Analysis of amino acids as their tert.-butyldimethylsilyl derivatives by gas—liquid chromatography and mass spectrometry

Mawhinney, Thomas P.; Robinett, R.S. Robin; Atalay, Asmare; Madson, Michael A.

doi:10.1016/s0021-9673(01)90333-4

Cited by 237 publications

(150 citation statements)

References 21 publications

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“…These leucine isotopomers have isotopic distributions of ions of increasing mass from their most abundant mass, the base mass M. The lesser abundant higher mass ions exist due to the natural occurrence of the less abundant stable isotopes, e.g. 13 C, 15 All mixtures are the sum of their components. Figure 2 shows how a mass spectrum of a mixture of unlabeled, singly, and doubly labeled isotopomers is the sum of the individual isotopic distributions for each isotopomer.…”

Section: Definitions Of Terms and Key Equations Of The Methodsmentioning

confidence: 99%

“…The 13 C natural abundance that is lost by the incorporation of 13 C labels into the molecule will be the difference between eq 12 and 13. We can subtract this difference between eq 12 and 13 from the observed (M+2)/M to get the corrected (M+2)/M: (14) Equation 14 can be simplified to give the useful form of the (M+2)/M 13 C correction: (15) The 13 C correction for the (M+2)/M isotope can be carried out in our matrix equation by adjusting the a 0 (2) values for each isotopomer containing 13 C labels: (16) The above 13 C correction is carried out on the measured isotopic distribution of the unlabeled isotopomer's M+1 and M+2 abundances using eq 11 and 16, respectively. The unlabeled isotopomer's isotopic distribution with the 13 C correction is used in the A matrix in eq 9:…”

Section: Definitions Of Terms and Key Equations Of The Methodsmentioning

confidence: 99%

“…We then determined the isotopic content of the same [1-13 C]leucine and [1,2-13 C 2 ]leucine materials using the t-butyldimethylsilyl (TBDMS) derivatives, as previously described, 15 leucine were mixed together, and the TBDMS derivatives were made. Unlabeled TBDMSleucine was also prepared separately.…”

Section: Overview Of Experimentsmentioning

confidence: 99%

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Determination of Complex Isotopomer Patterns in Isotopically Labeled Compounds by Mass Spectrometry

Jennings

Matthews

2005

Anal. Chem.

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A classic problem in analytical chemistry has been determination of individual components in a mixture without availability of the pure individual components. Measurement of the distribution of isotopomers in a labeled compound or mixture of labeled compounds is an example of this problem that is commonly encountered when stable isotopically labeled metabolites are used to determine in vivo kinetics and metabolism. We present a method that uses the measured mass spectral data of the unlabeled material to represent any and all combinations of isotopomer variations of that material and to determine abundances of these isotopomers. Although examples of the method are presented for gas chromatography-mass spectrometry, the method is applicable to any type of mass spectrometry data. The method also accounts for errors induced by mass spectrometer ionization and resolution effects. To demonstrate this method, we determined the isotopomer distributions of samples of 13 C-labeled leucine and glucose for both highly enriched isotopomers and labeled isotopomers present in low abundance against a natural isotopic abundance background. The method accurately and precisely determined isotopomer identity and abundance in the labeled materials without adding noise or error that was not inherent in the original mass spectral data. In examples shown here isotopomer uncertainties were calculated with relative standard errors of <1% from good quality mass spectral data.

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Section: Definitions Of Terms and Key Equations Of The Methodsmentioning

confidence: 99%

Section: Definitions Of Terms and Key Equations Of The Methodsmentioning

confidence: 99%

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Determination of Complex Isotopomer Patterns in Isotopically Labeled Compounds by Mass Spectrometry

Jennings

Matthews

2005

Anal. Chem.

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“…The percentage distribution of 15 N-labeling relative to the total amount of metabolite was determined using GC-MS. Amino acids were extracted into an organic phase of ethanol and benzene, dried under nitrogen, and reconstituted in N,N-dimethylformamide before they were derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide in the presence of 1% tertbutyldimethylchlorosilane modified after the method of Mawhinney et al 23 Mass spectrometric analysis was performed on a GC-MS system consisting of a Shimadzu GC-MS QP2010Plus mass spectrometer coupled to a Shimadzu GC2010 gas chromatograph (Shimadzu Corp., Japan). All labeling data were corrected for natural abundance of 15 N by subtracting the mass distribution of a standard containing the relevant metabolites.…”

Section: Gc-ms and Hplc Analysesmentioning

confidence: 99%

Brain Alanine Formation as an Ammonia-Scavenging Pathway during Hyperammonemia: Effects of Glutamine Synthetase Inhibition in Rats and Astrocyte—Neuron Co-Cultures

Dadsetan

Kukolj

Bak

et al. 2013

J Cereb Blood Flow Metab

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Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of 15 NH 4 þ in alanine during acute hyperammonemia. We observed a fourfold increased amount of 15 NH 4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to 15 NH 4 Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of 15 NH 4 into alanine together with increased 15 N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.

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“…Plasma samples and tissue extracts used for determination of percent 15 N-labeling of amino acids were derivatized with N-methyl-N-(tertbutyldimethylsilyl) trifluoroacetamide in the presence of 1% tert-butyldimethylchlorosilane 26 followed by GC-MS (gas chromatography coupled to mass spectrometry). Labeling measurements were corrected for natural isotopic abundance by subtracting the mass distribution of a standard and isotopic enrichment was calculated according to Biemann 27 and reported as percent labeling.…”

Section: Gas Chromatography Coupled To Mass Spectrometry and High Permentioning

confidence: 99%

Effect of Glutamine Synthetase Inhibition on Brain and Interorgan Ammonia Metabolism in Bile Duct Ligated Rats

Fries

Dadsetan

Keiding

et al. 2013

J Cereb Blood Flow Metab

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Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of 15 NH 4 þ into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of 15 NH 4 þ into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of 15 NH 4 þ into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of 15 N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

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Analysis of amino acids as their tert.-butyldimethylsilyl derivatives by gas—liquid chromatography and mass spectrometry

Cited by 237 publications

References 21 publications

Determination of Complex Isotopomer Patterns in Isotopically Labeled Compounds by Mass Spectrometry

Determination of Complex Isotopomer Patterns in Isotopically Labeled Compounds by Mass Spectrometry

Brain Alanine Formation as an Ammonia-Scavenging Pathway during Hyperammonemia: Effects of Glutamine Synthetase Inhibition in Rats and Astrocyte—Neuron Co-Cultures

Effect of Glutamine Synthetase Inhibition on Brain and Interorgan Ammonia Metabolism in Bile Duct Ligated Rats

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