Evaluation of [Formula: see text] enrichment values obtained with an oral breath test under conditions of impaired gastric functioning.

Gastric emptying can be assessed by an oral administration of a 13C labeled substrate and its response in the expiratory release of the oxidation product [Formula: see text]. Impaired gut function, reflected, for example, in an intolerance against enteral nutrition may delay or discontinue gastric emptying, potentially leading to multiple peaks in the time profile of expiration. The resulting profile cannot be analyzed by the usual data evaluation that is based on a 'beta exponential' (BEX) function. We developed a new approach that better reflects the underlying physiology. It allows a flexible time profile of gastric release and considers a transient [Formula: see text] retention in different compartments as well as an incomplete recovery of [Formula: see text] in the expiration. Parameters that describe the distribution/retention kinetics cannot be determined based on the same breath data that were used to estimate emptying. To enable the determination of the kinetic parameters, they were constrained to match published data using a Bayesian statistical analysis. The applicability of the new model was compared with BEX for healthy subjects. BEX fails to explain the observed data and, compared to the new approach, overestimates the speed of emptying. Predictive accuracy under impaired gastric motility was explored using synthetic data. Only the new approach can reproduce a multiphase absorption profile. When routine benchtop equipment was used for measurements, then the rate-limiting step for precision in the estimate of emptying is the quality in the a priori estimate for kinetic parameters rather than precision in measurements. Only about 80% of the absorbed [Formula: see text] has to be released by expiration. With these features, the new approach promises to widen the applicability of breath tests for gastric emptying.

Phenylalanine conversion to tyrosine: comparative determination with L-[ring-2H5]phenylalanine and L-[1-13C]phenylalanine as tracers in man.

The in vivo rate of conversion of phenylalanine to tyrosine (PheOH) can be estimated using combinations of stable isotope-labeled phenylalanine and tyrosine. We have compared in four healthy adult men the rates of phenylalanine conversion to tyrosine based on the following pairs of primed, continuous tracer infusions administered simultaneously: (1) L-[ring-2H5]phenylalanine and 2H2-tyrosine with a 2H4-tyrosine prime, and (2) L-[1-13C]phenylalanine and 2H2-tyrosine with a 1-13C-tyrosine prime. Phenylalanine oxidation was determined from measurement of 13CO2 excretion in expired air. Tracers were given for 8 hours, with subjects being in the postabsorptive state during the first 3 hours and in the fed state during the remaining 5 hours. Mean (+/- SD) rates (mumol.kg-1.h-1) of phenylalanine conversion to tyrosine for fasted and fed states, respectively, were 5.1 +/- 2.9 and 6.8 +/- 3.4 with 2H5-phenylalanine and significantly higher (P < .05) at 11.1 +/- 5.6 and 12.7 +/- 7.7 with 13C-phenylalanine as tracer. Phenylalanine oxidation was 9.9 +/- 2.0 and 13.5 +/- 2.6, respectively, for fasted and fed states, and these mean values did not differ (P > .1) from the rate of phenylalanine conversion to tyrosine determined using 13C-phenylalanine. These results indicate the need for caution in interpreting kinetic aspects of phenylalanine metabolism when based on isotopic data from multideuterated phenylalanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Splanchnic and whole body L-[1-13C,15N]leucine kinetics in relation to enteral and parenteral amino acid supply.

The effect of the route of administration of a complete amino acid solution (0.24 g.kg-1.h-1) on leucine (Leu) and alpha-ketoisocaproate (KIC) metabolism in the splanchnic region (Sp) was assessed in nine chronically catherized mongrel dogs receiving, for 6 h, amino acids by jugular vein (PN feeding). Results were compared with those obtained previously [Y. M. Yu, D. A. Wagner, E. E. Tredget, J.A. Waleszewski, J. F. Burke, and V. R. Young. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E36-E51, 1990] in eight dogs similarly studied but given amino acids by constant enteral feeding (EN). We used primed continuous intravenous infusions of L-[1-13C,15N]Leu and measurements of arteriovenous isotope and Leu balance across the gut, liver, and Sp to estimate parameters of whole body and organ Leu metabolism [Leu-N and Leu-C flux, Leu----KIC; KIC----Leu, Leu oxidation and rates of Leu appearance (B) from and disappearance (S) into proteins]. Whole body Leu kinetics were the same for both routes of amino acid administration. With PN, KIC----Leu, Leu----KIC, and total metabolic processing were lower than for EN in Sp, and overall Leu balance (S - B) was higher in Sp for EN. Leu appearance from protein breakdown in gut was higher with PN. The rate of KIC----Leu was higher in liver for EN. These findings reveal that route of amino acid (Leu) administration, under these acute feeding conditions, alters the processing and metabolic fate of Leu in Sp but that whole body parameters of Leu metabolism are stable. The implications of these metabolic findings in relation to the maintenance of intestinal function and integrity are discussed.

Determination of the isotope enrichment of one or a mixture of two stable labelled tracers of the same compound using the complete isotopomer distribution of an ion fragment; theory and application to in vivo human tracer studies.

Calculations of flux rates for stable isotope tracer studies are based upon enrichment values of an infused tracer. We propose the determination of enrichment values by gas chromatography/mass spectrometry, which is based on tracer mole fraction and mass spectrometer signals, normalized over the total signal of an ion fragment isotopomer distribution. The method accounts for overlap of the signals of one or two tracers and the tracee, high tracer mole fraction and incomplete labelling of the (infused) tracer. For the single and multiple tracer case a linear relationship between tracer mole fraction (from zero to one) and all normalized mass spectrometer signals is derived. This linearity over the entire range is demonstrated with a single (1-13C)glucose tracer and for mixtures of (1-13C)- and (3,3-2H2)tyrosine tracers. The linearity allows determination of the tracer mole fraction for two tracers, using multiple linear regression. The corresponding calibration can rely on measurements of the pure tracer and tracee compound, without weighing or check for chemical purity. This is compared with a calibration based on tracer/tracee mixtures. Estimates for the tracer mole fraction are slightly better if based on a calibration, using standard mixtures. In all cases the tracer mole fraction can be determined with high precision (coefficient of variation smaller than 5%) and high accuracy. For tyrosine it is demonstrated that the measurement of seven channels rather than three, for the main isotopomers, does not reduce the precision in the prediction of the tracer mole fraction. Equations are also derived to use the tracer mole fraction to estimate the endogenous production of the tracee under study conditions, assuming a steady state of the host metabolism.

A general model for analysis of the tricarboxylic acid cycle with use of [13C]glutamate isotopomer measurements.

A generalized steady-state model was developed for determining tricarboxylic acid cycle fractional fluxes from 13C nuclear magnetic resonance (NMR) data. The model relates the measured mole fractions of [13C]glutamate isotopomers to the fractional fluxes and predicted mole fractions of isotopomers of oxaloacetate (OAA) and acetyl-CoA. This model includes cycling between OAA and fumarate. Fractional fluxes are determined by fitting the model equations to NMR parameters by use of nonlinear least squares. Although only fractional fluxes can be determined from 13C-NMR data, when they are combined with mass spectroscopic measurements, absolute values can be derived. A specific metabolic system represented by published 13C-NMR data from extracts of hearts perfused with [13C]acetate, [13C]pyruvate (PYR), and [13C]acetate plus [13C]PYR was used to test the model. The intensities of predicted 13C-NMR splitting patterns were compared with observed values, and there was excellent agreement between observed and predicted signal intensities. With this model, important physiological parameters, including the OAA-derived fraction of inflow to PYR, PYR-derived fraction of inflow to acetyl-CoA, citrate-derived fraction of inflow to OAA, and PYR-derived fraction of inflow to OAA, can be determined.

TCA cycle flux estimates from NMR- and GC-MS-determined [13C]glutamate isotopomers in liver.

Infusion of 13C-labeled lactate into rabbits and the subsequent measurement of glutamate isotopomers by 13C nuclear magnetic resonance (NMR) spectroscopy enables one to calculate relative flow rates associated with the tricarboxylic acid (TCA) cycle, albeit with a lower precision than one would obtain using a perfused organ. Two factors contribute to the lower precision in the determination of relative flow rates for the in vivo system: 1) a poorly defined pyruvate input and 2) low levels of 13C-enriched oxaloacetate and acetyl-CoA isotopomers, which give rise to weaker glutamate isotopomer NMR signals. To help overcome these limitations, we introduce a procedure to 1) include experimental data from gas chromatography-mass spectrometry (GC-MS) and 2) account for the uncertainty in the labeling of the input to pyruvate by creating the labeling as a measurement that is subject to measurement error. The effects of the uncertainties in the input labeling, NMR data, and MS data are evaluated via a Monte Carlo method. The change in the precision of the relative fluxes for the cases of high/low NMR and high/low MS precision is given. An uncertainty in the lactate measurements of up to 10% does not add significantly to the imprecision of the relative flow rates.

Phenylalanine kinetics in healthy volunteers and liver cirrhotics: implications for the phenylalanine breath test.

Expired 13CO2 recovery from an oral l-[1-13C]phenylalanine ([13C]Phe) dose has been used to quantify liver function. This parameter, however, does not depend solely on liver function but also on total CO2 production, Phe turnover, and initial tracer distribution. Therefore, we evaluated the impact of these factors on breath test values. Nine ethyl-toxic cirrhotic patients and nine control subjects received intravenously 2 mg/kg of [13C]Phe, and breath and blood samples were collected over 4 h. CO2 production was measured by indirect calorimetry. The exhaled 13CO2 enrichments were analyzed by isotope ratio mass spectrometry and the [13C]Phe and l-[1-13C]tyrosine enrichments by gas chromatography-mass spectrometry. The cumulative 13CO2 recovery was significantly lower in cirrhotic patients (7 vs. 12%; P < 0.01), in part due to lower total CO2 production rates. Phe turnover in cirrhotic patients was significantly lower (33 vs. 44 micro mol. kg(-1). h(-1); P < 0.05). When these extrahepatic factors were considered in the calculation of the Phe oxidation rate, the intergroup differences were even more pronounced (3 vs. 7 micro mol. kg(-1). h(-1)) than those for 13CO2 recovery data. Also, the Phe-to-Tyr conversion rate, another indicator of Phe oxidation, was significantly reduced (0.7 vs. 3.0 micro mol. kg(-1). h(-1)).