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As part of an integrated assessment of multiple sector impacts produced by predicted changes in climate we have integrated a set of models, which provide predictions of fish populations under changing flow and temperature regimes. The core of the approach is the U. S. Fish and Wildlife Service Physical Habitat Simulation Model (PHABSIM). PHABSIM estimates habitat conditions based on flow, which are life stage specific. The output from PHABSIM is used to model fish populations, considering both flow and a temperature threshold, which affects spawning date. Water temperatures were modelled based on air temperature. The resulting assessment tool provides the means to evaluate the effect of multiple stressors produced by climate change scenarios. The model has been used to estimate smallmouth bass (Micropterus dolomieui) populations for representative reaches of the Mackinaw River, Illinois. The model has been used to illuminate population effects of changing flow and temperature under historical climate/weather conditions, as well as under climate change scenarios. The integrated models in the assessment tool have provided a useful addition to watershed management, improving our capacity to evaluate natural resources impact at temporal scales typical of climate change, and management response systems.

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We examined antepartum clinical characteristics along with measures of glucose tolerance, insulin sensitivity, pancreatic beta-cell function, and body composition in Latino women with gestational diabetes mellitus (GDM) for their ability to predict type 2 diabetes or impaired glucose tolerance (IGT) within 6 months after delivery. A total of 122 islet cell antibody-negative women underwent oral and intravenous glucose tolerance tests (OGTT; IVGTT), hyperinsulinemic-euglycemic clamps, and measurement of body fat between 29 and 36 weeks' gestation and returned between 1 and 6 months postpartum for a 75-g OGTT. Logistic regression analysis was used to examine the relationship between antepartum variables and glucose tolerance status postpartum. At postpartum testing, 40% of the cohort had normal glucose tolerance, 50% had IGT, and 10% had diabetes by American Diabetes Association criteria. Independent antepartum predictors of postpartum diabetes were the 30-min incremental insulin:glucose ratio during a 75-g OGTT (P = 0.0002) and the total area under the diagnostic 100-g glucose tolerance curve (P = 0.003). Independent predictors of postpartum IGT were a low first-phase IVGTT insulin response (P = 0.0001), a diagnosis of GDM before 22 weeks' gestation (P = 0.003), and weight gain between prepregnancy and the postpartum examination (P = 0.03). All subjects had low insulin sensitivity during late pregnancy, but neither glucose clamp nor minimal model measures of insulin sensitivity in the 3rd trimester were associated with the risk of IGT or diabetes within 6 months' postpartum. These results highlight the importance of pancreatic beta-cell dysfunction, detectable under conditions of marked insulin resistance in late pregnancy, to predict abnormalities of glucose tolerance soon after delivery in pregnancies complicated by GDM. Moreover, the association of postpartum IGT with weight gain and an early gestational age at diagnosis of GDM suggests a role for chronic insulin resistance in mediating hyperglycemia outside the 3rd trimester in women with such a beta-cell defect.

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A method is described for the calibration of an isotope ratio mass spectrometry working standard for 2H/1H analysis using highly purified (> 99.99% enriched) deuterium oxide (2H2O). Serial dilution of such a compound can be used to construct a standard curve for calibration purposes using the relationship [formula: see text] where APE is the atom percent excess. It was found that the determined rWR had a precision of +/- 1%. Most of the variation came from sample handling, which includes the serial dilution of the weighed deuterium oxide, the reduction of water to hydrogen gas and the introduction of the hydrogen gas into the mass spectrometer. The use of this calibration procedure allows the direct determination of the isotope ratio rSA, and APE from a standard curve over a wide range of enrichment. Unlike the reference standards V-SMOW and SLAP, the highly purified deuterium oxide is non-exhaustible and readily available. The highly purified deuterium oxide should be used in place of secondary standards to calibrate working references for 2H/1H analysis in biological studies.

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Fatty acid cycling by chain shortening/elongation in the peroxisomes is an important source of fatty acids for membrane lipid synthesis. Its role in the homeostasis of nonessential fatty acids is poorly understood. We report here a study on the cycling of saturated fatty acids and the effects of troglitazone in HepG2 cells in culture using [U-13C]stearate or [U-13C]oleate and mass isotopomer analysis. HepG2 cells were grown in the presence of 0.7 mmol/liter [U-13C]stearate or [U-13C]oleate, and in the presence and absence of 50 microM troglitazone for 72 h. Fatty acids extracted from cell pellets after saponification were analyzed by gas chromatography/mass spectrometry. Peroxisomal beta-oxidation of uniformly 13C-labeled stearate (C18:0) and oleate (C18:1) resulted in chain shortening and produced uniformly labeled palmitate (C16:0) and palmitoleate (C16:1). In untreated cells, 16% of C16:0 was derived from C18:0 and 26% of C16:1 from C18:1 by chain shortening. Such contributions were significantly increased by troglitazone to 23.6 and 36.6%, respectively (p < 0.001). Desaturation of stearate contributed 67% of the oleate, while reduction of oleate contributed little to stearate (2%). The desaturation of C18:0 to C18:1 was not affected by troglitazone. Our results demonstrated a high degree of recycling of C18:0 and C18:1 to C16:0 and C16:1 through chain shortening and desaturation. Chain shortening was accompanied by chain elongation in the synthesis of other long chain fatty acids. Troglitazone specifically increased recycling by peroxisomal beta-oxidation of C18 to C16 fatty acids, and the interconversion of long chain fatty acids was associated with reduced de novo lipogenesis.

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To determine the transport and utilization of dietary saturated, monounsaturated, and n-6 and n-3 polyunsaturated fatty acids for the developing brain and other organs, artificially reared rat pups were fed a rat milk substitute containing the perdeuterated (each 97 atom% deuterium) fatty acids, i.e., palmitic, stearic, oleic, linoleic, and linolenic, from day 7 after birth to day 14 as previously described. Fatty acids in lipid extracts of the liver, lung, kidney, and brain were analyzed by gas chromatography-mass spectrometry to determine their content of each of the deuterated fatty acids. The uptake and metabolism of perdeuterated fatty acid lead to the appearance of three distinct groups of isotopomers: the intact perdeuterated, the newly synthesized (with recycled deuterium), and the natural unlabeled fatty acid. The quantification of these isotopomers permits the estimation of uptake and de novo synthesis of these fatty acids. Intact perdeuterated palmitic, stearic, and oleic acids from the diet were found in liver, lung, and kidney, but not in brain. By contrast, perdeuterated linoleic acid was found in all these organs. Isotopomers of fatty acid from de novo synthesis were observed in palmitic, oleic, and stearic acids in all tissues. The highest enrichment of isotopomers with recycled deuterium was found in the brain. The data indicate that, during the brain growth spurt and the prelude to myelination, the major saturated and monounsaturated fatty acids in brain lipids are exclusively produced locally by de novo biosynthesis. Consequently, the n-6 and n-3 polyunsaturated fatty acids must be transported and delivered to the brain by highly specific mechanisms.

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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.

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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.

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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.

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Fractional biosynthesis of palmitate, stearate, and cholesterol was determined with deuterated water (2H2O) using mass isotopomer analysis in Hep G2 and MCA sarcoma cells in culture. The method employed differs from previous ones in that the number of deuterium atoms from 2H2O incorporated into newly synthesized molecules was determined and not assumed. After correction for background natural abundances, the isotopomer distribution due to deuterium incorporation in fatty acids and cholesterol was shown to follow a simple binomial distribution depending on the deuterium enrichment in water (p) and the maximum number of deuterium atoms incorporated per molecule (N). Under a wide range of 2H2O enrichments, N could be determined to be 17 for palmitate, 20 for stearate, and 20 for cholesterol by regression analysis or from a series of consecutive mass isotopomer ratios. The fraction derived from de novo synthesis was given by the ratio of the observed to the theoretical deuterium enrichment, which is the product (N x p). The new synthesized fraction of palmitate and stearate by Hep G2 cells for the length of the experiment was found to be 77 and 65%, respectively. These values were confirmed by experiments with [U-13C]glucose as the precursor. In MCA sarcoma cells grown in lipid-poor medium, the average values for fractional synthesis of palmitate, stearate, and cholesterol were 70, 35, and 70%, respectively. This approach should be generally applicable to the simultaneous determined of fractional synthesis of a number of compounds with either deuterium or 13C tracers. Its application is only limited by the accuracy of mass spectrometric analysis.

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The synthesis of a homonucleus polymer from its labeled precursor will lead to the formation of molecules with different masses. The distribution of these mass isotopomers is strictly a function of the enrichment of the 13C-labeled precursor, and can thus be used for the determination of the precursor enrichment and product dilution in the de novo synthesis of the polymer. We present here a study of the isotopomer pattern of a polymer of acetate in the form of glucose pentaacetate synthesized from 13C-enriched acetic anhydride. The molecular ion contains four acetyl units. Its synthesis is analogous to that of octanoic acid from acetyl coenzyme A. The process of obtaining the mass isotopomer distribution in the tetraacetyl moiety from the ion cluster of m/z 331 of glucose pentaacetate is illustrated. After correcting for the contribution of 13C natural abundance, the plot of the ratio of mass isotopomers (m4/m2) against the observed enrichment of the tetraacetate moiety yielded a straight line with a slope of 1.45. The ratio was not altered by dilution with pre-existing unenriched product, as predicted. The slope of the observed linear relationship agreed with the general formula (N-(j-1))/j for the ratio of any two consecutive mass isotopomers (mj/mj-1). Theoretical and practical aspects of determining precursor enrichment from isotopomer pattern in polymers are discussed.

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A theory of mass isotopomer analysis based on the well-known principle of isotope dilution mass spectrometry is reviewed. An algorithm for the determination of isotope incorporation into a metabolic substrate from a labeled precursor using mass isotopomer analysis is presented. The steps include the determination of the contribution of the derivatization reagent to the observed spectrum of the derivatized substrate and the correction of contribution from 13C natural abundance using multiple linear regression analysis. Examples of the application of this theory to determine the spectrum of the trimethylsilyl derivative of the 'pure unlabeled' or mononuclidic cholesterol, and the calculation of mass isotopomer distribution in cholesterol due to tracer incorporation using this 'pure unlabeled' spectrum, are also provided.

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Experimental determinations of glucose carbon recycling using 14C or 13C glucose tracer often underestimate true Cori cycle activity because of dilution and exchange of isotope tracer through the tricarboxylic acid (TCA) cycle. The term glucose isotope recycling therefore is used to distinguish recycling of isotope from recycling of glucose carbon, the actual quantity of circulating glucose recycled. Recently, per-labeled glucose ([U-13C6]glucose) has been used to estimate glucose appearance rate and glucose isotope recycling. Chemical structural information determined by mass isotopomer analysis has been used to correct for dilution of isotope through the TCA cycle. In this report, we present experiments in the study of glucose turnover and recycling using [U-13C6]glucose. Methods of single injection and continuous infusion of [U-13C6]glucose are compared. A formula for the calculation of a dilution factor using TCA cycle parameters is derived. In this study of six rabbits, glucose turnover rate ranged from 3.4 to 8.8 mg/kg/min, and glucose m + 3 mass isotopomer recycling from 7 to 12%. The rate of pyruvate carboxylation (Y) was comparable to that of citrate synthetase, having an average relative flux of 0.89. Applying the correction factor for tracer dilution to the observed mass isotopomer recycling, we determined glucose carbon recycling (or Cori cycle activity) to be 22-35% of hepatic glucose output.

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Hepatic glucose production (HGP) and glucose carbon recycling are traditionally estimated by the combined use of hydrogen and carbon-labeled glucose tracers. A single-isotope method such as that of Reichard et al. for the determination of HGP and glucose carbon recycling requires the determination of activities in different glucose carbons by chemical degradation. Since the 13C content in the glucose carbon skeleton can be determined from mass fragmentography, the use of 13C-labeled glucose and mass fragmentography can provide a single-isotope method for the quantification of the recycled carbons. Correction for the recycling makes it possible to determine the true HGP. In this study, (1-13C1)glucose and mass fragmentography were used for the determination of HGP and glucose carbon recycling in six colon cancer patients. Molar enrichment of the molecular ion (m/z 328 cluster of glucose aldonitrile pentaacetate) was used to determine 'uncorrected' HGP, which was 1.93 +/- 0.11 mg kg-1 min-1 (mean +/- s.e.m.). The difference in molar enrichment of the molecular ion C1-C6 (m/z 328) and the ion corresponding to C1-C4 fragment (m/z 242) was used to determine the contribution of recycled label carbon. After this correction, the 'corrected' HGP was 2.04 +/- 0.12 mg kg-1 min-1, which is not significantly different from the 'true' HGP rate of 2.05 +/- 0.15 mg kg-1 min-1 determined by using (6-3H)glucose. HGP determined from the enrichment of the molecular ion C1-C6 underestimates true HGP, as expected. The corrected HGPs correlate well with those from 6-3H method (r = 0.86, y = 1.06x - 0.12; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

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Starved rats were infused intragastrically via indwelling duodenal cannulae with glucose at a rate of 30 mg/min/kg. The infusate contained [U-13C]glucose at an enrichment of 32 or 17%. At the end of the infusion, after 160 min, glucose and lactate were isolated from arterial and portal blood and from liver, and liver glycogen was isolated and hydrolyzed to glucose. The enrichment in glucose and lactate and the isotopomer distribution in glucose of masses from 180 to 186 were determined by gas chromatography-mass spectrometry (GC-MS). From analysis of these data we determined (a) gluconeogenesis proceeds at half the basal rate in the presence of a large infused glucose load, (b) one-quarter of the hepatic pyruvate pool is derived from nonglucose carbon, (c) half of the labeled molecules in liver glycogen are of mass 186 from the infused glucose and half are of masses 181-183, (d) the contribution of the indirect path from pyruvate when corrected for synthesis from unlabeled pyruvate ranges from 55 to 65%, (e) the rate of pyruvate carboxylase averages 90% that of citrate synthase, and (f) the rate of exchange of oxaloacetate with fumarate is about three times the rate of flux in the Krebs cycle (four times in the "forward" direction), and the enrichment in carbon 1 of oxaloacetate was 2.3 times that in carbon 4. In the Appendix a method to obtain the isotopomer distribution of newly formed glucose and glycogen glucose is described. An algorithm to correct for the contribution of natural abundance of 13C and the presence of 12C in commercial [U-13C]glucose is presented. A novel mathematical analysis to obtain the parameters of the Krebs cycle from the isotopomer distribution is developed in the Appendix. Equations to calculate the relative rates of pyruvate carboxylase (y), and the equilibration of oxaloacetate with fumarate from the isotopomer distribution are derived. Mass isotopomer analysis provides a novel and powerful tool for the study of carbohydrate metabolism and the operation of the Krebs cycle.

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Patients with steroid sulphatase deficiency develop ichthyosis with accumulation of cholesterol sulphate in plasma and in the stratum corneum. The present study was undertaken to determine whether desulphation of the C19 steroid DHEAS is also impaired. The mean plasma concentrations of DHEA and androstenedione were significantly lower for patients than for controls (p less than 0.02 and 0.001) while the mean concentration of DHEAS was higher (p less than 0.002). Following intravenous administration of 3H-DHEAS, one patient failed to desulphate 3H-DHEAS as evidenced by an absence of urinary 3H-glucuronides. A second produced normal amounts of urinary 3H-glucuronides (indicative of desulphation capacity) in a baseline study but did not desulphate 3H-DHEAS following ampicillin treatment to alter gut microflora. A third patient had consistent sulphatase activity with and without ampicillin.

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We studied two families with an inherited deletion of the short arm of an X chromosome (Xp) in which affected male offspring have epiphyseal stippling in infancy (chondrodysplasia punctata), nasal hypoplasia, ichthyosis, and mental retardation. The presence of ichthyosis and the apparent pattern of X-linked recessive inheritance prompted investigation of the short arm of the X chromosome through studies of genetic markers and focused cytogenetic analysis. Biochemical studies suggested that there was a deletion of three genes previously mapped to the X-chromosome short arm, including the steroid sulfatase locus, the Xg locus, and the M1C2X locus. Prometaphase chromosomes demonstrated a deletion of Xp at p22.32 in the affected boys, in their obligate-carrier mothers, and in 11 of 25 women at risk as potential carriers. The women carrying the Xp deletion had normal gonadal function and fertility but were shorter than the noncarriers in their families (P less than 0.00001). These findings have implications for the genetic organization of this portion of the human X chromosome and demonstrate that small cytogenetic abnormalities may account for disorders with apparent mendelian patterns of inheritance.

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Steroid sulfatase deficiency is an inborn error of metabolism characterized during fetal life by decreased estriol production and postnatally by x-=linked ichthyosis. No consistent substrate abnormalities have been found beyond the perinatal period. Utilizing gas chromatography, we found that the cholesterol sulfate concentration was less than 350 micrograms/100 ml plasma in 9 normal adults, 2 subjects with ichthyosis vulgaris and 2 subjects with lamellar ichthyosis. Control red cell membranes had less than 300 micrograms/100 ml erythrocytes. Eight subjects (age 3 months-74 years) with steroid sulfatase deficiency had strikingly elevated cholesterol sulfate levels with means and ranges as follows: plasma - 3,300 micrograms/100 ml (2,700-4,000), red cell membranes- 7,500 (5,200-9,800) Cholesterol sulfate is known to effect membrane stability and the present observations may help to explain the pathogenesis of STS deficiency and x-linked ichthyosis.

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We report the successful detection of urinary biogenic amines by a procedure which involves isolation of the amines on CG-50 cation exchange resin, derivatization of the amines with fluorescamine while on the resin, followed by direct probe negative chemical ionization mass spectrometry screening of the resin extract. This isolation procedure allowed the detection of tyramine, octopamine, normetanephrine, 3-methoxytyramine and a variety of aliphatic amines. Norepinephrine, dopamine and serotonin were chemically reactive under these conditions and were not detectable at physiological concentrations.

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