Biomedical subjects

D L Hachey

Publications and source records attributed to D L Hachey.

At least 55 records · Page 3Linked to original sources

PubMed1993

Unique lipids of primate spermatozoa: desmosterol and docosahexaenoic acid.

Spermatozoa represent a tissue readily accessible for study after various exogenous perturbations. To characterize the lipid composition of monkey sperm and to establish a baseline from which dietary or pharmaceutical influences may then be evaluated, we collected semen samples from five rhesus monkeys by electroejaculation and analyzed the sperm for sterols, fatty acid composition, and the molecular species of the ethanolamine glycerophospholipids. Two sterols were identified: cholesterol, 41%, and desmosterol, 59% of total sterols. Desmosterol was found only in the free form. Cholesterol existed in three different forms: free, 60%; esterified, 20%; and sulfated, 20%. Docosahexaenoic acid (22:6, DHA) was almost the only n-3 fatty acid in sperm phospholipids, 24% of the total fatty acids. DHA was present mainly in phosphatidylcholine and phosphatidylethanolamine. Oleic and palmitic acids were the predominant monounsaturated and saturated fatty acids. The ethanolamine glycerophospholipids were separated into three subclasses: diacyl 49%, alkenylacyl 43%, and alkylacyl 8%. Thirteen molecular species were identified and quantified. The sn-1 position of these molecular species contained exclusively 16:0, 18:0, or 18:1. The sn-2 position contained n-3, n-6, and n-9, as well as saturated fatty acids. The molecular species having n-3 fatty acids in the sn-2 position contributed 43, 73, and 100% of the total in the diacyl, alkenylacyl, and alkylacyl subclasses, respectively. The presence of the unusual sterol, desmosterol, a cholesterol precursor not found in measurable quantities in any other tissue suggests an important functional and structural role for desmosterol in spermatozoa. The other unique lipids, cholesterol sulfate and the n-3 docosahexaenoic acid, may also have a significant role in the function of spermatozoa.

Animals↗

PubMed1992

Beta-adrenergic receptor-mediated functions in porcine adipose tissue are not affected differently by saturated vs. unsaturated dietary fats.

Young postweaning pigs were fed a high fat diet containing beef tallow (saturated fat) or corn oil (unsaturated fat). Adipose tissue was used to measure adipocyte size and number of cells per gram of tissue, ligand binding by beta-adrenergic receptors and lipolytic and palmitate esterification rates. Pigs fed the saturated fat diet had more saturated and monounsaturated fatty acids and less polyunsaturated fatty acid in the crude membrane fraction. Adipocytes were larger in pigs fed the saturated fat diet. There was no difference in the binding affinities of the receptors; more binding sites were expressed on a protein or cell basis and fewer sites were expressed per unit surface area in adipocyte ghosts isolated from pigs fed the saturated fat diet. Fatty acid esterification was greater in pigs fed saturated fat diets. Isoproterenol inhibition was marginal in both groups but tended to be greater in pigs fed saturated fat diets. The beta-adrenergic receptor-mediated lipolytic rates were not different; only the theophylline-stimulated rates tended to be greater in the saturated fat-fed group. Thus, a large increase in saturated fatty acid concentration of porcine adipose tissue membranes caused an increase in beta-adrenergic receptor number without any change in receptor affinity. These receptor changes were at best only marginally reflected in beta-adrenergic agonist-mediated functions.

Adipose Tissue↗

PubMed1992

VLDL apolipoprotein B-100, a potential indicator of the isotopic labeling of the hepatic protein synthetic precursor pool in humans: studies with multiple stable isotopically labeled amino acids.

Four adult men received a 48-h constant intravenous infusion of [2H4]lysine, [2H3]leucine, L-[ring-13C6]phenylalanine, and L-[1,2,3,-13C3]alanine. Subjects ingested hourly meals for two 12-h periods, separated to two 12-h fasting periods. The isotopic enrichments of free amino acids in venous plasma and in VLDL apolipoprotein B-100 (apoB)-bound amino acids, plasma alpha-keto isocaproic acid (alpha-KIC) and plasma pyruvic acid (PYR) were measured by negative chemical ionization gas chromatography-mass spectrometry. By 7 h of infusion, all four amino acids achieved an equilibrium isotopic enrichment (EIE) in plasma and in apoB. In the fed state, the EIE of the amino acids in apoB was lower than that in plasma free amino acids. The ratio EIE-apoB:EIE-plasma differed significantly among amino acids in the fed state (alanine 0.30; lysine 0.64; leucine 0.70; phenylalanine 0.81). In the postabsorptive state, the EIE-apoB:EIE-plasma ratio rose significantly compared with the fed state (alanine 0.38; lysine 0.73; leucine 0.94; phenylalanine 1.05). Plasma PYR and apoB-alanine were in isotopic equilibrium irrespective of nutritional state. The EIE-apoB-leucine:EIE-plasma-alpha-KIC ratio rose from 0.75 in the fed state to near 1 in the postabsorptive state. We conclude that the contribution of systemic amino acids to apoB-100 synthesis is sensitive to nutritional state, and that systemic essential amino acids seem to be preferentially incorporated into apoB.

Adult↗

PubMed1992

Measurement of human apolipoprotein B-48 and B-100 kinetics in triglyceride-rich lipoproteins using [5,5,5-2H3]leucine.

A primed-constant infusion of deuterated leucine was used in humans to determine the maximal level of enrichment at plateau of apolipoprotein (apo)B-48 and apoB-100 which are synthesized in the intestine and liver, respectively, and to compare the kinetics of these two proteins under identical conditions. Eight normal subjects (four post-menopausal females and four males) over the age of 40 were studied in the constantly fed state over a 20-h period by providing small hourly feedings of identical composition. [5,5,5-2H3]Leucine (10 mumol/kg body weight followed by 10 mumol/kg body weight per hour) was infused over 15 h intravenously. The enrichment of deuterated leucine in apoB-48 and apoB-100 triglyceride-rich lipoproteins isolated by ultracentrifugation (d less than 1.006 g/ml) was determined during the entire infusion period. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-48 was 3.96 +/- 1.41 tracer/tracee ratio (%) which was 39.7% of the plasma leucine enrichment level. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-100 was 7.23 +/- 1.17 tracer/tracee ratio (%) which was 72.5% of the plasma leucine enrichment level. Mean fractional secretion rates of triglyceride-rich lipoprotein apoB-48 and apoB-100 were 4.39 +/- 2.00 and 5.39 +/- 1.98 pools per day, respectively, with estimated residence times of 5.47 and 4.45 hours, respectively. The data indicate that in the fed state there is about a twofold difference in the plateau enrichment of an intestinally derived protein, as compared to one of hepatic origin, most likely attributable to differences in the enrichment of the intracellular leucine in the two organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1991

Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo.

The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.

Journal Article↗

PubMed1991

Determination of estradiol 2- and 4-hydroxylase activities by gas chromatography with electron-capture detection.

A highly sensitive assay has been developed for measuring the rate of formation of 2-hydroxyestradiol and 4-hydroxyestradiol from estradiol by microsomal preparations. Catechol estrogens were converted to heptafluorobutyryl esters, which were separated by capillary column gas chromatography and quantified using electron-capture detection. 2-Hydroxyestradiol 17-acetate was used as an internal standard. The identity of catechol estrogen derivatives was verified by gas chromatography-mass spectrometry using negative-ion chemical ionization. Estrogens were identified by negative molecular ions and/or by characteristic fragments. This procedure permits quantification of catechol estrogens at the subpicogram level. The assay was validated by comparing estrogen 2- and 4-hydroxylase activities in microsomes from hamster and rat liver with values reported previously.

Animals↗

PubMed1991

Isotopic determination of organic keto acid pentafluorobenzyl esters in biological fluids by negative chemical ionization gas chromatography/mass spectrometry.

A rapid, single-step procedure for the extraction and derivatization of organic alpha-keto acids from microliter quantities of human plasma has been developed. The keto acids were analyzed as the pentafluorobenzyl (PFB) ester by methane negative chemical ionization gas chromatography/mass spectrometry. The PFB esters possess excellent chromatographic properties and required no further derivatization to block the keto group. They fragment to produce intense carboxylate anions, often as the sole ion in the spectrum, and offer detection limits below 1 pmol. This derivative is suitable for isotopic analysis of organic keto acids because it does not introduce any additional isotopic complexity into the target molecule. Normal human plasma 4-methyl-2-oxopentanoic acid levels were 34.9 +/- 5.3 mumol.L-1 and could be determined with 1.1% precision by isotope dilution GC/MS. We have used this procedure to study leucine and 4-methyl-2-oxopentanoic acid metabolism by using stable isotopically labeled tracers in a variety of normal and abnormal conditions.

Fluorobenzenes↗

PubMed1991

Origin of intact lactoferrin and its DNA-binding fragments found in the urine of human milk-fed preterm infants. Evaluation by stable isotopic enrichment.

The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [13C]leucine and [15N2]lysine or [2H4]lysine. Mothers of infants selected for the study were infused i.v. with [13C] leucine and [15N2]lysine or [2H4]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNA-binding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each mother/infant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. The possible immunoregulatory functions of the absorbed intact, maternal lactoferrin are discussed.

DNA-Binding Proteins↗

PubMed1991

Measurement of in vivo cholesterol synthesis from 2H2O: a rapid procedure for the isolation, combustion, and isotopic assay of erythrocyte cholesterol.

A rapid preparative scale purification of erythrocyte free cholesterol has been developed for measurements of in vivo cholesterol synthesis from 2H2O. The quantity and purity of cholesterol obtained is suitable for combustion, zinc reduction of the water formed, and determination of deuterium isotopic content by gas isotope ratio mass spectrometry. The ability to detect and to quantitate a range of cholesterol synthesis rates is illustrated by measurements on young pigs receiving diets without and with added dietary cholesterol.

Animals↗

PubMed1991

Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins.

Procedures are presented for the separation and determination of the isotopic enrichment of multiple human apolipoproteins labeled in vivo with a stable isotope amino acid. The isotopic enrichments of plasma lysine and plasma apolipoproteins were monitored for 16 days after a single intravenous dose of [4,4,5,5-2H4]lysine (5 mg/kg body weight). The use of a multiply deuterated amino acid enabled the measurement of isotopic enrichments above background over the entire 16-day time course in all proteins. Individual apolipoproteins were separated on a specially designed gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis system cast in a conventional slab gel apparatus which resolved apoB-100, apoE, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III-1, and apoC-III-2 on a single gel. After staining with Coomassie blue, proteins bands (containing 5 to 30 micrograms of individual apolipoprotein) were excised from the gel. Amino acids were recovered from hydrolyzed gel slices, derivatized, and analyzed by gas chromatography-mass spectrometry for determination of lysine isotopic enrichments. The utility of the method is demonstrated using examples of apolipoproteins B-100, A-I, A-II, C-I, C-II, and C-III from either total plasma d less than 1.21 g/ml lipoproteins or selected lipoprotein subfractions. Lysine isotopic enrichments of proteins were generally determined with a precision of better than 5%. The isotopic enrichment profiles were consistent with literature reports of apolipoprotein metabolic kinetics based on the use of radioiodinated apolipoproteins. The procedures outlined can be used to separate and measure the isotopic enrichment of virtually any apolipoprotein from any chosen lipoprotein fraction. Thus, these procedures should find wide application in the study of apolipoprotein metabolic kinetics.

Adult↗

PubMed1990

Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics.

The production rates of apolipoprotein (apo)B-100 in very low density lipoprotein and in low density lipoprotein and apolipoprotein A-I in high density lipoprotein were determined using a primed-constant infusion of [5,5,5,-2H3]leucine, [4,4,4,-2H3]valine, and [6,6-2H2,1,2-13C2]lysine. The three stable isotope-labeled amino acids were administered simultaneously to determine whether absolute production rates calculated using a stochastic model were independent of the tracer species utilized. Three normolipidemic adult males were studied in the constantly fed state over a 15-h period. The absolute production rates of very low density lipoprotein apoB-100 were 11.4 +/- 5.8 (leucine), 11.2 +/- 6.8 (valine), and 11.1 +/- 5.4 (lysine) mg per kg per day (mean +/- SDM). The absolute production rates for low density lipoprotein apoB-100 were 8.0 +/- 4.7 (leucine), 7.5 +/- 3.8 (valine), and 7.5 +/- 4.2 (lysine) mg per kg per day. The absolute production rates for high density lipoprotein apoA-I were 9.7 +/- 0.2 (leucine), 9.4 +/- 1.7 (valine, and 9.1 +/- 1.3 (lysine) mg per kg per day. There were no statistically significant differences in absolute synthetic rates of the three apolipoproteins when the plateau isotopic enrichment values of very low density lipoprotein apoB-100 were used to define the isotopic enrichment of the intracellular precursor pool. Our data indicate that deuterated leucine, valine, or lysine provided similar results when used for the determination of apoA-I and apoB-100 absolute production rates within plasma lipoproteins as part of a primed-constant infusion protocol.

Adult↗

PubMed1989

Whole-body protein metabolism in lactating and nonlactating women.

The adaptive responses of body protein metabolism to lactation were characterized in women at 1, 5, and 12 mo postpartum and in nulliparous controls during a controlled diet of measured protein and energy intakes by nitrogen balance, a constant infusion of [13C]bicarbonate, and a primed constant infusion of [1-13C]leucine and [alpha-15N]-lysine. Dietary energy intakes in the lactating women were 27% greater than those in the nulliparous controls. Despite these differences, lactating women had significantly lower nitrogen balances compared with the nonlactating women (-4.0 +/- 37.8 vs. +44.7 +/- 30.8 mg.kg-1.day-1). No significant differences in amino acid flux, oxidation, or incorporation into protein were detected during fasting conditions in the two groups of women. However, significantly positive associations were noted between dietary intakes and the variables of protein metabolism in the lactating women. A more complete understanding of the mechanisms that regulate the disposition of dietary nutrients into maternal body stores or milk production will enhance the determination of nutrient requirements in lactating women.

Bicarbonates↗

PubMed1989

Human lactation. II: Endogenous fatty acid synthesis by the mammary gland.

We studied the effects of a diet that was low in fat, high in carbohydrate (CHO) on milk lipid composition and de novo endogenous fatty acid synthesis by the mammary gland in five lactating women. The women consumed either a low fat (LF) (5% fat, 80% CHO) diet or a high fat (HF) (40% fat, 45% CHO) diet. Fat synthesis was determined after an oral dose of 500 mg/kg D2O by measuring the incorporation of deuterium into C10:0 to C18:0 saturated fatty acids of milk fat and plasma triglycerides by gas chromatography-mass spectrometry. Synthesis of plasma C16:0 and C18:0 triglycerides was barely detectable while women consumed the HF diet, but was increased 6-fold during the LF diet. Medium chain fatty acids secreted by the mammary gland increased from 12.8% (HF diet) to 16.3% (LF diet) in milk fat from four of five subjects (p = 0.027). Medium chain fatty acid secretion, however, increased from 13.9% (HF diet) to 29.9% (LF diet) in one subject. The primary fatty acids synthesized during lactation were C10:0, C12:0, and C14:0 in the majority of women studied. The LF diet significantly increased the apparent synthesis of C14:0 (p = 0.05), whereas no changes were observed in C12:0, C16:0, or C18:0. One subject had highly enriched C16:0 and C18:0 fatty acids in her milk on the LF diet, which could have been the result of mammary synthesis or of transport and secretion of hepatically synthesized lipids.

Body Water↗

PubMed1989

Relationships among lactation performance, maternal diet, and body protein metabolism in humans.

The relationships between lactation performance and maternal diet and body protein metabolism were determined at 1, 5, and 12 months postpartum in lactating women who consumed a controlled diet of measured protein and energy. Milk production was measured by the 24-h test weighing procedure. Maternal body protein metabolism was evaluated by nitrogen balance and a primed, constant infusion of [1-13C]leucine and [alpha-15N]lysine. Milk production was associated positively with lysine flux (P less than 0.05, r = 0.59), leucine incorporation into body protein (P less than 0.05, r = 0.58), nitrogen intakes (P less than 0.05, r = 0.56), and energy intakes (P less than 0.01, r = 0.69). When adjusted for postpartum time, significant associations between total nitrogen concentrations in milk and nitrogen balance also were present (P less than 0.05, r = 0.77). These observations document associations among lactation performance, maternal diet, and the metabolic responses of body protein stores in well-nourished women and suggest strategies for the improvement of milk production in settings where nutrient insufficiency and malnutrition prevail.

Adult↗

PubMed1989

Incorporation of deuterium-labeled fatty acids into human milk, plasma, and lipoprotein phospholipids and cholesteryl esters.

Fatty acid metabolism and the contribution of dietary fatty acids to milk cholesteryl ester (CE) and phospholipid (PL) were investigated in normal lactating mothers. The approach used was to feed mixtures of triglycerides containing deuterium-labeled palmitic acid (16:0-2H2), oleic acid (18:1-2H6), and linoleic acid (18:2-2H4). Milk and plasma samples were collected for 72 hr. Triglyceride (TG), CE, and PL fractions from milk, plasma, and lipoprotein were isolated and analyzed by gas-liquid chromatography and mass spectrometry. Data for the milk CE and PL fractions showed a definite selectivity for incorporation of 16:0-2H2 and 18:1-2H6 relative to 18:2-2H4. Based on the ratios of the deuterated fatty acids incorporated into the milk CE and PL samples, their incorporation times and isotopic enrichment data, it appears that these fatty acids are supplied mainly by the TG derived from chylomicrons and very low density lipoproteins. Plasma and lipoprotein CE data showed a progressive increase in 18:2-2H4 content, with 16:0-2H2 and 18:1-2H4 remaining relatively constant over the collection period. Plasma and lipoprotein PL data showed a higher rate for incorporation of 18:2-2H4 than 16:0-2H2 and 18:1-2H6 over the course of the sampling period. Comparison to previous data from adult males indicates lactation does not have a major effect on the general metabolism of these fatty acids. An increase with time in the isotopic enrichment of 18:2-2H4 in the plasma and lipoprotein CE and PL samples was observed which is consistent with in vitro selectivities reported for lecithin:cholesterol acyltransferase and phosphatidylcholine acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol Esters↗

PubMed1988

Manipulation of maternal diet to alter fatty acid composition of human milk intended for premature infants.

Ten lactating mothers (five of preterm and five of term infants) 9-17 d postpartum consumed a 5% fat, 15% protein, and 80% carbohydrate diet for 5 d. Their milk was analyzed for fatty acid composition by gas chromatography. Significant increases in the sum of the absolute and relative concentrations of C10:0, C12:0, and C14:0 fatty acids and significant decreases in the absolute and relative concentrations of C18:0, C18:1, and C18:2 fatty acids were detected on day 4 in both groups (p less than 0.01). Women who delivered prematurely or at term responded similarly in early lactation to a low-fat, high-carbohydrate diet with an increase in the concentration of fatty acids less than 16 carbons in length. The magnitude of this response is highly variable and may be controlled by total energy balance as well as by individual endocrine responses.

Adult↗

PubMed1987

Human lactation: maternal transfer of dietary triglycerides labeled with stable isotopes.

A stable isotope tracer method was utilized to measure quantitatively the secretion of diet-derived fatty acids (FA) into human milk. A mixture of [2H6]tripalmitin, [2H18]-triolein, and [2H12]trilinolein was administered to three healthy, lactating women 22 to 30 years of age. Milk and blood samples were collected sequentially for 72 hr. The FA composition and concentration of total plasma, lipoprotein, and milk triglycerides were determined by gas-liquid chromatography (GLC) and the isotopic enrichment was determined by gas-liquid chromatography-mass spectrometry (GLC-MS). There were no statistically significant differences in mammary secretion of the individual fats, either by a single individual or between subjects. The mean secretion of fat by one breast was 5.11 +/- 1.26% of the dose (CV = 25%). There was a significant 6.0-hr delay between peak occurrence of the tracer in plasma and its occurrence in milk. The lipids are transported to the mammary gland primarily by the chylomicron and very low density lipoprotein triglycerides.

Adult↗

PubMed1985

Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry.

Methods have been developed for the simultaneous quantitative analysis of vitamin B6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry (GC/MS). The three resulting vitamers were analyzed by electron ionization GC/MS using a silicone capillary column. The methods have been applied to analysis of vitamin B6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.

Animals↗