follow the same trend. However, decreased oxidation could be supported by the fact that there is also a decreased glycine concentration at rest, the product of threonine catabolism. The reduced resting concentrations of tyrosine and cystine are most likely due to decreased synthesis from their precursors, phenylalanine and methionine, respectively, or increased use for either protein or secondary products of metabolism.
The increase in muscle aspartate, asparagine, and lysine content with prolonged exercise in both placebo and taurine trials is as expected with this type and intensity of effort (16). The maintained muscle glutamate with exercise following taurine supplementation combined with an elevated glutamine at the end of exercise on the taurine trial compared with a fall and no change on placebo could reflect increased glutamate/glutamine cycling. Trained subjects have previously been shown to have better maintenance of muscle glutamate and glutamine contents with prolonged exercise, and also demonstrated higher muscle taurine content compared with untrained subjects (16). Increased glutamate and glutamine contents in muscle have also been associated with lower ammonia accumulation during prolonged moderate-intensity exercise (17), which is again an expected training-induced adaptation.
We were unable to substantiate any of these potential links as muscle taurine content did not change in the present study, and we did not have enough muscle tissue to examine muscle IMP or ammonia accumulation during the exercise trials. In addition, the increased muscle alanine at the end of exercise following taurine supplementation compared with a decrease following placebo also reflected the difference noted in skeletal muscle amino acid responses to exercise between trained vs. untrained humans (16). An explanation for supplemental taurine inducing these changes in alanine, glutamate, and glutamine response to exercise is unclear.
The interaction effects on valine, leucine, and isoleucine (branched-chain amino acids) contents in response to exercise following taurine supplementation compared with placebo could suggest that further work exploring the link between taurine-induced amino acid manipulations, effects on cell volume regulation, effects on muscle protein synthesis, and the generation of TCA cycle intermediates would be of interest. Also, the observed higher muscle content of most of the indispensable amino acids (phenylalanine, histidine, isoleucine, leucine, lysine, methionine, threonine, and valine) at the end of exercise following taurine supplementation compared with placebo is a novel finding. This type of amino acid response has been attributed to increased muscle protein degradation when observed in both muscle and plasma and was shown to be exacerbated when the muscle had low glycogen content (41). However, in the present study the plasma amino acids were unchanged between trials and the muscle glycogen content was the same before and following exercise, suggesting that the findings are more the result of alterations in transport or metabolism of these amino acids, possibly reflecting reduced oxidation, or a change in cell volume regulation. Further work should examine what changes a period of taurine supplementation could induce on the transport and/or oxidation of amino acids during exercise.
In conclusion, taurine does not accumulate in skeletal muscle following 7 days of supplementation despite large acute changes in plasma taurine concentration following oral supplementation. The absence of any effects of taurine on storage or metabolism of carbohydrate appears to reflect the tight regulation of the skeletal muscle taurine pool and does not support previous findings in rodents of taurine-induced insulin-like actions. However, some skeletal muscle amino acid differences observed between trials suggests that taurine supplementation may impact on the mobilization, metabolism, or transport of indispensable amino acids in human skeletal muscle during exercise.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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. Brain Res 1023: 141–147, 2004.[CrossRef][Web of Science][Medline]This article has been cited by other articles:
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  C. A. Goodman, D. Horvath, C. Stathis, T. Mori, K. Croft, R. M. Murphy, and A. Hayes Taurine supplementation increases skeletal muscle force production and protects muscle function during and after high-frequency in vitro stimulation J Appl Physiol, July 1, 2009; 107(1): 144 - 154. [Abstract] [Full Text] [PDF]
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indicates ingestion of 1.66 g of taurine with a meal. *All time points from 0.5 h onward are significantly above baseline (0 h, P < 0.05). Values are means ± SE.
