High-precision continuous-flow measurement of delta13C and deltaD of atmospheric CH4.

We describe our development of a CH4 preconcentration system for use with continuous-flow gas chromatograph combustion isotope ratio mass spectrometry (GC/C/IRMS). Precision of measurement of delta13C-CH4 is 0.05/1000 (1sigma) on multiple 60-mL aliquots of the same ambient air sample. The same front-end on-line CH4 preconcentration system allows us to measure deltaD of CH4 by gas chromatography IRMS when the combustion furnace is replaced with a pyrolysis oven (GC/P/IRMS). Precision of measurement for deltaD-CH4 is 1.5/1000 (1sigma) using 120 mL of ambient air based on multiple aliquots of the same air sample. These are the first reported measurements of atmospheric CH4 using GC/P/IRMS methodology. Each isotope analysis can be made much more rapidly (30-40 min) than they could using off-line combustion of an air sample (1-6 h) followed by conventional dual-inlet IRMS measurements (12-20 min), while requiring much less total volume and retaining a comparable level of precision and accuracy. To illustrate the capabilities of our preconcentration GC/C/IRMS system, we compare the results of measurement of 24 background air samples made using both GC/C/IRMS and conventional vacuum line/dual-inlet IRMS methodology. The air samples were collected on a shipboard air sampling transect made across the Pacific Ocean in July 2000 and are part of an ongoing atmospheric CH4 research program. The average difference between the two methods of IRMS analyses on these 24 samples is 0.01 +/- 0.03/1000 (95% confidence interval) for delta3C-CH4. These are the first measurements to be reported of air samples directly intercompared for delta13C-CH4 using both GC/C/IRMS and dual-inlet IRMS measurement methodology. Measurement of deltaD-CH4 of these air samples is also presented as an illustration of the ability of this system to resolve small isotopic differences in remote air. High-precision measurement of delta13C and deltaD of atmospheric CH4 made using our coupled preconcentration GC/IRMS system will greatly improve our ability to utilize isotopic data in understanding spatial and temporal changes in atmospheric CH4 and the biogeochemistry of its sources and sinks.

Feasibility of analysing [13C]urea breath tests for Helicobacter pylori by gas chromatography-mass spectrometry in the selected ion monitoring mode.

BACKGROUND: The [13C]urea breath test for Helicobacter pylori is nonradioactive, as well as noninvasive, but few clinical laboratories have the expensive isotope ratio mass spectrometer used for analysis. METHODS: To demonstrate the feasibility of analysing [13C]urea breath tests with a gas chromatograph-mass spectrometer routinely used for drug testing, 13CO2 standards for breath tests and breath samples from patients in a multiple-blind study were analysed. The breath samples were also analysed by isotope ratio mass spectrometry, and the diagnoses were compared with biopsy results. RESULTS: The precision of the enrichment measurements by gas chromatography-mass spectrometry was 1.1 parts per thousand, and the calculated differences in enrichment between standard gases equaled the certified values. The sensitivity (94%), specificity (94%), and percentage agreement (94%) for diagnosis of Helicobacter pylori (n = 34) were as high or higher than for analysis of replicate breath samples by isotope ratio mass spectrometry and comparable to the values reported for diagnosis of the bacterium by other currently accepted tests. CONCLUSIONS: The study demonstrates that a gas chromatograph-mass spectrometer can be used to analyse [13C]urea breath tests, thus potentially lowering the cost of the test and increasing the number of laboratories that can perform the test.

Effect of exercise training on energy expenditure, muscle volume, and maximal oxygen uptake in female adolescents.

OBJECTIVES: American female adolescents are at high risk of a physically inactive lifestyle that likely leads to health problems later in life. We hypothesized that a brief program of endurance exercise training in female adolescents would result in increased energy expenditure and quantifiable structural and functional adaptations. STUDY DESIGN: Forty-four high school girls (aged 15 to 17 years, none were elite athletes) enrolled in a 5-day per week anatomy class for 5 weeks and were randomly assigned to control (n = 22) and training groups. All subjects participated in a 2-hour daily teaching program. During the remaining time (2 hours), the training group members underwent endurance-type training and control group subjects participated in a computer workshop. The intervention was assessed by (1) comparison of total energy expenditure between groups with the doubly labeled water technique, (2) determination of changes in thigh muscle volume by magnetic resonance imaging, and (3) determination of changes in maximal oxygen uptake by use of respiratory gas exchange responses. RESULTS: Total energy expenditure was significantly greater (15.3%) in the training group compared with the control subjects (p < 0.003). Five weeks of training led to a 4.3% +/- 1% increase in thigh muscle volume (p < 0.0002) and a 12.1% +/- 3.7% increase in maximal oxygen uptake (p < 0.004); there were no changes in the control group. The training effect was most pronounced in the least fit subjects. CONCLUSIONS: Exercise training programs for female adolescents can be successfully integrated into a high school summer curriculum. Quantifiable, substantial structural and functional responses occur with relatively short periods of training. Approximately 60% of the training response was related to factors independent of muscle size per se. These data may serve to better design physical activity programs for female adolescents.

In vivo study of the biosynthesis of long-chain fatty acids using deuterated water.

To determine the contributions of preexisting fatty acid, de novo synthesis, and chain elongation in long-chain fatty acid (LCFA) synthesis, the synthesis of LCFAs, palmitate (16:0), stearate (18:0), arachidate (20:0), behenate (22:0), and lignocerate (24:0), in the epidermis, liver, and spinal cord was determined using deuterated water and mass isotopomer distribution analysis in hairless mice and Sprague-Dawley rats. Animals were given 4% deuterated water for 5 days or 8 wk in their drinking water. Blood was withdrawn at the end of these times for the determination of deuterium enrichment, and the animals were killed to isolate the various tissues for lipid extraction for the determination of the mass isotopomer distributions. The mass isotopomer distributions in LCFA were incompatible with synthesis from a single pool of primer. The synthesis of palmitate, stearate, arachidate, behenate, and lignocerate followed the expected biochemical pathways for the synthesis of LCFAs. On average, three deuterium atoms were incorporated for every addition of an acetyl unit. The isotopomer distribution resulting from chain elongation and de novo synthesis can be described by the linear combination of two binomial distributions. The proportions of preexisting, chain elongation, and de novo-synthesized fatty acids as a percentage of the total fatty acids were determined using multiple linear regression analysis. Fractional synthesis was found to vary, depending on the tissue type and the fatty acid, from 47 to 87%. A substantial fraction (24-40%) of the newly synthesized molecules was derived from chain elongation of unlabeled (recycled) palmitate.

Isotopomer study of lipogenesis in human hepatoma cells in culture: contribution of carbon and hydrogen atoms from glucose.

Recent developments in the application of stable isotopes and mass spectrometry have permitted the estimation of precursor enrichment and fractional synthesis of the product through mass isotopomer analysis. Thus, the application of isotopomer analysis in studies with 2H- and 13C-labeled glucose may potentially overcome the limitations of traditional methods which can only estimate the fractional use of carbon and hydrogen from glucose for lipogenesis. To illustrate this approach, isotope incorporation and mass isotopomer distribution were determined in fatty acids and cholesterol from a hepatoma cell line (Hep G2) grown in media containing specific (C1 or C6) 2H- or 13C-labeled glucose. Using the binomial model, the respective precursor enrichment, and fractional synthesis of palmitate, stearate and cholesterol were determined using mass isotopomer distribution analysis. In 1 week, 80% of palmitate, 65.5% of stearate, and 50% of cholesterol molecules in the cell extract were derived from de novo synthesis. Under serum-free condition, glucose contributed about 80% of the carbon of the newly synthesized lipids. Using the relative isotope yield of [1-13C] and [6-13C]glucose and a standard formula, the contribution of the pentose pathway to glucose catabolism was calculated to be 4.7%. Fractional syntheses of palmitate, stearate, and cholesterol determined using [1-2H]glucose agreed well with values determined using 13C-labeled glucose. After correcting for the contribution of deuterium label from the glycolytic pathway, the deuterium from [1-2H]glucose contributed 4.7% of the total reducing equivalents for lipogenesis. Unlike radioisotope studies, the stable isotope approach provides information from the perspective of the product and insight into the economy of acetyl units and reducing equivalents which were otherwise not available.

In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.

The synthesis of palmitate, stearate, and cholesterol in liver and nervous tissues (brain, cord, and nerve) of Sprague-Dawley rats was determined using deuterated water (D2O) and mass isotopomer analysis. Rats were given 4% deuterium in their drinking water after each receiving an intraperitoneal priming dose. Animals were killed at 1, 2, 4, and 8 wk for deuterium enrichment in body water and determination of mass isotopomer distribution in lipids from various tissues. In 1 wk, the enrichment in the body water reached a plateau of 2.6%, which is 65% of that in the drinking water. We observed the maximum incorporation number (N) in all lipids to be higher than those previously observed, being 22, 24, and 30 for liver palmitate, stearate, and cholesterol, respectively, and N may vary among tissues. Using a single exponential model, we found the half-time (t1/2) and the plateau levels of the newly synthesized lipids of the nervous tissues (t1/2 values ranging from 5 to 28 days) to be different from those of the liver (t1/2 values < or = 4 days) in this relatively long-term study. Mass isotopomer distribution analysis and D2O can be used not only to quantitate the replacement rate of many lipids in various compartments but may also be used to elucidate the tissue-specific synthetic pathways from N.

Modification in amino acids of Dead Sea Scroll Parchments.

Fragments of Dead Sea Scroll Parchments were extracted for collagen and subjected to amino acid analysis. In modern parchment samples, 90% or more of the protein could be extracted in hot aqueous solution as collagen. In the ancient specimens, 70% or less was extractable. The hot-solution insoluble material was analyzed for collagen. In the soluble extract, the quantity of tyrosine, histidine, and methionine was reduced. Dityrosine was detected. The need to extend such studies is discussed.

The origin of palmitic acid in brain of the developing rat.

A rat milk substitute containing lower amounts of palmitic and oleic acid in the triacylglycerols in comparison to natural rat milk was fed to artificially reared rat pups from day 7 after birth to day 14. Pups reared by their mother served as controls. Free trideuterated (D3) palmitic acid [(C2H3)(CH2)14COOH, 98 atom % D] and free perdeuterated (D31) palmitic acid [C15(2)H31COOH, 99 atom % D] in equal quantity were mixed into the triacylglycerols of the milk substitute in an amount equal to 100% of the palmitic acid in the triacylglycerols. A control milk substitute contained unlabeled free palmitic acid in an amount equal to 100% of the palmitic acid in the triacylglycerols of the milk substitute. The objective was to determine if palmitic acid in the diet contributed significantly to the palmitic acid content of developing brain and other organs. The methyl esters of the fatty acids were analyzed by gas chromatography and the palmitic acid methyl ester was examined by fast atom bombardment mass spectrometry. The proportion of deuterated methyl palmitate as a percentage of total palmitate was determined; 32% of the palmitic acid in liver and 12% of the palmitic acid in lung were trideuterated and perdeuterated palmitic acid in approximately equal amounts. The brain, by contrast, did not contain the deuterated palmitic acid moiety. Quantitation of palmitic acid and total fatty acids revealed a significant accumulation in organs in the interval from 7 to 14 days of age. Under our experimental conditions, labeled palmitic acid does not enter the brain. Consequently, we conclude that the developing brain produces all required palmitic acid by de novo synthesis.

Isotopic relationships between saponifiable lipids and cellulose nitrate prepared from red, brown and green algae.

Stable carbon and hydrogen isotope ratios were determined for the saponifiable lipid fraction as well as the cellulose fraction (the latter after nitration to remove exchangeable hydrogens) of several species of red, brown and green algae from three locations. A significant correlation was observed between the hydrogen isotope ratios of cellulose nitrate and saponifiable lipid for red algae, but not for brown or green algae. Carbon-13/carbon-12 ratios for both fractions of red algae were in general lower than those observed for brown and green algae. The results reported here are consistent with the proposals that red algae evolved much earlier than and are metabolically different from the brown and green algae.