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PubMed1986

Radiometric method for the rapid detection of Leptospira organisms.

A rapid and sensitive radiometric method for detection of Leptospira interrogans serovar pomona and Leptospira interrogans serovar copenhageni is described. Stuart's medium and Middlebrook TB (12A) medium supplemented with bovine serum albumin, catalase, and casein hydrolysate and labeled with 14C-fatty acids were used. The radioactivity was measured in a BACTEC 460 (Johnston Laboratories). With this system, Leptospira organisms were detected in human blood in 2 to 5 days, a notably shorter time period than that required for the majority of detection techniques.

Humans↗

PubMed1993

Metabolism of a long-chain diacylglycerol by permeabilized A10 smooth muscle cells.

The regulatory effects of diacylglycerol (DAG) second messengers will be terminated by metabolism. A long-chain DAG, 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol (2-[14C]POG), was metabolized by cultured A10 smooth muscle cells after permeabilization by preincubation with 340 U/ml alpha-toxin from Staphylococcus aureus. In contrast to results with the cell-permeable DAG analogue, dioctanoyl-glycerol ([3H]diC8), no appreciable 2-[14C]POG degradation could be detected in control A10 cells not treated with alpha-toxin. With permeabilized A10 cells, 2-[14C]POG was mainly converted into lipolytic products of a lipase pathway, monoacylglycerol (MG) and fatty acid (FA); very little radioactivity was incorporated into triacylglycerol (TG) or phospholipid (PL) via reactions catalyzed by either DAG acyltransferase, cholinephosphotransferase, or DAG kinase. Similar results were obtained in experiments with 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol. The conversion of 2-[14C]POG into PL and TG was not enhanced by the addition of 1 mM ATP-MgCl2, 1 mM CDP-choline, or 1 mM oleoyl-CoA to the alpha-toxin-treated A10 cells. The formation of FA and MG by permeabilized A10 cells was inhibited by DAG lipase inhibitors, U-57,908 (50 microM) and tetrahydrolipstatin (1-25 nM). The predominant contribution of the lipase pathway to the metabolism of a long-chain DAG, 2-[14C]POG, by alpha-toxin-treated A10 cells is similar to results for the degradation of [3H]diC8 by intact A10 cells.

Animals↗

PubMed1969

Effect of diet and triiodothyronine on the activity of sn-glycerol-3-phosphate dehydrogenase and on the metabolism of glucose and pyruvate by adipose tissue of obese patients.

Lipogenesis and the metabolism of sn-glycerol-3-phosphate were studied in 23 fat biopsies from eight grossly obese patients. The first biopsy was obtained after a minimum of 12 days on a 3500 cal diet, the second biopsy after 2 wk on a 900 cal diet, and the third biopsy after an additional 2 wk on 900 cal supplemented with thiiodothyronine, 250 mug/day.Oxygen consumption and respiratory quotient declined during caloric restriction. Oxygen consumption was restored to the initial level during treatment with triiodothyronine, and the respiratory quotient rose somewhat.Lipogenesis from glucose and pyruvate was demonstrated in fat obtained from the first biopsy but could not be detected in the other biopsies. The incorporation of radioactivity from pyruvate into fatty acids was stimulated by the addition of glucose. Insulin stimulated lipogenesis in pieces of fat from the first biopsy, but isolated fat cells were unaffected by insulin. After caloric restriction no effects of insulin could be detected. The activity of both the cytoplasmic and mitochondrial sn-glycerol-3-phosphate dehydrogenase in homogenates of adipose tissue declined with caloric restriction. Treatment with triiodothyronine enhanced the activity of the mitochondrial sn-glycerol-3-phosphate dehydrogenase but did not affect the cytoplasmic enzyme.

Adipose Tissue↗

PubMed1977

Lactogenesis in explant cultures of mammary tissue from pregnant cows.

The hormonal requirements for lactogenesis were investigated using explant cultures of mammary tissue obtained from cows at 30-40 days prepartum. Hormones used were insulin, hydrocortisone, and prolactin, and parameters examined were radioactive acetate incorporation into fatty acids, secretory response ratings, and histological and ultrastructural analysis. Data indicated that insulin was essential for mammary epithelial cell survival, but insulin alone did not result in the intiation of milk synthesis. The culture of explants in a medium containing insulin plus hydrocortisone resulted in alterations in the cytology of alveolar cells but no induction of milk synthesis. Biosynthesis results and secretory response ratings indicated that the initiation of milk synthesis occurred when explants were cultured in insulin and prolactin; however, synthesis was limited and alveolar integrity was not well maintained. Results from all parameters demonstrated that the maximal lactogenic response was obtained when the culture medium contained insulin, hydrocortisone and prolactin. After 48 h of culture in this medium, the rate of acetate incorporation into fatty acids had increased 3-fold and the markedly distended alveolar lumina contained many fat droplets and abundant eosinophilic staining secretion. However, an unusal amount of casein-like micelles and especially lipid also accumulated in the alveolar cells. The accumulation of milk components within the epithelial cells may be related indirectly to the accumulation of products in the lumina or perhaps related to a difference in the hormonal requirement between the initiation of milk synthesis and initiation of milk secretion.

Acetates↗

PubMed1979

Covalent cross-linking of photosensitive phospholipids to human serum high density apolipoproteins (apoHDL).

Human serum high density apolipoproteins were reassociated with three different lecithin species substituted with radioactively labelled photosensitive azido fatty acids, bis([3H]-16-azidopalmitoyl)-, bis([3H]12-azidooleoyl)- and bis([3H]18-azidolinoleoyl)glycerophosphocholine. The lipoprotein particles were reconstituted from a mixture of azido-labelled phosphatidylcholine and non-labelled dioleoylglycerophosphocholine (1:9). Excess lipid was separated from the homogeneous particles by Bio-Gel A-5m. The molecular weight, stoichiometry, fluorescence and circular dichroism of the reconstituted particles were determined before and after photoactivation with covalent cross-linking of the phospholipids with the apoproteins. The physical parameters of the reconstituted lipoproteins remained unperturbed by the cross-linking reaction between the generated nitrenes and apolipoprotein A-I and A-II. Thus the hydrophobic interactions of the phospholipid molecules with the apoproteins have been proved for the first time by a chemical method.

Apolipoproteins↗

PubMed1987

Improved method for the determination of phospholipase A2 catalytic activity concentration in human serum and ascites.

An improved radiochemical method is presented for the selective determination of phospholipase A2 activity in human serum and ascites, using only commercially available reagents. The method can be applied to large quantities of samples. As substrate we used 1,2-dipalmitoyl-sn-glycero(3)phosphorylcholine and phosphatidylcholine containing tritiated palmitic acid in position 2 (1-palmitoyl,2-[9,10-3H]palmitoyl-sn-glycero(3)phosphorylcholine). The liberated fatty acids are extracted and radioactivity is detected in a liquid scintillation spectrometer. A preliminary reference range of human serum samples was established ranging up to 1.0 U/l. In sera of patients with acute pancreatitis we found activities up to 20 U/l. The correlation of phospholipase A2 activity with that of other enzymes and with the severity and complications of acute pancreatitis was investigated. A possible relationship between phospholipase A2 activities and pulmonary complications is discussed.

Ascites↗

PubMed1982

Incorporation of [U-14C] palmitate into lipids by the stable fly, Stomoxys calcitrans (L.).

The incorporation in vivo of [U-14C] palmitate into fat body lipids was studied in the blood feeding stable fly, Stomoxys calcitrans. Palmitate was rapidly esterified into triacylglycerol and after one-half h, nearly one third of the total recovered label was in the triacylglycerol fraction with a concomitant loss of radioactivity in the free fatty acids. The fat body, from one day sugar-water-fed flies incorporated more label into triacylglycerol than those from flies fed on blood. The present study indicates that fat body from sugar-water-fed stable flies retains a high lipogenic ability and that availability of proper precursors may be a limiting factor in lipogenesis. The incorporation of [14C] palmitate into triacylglycerol decreased during subsequent blood meals, except for an increased incorporation into triacylglycerol by the fat body of males after the fourth blood meal. Flies fed five blood meals showed the least incorporation of label into fat body lipids after [14C] palmitate injection. The capacity for incorporation of [14C] palmitate into fat body lipids was reduced in starved stable flies. In addition, the lipids isolated from starved flies showed reduced specific activities.

Adipose Tissue↗

PubMedDate not supplied

[Lipid metabolism and lipogenesis: application of stable isotopes].

Fat metabolism is regulated by several neuroendocrine and nutritional factors, which affect equilibrium between lipogenesis and lipolysis. Lipid utilization and fate in the organism can be assessed by in vivo and in vitro methods by measuring the rate of the different metabolic pathways (dynamic aspects), but also the net balance which may lead to fat accumulation or loss (static aspects). The quantitation of synthesis and breakdown reactions can be performed by using different tracers such as radioactive and stable isotopes. Fatty acid synthesis can be independently measured by the intravenous infusion of 13C acetate and application of the MIDA technique. In brief, this method uses probability analysis to measure the synthesis of biological polymers. It is based on the mathematical principle that the labeling pattern of a polymer synthesized from a stable-isotopically labeled precursor will conform to a predicted binomial or multinomial expansion. Thus, the isotopic enrichment of the precursor pool is calculated from measurements on the product alone. In the case of fatty acid synthesis, the proportions of excess (above natural background abundance) of single-labeled and double labeled (EM1 and EM2 species respectively) are a function of the probability (p) that the precursor subunits were isotopically labeled. Using this value of P for the isotopic enrichment of the acetylCoA pool, the theoretical 13C enrichment in the fatty acid if 100% were newly formed from this acetate pool is calculated. The actual isotopic enrichment is measured by gas chromatography-mass spectrometry (GCMS). This value divided by the theoretical maximum value equals the fraction of the fatty acid that is newly formed (f). The value for f represents dilution of de novo synthesized fatty acid by non-de novo sources. This method requires that newly synthesized (labeled) and preformed (unlabeled) mix in the liver and communicate with plasma VLDL over the period of the isotope infusion. It also assumes that the major de novo fatty acid is only a single fatty acid, with minor elongation and/or desaturation processes. Finally, the infused isotopic acetate should have no physiologically important effect. This methodology can be applied to assess lipogenesis in very different nutritional and physiopathological conditions such as diabetes, AIDS, obesity, etc.

Acetates↗

PubMed1990

Linoleate and linolenate desaturation by rat hepatoma cells.

Morris 7777 rat hepatoma cells in culture possess high delta 6 and delta 5 desaturase activities over linolenic acid added to the medium as albumin or alpha-fetoprotein complexes. After 2 hours incubation with [1-14C] linolenic acid (7 microM), around 40% of the radioactivity was recovered in other polyene fatty acids, mainly pentaenes. After 24 hours incubation with this substrate the polyene derivatives raised to more than 60%. However, [1-14C] linoleic acid was poorly converted to other polyene fatty acids. Linoleic acid up to 58 microM concentration in the medium do not inhibited linolenic acid desaturation. Long-term supplementation with 50 microM linoleic or linolenic acid, which modified the fatty acid profile of hepatoma lipids, enhanced the desaturase activities against linoleic acid. Desaturase activities were not affected by the fatty acid protein carrier, alpha-fetoprotein or albumin.

Animals↗

PubMed1976

A stable, radioactive substrate emulsion for assay of lipoprotein lipase.

A method is described for the assay of lipoprotein lipase, using a stable, radioactive substrate emulsion. Fatty acid-labeled trioleoylglycerol was emulsified by homogenization in glycerol with lecithin as detergent. This anhydrous emulsion was stable for at least six weeks. Substrate solutions for enzyme assay were prepared by diluting the emulsion with buffer containing serum and albumin. The fatty acid produced on hydrolysis was isolated in a one-step liquid-liquid partition system. Incubations with extracts of acetone powders from adipose tissue displayed characteristics of lipoprotein lipase activity, i.e., serum dependence and inhibition by NaCl and protamine. The method is rapid (less than 1 hour), sensitive and reproducible, and suitable for routine use.

Animals↗

PubMed1968

Metabolism of phospholipids and the characterization of fatty acids in bovine corpus luteum.

1. Phosphatidylcholine was the predominant phospholipid in bovine corpora lutea; it accounted for about 50% of the total phospholipid phosphorus. Phosphatidylethanolamine (13%) and ethanolamine plasmalogen (8-9%) were the next two major components. 2. After incubation of the tissue with [(32)P]orthophosphate the total radioactivity and specific radioactivity of phosphatidylinositol were higher than those of any other lipid. 3. Luteinizing hormone failed to increase significantly the incorporation of [(32)P]orthophosphate into total phospholipids from luteal tissue slices, but did stimulate progesterone synthesis and lactate production. 4. The proportion of oleate (18:1) in the neutral lipids and phospholipids was higher than that of any other fatty acid. 5. The proportion of unsaturated fatty acid in the tissue lipids exceeded 60%, and almost half of this was polyunsaturated. Arachidonate (20:4), docosatetraenoate (22:4) and docosapentaenoate (22:5) were the principal polyunsaturated fatty acids. 6. After incubation of luteal tissue with [1-(14)C]acetate, the greatest proportion of radioactivity in the fatty acids isolated from the total lipid fraction was in palmitate (16:0) and docosatetraenoate (22:4). Polyunsaturated fatty acids accounted for almost 50% of the (14)C radioactivity incorporated and this pattern was observed in phospholipids, triglycerides and free fatty acids.

Acetates↗

PubMed1966

Studies on lipogenesis in vivo. Effect of dietary fat or starvation on conversion of [14]glucose into fat ad turnover of newly synthsized fat.

1. Lipogenesis was studied in vivo by giving mice 250mg. meals of [U-(14)C]glucose and measuring the disposition and incorporation of label. About 48% of the (14)C dose was eliminated as (14)CO(2) in the first 2hr. At 60min. after administration, 1.0, 1.9 and 11.9% of the dose was recovered as liver glycogen, liver fatty acid and carcass fatty acid respectively. Of the [(14)C]glucose converted into fat in the epididymal pads about 90% was present as glyceride fatty acid and 10% as glyceride glycerol. 2. Hepatic synthesis of fatty acid was depressed by dietary fat to a much greater extent than was synthesis outside the liver. Both feeding with fat and starvation decreased the proportion of the label taken up by adipose tissue present as fat (triglyceride) and increased the proportion of triglyceride label present as glyceride glycerol. These results are consistent with the hypothesis that the primary action of both these conditions in decreasing fat synthesis is to inhibit synthesis of fatty acids. 3. Turnover of body fat labelled in vivo from [U-(14)C]glucose was estimated from the decline in radioactivity measured over the first 24hr. of the experiment. The half-life of liver and extrahepatic fatty acids (excluding epididymal fat) was 16hr. and 3 days respectively. In contrast, no measurable decrease in radioactivity of the fatty acids of epididymal fat was observed for 7 days after administration of the [U-(14)C]glucose.

Adipose Tissue↗

PubMed1976

The carbon pathway for lipogenesis in isolated adipocytes from rat, guinea pig, and human adipose tissue.

The synthesis of fatty acids from a variety of labeled substrates by isolated adipocytes of the rat, guinea pig, and human was investigated. The incorporation of radioactive glucose and pyruvate into triglyceride fatty acids was considerably lower in human than either rat or guinea pig adipose tissue. By contrast, the incorporation of palmitate into adipose tissue triglycerides was approximately the same in all three species. End carbon analysis of fatty acids isolated from adipocytes incubated with pyruvate-U-14C indicated that although the synthesis of fatty acids in human adipose tissue was extremely low compared to that of the rat and guinea pig, it represented de novo biosynthesis rather than chain elongation of existing fatty acids. It is suggested that in the human, fatty acids are synthetised de novo primarily in the liver. In adipose tissue, lipogenesis consists essentially of the esterification of fatty acids, obtained from plasma, into triglycerides.

Adipose Tissue↗

PubMed1990

Uptake, incorporation and metabolism of (3H)triolein in the isolated perfused rabbit heart.

The purpose of these experiments was to study the uptake and metabolism of exogenous triglyceride in the isolated perfused rabbit heart. When infused into the rabbit heart, [9,10-3H(N)]triolein was retained and incorporated into a lipid fraction that had the chromatographic mobility of authentic triolein. Incorporation of labeled triolein was not likely to be the result of a lipoprotein lipase-mediated lipolysis/resynthesis cycle, since: (i) The distribution of radioactivity following administration of [3H]oleic acid was markedly different from the distribution of radioactivity following the administration of [3H]triolein; (ii) heparin was administered to the rabbits at the time of sacrifice; and (iii) the hearts were perfused with a protein-free buffer for 20 min prior to the labelling period. When isoproterenol was administered to hearts labelled with [3H]triolein, there was an increased output of total radioactivity, composed of labelled free fatty acids, diacylglycerol and monoacylglycerol. In these same hearts, there was an increased output of glycerol in response to isoproterenol. However, following the administration of bradykinin or angiotensin II, neither the radioactivity nor the glycerol content of the perfusate was changed. These data suggest that [3H]triolein is selectively incorporated into the triglyceride pool of the isolated perfused rabbit heart. Furthermore, this [3H]triolein is available to hormonally-activated lipolytic enzymes.

Angiotensin II↗

PubMed1986

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes.

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

8,11,14-Eicosatrienoic Acid↗

PubMed2002

Effect of diabetes and contractile activity on incorporation of the plasma-borne fatty acids into skeletal muscle lipids.

There are only few data on skeletal muscle lipid metabolism in diabetes. The aim of the present study was to examine effect of streptozotocin diabetes on incorporation of the blood-borne 14C-palmitic acid into different fractions of skeletal muscle lipids at rest and during contractile activity. The experiments were carried out on male Wistar rats. Streptozotocin was administered intravenously. The control rats received saline. Seven days later, the rats were anaesthetized and the calf muscles of one hindleg were made to contract for 1 min by stimulation of the sciatic nerve with tetanic pulses (30 tetani/min). Thereafter, 14C-palmitic acid was administered intravenously and the stimulation was continued for 5 min. The contralateral resting leg served as a control. Samples of the soleus, red and white gastrocnemius were taken. Lipids were extracted with chloroform/methanol and separated into the following fractions by means of thin layer chromatography: phospholipids, mono-, di-, and triacylglycerols, free fatty acids, cholesterol and cholesterol esters. Radioactivity of each fraction was counted. It has been found, that the label was always incorporated mostly into the fraction of triacylglycerols and phospholipids. Diabetes increased radioactivity of each lipid fraction, both at rest and during contractile activity, comparing to the respective values in the control rats. It is concluded that transport of the blood-borne free fatty acids into the myocytes and their incorporation into different lipid fraction increases in acute diabetes.

Animals↗

PubMed1986

In vitro metabolism of mevalonic acid in the bovine retina.

Bovine retinas were incubated with 3RS-[5-3H]-mevalonic acid under conditions similar to those previously shown to support opsin biosynthesis in vitro. TLC of the total lipids indicated the formation of numerous radiolabeled components, including sterols, hydrocarbons, and "fatty acid-like material." The nonsaponifiable lipids were analyzed by TLC, GLC, and chromatography on columns of silicic acid-Super Cel, silica gel G-Super Cel-silver nitrate, and alumina-Super Cel-silver nitrate. The major nonsaponifiable components had the chromatographic properties of squalene and "methylated sterols" (i.e., C30, C29, and C28 monohydroxy sterols). Cholesterol represented no more than 1% of the total radioactivity in the nonsaponifiable lipid fraction. The "fatty acid-like material" was derivatized with diazomethane, and the resulting methyl esters were analyzed by GLC before and after catalytic hydrogenation. The radioactivity did not correspond to the normal fatty acids endogenous to the retina, but rather had the chromatographic properties of C15 and C20 isoprenoid acids. These results obtained with intact retinas are consistent with our previous observations concerning mevalonic acid metabolism in cell-free homogenates of bovine retinas.

Animals↗

PubMed1966

Metabolism of saturated 1-14C-labelled fatty acids in the silkworm Bombyx mori L.

1. Only a small percentage of 1-(14)C-labelled saturated fatty acids injected in the silkworm is respired as carbon dioxide. 2. The rate of utilization of fatty acids is low both at the larval and pupal stages. 3. The insect has the ability to elongate C(12) and C(16) saturated fatty acids and to desaturate C(18) saturated fatty acids. 4. Much of the administered radioactivity is found in the triglyceride fraction, followed by the phospholipid and diglyceride fractions. 5. Diglycerides seem to be the transport form of fatty acids. 6. The insect seems to metabolize both natural and unnatural fatty acids in the same manner.

Animals↗