Biomedical subjects

D L Hachey

Publications and source records attributed to D L Hachey.

At least 19 recordsLinked to original sources

PubMed2000

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography-mass spectrometry.

We report here the development and validation of an LC-MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C18 reversed-phase column using an acetonitrile-water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS-MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477-266), DMLOP (m/z 463-->252) and A-LOP (m/z 519-->266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3+/-1.50% (range: 70.7-73.7%) and 79.4+/-12.8% (64.9-88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.

Adult↗

PubMed2000

Formation of novel D-ring and E-ring isoprostane-like compounds (D4/E4-neuroprostanes) in vivo from docosahexaenoic acid.

Free radical-mediated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. We have reported that bioactive prostaglandin D2/E2-like compounds, termed D2/E2-isoprostanes, are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid. Docosahexaenoic acid, in contrast to arachidonic acid, is the most abundant unsaturated fatty acid in brain. We therefore questioned whether D/E-isoprostane-like compounds (D4/E4-neuroprostanes) are formed from the oxidation of docosahexaenoic acid. Levels of putative D4/E4-neuroprostanes increased 380-fold after oxidation of docosahexaenoic acid in vitro from 15.2 +/- 6.3 to 5773 +/- 1024 ng/mg of docosahexaenoic acid. Subsequently, chemical approaches and liquid chromatography electrospray ionization tandem mass spectrometry definitively identified these compounds as D4/E4-neuroprostanes. We then explored the formation of D4/E4-neuroprostanes from a biological source, rat brain synaptosomes. Basal levels of D4/E4-neuroprostanes were 3.8 +/- 0.6 ng/mg of protein and increased 54-fold after oxidation (n = 4). We also detected these compounds in fresh brain tissue from rats at levels of 12.1 +/- 2.4 ng/g of brain tissue (n = 3) and in human brain tissue at levels of 9.2 +/- 4.1 ng/g of brain tissue (n = 4). Thus, these studies have identified novel D/E-ring isoprostane-like compounds that are derived from docosahexaenoic acid and that are formed in brain in vivo. The fact that they are readily detectable suggests that ongoing oxidative stress is present in the central nervous system of humans and animals. Further, identification of these compounds provides a rationale for examining their role in neurological disorders associated with oxidant stress.

Animals↗

PubMed2000

Structural analysis of diacyl peroxides by electrospray tandem mass spectrometry with ammonium acetate: bond homolysis of peroxide-ammonium and peroxide-proton adducts.

Organic peroxides have significant implications in organic chemistry and biological processes. The weak O-O bond makes them extremely difficult to characterize by conventional analytical methods. Diacyl peroxides are one of the major radical sources in polymerization and organic synthesis. It is well known that diacyl peroxides are thermal labile and thus are not amenable to study by gas chromatography/mass spectrometry (GC/MS). Electrospray tandem mass spectrometry (ESI-MS/MS) has been applied to the structural analysis of diacyl peroxides by formation of ammonium adducts. Collision induced dissociation (CID) studies of the ammonium adducts of the peroxide [M + NH(4)](+) give collision energy dependent fragments. For most diacyl peroxides, homolysis of the peroxy bond predominates the fragmentation pathways of the peroxide-ammonium adducts. Deuterated substrates have been employed to provide evidence for typical fragmentation pathways. The CID studies were also used to locate the O-18 in some O-18 specifically labeled diacyl peroxides. For branched alkyl or alkoxy substrates, McLafferty rearrangement and decarboxylation become a major pathway. By comparison with some anhydride analogues, ESI-MS/MS can also be used to study this class of compounds.

Mass Spectrometry↗

PubMed2000

Metabolism of bradykinin In vivo in humans: identification of BK1-5 as a stable plasma peptide metabolite.

Studies investigating the role of bradykinin in disease states such as hypertension, sepsis, and asthma have been confounded by difficulties in measuring the concentration of this short-lived peptide. The purpose of this study was to determine a stable metabolite of bradykinin in the systemic circulation of humans. Bradykinin (containing trace concentrations of [(3)H]bradykinin) was administered i.v. into three human volunteers in increasing amounts up to a maintenance rate of 200 ng/kg/min until a total dose of 1 mg was given. Metabolic products were purified and identified by HPLC and by electrospray ionization mass spectrometry. Infused bradykinin was rapidly degraded, such that no exogenous bradykinin was detected in venous plasma sampled during infusion. BK1-5 (Arg-Pro-Pro-Gly-Phe), the 1-to-5 amino acid fragment of bradykinin, was identified as a major stable plasma metabolite of bradykinin. Plasma concentrations of BK1-5 correlated with dose of bradykinin infused and concentrations at the end of bradykinin infusion were 1510 to 4600 fmol/ml of blood. BK1-5 was cleared from blood with a terminal half-life of 86 to 101 min. Thus, in humans, bradykinin is rapidly degraded in vivo to BK1-5, a stable metabolite. Measurement of this metabolite could provide a tool to assess pathophysiologic and pharmacologic alterations in systemic bradykinin generation associated with human disease.

Adult↗

PubMed1999

Quantitation of monosaccharide isotopic enrichment in physiologic fluids by electron ionization or negative chemical ionization GC/MS using di-O-isopropylidene derivatives.

The aldonitrile pentaacetate and other derivatives lack ions in the electron ionization (EI) spectra possessing an intact hexose structure and thus must be analyzed by chemical ionization GC/MS in order to study multiple isotopomers. We report methods for quantitation of hexose di-O-isopropylidene acetate (IPAc) or pentafluorobenzoyl (PFBz) esters. These were prepared in a two-step procedure using inexpensive reagents that do not adversely impact the isotopomer structure of the sugar. The acetate derivative possesses an abundant [M - CH3] ion in the EI spectrum which is suitable for quantitative analysis of isotopomers. The negative chemical ionization (NCI) spectrum of the corresponding pentafluorobenzoyl derivative has a dominant molecular anion. Moreover, the PFBz derivative is about 100-fold more sensitive than the acetate, which offers some advantages for analysis of minor hexoses found in plasma. Isotopic calibration curves of [U-13C]glucose are linear over the 0.1-60% tracer/tracee range tested. The useful range for isotopic tracer studies is 25-2500 pmol for EI analysis of the acetate derivative and 0.1-55 pmol for NCI analysis of PFBz derivative (sample amount injected). For most studies where sample size is not limited, EI-GC/MS analysis of the IPAc derivative is preferred. NCI-GC/MS analysis is reserved when sample size is limiting or when studies involve hexoses other than glucose that are normally present at low concentration.

Alkenes↗

PubMed1999

Neocytolysis contributes to the anemia of renal disease.

Neocytolysis is a recently described physiological process affecting the selective hemolysis of young red blood cells in circumstances of plethora. Erythropoietin (EPO) depression appears to initiate the process, providing the rationale to investigate its contributions to the anemia of renal disease. When EPO therapy was withheld, four of five stable hemodialysis patients showed chromium 51 (51Cr)-red cell survival patterns indicative of neocytolysis; red cell survival was short in the first 9 days, then normalized. Two of these four patients received oral 13C-glycine and 15N-glycine, and there was a suggestion of pathological isotope enrichment of stool porphyrins when EPO therapy was held, again supporting selective hemolysis of newly released red cells that take up the isotope (one patient had chronic hemolysis indicated by isotope studies of blood and stool). Thus, neocytolysis can contribute to the anemia of renal disease and explain some unresolved issues about such anemia. One implication is the prediction that intravenous bolus EPO therapy is metabolically and economically inefficient compared with lower doses administered more frequently subcutaneously.

Adult↗

PubMed1999

Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment.

Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH(3)I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF(3)/methanol. The TLC method was found to be less reliable than the new CH(3)I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH(3)I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction. We conclude that the CH(3)I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC.

Chromatography↗

PubMed1998

The interaction between nutritional status and growth hormone in young cattle: differential responsiveness of fat and protein metabolism.

The effect of dietary intake level on in vivo plasma leucine and plasma palmitate flux rates and on the response to a bolus injection of bovine growth hormone (GH) was investigated in six young steers. Animals were fed on a pelleted diet of dried grass-barley (0.7:0.3, w/w) in quantities sufficient to supply 0.8, 1.2, 1.6, 2.0, 2.4 or 2.65 x maintenance energy requirement, offered in hourly portions. Continuous intravenous infusions of [1-13C]leucine or [1-13C]palmitate were used to determine the flux of amino acid and fatty acid through the plasma pool before, immediately (1-3 h) after and 22-24 h after a subcutaneous injection of bovine GH (0.55 mg/kg body weight). Hourly blood samples were taken for 27 h to monitor the temporal responses of circulating hormones and metabolites following GH administration. The animal on the lowest plane of nutrition had elevated plasma GH and reduced insulin-like growth factor-1 concentrations compared with those fed on higher intake levels. Plasma leucine flux and leucine concentration increased with intake while palmitate flux and plasma non-esterified fatty acid (NEFA) concentrations were inversely related to intake. Leucine flux rate decreased in the animals fed on the two highest intake levels in response to GH 22-24 h after administration, but plasma leucine concentrations were reduced in all animals at this time. Only the animal fed on the lowest intake level showed an immediate response to GH (within 3 h of administration) with increased palmitate flux and plasma NEFA concentrations but a lipolytic response was apparent in other animals 22-24 h post-administration although the magnitude of the response was markedly reduced at high intakes. We conclude that lipid and protein metabolism are differentially responsive to GH and nutritional status.

Animals↗

PubMed1998

Cholesterol-lowering effect of soy protein in normocholesterolemic and hypercholesterolemic men.

Cardiovascular heart disease is a major health problem in the United States. Elevated blood cholesterol has been shown to significantly increase the risk of cardiovascular heart disease. The National Cholesterol Educational Program (NCEP) Step I diet, which restricts fat and cholesterol intakes, is usually recommended as the initial treatment to lower blood cholesterol. Soy protein has been shown to be hypocholesterolemic, particularly in hypercholesterolemic subjects. However, the hypocholesterolemic effect of soy protein in subjects with a blood total cholesterol concentration <5.17 mmol/L is not clear. To determine whether soy protein could enhance the hypocholesterolemic effect of the NCEP Step I diet, 13 normocholesterolemic and 13 hypercholesterolemic men aged 20-50 y were enrolled in a randomized, 2-part, crossover study. Subjects were fed either an NCEP Step I soy-protein diet or an NCEP Step I animal protein diet for 5 wk. After a washout period of 10-15 wk, the subjects were fed the alternate diet for 5 wk. The hypocholesterolemic effect of soy protein was found to be independent of age, body weight, pretreatment plasma lipid concentrations, and sequence of dietary treatment. Regardless of plasma lipid status, the soy-protein diet was associated with a statistically significant decrease in the plasma concentrations of LDL cholesterol (P = 0.029) as well as the in the ratio of plasma LDL cholesterol to HDL cholesterol (P = 0.005). Our results indicate that soy protein enhances the hypocholesterolemic effect of the NCEP Step I diet in both normocholesterolemic and hypercholesterolemic men.

Adult↗

PubMed1997

New insights into the metabolism of long chain polyunsaturated fatty acids during infancy.

Data obtained with stable isotope methodology have demonstrated that preterm and term infants can convert LA and ALA, respectively, to AA and DHA. In addition, they have clarified the pathways by which infants convert LA and ALA to LCPUFA and have demonstrated the importance of factors such as the dietary LA/ALA ratio and postnatal age on biosynthesis of AA and DHA. Further work is needed to clarify the role of other influential factors on endogenous synthesis of LCPUFA and to determine the absolute amounts of endogenous LCPUFA synthesis. Such data are necessary to define more precisely the LCPUFA requirements of growing infants.

Dietary Fats↗

PubMed1997

Intermediates in endogenous synthesis of C22:6 omega 3 and C20:4 omega 6 by term and preterm infants.

An alternative pathway of omega 3 and omega 6 fatty acid metabolism has been described in isolated rate hepatocytes and human fibroblasts. This alternative pathway, which is independent of delta 4 desaturation, involves elongation of C22 5 omega 3 and C22:4 omega 6 to C24 fatty acids, delta 6 desaturation of the C24 fatty acids and subsequent beta oxidation of the desaturated products to C22:6 omega 3 and C22:5 omega 6. To determine whether this alternative pathway is operative in the human infant and also to obtain additional information concerning endogenous conversion of C18:3 omega 3 and C18:2 omega 6 to longer chain more unsaturated fatty acids, presence of [M + 18] isotopomers of omega 3 and omega 6 fatty acids in the plasma phospholipid fraction of term and preterm infants after administration of [U-13C]18:3 omega 3 and [U-13C]18:2 omega 6 was determined by negative chemical ionization gas chromatography/mass spectrometry. [M + 18] isotopomers of the following omega 3 fatty acids were detected: C18:3, C18:4, C20:3, C20:4, C20:5, C22:4, C22:5, C22:6, C24:4 (two infants only), C24:5, and C24:6. [M + 18] isotopomers of omega 6 fatty acids detected included only C18:2, C18:3, C20:2, C20:3, and C20:4, but sensitivity was insufficient to detect [M + 18] isotopomers of C22 and C24 omega 6 fatty acids. Presence of [M + 18] isotopomers of C24:5 omega 3 and C24:6 omega 3 indicates that these fatty acids were synthesized endogenously from C18:3 omega 3. This plus the in vitro data strongly suggests that infants use the recently described alternative pathway in endogenous synthesis of C22:6 omega 3. However, involvement also of delta 4 desaturation cannot be excluded. Detection of [M + 18] isotopomers of C20:3 omega 3, C20:2 omega 6, and C22:4 omega 3 suggests that C18:3 omega 3, C18:2 omega 6, and C20:4 omega 3 are elongated as well as desaturated. The specific fate of these elongation products and their importance in endogenous synthesis of omega 3 and omega 6 long chain polyunsaturated fatty acids remain to be determined.

Arachidonic Acid↗

PubMed1997

Torpedo maculopathy.

BACKGROUND: Congenital nevi of the retinal pigment epithelium (RPE) may manifest variable degrees of pigmentation. These nevi, which are almost always asymptomatic, can be either solitary or grouped. Torpedo maculopathy is a recently described congenital RPE nevus. METHODS: A review of congenital nevi of the RPE is presented to include torpedo maculopathy. RESULTS: Torpedo maculopathy is a solitary congenital RPE nevus; it is oval, variably pigmented, and located in the temporal macula. Diagnosis of this lesion is made on the basis of its characteristic shape and location. The etiology may be related to alterations in the choroidal vasculature in the macular area during the embryologic development of the eye. Because of the benign nature of the nevus, yearly evaluations are recommended. CONCLUSIONS: Classification of congenital nevi of the RPE is still evolving. As more is learned, a better system of organizing these lesions will be developed.

Diagnosis, Differential↗

PubMed1996

Quantification of cholesterol tracers by gas chromatography--negative ion chemical ionization mass spectrometry.

Because of its high sensitivity, gas chromatography negative ion chemical ionization mass spectrometry (GC-NCI-MS) is a potentially valuable analytical tool for the study of cholesterol metabolism. Of several derivatives prepared for potential use in tracer studies pentafluorobenzoyl cholesterol was selected because it formed rapidly at ambient temperature and was stable for long periods, could be detected at a level of 1 fmol, and yielded a mass spectrum in which the molecular ion was the principal component. Hexadeuterated cholesterol tracer ([26,26,26,27,27,27-2H6]cholesterol) could be detected in dilutions up to 2700 in unlabeled cholesterol by selected ion monitoring with a coefficient of variation averaging 3.2%. In seven normal subjects tracer cholesterol was infused intravenously and plasma cholesterol enrichment was determined after 4 h. The measured rapidly miscible cholesterol pool was 391.0 +/- 38.6 mg cholesterol/kg. Negative ion mass spectrometry of pentafluorobenzyol cholesterol will facilitate analysis of both small amounts of natural cholesterol and labeled cholesterol in applications where sensitivity is critical.

Benzoates↗

PubMed1996

Effect of dietary alpha-linolenic acid intake on incorporation of docosahexaenoic and arachidonic acids into plasma phospholipids of term infants.

The fractional conversion rates of plasma phospholipid alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) to docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6), respectively, and the fractional rates of incorporation of 22:6n-3 and 20:4n-6 into plasma phospholipids were determined in 27 healthy 3-wk-old term infants who had received formulas with approximately 16% of fat as 18:2n-6 and 0.4% (n = 6), 1.0% (n = 11), or 3.2% (n = 10) as 18:3n-3 from birth. The infants were given a single dose of both [U-13C] 18:2n-6 and [U-13C] 18:3n-3 with a feeding, and blood samples were collected 8, 12, and 24 h afterward for determination of the isotopic enrichments of the [M + 18] isotopomers of plasma phospholipid fatty acids by negative chemical ionization gas chromatography/mass spectrometry. A simple precursor/product compartmental model was used to estimate fractional rates of conversion and incorporation. All infants converted 18:3n-3 to 22:6n-3 and 18:2n-6 to 20:4n-6. Although the fractional rate of conversion of 18:3n-3 to 22:6n-3 did not differ among groups, the fractional rate of incorporation of 22:6n-3 into the plasma phospholipid fraction was greater in infants who received 3.2% vs. 0.4% or 1.0% 18:3n-3 (4.1 +/- 2.2 vs. 1.6 +/- 1.5 or 2.0 +/- 1.0% of the plasma phospholipid 22:6n-3 pool daily). The fractional rate of conversion of 18:2n-6 to 20:4n-6 was less in infants who received the 3.2% 18:3n-3 intake (0.4 +/- 0.3% of the plasma phospholipid 18:2n-6 pool daily vs. 1.1 +/- 0.7% and 0.8 +/- 0.5% in those who received 0.4 and 1.0% 18:3n-3, respectively). The fractional rate of incorporation of 20:4n-6 into plasma phospholipid also was less in the 3.2% vs. the 0.4 and 1.0% 18:3n-3 groups (2.7 +/- 1.4% vs. 5.9 +/- 2.6 and 4.4 +/- 1.7%, respectively, of the plasma phospholipid 20:4n-6 pool daily).

Arachidonic Acid↗

PubMed1996

The duration of medium-chain triglyceride feeding determines brush border membrane lipid composition and hydrolase activity in newly weaned rats.

To better understand the effect of dietary fat on intestinal brush border (BB) membranes in the young animal, we compared the effect of dietary medium-chain triglycerides (MCT) with that of monounsaturated and saturated long-chain triglycerides (LCT) on jejunal brush border membrane lipid composition and hydrolase activity in newly weaned rats. Twenty-day-old rat pups were divided into three groups and were weaned to diets containing 14% MCT + 6% soybean oil, 18% olive oil + 2% soybean oil, or 14% tallow + 6% soybean oil, and fed for 40 h or for 33 d. The diets were isonitrogenous and contained similar amounts of cholesterol and polyunsaturated fatty acids. Within 40 h, the fatty acid compositions of the brush border membranes were significantly different among treatment groups. These differences were maintained in rats fed for 33 d. No medium-chain fatty acids, but significantly greater amounts of polyunsaturated fatty acids, especially 18:2(n-6) and 20:4(n-6), were found in the brush border membranes of rats fed the medium-chain triglyceride diet. The cholesterol and phospholipid concentrations in the membranes were highest in rats fed the medium-chain triglyceride diet for 33 d. Rats fed that diet for 40 h had generally higher leucine aminopeptidase, sucrase and maltase activities compared with rats fed the olive oil or tallow diets. However, after 33 d of feeding, the differences between dietary treatment groups disappeared. This study demonstrates that, in the newly weaned rat pup, dietary medium-chain triglycerides and long-chain triglycerides rapidly affect the fatty acid composition of the brush border membrane. However, the changes in the hydrolase activities associated with the changes in the lipid composition of the membranes are transient.

Aging↗

PubMed1995

Thyroid hormone influences the maturation of apolipoprotein A-I messenger RNA in rat liver.

Chronic administration of thyroid hormone (T3) increases apolipoprotein (apo) A-I gene expression in rat liver. That transcriptional activity of the apoA-I gene is reduced to 50% of control, whereas abundance levels of nuclear and total cellular apoA-I mRNA are increased 3-fold, implies more effective apoA-I mRNA maturation. To study hormonal effects on apoA-I RNA processing, we quantified mRNA precursors in control and T3-treated rats (50 micrograms/100 g body weight for 7 days). Northern blotting, amplification of reverse-transcribed RNA, and ribonuclease protection assays showed that the splicing pathway is branched, in that either intron 1 or intron 2 is removed first from the primary transcript, whereas intron 3 is removed last. In T3-treated rats, abundance levels of the primary transcript, the intron 1-containing precursor devoid of intron 2, the intron 2-containing precursor devoid of intron 1, the intron 3-containing precursor lacking both introns 1 and 2, and nuclear mRNA were 65, 183, 78, 195, and 268% of controls. Compared with control rats, the half-life of the intron 1-containing precursor, measured after injection of actinomycin D, was increased 2-fold in T3-treated rats. In contrast, half-lives of the primary transcript and the intron 2-containing precursor were similar in control and T3-treated rats. Ribonuclease protection assays revealed an RNA species extending from the transcription start site close to the 3' end of intron 1. The abundance of this RNA fragment, probably representing a degradation product, was 2.5-fold higher in control than in T3-treated animals (p < 0.001). Sequences of apoA-I mRNA precursors were identical in control and T3-treated rats which excluded hormonal effects on splice-site selection or post-transcriptional editing of apoA-I transcripts. Compartmental modeling of apoA-I mRNA processing suggested that chronic thyroid hormone administration enhances apoA-I mRNA maturation more than 7-fold by protecting the intron 1-containing precursor devoid of intron 2 from degradation and by facilitating the splicing of intron 1 from this precursor.

Animals↗

PubMed1995

Determination of multiply labeled serine and glycine isotopomers in human plasma by isotope dilution negative-ion chemical ionization mass spectrometry.

A gas chromatography/negative-ion chemical ionization mass spectrometry method is presented to measure the isotopic enrichment of multiple serine and glycine isotopomers. The amino acid N-heptafluorobutyryl n-propyl ester derivatives were used. The method had good analytical linearity between 10 and 800 microg mL-1 and both precision and accuracy were 5% for plasma amino acids. Sensitivity permitted analysis of 100 pg amino acid on column. The method was applied to metabolic studies of the serine to glycine interconversion in humans. (alpha-15N)serine and (1,2-13C2)glycine were given as a single intravenous bolus to six healthy male subjects. Plasma concentration of glycine and serine were determined after addition of (alpha-15N,1,2,3-13C3) serine and (alpha-15N,1,2-13C2)glycine as internal standards to 500 microL of plasma. Since glycine and serine are rapidly interconverted by hepatic serine hydroxymethyl transferase, the resultant tracer spectrum requires deconvolution of the enrichment of four isotopomers of each amino acid. Deconvolution of the ion abundance ratios to yield tracer-to-tracee ratios for each isotopomer was done using Brauman's least squares approach.

Gas Chromatography-Mass Spectrometry↗

PubMed1995

Cholesterol synthesis and absorption by 2H2O and 18O-cholesterol and hypocholesterolemic effect of soy protein.

The kinetic behavior of orally ingested 18O-cholesterol was compared with that of orally ingested 13C5-cholesterol in two normocholesterolemic men and the hypocholesterolemic effect of soy protein was demonstrated in 12 hypercholesterolemic men. Our results indicated no difference in the metabolism of orally ingested 18O- and 13C5-cholesterol. The use of 18O-cholesterol and 13C5-cholesterol also allowed simultaneous estimation of fractional rates of cholesterol synthesis using the 2H2O method, which were calculated to be 5.76 and 8.17%/d for the two normocholesterolemic subjects. The percent reduction in plasma cholesterol levels were found to be greater when the hypercholesterolemic men were placed on a soy protein diet than on an animal protein diet.

Absorption↗