Biomedical subjects

D L Hachey

Publications and source records attributed to D L Hachey.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1995

Impact of age on the metabolism of VLDL, IDL, and LDL apolipoprotein B-100 in men.

Levels of plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL) constituents increase with age. In an attempt to further define the mechanisms responsible for these changes, kinetic studies of VLDL and LDL apolipoprotein (apo) B-100 were carried out in 19 normolipidemic male subjects with plasma total cholesterol and triglyceride levels below the 90th percentile whose ages ranged from 24 to 73 years. Subjects were maintained on standardized diets consisting of 47-49% of calories as carbohydrate, 15% protein, and 36-40% fat (15-17% saturated, 15-17% monounsaturated, 6% polyunsaturated) with 150 mg cholesterol/1000 kcal. At the end of the diet period, the metabolism of apoB-100 within VLDL, intermediate density lipoprotein (IDL), and LDL was studied in the fed state using a primed-constant infusion of [2H3]leucine. Data were fit to a multicompartmental model to determine residence times and production rates of apoB-100 in each fraction. There were significant positive correlations between age and VLDL, IDL, and LDL apoB-100 concentrations (r = 0.50, 0.62, and 0.69; P = 0.03, 0.004, and 0.001, respectively). There was a positive correlation between age and the production rate of VLDL apoB-100 (r = 0.50, P = 0.03), but there was no significant relationship between age and either IDL or LDL apoB-100 production rates. Age was also positively correlated with the residence time of LDL apoB-100 (r = 0.68 P = 0.001). Our data suggest that the age-associated increase in VLDL apoB-100 is due to an increased production rate of this constituent, whereas the age-associated increase in LDL apoB-100 is due to an increased residence time of these particles in plasma.

Adult↗

PubMed1994

Microanalysis of cholesterol, phospholipid, and medium- and long-chain fatty acids in biologic materials.

We have developed a procedure for the microanalysis of the cholesterol and phospholipid concentrations and the fatty acid (including medium-chain fatty acids) composition of total lipid and phospholipid. One-milligram lipid samples were extracted from jejunal brush border membranes isolated from rats fed a diet containing 14% medium-chain triglycerides. The samples were analyzed by gas chromatography using an autosampler. We were able to identify 17 fatty acids in the total lipid and in the phospholipid fractions. The cholesterol and phospholipid contents of the membranes were 270 +/- 21 and 298 +/- 35 nmol.mg protein-1, respectively; thus the cholesterol/phospholipid ratio was 0.92 +/- 0.05, a value typically reported for the rat. Medium-chain fatty acids were not detected in the brush border membranes. Therefore, to demonstrate that medium-chain fatty acids were not lost during analysis, we analyzed the rat diet, and brush border membrane samples to which medium-chain triglycerides had been added. Recovery of medium-chain fatty acids from brush border membrane samples was 95.7 +/- 2.6%. The percentage of medium-chain fatty acids detected in the diet was not significantly different from the percentage when the diet was prepared. In summary, our procedure enabled a complete lipid analysis of very small biologic specimens.

Animals↗

PubMed1994

Stable isotopes for measurement of nutrient dynamics during pregnancy and lactation.

Only a few of the advantages of stable isotopes have been illustrated here. However, two valuable concepts have emerged in recent years; (1) the use of multiple isotopomers for comprehensive studies of a nutrient system, and (2) the use of multiple tracers for parallel investigations of different metabolic systems. These concepts provide flexibility in the conduct of clinical research, however, precautions are necessary to employ them properly. Nearly every example described here can be traced back to a pioneering study by Schoenheimer and Rittenberg. Certainly we do them faster, and more precisely, and with a deeper understanding of the biology, but the credit for this wonderful tool belongs to the original visionaries.

Animals↗

PubMed1994

An improved HPLC method to purify erythrocyte cholesterol for estimation of in vivo cholesterol synthesis using the deuterium method.

We report on an improved HPLC method to purify erythrocyte cholesterol for use in measuring in vivo cholesterol synthesis from 2H2O. The new procedure uses a smaller compression cartridge column at a flow rate of 3 mL/min. The new method reduces by almost tenfold the amount of solvent required to maintain the analytical purity of the isolated cholesterol.

Animals↗

PubMed1994

Benefits and risks of modifying maternal fat intake in pregnancy and lactation.

The National Cholesterol Education Program recommends that healthy Americans aged > 2 y reduce energy intake to maintain ideal body weight, saturated fat to 10% of energy, fat intake to 30% of energy, and cholesterol consumption to < 300 mg/d. Although these guidelines exclude pregnant or lactating women, nursing infants, and very young children, women with gestational diabetes, preeclampsia, and familial hyperlipidemias may benefit from them. In a normal pregnancy, serum cholesterol and triglycerides rise 25-40% and 200-400%, respectively. Multiparous middle-aged women may have an increased incidence of angina and cholesterol gallstones from the hypercholesterolemia of pregnancy. Few studies support the safety of maternal low-fat diets for the developing fetus or demonstrate benefits to the mother. Polyunsaturated fatty acids lower serum lipids, and n-3 fatty acids may improve some obstetric complications. Arachidonic acid (20:4) and docosahexaenoic acid (22:6) may benefit the psychomotor and visual development of children.

Adult↗

PubMed1994

Jejunal brush border hydrolase activity is higher in tallow-fed pigs than in corn oil-fed pigs.

We tested the effect of dietary fat on the lipid composition and hydrolase activity of jejunal brush border membranes in piglets. Eighteen 5-wk-old piglets were divided into three groups and for 4 wk fed either an unsaturated low fat diet (3.2% corn oil), an unsaturated high fat diet (17.2% corn oil) or a saturated high fat diet (2.2% corn oil + 15% tallow). Brush border membranes were prepared from the jejunal mucosa and analyzed for cholesterol, phospholipid and fatty acids. The activities of sucrase-isomaltase, lactase-phlorizin hydrolase, maltase-glucoamylase, aminopeptidase and alkaline phosphatase were measured. Lactase-phlorizin hydrolase isoforms were immunopurified and separated by SDS-PAGE, and their relative proportions were measured by densitometry. The activities of the disaccharidases and alkaline phosphatase, but not aminopeptidase, were greater in animals fed the saturated high fat diet than in animals fed the unsaturated high fat diet. The fatty acid composition of the membranes generally reflected the composition of the diet. Correlation analysis demonstrated that the phospholipid, fatty acid and cholesterol compositions of the membranes were associated with the differences in brush border hydrolase activity.

Animals↗

PubMed1994

Dietary cholesterol inhibits whole-body but not cerebrum cholesterol synthesis in young pigs.

We studied the effect of dietary cholesterol on tissue cholesterol synthesis in 10 genetically lean pigs using the 2H2O incorporation method. Five pigs were fed a diet containing 0% cholesterol (controls) and five were fed a diet containing 0.5% cholesterol (cholesterol-fed) from d 1 to d 54 of age. From d 30 to d 54, they received 2H2O and cholesterol synthesis was determined by measuring deuterium enrichment in erythrocyte cholesterol. All pigs were killed on d 54 and liver, ileum, jejunum, kidney, adipose tissue, muscle, and cerebrum were collected for measurement of free and esterified cholesterol and of the deuterium content of tissue free cholesterol. In vivo cholesterol synthesis was inhibited by dietary cholesterol as reflected by a decrease in fractional synthesis rate from 16.8 +/- 1.7%/d in controls to 2.3 +/- 1.1%/d in cholesterol-fed pigs. Cholesterol feeding raised cholesterol ester concentration in plasma, liver, and other tissues. Deuterium enrichment of free cholesterol in tissues, such as liver, was reduced from 11.98 +/- 2.82 milli atom percent excess in controls to 1.83 +/- 1.14 in cholesterol-fed pigs. Greater cholesterol intake did not inhibit cholesterol synthesis in the cerebrum. Deuterium enrichment levels of cholesterol in plasma and liver were similar to those in other tissues except for brain, suggesting an equilibration of cholesterol between the liver and most extrahepatic tissues. The control of cholesterol synthesis in brain appears to be independent of that in liver and other tissues of the body.

Animals↗

PubMed1994

Insulin, cortisol and thyroid hormones modulate maternal protein status and milk production and composition in humans.

The partitioning of dietary and endogenous nutrients during lactation is not well understood. To examine associations between plasma hormone and substrate profiles and indices of either maternal body protein metabolism or lactational performance, we measured plasma insulin, cortisol, prolactin, thyroxine, triiodothyronine, individual amino acid, blood urea nitrogen, and prealbumin concentrations in lactating and nulliparous women in the postabsorptive state. We related these measurements to the subjects' nitrogen balance, urinary 3-methylhistidine excretion, [1-13C]leucine metabolism and milk production. Insulin concentrations showed significant positive relationships with nitrogen balance and prealbumin concentrations; cortisol levels showed a significant negative relationship with nitrogen balance and a significant positive relationship with leucine incorporation into protein. Thyroid hormone concentrations showed significant positive relationships with urinary 3-methylhistidine excretion, leucine incorporation into protein, and milk production. Proline concentrations were associated positively with nitrogen balance and negatively with leucine incorporation into protein, whereas glutamate-glutamine concentrations showed positive associations with leucine oxidation and milk nitrogen concentrations. We propose that insulin and cortisol modulate the channeling of nutrients between anabolic and anti-anabolic aspects of maternal body protein metabolism, whereas thyroid hormones and cortisol modulate nutrient partitioning toward milk production and visceral protein synthesis. We suggest that some nonessential amino acids (proline, glutamate-glutamine) may become limiting during lactation because of their unique contributions to milk protein synthesis.

Adult↗

PubMed1994

Effects of infant nutrition on cholesterol synthesis rates.

Nutrient effects on cholesterol fractional synthesis rates (FSR) in infancy by stable isotope determination have not been studied. We hypothesized that FSR is significantly reduced with high dietary cholesterol and phytoestrogen intake and increased with low dietary cholesterol and phytoestrogen intake. We prospectively studied 33 term male infants exclusively fed human milk (high cholesterol, low phytoestrogen, n = 12), cow milk-based formula (low cholesterol, low phytoestrogen, n = 8), soy milk-based formula (zero cholesterol, high phytoestrogen, n = 7), or soy milk-based formula modified to contain cholesterol (low cholesterol, high phytoestrogen, n = 6) during the first 4 mo of life. Cholesterol FSR was determined from rate of incorporation of deuterium into erythrocyte membrane cholesterol, and urinary isoflavone excretion (an index of dietary phytoestrogen exposure) was measured by gas chromatography-mass spectrometry. Significant differences in cholesterol FSR were found. FSR (%/d) was lowest in human milk (2.62 +/- 0.38), highest in soy milk-based formula (9.40 +/- 0.51), and intermediate in cow milk-based and modified soy milk-based formula (6.90 +/- 0.48 and 8.03 +/- 0.28, respectively), p < 0.0001. Cholesterol FSR was significantly lower in modified soy milk-based compared with soy milk-based formula, p < 0.05. We also show for the first time that dietary phytoestrogens are absorbed and excreted by the infant fed soy protein-based formula. Urinary isoflavone excretion was inversely related to cholesterol FSR, but it was not significantly related to serum cholesterol concentration. We conclude that the type of infant nutrition and dietary cholesterol are major factors influencing cholesterol fractional synthesis rates in infancy.

Adaptation, Physiological↗

PubMed1993

In vivo sucrase-isomaltase and lactase-phlorizin hydrolase turnover in the fed adult rat.

We estimated in vivo turnover rates of sucrase-isomaltase and lactase-phlorizin hydrolase in adult rats. Fed animals received a primed continuous infusion of phenylalanine (300 microCi, 150 mumol Phe/100 g of body weight for 30 s, then 7.5 microCi, 3.75 mumol Phe/min for 10 to 140 min). Sucrase-isomaltase and lactase-phlorizin hydrolase were immunoprecipitated from jejunal mucosal membranes; isoforms were separated by SDS-polyacrylamide gel electrophoresis. Endoglycosidase H digestions and (for lactase-phlorizin hydrolase) N-terminal amino acid sequencing were performed on all isoforms. Specific radioactivity of prosucrase-isomaltase and prolactase-phlorizin hydrolase isoforms reached isotopic equilibrium by 60 and 90 min, respectively. Specific radioactivity of brush border sucrase and lactase did not reach steady state. The isotope kinetic, N-terminal amino acid sequencing, and endoglycosidase H digestion data suggested that one of the high molecular weight lactase isoforms is a dimer of mature lactase. Compartmental modeling of specific radioactivity demonstrated that mean intracellular residence time is 59 min for prosucrase-isomaltase isoforms and 68 min for prolactase-phlorizin hydrolase isoforms. Mean residence time in the brush border was 5.8 h for sucrase and 7.8 h for lactase. Fractional synthesis rates were 414%/day for sucrase and 307%/day for lactase. Thus, in vivo brush border sucrase and lactase turn over at similar rates in the adult rat.

Amino Acid Sequence↗

PubMed1993

Body weight gain is correlated with serum cholesterol at 8 weeks of age in pigs selected for four generations for low or high serum cholesterol.

We determined the relationship of BW at birth, weaning (4 wk of age), and 8 wk of age to serum total cholesterol (C), high-density lipoprotein-cholesterol (HDL-C), and triglycerides (TG) at 8 wk of age in pigs, from the fourth generation that had been selected for low (10 litters, 75 pigs, LC) or high (10 litters, 63 pigs, HC) C at 8 wk of age. Mean C concentration at 8 wk of age was 81 +/- 30 mg/dL for LC groups and 136 +/- 19 mg/dL for HC groups. Serum C, HDL-C, and TG concentrations were not correlated with birth weight, suggesting that the physiological factors that may cause reduced weight gain in older animals are not operative in newborn pigs. All three constituents were correlated (P < .05) with BW at weaning and at 8 wk. However, only 4% of the variation in weight at weaning and 7% at 8 wk could be explained by a relationship with serum TG. There was a positive correlation between C and BW at 8 wk (r = .46, P < .05), which was apparent within the subgroups of LC and HC females and LC males (r = .46, .48, .68, respectively); the correlation was low (r = .26) in HC males.

Animals↗

PubMed1993

Effect of dietary cholesterol on cholesterol synthesis in breast-fed and formula-fed infants.

The fractional synthesis rate (FSR) of cholesterol was measured in 6 breast-fed and 12 formula-fed infants (ages 4 to 5 months) using the 2H2O method. The breast-fed infants had higher cholesterol intakes (18.2 +/- 4.0 vs. 3.4 +/- 1.8 mg/kg per day, P = 0.001), plasma total cholesterol (183 +/- 47 vs. 112 +/- 22 mg/dl, P = 0.013), and plasma low density lipoprotein (LDL)-cholesterol (83 +/- 26 vs. 48 +/- 16 mg/day, P = 0.023) than the formula-fed infants (6.9 +/- 2.6 vs. 2.1 +/- 0.6%/day, P < 0.001). Among all infants, there was a significant inverse relationship (P = 0.002, r = 0.66) between the FSR of cholesterol and dietary cholesterol intake. Our findings indicate that the greater cholesterol intake of the breast-fed infants was associated with elevated plasma LDL-cholesterol concentrations and that cholesterol synthesis in human infants may be efficiently regulated via HMG-CoA reductase when infants are challenged with high intakes of dietary cholesterol.

Breast Feeding↗