1Institut National de la Santé et de la Recherche Médicale U290, Hôpital Lariboisière-Saint-Lazare, 75475 Paris; 2Institut National de la Santé et de la Recherche Médicale U539, Centre de Recherche en Nutrition Humaine, 44093 Nantes, France; and 3Department of Physiology, University of Antananarivo School of Medicine, Antananarivo, Madagascar
Submitted 18 July 2003 ; accepted in final form 27 February 2004
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ABSTRACT |
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constant infusion; bolus injection technique
Most of the knowledge of the dynamics of taurine metabolism, however, is derived from the measurement of taurine concentration, which provides little information as to the mechanisms leading to taurine depletion and repletion (11, 35, 45, 48, 50).
In theory, the use of tracer dilution techniques should provide further insight into these mechanisms and help in designing strategies for taurine supplementation as well. Although tracer methodology has been applied extensively to the exploration of various amino acids, including methionine and cysteine (37), very few data have appeared with regard to in vivo taurine kinetics. The first estimates of taurine kinetics were reported from compartmental analysis using either [35S]taurine bolus injection in humans (44) or bolus injection of [35S]taurine and [13C]taurine in rhesus monkeys (33). In recent years, we developped a gas chromatography-mass spectrometry (GC-MS) assay to determine both the concentration and the stable isotope enrichment of taurine in blood and obtained preliminary estimates of taurine kinetics in human subjects by use of [1,2-13C2]taurine tracer (31). The aims of the present study were therefore 1) to expand on these preliminary data and assess the turnover rate of circulating taurine and the size of taurine tracer-miscible pools in healthy adults in the fasting state and 2) to determine whether the administration of labeled taurine as an intravenous bolus injection would yield kinetic parameters comparable to those obtained with constant infusion.
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METHODS |
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A priming dose equivalent to 1 h of tracer infusion rate was chosen for several reasons: 1) assuming that taurine would be distributed throughout body water, taurine volume of distribution would be equivalent to total body water (0.6 liter/kg), and assuming the average taurine concentration in body tissues to be close to that measured in plasma (50 µmol/l), we estimated body taurine pool at 0.6 x 50 = 30 µmol/kg, so that a 3 µmol/kg labeled taurine prime would enable us to instantaneously achieve a taurine enrichment
10%; and 2) in a first set of preliminary studies using a bolus injection, we had obtained an estimate of
2.7 mmol (37 µmol/kg) for the "rapid" taurine pool, and because we aimed at a plateau enrichment
8% during the primed, continuous infusion, we therefore elected to use 3 µmol/kg as a priming dose.
The priming dose was injected over 12 min and immediately followed by a continuous infusion using a syringe pump (PerfusorV, B Braun, Melsungen, Germany) at a rate of 6 ml/h. Blood samples were drawn at 0, 10, 20, and 30 min after the bolus and every 30 min thereafter until 480 min. Expired air was collected at hourly intervals throughout the study for 13CO2 determination.
In the second set of studies (protocol B), five subjects were studied on two separate occasions while receiving [1,2-13C2]taurine 1) as a primed continuous infusion on one day and 2) as a single intravenous bolus injection on the other day. The two studies were 1 wk apart and in random order.
For the primed, continuous infusion study, [13C2]taurine was administered at a rate of 3 µmol·kg1·h1 via a peripheral venous line, and a catheter placed in a dorsal vein of the contralateral hand was used for blood sampling. During the blood sampling period, the hand was kept in a radiant warmer at 60°C to obtain arterialized venous blood at 0, 240, 270, 300, 330, and 360 min (5).
On the day of the bolus injection study, baseline blood samples were obtained, immediately followed by the intravenous injection of 3 µmol/kg [13C2]taurine over 1 min. Immediately thereafter, the intravenous line was abundantly flushed with >25 ml of saline, and blood was sampled from the same venous line at 2, 5, 8, 10, 15, 30, 45, 60, and 120 min.
Materials. [13C2]taurine (99% atom 13C) was purchased from MassTrace (Woburn, MA). Tracer solutions were prepared using aseptic technique by dissolving accurately weighed amounts of labeled taurine in known volumes of sterile, 0.9% saline, passed through a 0.22-µm Millipore filter, and stored at 4°C until the infusion. All infusates were verified to be pyrogen free by the Pharmacie Centrale des Hôpitaux (Paris, France) using the limulus lysate method.
Breath CO2 analysis. To determine the 13C-to-12C ratio in expired CO2, aliquots of expired air were collected in 10-ml Vacutainer tubes at 20, 1, and 0 min (baseline) and again at 30-min intervals throughout the study and were analyzed by gas chromatography-isotope ratio mass spectrometry (Europa VG).
Blood taurine analysis. For the determination of plasma taurine enrichment, blood samples were immediately separated by centrifugation at room temperature at a minimum rotary speed of 800 g for 10 min. The residual plasma was centrifuged a second time at 300 g for 10 min to remove platelets. Platelet-free plasma samples were then stored at 80°C until the day of analysis. The method for preparing the N-pentafluorobenzoyl-di-n-butylamine derivative of taurine has been previously described in detail (31). All solvents and reagents used in the analysis of plasma taurine were, at minimum, of HPLC grade purity.
Both taurine concentration and isotopic enrichment were determined by electron impact (70 eV) GC-MS using a Hewlett-Packard 5973 (Palo Alto, CA) instrument. Aliquots were injected in triplicate into a GC (model 6890, Hewlett-Packard, Fullerton, CA) equipped with an OV1 fused silica capillary column (30 m x 0.32 mm ID, 0.25 µm film thickness). Ions at mass-to-charge ratios (m/z) = 302 and 304, representing natural and [13C2]taurine, respectively, were selectively monitored. A standard curve obtained by diluting natural taurine with graded amounts of [13C2]taurine was run before each sample series. When the observed 304-to-302 ion current ratios were plotted as a function of expected [13C2]taurine-to-natural taurine mole ratios, linear regression coefficients of >0.99 were consistently observed along with slopes of 0.820.92. Taurine concentration was assessed in the full-scan mode by monitoring masses ranging from m/z = 100350 using methyltaurine as internal standard.
Body compostion analysis. Body composition was measured using a two-frequency (5 kHz, 1 MHz) bioelectrical impendance analyzer (IMP B01, Cachan, France) to estimate fat-free mass, i.e., lean body mass.
Calculations.
The intracellular taurine concentration in "total blood cells" ([Tau]tbc), defined as the fraction of blood volume not accounted for by plasma, was estimated as
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For unprimed infusion, the rise of tracer enrichment [Ep, mole% excess (MPE)] to plateau was fitted to an exponential curve using a nonlinear regression
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For the bolus tracer intravenous injection study, the area under the curve (AUC, µmol·l1·h) sustended by plasma [13C2]taurine concentration was calculated by manual trapezoidal integration or Tai's formula (46). Taurine metabolic clearance rate (MCR, l·h1·kg1) was calculated as MCR = d/AUC, where d is the amount of labeled taurine injected (µmol/kg), and taurine Ra was calculated as
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Taurine pool determination was performed using a two-compartment model according to Sturman (43).
Statistics. Data are presented as means ± SD. Data comparisons were performed using SSPSS software (SPSS, Chicago, IL). Statistical significance was set at P < 0.05.
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RESULTS |
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At all time points, whole blood taurine concentrations were higher than plasma taurine (P < 0.01), due to considerably higher intracellular taurine levels, as estimated using hematocrit (Eq. 2). Regardless of the infusion rate and prime, isotopic enrichment remained considerably lower in whole blood than in plasma taurine (P < 0.02; Table 2). As shown in Fig. 1, a steady state was nevertheless observed in whole blood enrichment over the last hour of isotope infusion. The estimated total blood cell enrichment/plasma taurine enrichment ratio was 0.150.29 (95% confidence limit) between 300 and 480 min.
Regardless of the infusion protocol, no detectable 13C enrichment could be found in CO2 in expired air collected throughout the study period.
Protocol B: primed continuous infusion. In view of results from the pilot studies, five additional subjects received a 6-h primed continuous infusion of [13C2]taurine infusion at a rate of 3.1 ± 0.2 µmol·kg1·h1. Near-steady state was observed between 240 and 360 min, as attested by 1) coefficients of variation ranging between 6.2 and 2.1% (Table 3) in plasma enrichment and 2) slopes of regression curves not different from zero when enrichment was plotted as a function of sampling time. Yet the use of the 240- to 360-min sampling period resulted in a slightly but significantly (P = 0.03) lower calculated Ra than the use of the 300- to 360-min period (Table 3). Although the difference in flux values for the two tested periods was small (only 2 µmol·kg1·h1) and, most likely, of little biological significance, the steady state achieved during the 6th h of tracer infusion (300360 min) was of better quality, since in every single subject the CV in plateau enrichment was lower than that during the corresponding 5th h: on average, the CV was 2.1% during the 6th h, vs. 6.2% during the 5th h of tracer infusion (Table 3).
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Bolus injection study. The time course of 13C2 enrichment in plasma taurine after a single intravenous bolus of 3.02 ± 0.07 µmol/kg [13C2]taurine is depicted in Fig. 2 for four of the same five subjects. Because the very early sampling points were missed for subject 5, the Ra calculation could not be performed for that subject. There was no significant rise in the total (unlabeled + labeled) concentration of plasma taurine following the pulse dose, and for each subject, taurine concentration remained relatively stable between 2 and 120 min.
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DISCUSSION |
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Little is known about taurine kinetics in humans. Using [1,2-13C2]taurine, Vinton et al. (50) reported a taurine Ra 5.6 µmol·kg1·h1 in five healthy volunteers, one-sixth of the taurine flux measured in the current study. Differences in study design could have contributed to the discrepancy. Whereas Vinton et al. studied fed subjects, we kept our subjects in the postabsorptive state to avoid any potential effect of exogenous taurine on endogenous taurine production. In addition, we infused tracer for 6 h, compared with 21 days in the studies of Vinton et al., so tracer recycling cannot be excluded in the latter. Putative tracer recycling would result in higher enrichments in plasma taurine and, consequently, lower estimates of taurine Ra. Whereas protein breakdown cannot release label since taurine is not incorporated into protein (26), taurine conjugates to bile acids in liver and, once excreted into bile, is reabsorbed from intestinal lumen (21). Enterohepatic taurine cycling is therefore a potential source of tracer recycling and must increase with the two- to sixfold rise in bile flux associated with the fed state (1). We failed to detect any evidence of tracer recirculation, as judged by the lack of a secondary rise in enrichment during the last of 2 h of infusion; we therefore assume that recycling was minimal in our fasted subjects. We cannot, however, rule out the possibility that isotope enrichment would have continued to increase slowly, had we extended infusion time.
Moreover, achievement of plateau in plasma does not predict whether steady state had been reached inside cells where the bulk of taurine is located (2). We observed a much lower labeled taurine enrichment in total blood cells compared with plasma. Although "total blood cell" obviously is a loose term for a heterogeneous compartment that lumps taurine-rich (e.g., neutrophils and platelets) and taurine-poor cells such as erythrocytes (25, 26, 40, 51), lack of tracer equilibration between blood cells and plasma nevertheless suggests compartmentation between extra- and intracellular taurine.
Compared with the fluxes observed in the present report (32 µmol·kg1·h1), arteriovenous (a-v) gradients assessed across various vascular beds (9, 10, 16, 36) yield much lower estimates of taurine interorgan exchange: muscle releases 2.2 µmol·kg1·h1 taurine (36), and splanchnic bed (9) and brain (10) take up 1.9 and 1.3 µmol·kg1·h1 taurine, respectively, assuming blood flows of 1,200 ml/min in liver (9) and 2.2 and 60 ml·min1·100 g1 in muscle (36) and brain (39), respectively. Yet both approaches have intrinsic limitations. The a-v concentration differences are often of the same magnitude as measurement error, and determination of blood flow across organ beds is technically difficult. Moreover, a-v gradient methods measure net organ balance, so that simultaneous release and uptake in the same tissue can be missed. On the other hand, isotope dilution relies on enrichments measured in circulating taurine, although taurine may be produced and utilized inside cells without exchanging with plasma. The Ra obtained may thus reflect interorgan taurine exchange rather than whole body taurine production rate.
In the current study, estimates of taurine Ra derived from constant tracer infusion were consistently lower than those provided by bolus injection (Table 4), using the AUC describing the time course of [13C2]taurine level between 2 and 120 min. These cut-off times were chosen because of the difficulty in detecting isotope enrichments below 0.1 MPE by GC-MS and may have led to an underestimation of the AUC, resulting in higher estimates of taurine Ra. In addition, as the same intravenous line was used for bolus injection and for sampling, this technique could lead to "contamination" of initial blood samples by excess amounts of tracers. This source of error was quantitated in a recent study, albeit for a different substrate. When Boles Ponto et al. [Ponto et al. (35a)] infused 2-deoxy-2-[18F]fluoroglucose (18F-DFG) through a forearm vein and sampled blood from both the same line and a contralateral forearm vein, they found the 18F-DFG level to be, on average, 2% higher when sampling on the infused line, i.e., when using a 10-ml saline flush and discarding the initial 1-ml blood sampled. We believe the level of "contamination" was still less in the present studies, because 1) samples were obtained after a larger, 25-ml saline flush, and we discarded the initial 5 ml of blood; 2) we (data not shown) and others (14) have routinely used the same technique for intravenous glucose tolerance tests, a technique in which large doses of glucose are injected over 1 min and the same intravenous line is used for measuring blood glucose and insulin levels at 3 and 5 min after injection, and in the latter studies flushing of the intravenous line was sufficient to avoid contamination from the concentrated dextrose solution; and 3) if contamination had nevertheless occurred, it would have resulted in artificially high [13C2]taurine enrichment in the initial plasma samples obtained, which, in turn, would have increased the AUC describing the course of [13C2]taurine over time and, consequently, resulted in artificially decreased taurine Ra. This was obviously not the case, since the bolus injection technique yielded higher flux values than did the continuous infusion technique. Taurine Ra values obtained from [13C2]taurine continuous infusion are nevertheless close to the 2025 µmol·kg1·h1 obtained by monitoring the decay of taurine specific activity for 48 h after a bolus of [35S]taurine in humans (44) or monkeys (33). Taken together, these studies suggest that, whereas bolus injection may be appropriate for radioactive taurine, a continuous-infusion approach is preferable when stable isotopes are used.
Regardless of the methodological uncertainties, taurine Ra is lower than most amino acid Ra values assessed to date (3). The appearance of [18O]taurine after inhalation of 18O2 established the ability of humans to synthesize taurine de novo (24). Conversion of cysteine to taurine is the sole source of endogenous taurine and is, however, limited, due to 1) the low activity of cysteine sulfinate decarboxylase, the rate-limiting step in taurine synthesis (8, 26, 27); 2) the low Ra of cysteine (45 µmol·kg1·h1); and 3) the fact that two other major pathways, cysteine incorporation into body protein (22) and glutathione (12), compete for cysteine utilization (37, 42).
Regarding taurine disposal (Rd), irreversible taurine losses of 0.1 and 5.428.5 µmol·kg1·day1 have been reported in feces (47) and urine (38), respectively. The quantitative contribution of hepatic taurine conjugation to taurine utilization can be extrapolated from published data (15, 19, 41). Glycine is another amino acid moiety used for bile acid conjugation (18). Because 1) overall glycine Ra approximates 150 µmol·kg1·h1 (7) and 2) 2.3% of glycine flux is turning over for bile acid, then 3.4 µmol·kg1·h1 glycine are used for bile conjugation in an adult human (1719). Assuming a ratio of glycine to taurine of
3.5 in bile acids (17, 19, 21), bile acid conjugation may therefore only account for
1 µmol·kg1·h1, or <2% the taurine Rd measured in the current study. The fate of the bulk of taurine therefore remains unresolved.
One might argue that taurine is a "metabolic dead end," implying that, once it enters cells, it remains there, and the large intracellular taurine pool may be a vast "taurine sink." Taurine residence time in muscle was estimated at 427 h in healthy men, nine times that of glutamine (29). The low fractional turnover of taurine might account for the slow isotopic equilibration, as it took 4 h of a constant infusion of [13C2]taurine to reach isotopic equilibrium. A delay in tracer equilibration across cell membranes invariably occurs when a substrate has a large intracellular pool, such as was observed with [15N]glutamine in the skeletal muscle glutamine pool (49).
The two-component taurine pools obtained from the current data agree with previous reports (44, 50), and the small component pool size found in four subjects explored by bolus injection (2.78.0 mmol; Table 5) is in the same range as the tracer-miscible pool determined using continuous infusion (38 µmol/kg body wt, i.e., 2.6 mmol). Assuming extracellular fluid to be 0.2 l/kg with a taurine content of 50 µmol/l, we can estimate extracellular taurine pool at
10 µmol/kg. The extracellular taurine pool thus accounts for only 26% of tracer-miscible pool (10/38), and the rest of the taurine tracer-miscible pool must be accounted for by a fraction of intracellular taurine that exchanges with plasma. These pool sizes are far from whole body taurine content, since muscle alone contains >287 mmol (2), and large amounts of taurine are found in retina, interstitial spaces, or bound with intracellular peptides (22, 30). It is therefore safe to conclude that both taurine Ra and tracer-miscible pools represent a tiny fraction of whole body taurine.
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Taken in aggregate, data from the current studies using [13C]taurine, and from earlier studies, shed some light into the peculiar nature of taurine metabolism in humans. Like glutamine, taurine is highly concentrated in intracellular space (2, 7) and compartmentalized between extra- and intracellular milieus. Yet, contrary to glutamine, taurine has a slow turnover and exchanges very little with plasma (7, 22, 26, 44). In addition, whereas the active interorgan exchange of glutamine is known to serve as a shuttle of carbon and nitrogen between skeletal muscle and splanchnic bed (34), the physiological function of interorgan taurine exchange, if any, remains to be found. As a matter of fact, although taurine has major physiological functions in brain, retina, liver, and other tissues, the taurine needed for these functions may arise from de novo synthesis in situ rather from taurine taken up from systemic circulation. The precise significance of interorgan taurine exchange, measured using isotope dilution methods, therefore remains to be determined.
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GRANTS |
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ACKNOWLEDGMENTS |
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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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REFERENCES |
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