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

1997 ◽  
Vol 272 (6) ◽  
pp. C2049-C2062 ◽  
Author(s):  
J. A. Vogt ◽  
D. M. Yarmush ◽  
Y. M. Yu ◽  
C. Zupke ◽  
A. J. Fischman ◽  
...  
Keyword(s):  
Tca Cycle ◽  
Gas Chromatography Mass Spectrometry ◽  
Flow Rates ◽  
13C Nuclear Magnetic Resonance ◽  
Two Factors ◽  
Subsequent Measurement ◽  
System 1 ◽  
Relative Flow

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.

IDENTIFICATION AND DETERMINATION OF NEUTRAL STEROID SULPHATES IN HUMAN FOETAL ADRENAL AND LIVER TISSUE

1970 ◽  
Vol 64 (2) ◽  
pp. 273-286 ◽  
Author(s):  
I. Huhtaniemi ◽  
T. Luukkainen ◽  
R. Vihko
Keyword(s):  
Mass Spectrometry ◽  
Liver Tissue ◽  
Keto Group ◽  
Gas Chromatography Mass Spectrometry ◽  
Gas Liquid Chromatography ◽  
Silyl Ethers ◽  
Adrenal Tissue ◽  
Neutral Steroid

ABSTRACT The endogenous neutral steroids in adrenal and liver tissue obtained from foetuses of 12–17 weeks gestation were investigated. After extractions, the steroids were fractionated into a monosulphate and a disulphate fraction on Sephadex LH-20. The conjugates were cleaved by solvolysis, and purified on silicic acid and their trimethyl silyl ethers were analysed by gas-liquid chromatography and gas chromatography-mass spectrometry. Only neutral steroid monosulphates were found. The following compounds were identified in the monosulphate fraction of adrenal tissue: dehydroepiandrosterone, 16α-hydroxydehydroepiandrosterone, pregnenolone and 17α-hydroxypregnenolone, and of liver tissue; dehydroepiandrosterone, 16α-hydroxydehydroepiandrosterone, androst-5-ene-3β,16α,17β-triol, pregnenolone, 17α-hydroxypregnenolone and 16α-hydroxypregnenolone. The identifications and the results of the quantitative determinations of endogenous neutral steroid sulphates suggest that pregnenolone is synthesised in vivo in the foetal adrenal and converted to 17α-hydroxypregnenolone and dehydroepiandrosterone sulphates. The latter compound is further transformed to 16α-hydroxydehydroepiandrosterone sulphate. No 16α-hydroxylation of pregnenolone was observed in adrenal tissue, whereas liver tissue seems to form 16α-hydroxypregnenolone. Reduction of a 17-keto group to 17β-hydroxyl and a 20-keto group to 20α-hydroxyl seems to take place in liver but not in adrenal tissue.

scholarly journals Simultaneous Determination of the Rates of the TCA Cycle, Glucose Utilization, α-Ketoglutarate/Glutamate Exchange, and Glutamine Synthesis in Human Brain by NMR

1995 ◽  
Vol 15 (1) ◽  
pp. 12-25 ◽  
Author(s):  
Graeme F. Mason ◽  
Rolf Gruetter ◽  
Douglas L. Rothman ◽  
Kevin L. Behar ◽  
Robert G. Shulman ◽  
...  
Keyword(s):  
Human Brain ◽  
Glucose Consumption ◽  
Tca Cycle ◽  
High Rate ◽  
Cycle Rate ◽  
The Tca Cycle ◽  
Glutamine Synthesis ◽  
Physiological Values

13C isotopic tracer data previously obtained by 13C nuclear magnetic resonance in the human brain in vivo were analyzed using a mathematical model to determine metabolic rates in a region of the human neocortex. The tricarboxylic acid (TCA) cycle rate was 0.73 ± 0.19 μmol min−1 g−1 (mean ± SD; n = 4). The standard deviation reflects primarily intersubject variation, since individual uncertainties were low. The rate of α-ketoglutarate/glutamate exchange was 57 ± 26 μmol min−1 g−1 ( n = 3), which is much greater than the TCA cycle rate; the high rate indicates that α-ketoglutarate and glutamate are in rapid exchange and can be treated as a single combined kinetic pool. The rate of synthesis of glutamine from glutamate was 0.47 μmol min−1 g−1 ( n = 4), with 95% confidence limits of 0.139 and 3.094 μmol min−1 g−1; individual uncertainties were biased heavily toward high synthesis rates. From the TCA cycle rate the brain oxygen consumption was estimated to be 2.14 ± 0.48 μmol min−1 g−1 (5.07 ± 1.14 ml 100 g−1 min−1; n = 4), and the rate of brain glucose consumption was calculated to be 0.37 ± 0.08 μmol min−1 g−1 ( n = 4). The sensitivity of the model to the assumptions made was evaluated, and the calculated values were found to be unchanged as long as the assumptions remained near reported physiological values.

Growth of Paracoccus denitrificans on [2, 3- 13 C]succinate and [1, 4- 13 C]succinate IIl. Biosynthetic pathways

1988 ◽  
Vol 233 (1270) ◽  
pp. 1-15 ◽  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Gas Chromatography ◽  
Paracoccus Denitrificans ◽  
Tca Cycle ◽  
Gas Chromatography Mass Spectrometry ◽  
Biosynthetic Pathways ◽  
The Tca Cycle ◽  
Phosphoenol Pyruvate

The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2, 3- 13 C]succinate or [1, 4- 13 C]succinate was investigated by using gas chromatography–mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo . The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C 4 , C 5 and C 6 sugars without the use of fructose-1, 6-bisphosphate aldolase (which has not been detected in P. denitrificans ) is proposed. The 13 C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo .

Gas Chromatographic and Mass Spectrometric Determination of Hemoglobin Adducts of 1,3-Dinitrobenzene and 1,3,5-Trinitrobenzene in Shrew Cryptotis Parva

1999 ◽  
Vol 18 (5) ◽  
pp. 317-325 ◽  
Author(s):  
Steven R. Myers ◽  
Maria T. Pinorini-Godly ◽  
Tirumuru V. Reddy ◽  
F. Bernard Daniel ◽  
Gunda Reddy
Keyword(s):  
Acid Hydrolysis ◽  
Surface Waters ◽  
Environmental Contaminants ◽  
Gas Chromatography Mass Spectrometry ◽  
Hemoglobin Adducts ◽  
Organic Solvent Extraction ◽  
Basic Hydrolysis

1,3-Dinitrobenzene (DNB) and 1,3,5-trinitrobenzene (TNB) are used primarily in explosive compositions and munitions and have been detected as environmental contaminants of surface waters as well as ground waters near production waste disposal sites. Hemoglobin (Hb) adducts have recently been proposed as biological markers of exposure assessment for various environmental compounds, including nitroaromatics. In the present study, we have investigated the formation of DNB and TNB hemoglobin adducts in vivo and in vitro in the blood of shrew (Cryptotis parva). DNB and TNB hemoglobin adducts were detected by gas chromatography/mass spectrometry (GC/MS) after either basic (0.1 N NaOH) or acid (2 N HCl) hydrolysis followed by organic solvent extraction and derivatization of the corresponding amines. The levels of DNB-Hb adducts detected after basic hydrolysis (238.7 & pm; 50.2 pg/mg Hb) are higher than the corresponding levels detected after acid hydrolysis (52.5 & pm; 16.2 pg/mg Hb). For the TNB-Hb the levels after acid hydrolysis (132.2 & pm; 37.8 pg/mg Hb) are higher than the levels detected after basic hydrolysis (44.7 & pm; 15.3 pg-mg Hb). These results demonstrate the effectiveness of the hemoglobin adduct model for monitoring exposure to nitroaromatics.

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