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Biomedical subjects

R T Holman

Publications and source records attributed to R T Holman.

At least 19 recordsLinked to original sources
PubMed1995

Isolation and characterization of rat cholangiocyte vesicles enriched in apical or basolateral plasma membrane domains.

Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, are composed of plasma membranes with discrete apical (lumenal) and basolateral domains. While these domains are thought to contain different transporters, exchangers, channels, and receptors, no methodology currently exists for the isolation of these different membrane compartments. Thus, our aim was to develop a technique to isolated plasma membranes from cholangiocytes enriched in apical or basolateral domains. We isolate a cholangiocyte-enriched population of cells from rats 3 weeks after bile duct ligation (BDL), a maneuver which stimulates selective cholangiocyte proliferation. Using isopycnic centrifugation on linear sucrose gradients, we prepared a mixed cholangiocyte plasma membrane (MCPM) fraction from which we further generated separate apical and basolateral cholangiocyte plasma membrane (ACPM and BCPM, respectively). We characterized these fractions by specific marker enzyme assays, transmission electron microscopy (TEM), lipid analysis, anisotropy measurements, one- and two-dimensional gel electrophoresis, and quantitative immunoblots of the cystic fibrosis transmembrane conductance regulator (CFTR). Marker enzyme assays and TEM revealed that the MCPM fraction was essentially devoid of other organelles but was enriched approximately 70-fold in phosphodiesterase I, a general plasma membrane marker; the ACPM and BCPM were appropriately enriched in the respective apical and basolateral markers. TEM of ACPM and BCPM revealed homogeneous preparations of vesiculated membranes without contamination by other organelles. Lipid analysis, one- and two-dimensional gel electrophoresis, CFTR immunoblots, and anisotropy measurements showed unequivocal differences in lipid and protein composition and in fluidity between the ACPM and BCPM domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Patients with anorexia nervosa demonstrate deficiencies of selected essential fatty acids, compensatory changes in nonessential fatty acids and decreased fluidity of plasma lipids.

The objective of this study was to assess the essential fatty acid status of patients with anorexia nervosa. Blood was collected from eight fasting female anorexia nervosa patients with a mean of 81% ideal body weight. Fatty acid composition of phospholipids, nonesterified fatty acids, triglycerides and cholesteryl esters of plasma were determined by capillary gas chromatography to indicate polyunsaturated fatty acids status compared with 19 healthy female adults < 25 y old. Subjects with anorexia nervosa showed polyunsaturated fatty acid deficiencies in plasma phospholipids different from simple nutritional essential fatty acid deficiency or chronic malnutrition. The phospholipid profile showed significantly lower (n-6) and (n-3) elongation and desaturation products, and elevated short-chain saturated, short-chain monounsaturated, branched-chain and odd-chain fatty acids. These elevations indicate enhancement of biosynthesis of alternative fatty acids that only partially compensated for the loss of polyunsaturated fatty acids in providing membrane "fluidity." Calculated mean melting point of the fatty acids of phospholipids in patients with anorexia nervosa was elevated 7.7 degrees C above normal values. These results demonstrate that patients with anorexia nervosa have deficiencies of selected essential fatty acids, compensatory changes in nonessential fatty acids and decreased fluidity of plasma lipids.

Adolescent↗
PubMed1994

Serum fatty acid profiles in cystic fibrosis patients and their parents.

Fatty acid compositions of the major serum lipid classes from 43 cystic fibrosis (CF) homozygotes (CF patients), 36 obligate heterozygotes (parents of CF patients) and 34 controls were determined by capillary gas chromatography. Fatty acid compositions of the homozygote CF patients were skewed in the direction of relative essential fatty acid deficiency in comparison with the controls. Less pronounced, but similar deviations from normal, were observed in the heterozygotes. Homozygotes with normal fatty acid compositions and heterozygotes with considerably disturbed fatty acid profiles were found.

Adolescent↗
PubMed1994

Diets rich in lean beef increase arachidonic acid and long-chain omega 3 polyunsaturated fatty acid levels in plasma phospholipids.

Diets rich in meat are claimed to contribute to the high tissue arachidonic acid (20:4 omega 6) content in people in Westernized societies, but there are very few direct data to substantiate this assertion. Because meat contains a variety of long-chain polyunsaturated fatty acids (PUFA) that are susceptible to oxidation, we initially examined the effect of cooking on the long-chain PUFA content of beef, and then determined the effect of ingestion of lean beef on the concentration of long-chain PUFA in plasma phospholipids (PL). First, we examined the effect of grilling (5-15 min) and frying (10 min) different cuts of fat-trimmed lean beef on the long-chain PUFA content. Second, we investigated the effect of including 500 g lean beef daily (raw weight) for 4 wk on the fatty acid content and composition of plasma PL in 33 healthy volunteers. This study was part of a larger trial investigating the effect of lean beef on plasma cholesterol levels. In the first two weeks, the subjects ate a very low-fat diet (10% energy) followed by an increase in the dietary fat by 10% each week for the next 2 wk. The added fat consisted of beef fat, or olive oil (as the oil or a margarine) or safflower oil (as the oil or a margarine). This quantity of beef provided 60, 230, 125, 140 and 20 mg/d, respectively, of eicosatrienoic acid (20:3 omega 6), 20:4 omega 6, eicosapentaenoic acid (20:5 omega 3), docosapentaenoic acid (22:5 omega 3) and docosahexaenoic acid (22:6 omega 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Human subcutaneous adipose tissue shows site-specific differences in fatty acid composition.

Adipose tissue was obtained from six women undergoing liposuction twice at 6-mo intervals. Samples obtained bilaterally from abdomen, inner thigh, and outer thigh had fatty acids quantified by gas chromatography. There were no important differences between sides or over time. The saturates 14:0, 16:0, 18:0, and 20:0 were higher in abdominal adipose than in outer thigh (P < 0.002 for all); 16:1 and 18:1 omega 9 were lower in abdomen vs outer thigh (P < 0.01), whereas 18:1 omega 7 and 20:1 omega 9 were unchanged. Polyunsaturates 18:2 omega 6, 20:3 omega 6, and 20:4 omega 6 were higher in outer thigh than in abdomen (P < 0.06), and inner thigh values were intermediate. These changes in fatty acid composition resulted in lower mean triglyceride melting points from abdomen to inner thigh to outer thigh, and suggest that temperature may influence the selection process determining the variation in adipose fatty acid composition with anatomical location. Because the site-specific differences included essential fatty acids, selective uptake as well as potential differences in in situ fatty acid modification are indicated.

Abdomen↗
PubMed1991

Fatty acid composition of hepatic triglycerides in Reye's syndrome: implications for hepatic desaturase abnormalities.

Serum NEFA profiles in Reye's syndrome are reportedly unique with a disproportionate percent made up of polyunsaturated fatty acids some of which are not ordinarily found in the serum. This pattern is also reflected in the serum triglyceride composition as well. As the liver is probably the sole source of the serum triglyceride in Rye's syndrome because patients are vomiting or in coma, the fatty acid acid composition of the liver triglyceride was examined for insight regarding the lipid abnormalities in this disease. Palmitic acid (16:0) and the sum of all the saturated fatty acids in the liver triglycerides were significantly decreased whereas the sum of the monoenoic fatty acids and the products of delta 9 desaturase activity were increased in Reye's samples. When these data were compared to the fatty acid composition of the serum triglyceride from a separate cohort of Reye's and control subjects, certain inferences regarding hepatic delta 9, delta 6, delta 5, and delta 4 desaturase activities and the elongases can be drawn from the liver and serum triglyceride fatty acid profiles which are unique. Collectively, these data reflect considerable intrahepatic fatty acid desaturation and elongation activity and/or acyl transfer from lipid to lipid of various polyunsaturated fatty acids in Reye's syndrome.

Adolescent↗
PubMed1991

Unusual isomeric polyunsaturated fatty acids in liver phospholipids of rats fed hydrogenated oil.

Linoleic acid (18:2 omega 6) and linolenic acid (18:3 omega 3) are precursors of two series of essential fatty acids (EFA) formed by alternate desaturations and elongations. In EFA deficiency (EFAD), oleic acid (18:1 omega 9) and palmitoleic acid (16:1 omega 7) undergo the same reactions to form polyunsaturated fatty acids (PUFA) of other structures. Partially hydrogenated soybean oil (PHSO) contains isomeric 18:1 acids that can be converted to unusual isomers of 18:2 by liver microsomes. To test whether 18:2, 20:3, and 20:4 of unusual structure occur in phospholipids as a consequence of EFAD or ingestion of PHSO, rats were fed corn oil, an EFA-deficient diet, or PHSO to provide isomeric 18:1 acids. At 2.5 months the phospholipids were isolated from livers and converted to methyl esters, and the 18:2, 20:2, 20:3, and 20:4 fractions were isolated. The 18:2 and 20:2 fractions were ozonized, and, by using a computer solution of simultaneous equations, the structures and proportions of each isomer were calculated. The 20:3 and 20:4 fractions were analyzed by ozonolysis and capillary gas chromatography. When corn oil was fed, the major isomer in each group was 9,12-18:2, 11,14-20:2, 8,11,14-20:3, and 5,8,11,14-20:4. Patterns in EFAD- and PHSO-fed groups were more diverse, with large proportions of unusual isomers. Feeding EFA-deficient diet and PHSO induced measurable amounts of unusual PUFA at each step of the cascade, and these PUFA may compete in metabolism of normal PUFA and are substrates for oxidative formation of autacoids of unknown structures and function.

Animals↗
PubMed1991

Deficiency of essential fatty acids and membrane fluidity during pregnancy and lactation.

In a group of 19 normal pregnant women, plasma lipids were extracted, phospholipids were isolated, and the fatty acid (FA) compositions were measured by capillary gas chromatography. Blood samples were taken at 36 wk, at labor, and at 6 wk postpartum. The FA profiles showed deficiencies of omega 6 and omega 3 FA (omega indicating the length of the terminal saturated chain), the latter more severe, at all three times. Mean melting point (MMP) was calculated for each sample as an index of "fluidity" based upon all FA present. MMP varied linearly with total polyunsaturated FA and with double bond index, current measures of "fluidity" and essential FA status. MMP was elevated 9-11 degrees C in plasma phospholipids of women during pregnancy and labor and postpartum. Lactating mothers showed less recovery from the deficiencies than did the nonlactating mothers, but neither approached normal at 6 wk. The changes seen in phospholipid profiles suggest a significant transfer of omega 3 and omega 6 polyunsaturated FA from the mother to the fetus. These FA are essential for normal fetal growth and development; their relative deficiency in maternal circulation suggests that dietary supplementation may be indicated.

Adult↗
PubMed1991

Obesity and weight loss alter serum polyunsaturated lipids in humans.

Serum omega 6 (n-6) fatty acids were assessed in 12 obese women during an outpatient very-low-calorie diet (VLCD). Ten subjects (S10) achieved a mean weight loss of 17 kg over 3-5 mo (initial weight-for-height 157%). Serum was obtained before (baseline) and monthly during the VLCD and from five of them (S5) after 2-3 mo of weight stability (refed) at 21 kg of loss. At baseline for S10, the serum phospholipid (PL) 20:4 omega 6 was 9.16 wt% and differed from normal (12.81 wt%) by P less than 0.0001, but cholesterol ester (CE) 20:4 omega 6 did not differ from normal. During 3 mo of VLCD, the S10 serum PL and CE 18:2 omega 6 fell (P less than 0.005 and 0.0001, respectively). Serum PL 20:4 omega 6 rose to normal during VLCD months 1-3 (P less than 0.01) while the serum CE 20:4 omega 6 rose above normal (P less than 0.0002). During the VLCD, S5 results paralleled S10. However when refed, S5 PL and CE 18:2 omega 6 and 20:4 omega 6 all reverted to baseline (PL 20:4 omega 6 below normal, P less than 0.001). Serum PL 20:4 omega 6 is low in moderate obesity, corrects to normal during a VLCD, but regresses to the predict abnormality after weight loss.

Adult↗
PubMed1991

Essential fatty acid status and fluidity of plasma phospholipids in cystic fibrosis infants.

The fatty acid (FA) patterns of cord serum phospholipids (PLs) were examined in 4 cystic fibrosis (CF) newborns, 8 non-CF siblings, and 22 normal control subjects. Plasma from 27 newly diagnosed CF infants and 38 normal infants aged less than 2 y were studied for comparison. CF cord-blood PLs had patterns similar to those of CF siblings and to normal newborns, but the pattern for CF did not shift toward adult patterns during infancy as did patterns for normal infants. CF infants at diagnosis exhibited a deficiency pattern in which 18: 2 omega 6, 20:4 omega 6, 22:4 omega 6, an omega 3 polyunsaturated fatty acids (PUFAs) were significantly subnormal and 18:3 omega 6, total saturated fatty acids, and total monounsaturated fatty acids were significantly elevated compared with normal infants. In PLs in CF infants, although mean chain length of FAs was low, mean melting point was elevated 2.4 degrees C and double-bond index was low (both P less than 0.001), implying a significantly lessened fluidity. Nutritional supplements of both omega 6 and omega 3 PUFAs are cated.

Aging↗
PubMed1991

Omega 3 polyunsaturated fatty acids as modulators of cellular function in the critically ill.

Continued improvement in outcome in ICU patients with the hypermetabolism-organ failure syndrome require a better understanding of the disease process. Current research is focusing on altered regulation at the cell membrane and nuclear levels. Cell culture models have provided insight into one possible mechanism, that of altered cell-cell communication with dysregulation of the associated parenchyma. Alteration of the PUFA content of the membrane of macrophages with omega 3 PUFA can be easily induced and maintained, and can alter cell membrane physical characteristics and how the membrane responds to LPS-stimulated prostanoid release. There is also an associated alteration in the release of monokine. These changes are associated with improvements in T lymphocyte response to antigen and in outcome from septic peritonitis. The precise mechanisms through which these effects occur are the subject of investigations, as are the clinical implications. Nonetheless, nutrient pharmacology with omega 3 PUFA may be a promising area of research that will have clinical applicability in ICU patients.

Cell Membrane↗
PubMed1990

Alteration of the cellular fatty acid profile and the production of eicosanoids in human monocytes by gamma-linolenic acid.

We administered borage seed oil (9 capsules/day) for 12 weeks to 7 normal controls and to 7 patients with active rheumatoid arthritis. The therapy provided 1.1 gm/day of gamma-linolenic acid (GLA). GLA administration resulted in increased proportions of its first metabolite, dihomo-gamma-linolenic acid (DGLA), in circulating mononuclear cells. The ratios of DGLA to arachidonic acid and DGLA to stearic acid increased significantly in these cells. Significant reductions in prostaglandin E2, leukotriene B4, and leukotriene C4 produced by stimulated monocytes were seen after 12 weeks of GLA supplementation. The antiinflammatory effects of GLA administration observed in animal models, and the apparent clinical improvement experienced by 6 or 7 rheumatoid arthritis patients given borage seed oil in this open, uncontrolled study may be due in part to reduced generation of arachidonic acid oxygenation products.

Administration, Oral↗
PubMed1990

Reduced adipose 18:3 omega 3 with weight loss by very low calorie dieting.

The human undergoing rapid and sustained weight loss by very low calorie dieting (VLCD) derives the majority of daily energy needs from adipose fatty acids. To evaluate the rates of metabolic utilization of individual fatty acids in humans, two groups of adult women outpatients were studied during major weight loss by VLCD. The diets used were either food or formula, providing the recommended dietary allowance for minerals and vitamins, with fat contents of 2-20 g/d. Group 1 consisted of 10 subjects [initial body mass index (BMI) 32.7, 157% of ideal body weight (IBW)] with a mean loss of 17.7 kg in 3-5 months. Group 2 consisted of 14 subjects (initial BMI 36.7, 167% of IBW) with a mean loss of 25.6 kg in 4-5 months. Adipose tissue biopsies were obtained by needle aspiration from Group 1 before and after weight loss and from Group 2 before, at the midpoint, and after weight loss. With weight loss in Group 1, the adipose tissue content of 18:1 omega 9, 18:2 omega 6, and 20:4 omega 6 did not change, but 18:3 omega 3 fell (0.67 to 0.56 wt%, p less than 0.0001) as did 20:5 omega 3 (0.08 to 0.05, p less than 0.01). Adipose tissue 22:6 omega 3 rose from 0.03 to 0.07 (p less than 0.01). In Group 2, only 18:3 omega 3 showed a change, falling from 0.71 to 0.69 to 0.59 wt% across weight loss (p = 0.03 by analysis of variance). We conclude that the major fatty acids are oxidized in proportion to their composition in adipose triglyceride.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1990

Influence of hyperalimentation on rat hepatic microsomal fluidity and function.

Total parenteral nutrition with an amino acid-glucose solution has previously been shown to decrease rat hepatic drug metabolism compared with drug metabolic activity observed in rats receiving the same solution enterally and chow-fed animals. Because changes in membrane fluidity and lipid composition are reported to influence activity of a number of liver enzymes, effects of parenteral and enteral nutrition on hepatic microsomal membrane fluidity and lipid composition were assessed and compared with hepatic mixed-function oxidase activity. Both parenteral and enteral hyperalimentation produced a significant decrease in microsomal membrane fluidity (fluorescence anisotropy = 0.155 +/- 0.003 in both experimental groups versus 0.129 +/- 0.003 for microsomes from chow-fed animals). However, meperidine demethylase activity was significantly decreased compared with chow-fed experiments only in hepatic microsomes from parenterally hyperalimented animals, whereas ethoxyresorufin deethylase activity was significantly reduced only in the enteral-nutrition group. Inclusion of lipid in the parenterally administered hyperalimentation solution normalized microsomal membrane fluidity and lipid profile to those of chow-fed animals but did not increase hepatic meperidine demethylation. Both parenteral and enteral nutrition produce significant changes in physical state and lipid composition of rat hepatic microsomal membranes, but these changes are not responsible for the altered hepatic drug metabolism observed during hyperalimentation.

Animals↗
PubMed1990

Reduced arachidonate in serum phospholipids and cholesteryl esters associated with vegetarian diets in humans.

Lipid fractions such as phospholipids (PLs), cholesteryl esters (CEs), and free fatty acids (FFAs) represent source pools for eicosanoid synthesis. To determine whether dietary habits affect the enrichment of 20:4n-6 in these precursor pools, we studied humans with partial or complete arachidonate restriction resulting from chronic avoidance of animal fat and tissue. Fasting serum was obtained from omnivorous control subjects (Omni, n = 100), semivegetarians (Semiveg, n = 16), and vegetarians (Veg, n = 25). PLs, CEs, FFAs, and triglyceride (TG) fatty acids were quantitated by thin-layer and gas chromatography. Serum 20:4n-6 was lower in the PL fraction in both Veg (p less than 0.01) and Semiveg groups (p less than 0.05) than in the Omni group and lower in the CE fraction in the Veg group (p less than 0.05). Serum 18:2n-6 did not differ between groups for any serum lipid fraction. 18:3n-3 was elevated in PLs and CEs of both Veg (p less than 0.05 and 0.01) and Semiveg groups (p less than 0.05 and 0.01) compared with the Omni group but did not result in differences in 20:5n-3 in PLs or CEs between diet groups. The lower concentration of 20:4n-6 in serum PLs and CEs of the Veg group indicates that dietary arachidonic acid enriches its circulating pool in humans; however, 20:5n-3 is not similarly responsive to dietary restriction.

Adult↗