Mole Percent Excess Research Articles

Determination of very low stable isotope enrichments of [2H5]-phenylalanine in chicken liver using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Stable isotope labeled amino acids are frequently used to examine nutritive effects on protein synthesis. This technique is characterized by tracing the incorporation of the label into newly synthesized proteins. In the present investigation, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the determination of very low enrichment of protein-bound l-[2H5]-phenylalanine ([2H5]-phe) in chicken liver. The LC–MS/MS measurements were carried out in positive atmospheric pressure chemical ionization (APCI) mode. Two mass transitions each for [2H5]-phe (171.1/125.1 and 171.1/106.1) and l-phenylalanine (phe) (166.1/91.1 and 166.1/93.1) were chosen for quantification and qualification. Due to the high excesses of phe, less sensitive transitions were chosen in the case of phe. The separation was carried out on a phenyl-hexyl column using 0.1% formic acid as eluent A and methanol as eluent B. The method was calibrated with calibration standard solutions in the range of 0.01–0.5 mole percent excess (MPE). Linear regression analysis led to coefficients of determination (r2) greater than 0.9995. The method was applied on liver samples from experiments investigating nutritive effects on tissue protein synthesis in broiler chickens. These samples were analyzed with a gas chromatography–mass spectrometry (GC–MS) method and reanalyzed with the developed LC–MS/MS method one year later. Compared to GC–MS, the main advantages of the LC–MS/MS method are its higher selectivity as well as the elimination of the need to convert and derivatize the samples prior to measuring.

Acute resistance exercise augments integrative myofibrillar protein synthesis

The purpose of this study was to determine whether an acute bout of high-intensity resistance exercise (RE) would augment integrative mixed muscle and myofibrillar protein fractional synthesis rates (FSRs) when total energy and macronutrient intake was controlled. Twelve healthy young men were studied over 24 hours and performed an acute bout of exhaustive (5 sets until volitional failure of their 85% 1-repetition maximum) unilateral leg press and knee extension exercise, such that one leg was exercised (EX) and the other served as a control (CON). 2H 2O (70%) was provided to measure mixed muscle and myofibrillar FSR, and muscle biopsies (vastus lateralis) were collected from the EX and CON legs 16 hours following the RE session. 2H-labeling of body water over the course of the experiment was 0.32 ± 0.01 mole percent excess. Interestingly, integrative mixed muscle FSR (percent per hour) was similar between the CON (0.76% ± 0.08%) and EX (0.69% ± 0.06%) legs. In contrast, upon determination of myofibrillar FSR, there was an RE effect (EX, 0.94% ± 0.16% vs CON, 0.75% ± 0.08%; P < .05). High-intensity RE without prior training impacts integrative myofibrillar 24-hour FSR, perhaps without altering total responses.

The production of intrinsically labeled milk and meat protein is feasible and provides functional tools for human nutrition research

Administration of labeled, free amino acids does not allow direct assessment of in vivo dietary protein digestion and absorption kinetics. Consequently, dietary protein sources with labeled amino acids incorporated within their protein matrix are required. The aim of the present study was to produce intrinsically l-[1-13C]phenylalanine-labeled milk and meat protein that would permit in vivo assessment of postprandial protein digestion and absorption kinetics in humans. One lactating dairy cow was continuously infused with 420 μmol of l-[1-13C]phenylalanine/min for 96h, with plasma and milk being collected before, during, and after isotope infusion. Twenty-four hours after infusion, the cow was slaughtered to produce intrinsically labeled meat. Levels of l-[1-13C]phenylalanine enrichment as high as 40 mole percent excess (MPE) in milk and 1.5 MPE in meat protein were achieved. In a subsequent human proof-of-principle experiment, 2 healthy young males (25±1 yr; 66.2±5.2kg) each ingested 135g of l-[1-13C]phenylalanine intrinsically labeled minced beef, after which plasma samples were collected at regular time intervals. Plasma l-[1-13C]phenylalanine enrichments increased during the first 90min following beef ingestion, reaching peak plasma enrichment levels of 0.61±0.04 MPE. Whole-body net protein balance, assessed by continuous infusion of l-[ring-2H5]phenylalanine and l-[ring-2H2]tyrosine, was higher in the postprandial period compared with basal values (6.4±0.1 vs. −4.5±0.1μmol/kg per h). In conclusion, the production of intrinsically l-[1-13C]phenylalanine-labeled milk and meat protein is feasible and provides functional tools to investigate in vivo protein digestion and absorption kinetics in humans.

Isotopomics: A Top-Down Systems Biology Approach for Understanding Dynamic Metabolism in Rats Using [1,2-13C2] Acetate

Isotope labeled tracers are commonly used to quantify the turnover rates of various metabolic intermediates and yield information regarding physiological regulation. Studies often only consider either one nutritional state (fasted or fed) and/or one question (e.g., measure of lipid or protein turnover). In this article, we consider a novel application combining the global approach of metabonomics with widespread stable isotope labeling as a way of being able to map metabolism in open mammalian systems, an approach we call "isotopomics". A total of 45 15-week-old male Zucker rats were administrated different amounts (from 0.5 to 8 mmol/kg) of sodium [1,2-(13)C(2)] acetate. Plasma samples taken at 1, 4, and 24 h were analyzed with (13)C nuclear magnetic resonance (NMR) and gas chromatography/mass spectrometry (GC/MS) to measure (13)C isotopic enrichment of 39 plasma metabolites across a wide range of compound classes (amino acids, short-chain fatty acids, lactate, glucose, and free fatty acids). Isotopic enrichment from 0.1-7.1 mole percent excess (MPE) for the highest dose could be reliably measured in 16 metabolites, and the kinetics of their (13)C isotopic enrichment are reported. Clustering metabolites based on (13)C kinetic curves enabled highlighting of time dependent patterns of (13)C distribution through the key metabolic pathways. These kinetic and quantitative data were reported into a biochemical map. This type of isotopomic approach for mapping dynamic metabolism in an open system has great potential for advancing our mechanistic knowledge of how different interventions and diseases can impact the metabolic response of animals and humans.

The production of intrinsically labeled milk protein provides a functional tool for human nutrition research

Oral or intravenous administration of labeled, free amino acids does not allow the direct assessment of protein digestion and absorption kinetics following dietary protein intake. Consequently, dietary protein sources with labeled amino acids incorporated within the protein are required. The aim of this study was to produce milk proteins intrinsically labeled with l-[1-13C]phenylalanine that would allow the assessment of protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to dietary protein intake in vivo in humans. Two Holstein cows (body weight of 726±38kg) were continuously infused with l-[1-13C]phenylalanine at 402 μmol/min for 44 to 48h, during and after which plasma samples and milk were collected. After milk protein separation, casein was used in a subsequent human proof-of-principle experiment. Two healthy males (aged 61±1 yr; body mass index of 22.4±0.1 kg/m2) ingested 35g of casein highly enriched with [1-13C] phenylalanine. Plasma samples were collected at regular intervals, and skeletal muscle biopsies were collected before and 6h after casein ingestion. In the initial experiment, a total of 5.83kg of l-[1-13C]phenylalanine-enriched milk protein (casein enrichment was 29.4 molar percent excess) was collected during stable isotope infusion in the cows. In the proof-of-principle study, ingestion of 35g of intrinsically labeled casein resulted in peak plasma l-[1-13C]phenylalanine enrichments within 90min after protein ingestion (9.75±1.47 molar percent excess). Skeletal muscle protein synthesis rates calculated over the entire 6-h period averaged 0.058±0.012%/h. The production of intrinsically labeled milk protein is feasible and provides dietary protein that can be used to investigate protein digestion and absorption and the subsequent muscle protein synthetic response in vivo in humans.

Simultaneous measurement of 13C‐ and 15N‐isotopic enrichments of threonine by mass spectrometry

Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic enrichments of differently labelled chemical compounds (e.g. (13)C and (15)N in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic enrichments of doubly labelled threonine ([U(13)C] and (15)N) with isotopic enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. To calculate both isotopic enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. Moreover, the accuracy of GC/MS (13)C-isotopic enrichment measured was validated by analyzing the same plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Statistical analysis showed that both techniques gave the same results (ANOVA, P >0.05). This new GC/MS method offers the possibility to measure (13)C- and (15)N-isotopic enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS.

Piezoelectric and ferroelectric properties of Bi-compensated (Bi1/2Na1/2)TiO3–(Bi1/2K1/2)TiO3 lead-free piezoelectric ceramics

( Bi 1 / 2 Na 1 / 2 ) TiO 3 – ( Bi 1 / 2 K 1 / 2 ) TiO 3 (BNT-BKT) ceramics as a promising candidate for lead-free piezoelectric ceramics were studied with a special emphasis on the compositional dependence of piezoelectric and ferroelectric properties. One mole percent excess Bi was added to compensate for the volatilization of Bi3+ ions during sintering, which was found to be effective in improving the piezoelectric properties of the resultant BNT-BKT ceramics. The piezoelectric, ferroelectric, and dielectric properties of the Bi-compensated BNT-BKT ceramics were investigated and discussed in relation to the morphotropic phase boundary that is close to the composition range of 20–24 mol % BKT. The maximum piezoelectric constant d33 (207 pC/N) and electromechanical coupling factor kp (35%) were obtained at the compositions of 23% and 20%, respectively.

Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C–valine isotopic ratios in complex biological samples

On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio.

Effects of the Fusarium toxin deoxynivalenol from naturally contaminated wheat given subchronically or as one single dose on the in vivo protein synthesis of peripheral blood lymphocytes and plasma proteins in the pig

Trichothecenes, such as deoxynivalenol (DON), are known to inhibit the protein synthesis in vitro by binding at the 60S subunit of eukaryotic ribosomes. Therefore, cells and tissues with high protein turnover, such as lymphocytes and the liver (albumin and fibrinogen synthesis), were suggested to react most sensitively to DON. However, to the author’s knowledge this observation was not proven in vivo in pigs, which were regarded as the farm animals most susceptible to DON. A total of 31 castrated male, crossbred German Landrace × Pietrain pigs weighing approx. 40 kg were fed a DON contaminated diet (5.7 mg/kg) either acutely (one single dose) or subchronically (⩾4 weeks) or a control diet (0.1 mg/kg). In addition, one group received an intravenous injection of 53 μg DON/kg LW. One hour after feeding, a “flooding dose” of the stable isotope l-[ 2H 5]-phenylalanine (125 mg/kg LW) was given and frequent blood samples (permanent catheter) were collected over a 60 min period. The molar percent excess (MPE) of plasma free and protein-bound phenylalanine were measured by GC/MS. No differences could be observed in the plasma concentrations of total protein, albumin, fibrinogen and serum enzymes between the groups. On the other hand, fractional synthesis rates (FSR, %/d) of albumin were significantly decreased by 43%, 45% and 26% and FSR of lymphocytes declined by 27%, 19% and 24%, whereas fibrinogen was not significantly affected after subchronic or one single oral and intravenous DON exposure, respectively. Additionally, the absolute synthesis rate (ASR, g/d) of albumin and the proportion of albumin to total body protein synthesis were reduced in the same manner, whereas the albumin secretion time ranged between 6.8 and 34.4 min and was not affected by treatment. In conclusion, the flooding dose technique appeared to be suitable for distinguishing DON-related effects on the protein synthesis, while determination of plasma protein concentrations seemed not to be an appropriate parameter.

2H/H] Isotope ratio analyses of [2H5]cholesterol using high-temperature conversion elemental analyser isotope-ratio mass spectrometry: determination of cholesterol absorption in normocholesterolemic volunteers.

This paper validates the use of high-temperature conversion elemental analyser isotope-ratio mass spectrometry (TC-EA/IRMS) for measuring the [(2)H/H] enrichment of plasma [(2)H(5)]cholesterol. From a molecular point of view, the free cholesterol is initially separated from plasma by thin-layer chromatography (TLC) and then injected onto the TC-EA reactor which converts cholesterol molecules into CO and H(2) gases. The slope of the curve of the experimental mole percent excess (MPE((exp.))) versus MPE((theor.)) was very close to 1, demonstrating that no significant isotopic fractionation was observed during all processing of the samples (i.e., isolation of plasma free cholesterol by TLC and pyrolysis in the TC-EA reactor). Excellent linearity (r(2) = 0.9994, n = 4) of delta ( per thousand ) of [(2)H/H] isotopic measurements versus mole percent (MP) was assessed over the range 0 to 0.1 MP. The precision of the [(2)H/H] measurement, evaluated with two calibration points processed with TLC, was delta(2)H(V-SMOW) = -192.5 +/- 3.4 per thousand and delta(2)H(V-SMOW) = -136.9 +/- 2.9 per thousand. The standard deviations of the within-assay and between-assay repeatabilities of the analytical process, evaluated using the quality control (QC) of plasma samples, were 4.6 and 6.1 per thousand, respectively. Plant sterols are known to reduce cholesterol absorption and therefore were used as a positive control in a clinical study performed with normocholesterolemic volunteers. This present method produces biological results consistent with those already reported in the literature.

Improved Method for Gas Chromatographic–Mass Spectrometric Analysis of 1-13C-labeled Long-Chain Fatty Acids in Plasma Samples

Gas chromatographic-mass spectrometric (GC/MS) tracking of stable-isotope-labeled substrates is useful in metabolic studies. However, GC/MS analysis of long-chain fatty acid methyl esters yields results that mostly depend on their concentration in the system. We describe a protocol aimed to obviate this and other drawbacks in plasma [1-(13)C]palmitic and [1-(13)C]oleic acid measurements. Lipoproteins were separated by sequential ultracentrifugation. Free or esterified heptadecanoic acid was used as internal standard. Fatty acids were derivatized to trimethylsilyl (TMS) esters. GC separation was in isothermal mode at 210 degrees C for 27 min. For both TMS-palmitate and TMS-oleate, M and [M + 1] signals were simultaneously acquired with a dual acquisition program in single-ion monitoring mode. Calibration mixtures containing increasing amounts of labeled fatty acids were prepared gravimetrically to construct calibration curves for isotopic enrichment. Likewise, five calibration curves (for increasing concentrations) were constructed for each fatty acid; this allowed selection of the most appropriate curve for the concentration in a plasma sample. Oleic acid-TMS ester was clearly separated from that of its stereoisomer, elaidic acid. Within a 10-fold concentration range, the isotopic ratio was independent on the amount of the analyte in the sample, with a maximum uncertainty of 0.34% in terms of molar percent excess. In addition, the within- and between-day imprecision (CV) of the method was <1%. Results obtained with this method are independent of concentration and sufficiently precise for tracking 1-(13)C-labeled palmitic and oleic acids in biological samples

Role of glucose and glutamine synthesis in the differential recovery of 13CO 2 from infused [2- 13C] versus [1- 13C] acetate

Carbon exchange in the Krebs cycle may result in underestimation of substrate oxidation measured with 13C-labeled substrates, since carbon labeled in position 2 of acetyl-coenzyme A (CoA) could be incorporated into glucose (via gluconeogenesis) and glutamine. Five healthy volunteers were therefore infused with [1- 13C] and [2- 13C] acetate at a rate of 0.5 μmol · kg −1 · min −1 for 165 minutes on two different occasions in randomized order. Whole body acetate turnover did not differ between the two tracers: 7.9 ± 0.3 and 7.5 ± 0.6 μmol · kg −1 · min −1 (nonsignificant [NS]) for [1- 13C] and [2- 13C] acetate, respectively. Isotopic 13C enrichment was higher in expired CO 2 (0.177 ± 0.021 v 0.089 ± 0.009 atom percent excess [APE], P < .01) and lower in glucose (0.074 ± 0.017 v 0.291 ± 0.061 mole percent excess [MPE], P < .01) for [1- 13C] acetate compared with [2- 13C] acetate, respectively, at the end of the infusions. Glutamine isotopic enrichment was slightly but not significantly higher when infusing [1- 13C] acetate versus [2- 13C] acetate (0.348 ± 0.038 v 0.495 ± 0.069 MPE, NS, respectively). At the end of the experiment, the recovery of 13CO 2 from [1- 13C] acetate was 44.8% ± 2.7%, and from [2- 13C] acetate, 22.6% ± 1.3%. A significant correlation was observed between the differences in 13C enrichment of CO 2 for the two tracers and glucose (ΔCO 2 = 0.424 · Δglucose + 0.001, R 2 = .9856, P = .0007) or glutamine (ΔCO 2 = 0.621 · Δglutamine + 0.004, R 2 = .9573, P = .0038) during the infusion. These results suggest that (1) although gluconeogenesis appears to be more responsible than glutamine for the differential recovery of [2- 13C] versus [1- 13C] acetate, other secondary pathways are probably also implicated; and (2) different recovery correction factors should be applied when measuring substrate oxidation with a stable isotope tracer depending on the expected position of 13C in acetyl-CoA.

Whole-Body, Peripheral and Intestinal Endogenous Acetate Turnover in Dogs Using Stable Isotopes ,

Acetate metabolism supplies about 10% of energy requirements in food-deprived nonruminant animals. This study used a stable isotope dilution method to investigate the fate of acetate in 24-h food-deprived dogs free of colonic fermentation. Three dogs received intravenous bolus injections of 40 or 70 μmol/kg of [1-13C] acetate, and carotid blood was then sampled during a 15-min period to estimate the acetate distribution volume. Ten dogs received intravenous [1-13C] acetate infusions of 1.05 ± 0.02 or 2.10 ± 0.10 μmol/(kg⋅min) for 120 or 200 min after a prime of 200 or 70 μmol/kg, respectively. Cephalic venous and carotid arterial blood were sampled for all dogs, and portal blood for five. Acetate distribution volume was 0.27 ± 0.16 L/kg (mean ± SEM). The concentrations of acetate in arterial (144 ± 17 μmol/L), venous (155 ± 20 μmol/L) and portal plasma (131 ± 16 μmol/L) were not significantly different during infusion, whereas isotopic enrichments [mole percent excess (MPE): labeled acetate/all acetate molecules] in portal (1.2 ± 0.2 MPE) and venous plasma (1.7 ± 0.3 and 2.6 ± 0.7 MPE) were lower than in arterial plasma for both infusion rates (4.9 ± 0.6 and 7.6 ± 0.8 MPE, respectively, P< 0.005). Whole-body acetate turnover was 24.4 ± 2.4 μmol/(kg⋅min). Fractional acetate extractions for forelimb and intestine were 62 ± 7 and 72 ± 6%, respectively, and the production for each organ was 0.3 and 1.1 μmol/(kg⋅min) respectively, similar to that of utilization (P> 0.05). It is concluded that the forelimb and intestine produce and utilize acetate as an energy source in 24-h food-deprived dogs free of colonic fermentation.

Effects of hyperinsulinemia and hyperglycemia on lactate release and local blood flow in subcutaneous adipose tissue of healthy humans.

To determine the effects of hyperglycemia and hyperinsulinemia on lactate production by adipocytes, healthy volunteers were studied during three experimental protocols. In protocol 1, the changes in interstitial lactate concentrations were measured by microdialysis (sc tissue) after oral glucose administration. The plasma lactate concentration increased by 39.4 +/- 6.0%, and the dialysate lactate concentration increased by 117.9 +/- 16.3%. In protocol 2, a 2.5-h hyperinsulinemic euglycemic clamp and somatostatin infusion were performed. The plasma and dialysate lactate concentrations increased by 27.1 +/- 5.5% and 146.8 +/- 44.5%, respectively. In addition, [U-13C]glucose was infused through the probe, and dialysate lactate was enriched in 13C at 2.5 +/- 0.3 molar percent excess basally and at 3.4 +/- 0.3 molar percent excess during the clamp (P < 0.05 vs. basal). [13C]Urea was also infused through the probe, and the outflow to inflow ratio of [13C]urea was used as an index of local blood flow. It decreased by 10.2 +/- 3.6% (P < 0.001) at the end of the hyperinsulinemic euglycemic clamp, indicating an increase in blood flow. In protocol 3, a hyperglycemic clamp (10.0 mmol/L) at the basal insulin concentration was performed. It increased the dialysate lactate concentration by 43.5 +/- 15.9% and did not alter the plasma lactate concentration or local blood flow. It is concluded that hyperinsulinemia and, to a lesser extent, hyperglycemia stimulate glucose conversion into lactate in adipocytes. Hyperinsulinemia, but not hyperglycemia, also increases adipose tissue blood flow.

Use of gas chromatography/isotope ratio‐mass spectrometry to study triglyceride metabolism in humans

The study of triglyceride (TG) metabolism using stable isotope tracers would be facilitated by being able to detect low 13C enrichment. To meet this goal, we developed a gas chromatography/isotope ratio-mass spectrometry technique to measure the enrichment of palmitate in nonesterified fatty acids (NEFA) and TG as its methyl derivative. This method allows accurate and reproducible measurements of enrichment as low as 0.009 mole percent excess (MPE), in a range between 0-0.65 MPE. The usefulness of this method is shown by two studies of lipid metabolism in human beings. First, we studied the metabolic fate of an oral TG load labeled with [1,1,1-13C3]tripalmitin. Labeled palmitate appeared concurrently in plasma NEFA and TG, and four hours after the load, the labeling was higher in NEFA than in TG (MPE NEFA: 1.53 +/- 0.31 vs. MPE TG: 0.78 +/- 0.06, P < 0.05). In a second study, the hepatic reesterification of NEFA was estimated by measuring the appearance of infused [1-13C]palmitate in circulating TG. The estimated contribution of plasma NEFA to circulating TG increased to a maximum of 22%. Thus, gas chromatography/isotope ratio-mass spectrometry appears to be a useful tool for future studies of lipid metabolism in humans.

A new stable isotope tracer technique to assess human neonatal amino acid synthesis

The amino acid (AA) synthetic ability and requirements of human infants are undefined. A stable isotope tracer technique was employed in neonates to assess conversion of uniformly labeled 13C glucose into biochemically nonessential AA (NEAA). Ten neonates (5 males, 5 females) were studied at a mean age of 7 ± 2.0 (SEM) days. The mean gestational age was 35.5 ± 1.1 weeks, and the mean weight at time of study was 2,191 ± 181 g. Six infants were fed enterally, and four received only intravenous 10% dextrose (D10W). Blood samples were obtained before, and 30, 60, and 120 minutes after an orogastric bolus of D-[U- 13C]glucose (100 mg/kg). The conversion of glucose carbon into seven NEAA was assessed by measuring their isotopic enrichments in plasma, using gas chromatography/mass spectrometry ( GC MS ), and was expressed as mole percent excess (MPE), with detectable MPE defined as ≥0.2. The isotopic enrichment of plasma glucose also was measured using GC MS . Free plasma AA concentrations were assayed using an automated AA analyzer and expressed in micromoles per liter. The mean glucose enrichment was 9.33 ± 1.8 MPE (range, 5.82 to 13.48). Detectable 13C-labeling of the NEAA was observed as follows: Glu in 100% of infants; Gly, 100%; Ala, 90%; Ser, 80%; Asp, 70%; Cys, 60%; and Pro, 60%. Detectable Pro enrichment was observed in none of three premature infants on D10W. Free plasma Cys concentration was markedly lower than normal (19.8 v 86 μmol/L). The number of isotopically enriched NEAA in the plasma of premature infants was 5.1 ± 0.5, compared with 6.7 ± 0.3 in that of full-term infants ( t = 2.69, p < .05). Test NEAA synthesis was correlated with the following clinical variables: study weight ( r = .68), gestational age ( r = .30), study age ( r = .24), enteral feeding ( r = .24), and gender ( r = 0). By stepwise multiple linear regression analysis, only study weight independently predicted NEAA enrichment ( F = 5.72, P < .05). These results show that a new stable isotope tracer technique can be employed safely and effectively for the study of human neonatal amino acid metabolism. Furthermore, the data suggest that low-weight neonates are unable to synthesize Cys and Pro, AA considered to be biochemically nonessential in adults. Optimal nutritional strategies for neonates may need to address these apparent deficiencies.

Decreased cysteine and proline synthesis in parenterally fed, premature infants

Little is known about the amino acid (AA) biosynthetic capacity and requirements of premature infants. This study assessed the synthesis of seven biochemically nonessential AA from a universal precursor, glucose, in stable, parenterally fed, premature neonates. Seven infants (six boys, one girl) were studied at a mean age of 6.3 ± 0.6 (SEM) days; mean gestational age was 29.7 ± 1.3 (SEM) weeks, and mean birth weight was 1,222.8 ± 176.5 (SEM) grams. All infants were parenterally fed a mixture of 7.5% to 12.5% dextrose and 2.2% Trophamine, with or without lipid. Mean caloric intake was 93 ± 8.4 (SEM) kcal/kg/d, and total AA intake was standardized at 2.86 g/kg/d AA, plus supplemental cysteine (30 mg/g AA/d). Each infant received a 4-hour continuous, unprimed intravenous infusion of a stable isotope tracer of D-[U 13C] glucose (200 mg/kg). Blood samples were obtained before and at the end of the infusion. Conversion of the glucose tracer into seven biochemically nonessential AA (cysteine [Cys], proline [Pro], aspartate [Asp], serine [Ser], glutamate [Glu], alanine [Ala], and glycine [Gly]) was assessed by measuring their isotopic enrichment in plasma, using gas chromatography/mass spectrometry ( GC MS ), and expressed as mole percent excess (MPE) (mean ± SEM). The isotopic enrichment of plasma glucose was also measured using GC MS . Free plasma AA concentrations (mean ± SD) were measured using an automated amino acid analyzer. Mean MPE for M + 1, M + 2 and M + 3 Cys, and for M + 1 and M + 3 Pro were not significantly different from 0; M + 2 Pro barely achieved statistical significance ( P = .048). In contrast, there was significant ( P < .05) enrichment of all isotopomers of all the other test AA except M + 2 Ala ( P = .06). The mean free plasma Cys concentration (29.2 ± 13.4 μmol/L) was five-fold lower than the published value for term infants (153 ± 25.5 μmol/L), despite supplementation. Detectable enrichment of all glucose isotopomers confirmed precursor incorporation into the glycolytic/gluconeogenic pathways; the M + 6 isotopomer (precursor) enrichment was 6.87 ± 0.53 MPE. These results provide in vivo evidence to suggest that Cys and Pro are essential amino acids in parenterally fed, premature neonates.

Determination of taurine metabolism by measurement of 15N-enriched taurine in cat urine by gas chromatography—mass spectrometry

To understand the biological function of taurine, a study of taurine kinetics in the cat was undertaken. This paper describes a method developed for the accurate determination of 15N-taurine enrichment in cat urine by gas chromatography—mass spectrometry. 15N-Taurine was given to six animals as an oral bolus dose of 20 mg/kg body weight, and the urine was pooled on a daily basis. The hydrolysed or non-hydrolysed urine samples (for total and free taurine, respectively) were directly derivatized without further purification. The N-pentafluorobenzoyl di- n-butyl amide derivative obtained was analysed, and the fragment [M — (di- n-butyl amide)] +, carrier of the labelled nitrogen atom, was selectively recorded at m/z 302 ( 14N-taurine) and m/z 303 ( 15N-taurine). Calibration curves prepared in hydrolysed and non-hydrolysed urine samples spiked with 15N-taurine gave similar slopes to the calibration curve prepared in water. The average coefficient of variation observed for the mole percent excess in the non-hydrolysed samples was 1.22% ( n = 92) and for the hydrolysed urine 1.00% ( n = 98). There was no significant difference between free and total taurine enrichment. The half-life of taurine in cat body was found to be 29.3 ± 2.9 h and 35.0 ± 1.4 h for free and total taurine, respectively (non-significant). The taurine body pool, calculated by extrapolation of the curve to zero time, had a value of 137 ± 22 ng/kg and 157 ± 11 mg/kg for free and total taurine, respectively.

An evaluation of fetal glucogenesis in intrauterine growth-retarded pregnancies

The presence of fetal glucogenesis was evaluated in nine patients with pregnancies complicated by intrauterine growth retardation (IUGR) at the time of fetal blood sampling (FBS) between 29 and 35 weeks of pregnancy. Eight were singleton pregnancies and one was a twin pregnancy in which blood samples were obtained from both twins. A maternal primed-constant infusion of d(U- 13C]glucose was performed, was the presence of fetal glucogenesis was assessed by a comparison of steady-state maternal and fetal glucose enrichments. No significant difference was present between maternal and fetal molar percent excess ([MPE] P = .97), and the mean fetal to maternal ( FM M ) MPE ratio (0.99 ± 0.01) was not significantly different from 1 ( P = .76). F M MPE ratio was independent of the time of FBS and of umbilical venous glucose and lactate concentrations. Thus fetal glucogenesis is not demonstrable in a group of fairly severe growth-retarded fetuses after an overnight fast with this relatively noninvasive approach.

Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans

Measurement of hepatic fatty acid (FA) and cholesterol synthesis has been limited by lack of access to the precursor pool, cytosolic acetyl-CoA. We present a method for inferring the enrichment of the true hepatic lipogenic precursor pool in humans using the frequency distribution of mass isotopomers within enriched circulating polymers of acetyl-CoA [very low-density lipoprotein (VLDL)-palmitate, VLDL-stearate]. Human subjects were infused intravenously (n = 16) with [1-13C]- or [2-13C]acetate. Oral sulfamethoxazole (SMX) was administered concurrently, and the acetylated conjugate (SMX acetate) was used to estimate independently the hepatic cytosolic acetyl-CoA enrichment. Isotopomer frequencies in VLDL-FA were determined by gas chromatography-mass spectrometry, whereas high-performance liquid chromatography-mass spectrometry was used to measure enrichments in SMX acetate. Based on the excess M2/excess M1 ratio in VLDL-FA, calculated acetyl-CoA enrichments were 5.59 +/- 0.33 molar percent excess (MPE), whereas SMX acetate enrichments were 5.38 +/- 0.31 MPE (the 2 methods were not significantly different). Mass isotopomer-calculated and SMX acetate-measured estimates of acetyl-CoA enrichments correlated very closely in individual subjects (r2 = 0.93; P less than 0.0001). De novo hepatic lipogenesis can be measured using isotopomer-calculated precursor enrichments compared with measured incorporation in specific isotopomers of VLDL-FA. In summary, excess isotopomer frequencies in secreted lipids provide a non-invasive technique for estimating hepatic cytosolic acetyl-CoA enrichments in humans in vivo and correlate closely with enrichments observed using the xenobiotic probe technique. Isotopomeric distributions represent a new strategy for accurate measurement of macromolecule synthesis that may be applicable to other classes of molecules besides lipids.