Effect of reproductive states on lipid mobilization and linoleic acid metabolism in mammary glands

Effects of pregnancy and lactation on lipid metabolism in mouse mammary fat pads and nonmammary adipose tissues have been studied. In order to address the question whether the influence of hormonal milieu on lipid metabolism in mammary epithelial cells during pregnancy and lactation is the same as in fat cells, we have studied the mobilization of lipids and metabolism of fatty acids in the intact mammary glands, parenchyma-free mammary fat pads and in the perimetrial fat tissues of virgin, pregnant and lactating mice. Compared to parenchyma-free mammary fat pads, the perimetrial adipose tissues accumulated 5-fold higher levels of triglycerides during pregnancy. Mammary fat cells maintained overall lipid levels during pregnancy and lactation (16–20 μg/fat pad). In contrast, lactation depleted total lipid stores from 108 ± 5 to 24 ± 4.5 μg/fat pad in perimetrial fat pads. Results of comparative analysis of fatty acid composition of mammary fat pads, with and without epithelial tissue, from virgin and lactating mice showed stimulation of 18∶2ω6 metabolism leading to 130% increase in the ratio 20∶4ω6 to 18∶2ω6 in the epithelial compartment. Pregnancy and lactation resulted in the elevation of 20∶4ω6 levels probably due to a 4-fold increase in Δ5 desaturase activity and a decrease in oxidative degradation of 18∶2ω6. These results suggest that, unlike other adipose tissues, the metabolic pathways in mammary fat cells are not dedicated to sequestration and accumulation of dietary lipids during pregnancy. Lactation favors mammary epithelial cell-stimulated production of precursors of eicosanoids which are known to have agonist-like effect on mammary epithelial cells.     

Composition and biosynthesis of fatty acids inPyramimonas grossii

The green algaPyramimonas grossii orginating in the coastal waters of the Atlantic Ocean Argentina was subcultured until a monoalgal culture was obtained. The fatty acid composition of the alga grown in a mineral medium at 12 C was determined by gas liquid chromatography (GLC) on 2 columns. The major fatty acids were oleic, linoleic, palmitic and α-linolenic acids, but the 20-carbon polyunsaturated acids, 20∶4ω6 and 20∶5ω3, respectively, belonging to the linoleic and α-linolenic series, were also found. Incubation with [¹⁴C] oleate, [¹⁴C] acetate, [¹⁴C] linoleate and [¹⁴C] α-linolenate suggests that linoleate is not directly converted to α-linolenate. [¹⁴C] Acetate was easily converted to palmitic, palmitoleic and oleic acids. However, after 48 hr of incubation, only traces of radioactivity were detected in linoleic acid and no label was found in α-linolenic acid.     

Inhibition of the hormone-sensitive lipase in adipose tissue by long-chain fatty acyl coenzyme A

The effects of free fatty acids and fatty acyl esters of coenzyme A and carnitine on the activity of a hormone-sensitive lipase preparation made from pigeon adipose tissue were determined. Oleic acid (100 μM) resulted in a 40% inhibition of lipase activity A more potent inhibition of lipase activity was seen with long-chain fatty acyl CoA compounds. The concentration required for half-maximal inhibition with oleoyl CoA and palmitoyl CoA was 25–40 μM, whereas palmitoyl carnitine stimulated lipase activity. Activated lipase preparations (preincubated with Mg²⁺, ATP, cyclic AMP and protein kinase) were 4–6 times more sensitive to inhibition by oleoyl CoA than were nonactivated preparations. An increase in cellular levels of fatty acyl coenzyme A could, therefore, contribute to the feedback inhibition of lipolysis in adipose tissue.     

Effect of tumor necrosis factor-α on triglyceride and phospholipid content and fatty acid composition of liver and carcass in rats

We have previously shown that a continuous infusion of tumor necrosis factor-α (TNF-α) in rats results in an increase in plasma triglyceride (TG), liver protein and DNA, and at the same time a reduction in muscle protein. However, there is no information on the associated effects of TNF-α on liver and muscle lipids. The present study, therefore, determined the effect of TNF-α on the TG and phospholipid (PL) content and their fatty acid composition, in the liver and carcass of rats and correlated with the plasma levels of insulin, corticosterone, and catecholamaines. Total parenteral nutrition that met the daily nutrient requirements was continuously infused for 10 d through a central vein catheter in two groups of rats, one receiving infused TNF-α (100 μg/kg/d) and a control group. Hepatic TG and PL, expressed either as mg/g dry weight or total organ content, were significantly increased in the TNF-α-infused group compared with controls. Livers of TNF-α infused rats contained significantly less saturated and monounsaturated fatty acids and significantly more polyunsaturated fatty acids (PUFA) of the ω3 and ω6 series than controls. The carcass, on the other hand, contained significantly more monounsaturated and significantly less polyunsaturated fatty acids of the ω6 series than controls. There were no changes in corticosterone level. Although plasma glucose levels were comparable between control and TNF-α infused rats, the latter had a marked increase in insulin levels, demonstrating insulin resistance. In addition TNF-α infused rats had raised norepinephrine levels. The fall in carcass and rise in liver TG and PL are consistent with the presence of insulin resistance and raised norepinephrine levels. However, the increase in PUFA content of the liver, especially the increase in 20∶4ω6 levels in liver PL and TG, is likely to be the effect of TNF-α and suggests increased activity of Δ5-desaturase.     

Lipid and fatty acid characterization and metabolism in the sea anemonePhymactis clematis (Dana)

Neutral lipid, phospholipids and fatty acids of the sea anemonePhymactis clematis from the south-west Atlantic were characterized and quantified in spring and autumn. Neutral lipids predominated over phospholipids in both seasons. Triacylglycerol and diacylglycerol ethers were the major lipids. In spring, an increase of esterified sterols was noted. The major fatty acids found were 22∶5ω3, 20∶5ω3 and 16∶0. The sea anemones were also incubated in vivo with either [1-¹⁴C]linoleate or [1-¹⁴C] α-linolenate for 2 hr. Isotope incorporation into lipids and their transformations into higher fatty acids were examined. Both precursors were incorporated into the lipids, mainly in triacylglycerols and mono-acylglycerols, while α-linolenate was also incorporated into phospholipids. The radioactive linoleate was elongated to 20∶2, 22∶2 and 24∶2 fatty acids, but not desaturated to 18∶3ω6. α-Linolenate was desaturated by Δ6 desaturase to 18∶4ω3. The specificity of Δ6-desaturase is discussed.     

Effect of dietary fat supplementation on the composition and positional distribution of fatty acids in ruminant and porcine glycerides

Dietary fats which were protected from ruminal metabolism were fed to ruminants, and the constituent fatty acids subsequently appeared in the glycerides of tissues and secretory products. These dietary fat induced alterations in tissue lipid composition were particularly apparent when the fat source was enriched with linoleic acid. Similarly, when pigs were fed linoleic-enriched fats, the linoleic acid was incorporated into the adipose tissue triglycerides. Stereospecific analyses were carried out on triglycerides from various tissues and secretory products obtained from animals fed control or linoleate-enriched diets. The analysis of adipose tissue triglycerides showed that linoleate and oleate were preferentially esterified to positions 2 and 3 (cattle and sheep), and positions 1 and 3 (pigs). Of the other major adipose tissue fatty acids, palmitate was preferentially esterified at position 1 (ruminants) and position 2 (pigs), and stearate was preferentially esterified at positions 1 and 3 (ruminants), and position 1 (pigs). Stereospecific analysis of high mol wt milk triglycerides showed that linoleate was either evenly distributed on all three positions (goats), or predominantly on position 3 (cows). Furthermore, the incorporation of this linoleate did not markedly alter the positional specificity of the other major milk triglyceride fatty acids. Of these fatty acids, the short and medium chain length acids (butyratelaurate) were mainly on position 3, myristate and palmitate on positions 1 and 2, and stearate and oleate evenly distributed. Thoracic duct lymph triglycerides from sheep tended to show preferential incorporation of linoleate at position 3, palmitate at position 2, and stearate at position 1 and 3; oleate, on the other hand, tended to be evenly distributed on all three positions of the lymph triglyceride. The stereospecific arrangement of fatty acids in sheep liver triglycerides was similar to that of lymph triglycerides, and this may reflect the uptake of intact or partially hydrolysed chylomicron and/or very low density lipoprotein triglycerides by the liver. There were also some analogies in the stereospecific arrangement of fatty acids on ruminant lymph and milk triglycerides and this may reflect an incomplete hydrolysis of chylomicron and/or very low density lipoprotein triglycerides prior to uptake by the mammary gland. An unusual feature of lymph from sheep fed linoleate was the presence of phospholipids which contained large amounts of linoleate in ca. equal proportion at both positions 1 and 2 of the phospholipid molecule.     

Metabolic control in isolated brown fat cells

Experiments with brown fat cell preparations from the adult hamsters are described. The mitochondria of brown adipose tissue were shown to have a classical electron transport system. The basal respiration of brown fat cells was demonstrated to be coupled to oxidative phosphorylation. Evidence is presented for partial uncoupling of oxidative phosphorylation as a mechanism for controlling respiration during norepinephrine stimulation. Exogenously added fatty acids were found to mimick the norepinephrine stimulation of respiration. Norepinephrine and cyclic AMP were shown to have no effect on brown fat mitochondria. Experiments with labeled oleate showed that the triglyceride reesterification cycle does not control respiration in brown adipose tissue. One of nine papers to be published from the Symposium “Brown Adipose Tissue,” presented at the AOCS-AACC Joint Meeting, Washington, D.C., March 1968.     

Uptake of blood triglyceride by various tissues

Triglycerides are transported in the blood in chylomicrons and very low density lipoproteins. Electron microscopic studies indicate that these particles, which range in diameter from 0.03–0.6 μ, cannot cross the capillary endothelium in most tissues. There is now considerable evidence that the triglycerides are hydrolyzed to free fatty acids (FFA) during uptake and that this process is catalyzed by lipoprotein lipase. The enzyme is found in nearly all tissues that utilize circulating triglyceride, and the level of activity, in individual tissues, varies with nutritional and physiological states that affect triglyceride uptake, such as fasting, diabetes and pregnancy. Studies in perfused adipose tissue with doubly labeled chylomicrons showed that hydrolysis occurs outside of the blood stream. Two-thirds of the fatty acids are incorporated into tissue triglyceride and the rest are release as FFA, with glycerol, to the blood. Infusion of heparin causes immediate release of lipoprotein lipase activity to the blood and decreases the amount of chylomicron-triglyceride hydrolyzed by the tissue. Electron microscopic cytochemical studies showed that hydrolysis of blood glycerides by lipoprotein lipase in adipose tissue occurs within the capillary endothelial cells and in the subendothelial space near the pericytes, but not in the capillary lumen or near the fat cells. The results indicate that the fatty acids of chylomicrons cross the capillary endothelium as glycerides and FFA, within a membrane-bounded system, and cross the extravascular space to the fat cells as FFA. Presented at the AOCS Meeting, Atlantic City, October 1971.     

Hydrolysis of triglycerides in the isolated perfused rat lung

The purpose of this study was to investigate the hydrolysis of saturated and unsaturated triglycerides by lung lipoprotein lipase and to measure the incorporation of triglyceride fatty acids into lung tissue lipids. Lipolytic activity was studied in the isolated ventilated rat lung, perfused for 100 min in a recycling system with Krebs Ringer bicarbonate containing bovine serum albumin, 5.6 mM glucose, and 1.5 or 10 mM triglyceride. Saturated triglycerides were hydrolyzed at significantly (p<0.05) lower rates than unsaturated triglycerides; tricaprylin, trimyristin and tripalmitin were hydrolyzed at 8.1+1.8, 5.4+1.5 and 9.5+1.8 μmol free fatty acids/g dry wt/100 min, respectively, whereas triolein and trilinolein were hydrolyzed at 20.2+1.8 and 20.6+0.3 μmol free fatty acids/g dry wt/100 min, respectively. The polyunsaturated triglycerides, trilinolein and triarachidonin were hydrolyzed at even higher rates (44.3+3.0 and 50.9+5.4 μmol free fatty acids/g dry wt/100 min, respectively). Intralipid infused at a concentration of 10 mM triglyceride was hydrolyzed at a significantly higher rate than at 1.5 mM triglyceride (58+6.3 μmol free fatty acids/g dry wt/100 min vs 16.6+1.7 μmol free fatty acids/g dry wt/100 min, respectively). Labeled unsaturated triglycerides were broken down at significantly higher rates than labeled saturated triglycerides. Incorporation of triglyceride-fatty acid into lung lipid was greater into neutral lipids than into phospholipids. The data suggest that (a) the factors that appear to affect lung lipoprotein lipase activity are composition and concentration of circulating triglyceride, (b) uptake of fatty acids into the tissue was proportional to the rate of hydrolysis of the emulsion, and (c) triglyceride-fatty acids could therefore be used by the lung for metabolic needs. The data presented in part at the Annual Meetings of the American Physiological Society, Atlanta, GA, April 1981, and the American Thoracic Society, Detroit, MI, May 1981, and published in abstract form-Fed. Proc. 40, 621 (1981), andAm. Rev. Respir. Dis. 123, 219 (1981).     

Positional specificity of gastric hydrolysis of long-chain n−3 polyunsaturated fatty acids of seal milk triglycerides

Long-chain n−3 polyunsaturated fatty acids (n−3 PUFA) of marine oils are important dietary components for both infants and adults, and are incorporated into milks following maternal dietary intake. However, little is known about the hydrolysis of these PUFA from milk triglycerides (TG) by lipases in suckling young. Seals, like humans, possess gastric lipase; however, the milk lipids of seals and sea lions are almost devoid of the readily hydrolyzable medium-chain fatty acids, and are characterized by a large percentage (10–30%) of n−3 PUFA. Gastric hydrolysis of milk lipids was studiedin vivo in suckling pups of three species (the California sea lion, the harp seal and the hooded seal) in order to elucidate the actions and specificity of gastric lipases on milk TG in relation to fatty acid composition and TG structure. Regardless of milk fat content (31–61% fat) or extent of gastric hydrolysis (10–56%), the same fatty acids were preferentially released in all three species, as determined by their relative enrichment in the free fatty acid (FFA) fraction. In addition to 16∶1 and 18∶0, these were the PUFA of 18 carbons and longer, except for 22∶6n−3. Levels of 20∶5n−3 were most notably enriched in FFA, at up to five times that found in the TG. Although 22∶6n−3 was apparently also released from the TG (reduced in the diglyceride), it was also notably reduced in FFA. Positional analysis of milk TG based on the products of Grignard hydrolysis revealed that these PUFA, including 22∶6n−3, were preferentially esterified at the α-position of the TG, and that the fatty acids not released during gastric hydrolysis were located at thesn-2 position. The extreme reduction of 22∶6n−3 and enrichment of 20∶5n−3 in FFA is discussed. Results from this study are consistent with reports that gastric lipase acts stereo-specifically to release fatty acids at the α-positions (sn−3,sn−1). We conclude that the n−3 PUFA in milk are efficiently hydrolyzed by gastric lipase and that this has important implications for digestion of milks enriched in PUFA by neonates in general. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990; part of this work is from the doctoral dissertation by S.J.I., University of Maryland, 1988.     

Effect of dietary fish oil on the rate of very low density lipoprotein triacylglycerol formation and on the metabolism of chylomicrons

The mechanism by which ω3 fatty acids lower plasma triacylglycerol levels was investigated. Rats were fed fish oil, olive oil (10% fat by weight) or a nonpurified diet 4% fat by weight) for 15 days. Lipoprotein lipase was inhibited by intra-arterial administration of Triton WR 1339 to estimate hepatic triacylglycerol output. Rats fed the olive oil diet showed a higher rate of triacylglycerol formation than rats fed the ω3 fatty acid diet or the low-fat diet. All three groups showed identical rates of removal from plasma of intraarterially administered artificial chylomicrons that had simultaneously been labeled with cholesteryl [1-¹⁴C]oleate and [9,10(n)-³H]triolein. Liver radioactivity and total fat content were lowest in rats fed the fish oil diet, indicating that ω3 fatty acids were preferentially metabolized in liver. Chylomicrons obtained from donor rats fed either fish oil containg [¹⁴C]cholesterol or olive oil containing [³H]cholesterol were removed at similar rates when infused together intraarterially into recipient animals. A slower formation of plasma very low density lipoprotein triacylglycerols in rats fed fish oil is probably due to a faster rate of oxidation of the fatty acid chains in the liver resulting in decreased plasma triacylglycerol concentrations.     

Fatty acid content of marine oil capsules

The use of dietary ω3 fatty acid capsules has been associated with a decrease in plasma triglyceride levels. In addition, populations consuming diets rich in fish appear to have a decreased incidence of cardiovascular disease. Eicosapentaenoic acid (EPA, 20∶5ω3) and docosahexaenoic acid (DHA, 22∶6ω3) are major fatty acids in fish oils. It is believed that fish oils exert their biolotic effect through these fatty acids. Many individuals are currently taking fish oil capsules to lower lipids, increase bleeding time, and possibly decrease cardiovascular risk. These capsules also have been classified as food additives with less stringent controls on content. We assessed the fatty acid, cholesterol, and vitamin A and E content of eight commercially available capcules along with cod liver oil. The content of EPA was found to range from 8.7–26.4% (wt%) with a mean of 17.3% (82.4% of labeled content), and that of DHA from 8.9–17.4% with a mean of 11.5% (90.0% of labeled content) as assessed by capillary column gas-liquid chromatography. The mean content of the polyunsaturated ω3 fatty acids was 31.9%, and that of the ω6 fatty acids was 1.4%. The content of saturated fatty acids was 32,0%, and that of monounsaturated fatty acids was 25.1%. Cholesterol content was low, with a range of 0.7–8.3 mg/g, the α-tocopherol range was 0.62–2.24 mg/g, and the range of retinyl esters was 0.4–298.4 μg/g. Cod liver oil had substantially more retinyl esters (2450.1 μg/g) than did fish oil capsules. Our data serve as an independent guide to fish oil capsule fatty acid content upon single lot analysis, and indicate that these capsules contain as much saturated fat as they contain ω3 fatty acids.     

Cigarette smoke affects lipolytic activity in isolated rat lungs

Isolated perfused rat lungs liberated fatty acids at a rate of 15 μmol/hr during perfusion of triglyceride-rich medium through the pulmonary vascular bed. About 80% of this activity seemed to result from lipoprotein lipase and 20% to hormone-sensitive lipase. Ventilation of the lungs with cigarette smoke instead of air during the perfusion reduced fatty acid liberation by 23%. Pre-exposure of rats to cigarette smoke for either 1 or 10 days did not cause significant changes in lung lipolytic activity compared to sham-exposed controls.     

Application of new methods and analytical approaches to research on polyunsaturated fatty acid homeostasis

New methods and analytical approaches are important to challenge and/or validate established beliefs in any field including the metabolism of polyunsaturated fatty acids (PUFA; polyunsaturates). Four methods that have recently been applied toward obtaining a better understanding of the homeostasis of PUFA include the following: whole-body fatty acid balance analysis, magnetic resonance imaging (MRI), ¹³C nuclear magnetic resonance (NMR) spectroscopy, and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Whole-boby balance studies permit the measurement of both the percentage of oxidation of linoleate and α-linolenate and their conversion to long-chain PUFA. This method has shown that β-oxidation to CO₂ is normally the predominant metabolic fate of linoleate and α-linolenate. Furthermore, models of experimental undernutrition in both humans and animals show that β-oxidation of linoleate and α-linolenate markedly exceeds their intake, despite theoretically sufficient intake of linoleate or α-linolenate. Preliminary results suggest that by using MRI to measure body fat content, indirect whole-body linoleate balance can be done in living humans, ¹³C NMR spectroscopy provided unexpected evidence that linoleate and α-linolenate were metabolized into lipids synthesized de novo, an observation later quantified by tracer mass balance done using GC-C-IRMS. This latter method showed that within 48 h of dosing with ¹³C-α-linolenate, >80% underwent β-oxidation to CO₂ by suckling rats, whereas 8–9% was converted to newly synthesized lipids and <1% to docosahexaenoate. Further application of these recently developed methods in different models should clarify the emerging importance of β-oxidation and carbon recycling in PUFA homeostasis in mammals including humans.     

Modification of Palm Oil for Anti-Inflammatory Nutraceutical Properties

Palm oil is one of the most important edible oils in the world. Its composition (rich in palmitate and oleate) make it suitable for general food uses but its utility could be increased if its fatty acid quality could be varied. In this study, we have modified a palm olein fraction by transesterification with the n-3 polyunsaturated fatty acids, α-linolenate or eicosapentaenoic acid (EPA). Evaluation of the potential nutritional efficacy of the oils was made using chondrocyte culture systems which can be used to mimic many of the degenerative and inflammatory pathways involved in arthritis. On stimulation of such cultures with interleukin-1α, they showed increased expression of cyclooxygenase-2, the inflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1α and IL-1β and the proteinase ADAMTS-4. This increased expression was not affected by challenge of the cultures with palm olein alone but showed concentration-dependent reduction by the modified oil in a manner similar to EPA. These results show clearly that it is possible to modify palm oil conveniently to produce a nutraceutical with effective anti-inflammatory properties.     

Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids

Lipase hydrolysis was evaluated as a means of selectively enriching long-chain ω3 fatty acids in fish oil. Several lipases were screened for their ability to enrich total ω-3 acids or selectively enrich either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). The effect of enzyme concentration, degree of hydrolysis, and fatty acid composition of the feed oil was studied. Because the materials that were enriched in long-chain ω3 acids were either partial glycerides or free fatty acids, enzymatic reesterification of these materials to triglycerides by lipase catalysis was also investigated. Hydrolysis of fish oil by eitherCandida rugosa orGeotrichum candidum lipases resulted in an increase in the content of total ω3 acids from about 30% in the feed oil to 45% in the partial glycerides. The lipase fromC. rugosa was effective in selectively enriching either DHA or EPA, resulting in a change of either the DHA/EPA ratio or the EPA/DHA ratio from approximately 1:1 to 5:1. Nonselective reesterification of free fatty acids or partial glycerides that contained ω3 fatty acids could be achieved at high efficiency (approximately 95% triglycerides in the product) by using immobilizedRhizomucor miehei lipase with continuous removal of water.     

Ratios of polyunsaturated fatty acids to α-tocopherol in tissues of rats fed corn or soybean oils

This study was part of a larger experiment designed to assess the vitamin E adequacy of corn and soybean oils in relation to their polyunsaturated fatty acids (PUFA). Young male rats were fed a semipurified diet containing 20% corn or soybean oil and adequate selenium. After 8 and 12 weeks, animals were sacrificed, and 7 tissues analyzed for α- and γ-tocopherols and for fatty acids. Calculations were made of the molar ratios of total polyunsaturated fatty acids/α-tocopherol, and also of all polyunsaturated fatty acids, except linoleate, designated polyunsaturated fatty acids>18∶2, to α-tocopherol. It is proposed that the latter ratio may have more significance, physiologically, than when linoleic acid also is considered. Tissues from rats fed corn oil had slightly more favorable (lower) ratios than did tissues from rats fed soybean oil. In both groups, the molar polyunsaturated fatty acids>18∶2/α-tocopherol ratio was lowest for heart and lung, intermediate for muscle and testis, and highest for liver, kidney, and adipose tissue. Since both corn and soybean oils provide adequate vitamin E as determined by several biochemical and physiological parameters, adequate molar ratios of polyunsaturated fatty acids>18∶2/α-tocopherol were: lung, 400; heart and leg muscles, 700; testis, 1100; liver and kidney, 1500–2000; and adipose tissue, 2000.     

Studies on the specificity of a lipase system fromGeotrichum candidum

The lipase system fromGeotrichum candidum preferentially hydrolyzed oleic acid, regardless of position, from the four possible racemic triglycerides containing oleate and palmitate. The rate of hydrolysis of these glycerides was most rapid when the substrate contained two moles of oleate. This acid was also preferentially released from a series of triglycerides containing oleate and two moles of a saturated fatty acid. The chain length of the latter did not alter the specificity for oleate. Equimolar quantities of oleic and linoleic acids were released when triolein and trilinolein (equimolar mixture) were hydrolyzed by this lipase. No differentiation between oleate and palmitoleate was observed when racemic glyceryl 1-palmitoleate-2,3-dioleate was the substrate. However, only 7.2 M%cis-vaccenic acid was released from glyceryl 1-cis-vaccenate-2,3-dioleate and 5.4 M% petroselinic acid from glyceryl 1-palmitoleate-2,3-dipetroselinate. It therefore appears that the enzyme may be specific forcis-9-unsaturation as well as forcis-9,cis-12-unsaturation. When specificity was assumed, the fatty acid compositions of the diglycerides obtained from digestions withG. candidum were close to theoretical. Scientific contribution No. 256, Agricultural Experiment Station, University of Connecticut, Storrs.     

Control of metabolism in brown adipose tissue

Cells and mitochondria were isolated from brown adipose tissue of the adult hamster. Isolated mitochondria did not show respiratory control. Reversed electron transport was demonstrated and the oxidation rates of various substrates were compared. α-Glycerophosphate gave the highest oxidative rate with isolated mitochondria. The low basal respiration of isolated brown fat cells could be stimulated by catecholamines, oleate, succinate,a-glycerol phosphate and uncoupling agents. Only norepinephrine or oleate induced respiration was sensitive to inhibition by oligomycin, but this inhibition could not be released by uncoupling agents. Neither atractyloside nor (+) decanoylcarnitine were found to affect respiration, suggesting that mitochondrial nucleotide exchange is slow and that fatty acid oxidation might be carnitine independent. In resting brown fat cells, ATP amounts to 75% of the total adenine nucleotides. NE or oleate caused a small decrease of ATP and a corresponding increase of ADP. Oligomycin caused a partial depletion of ATP content, but subsequent NE addition increased ATP back to control values. This effect was abolished by arsenite. Similarly, uncoupling agents diminished the ATP level which was increased only slightly by NE. Arsenite alone decreased ATP levels to a small extent but a rapid depletion occurred upon subsequent NE addition while respiration was inhibited. Thus, substrate level phosphorylation may be the major energy producing reaction for the generation of ATP and GTP for the activation of fatty acids. Norepinephrine addition to brown fat cells caused an oxidation of pyridine nucleotides, a reduction of flavoproteins and an oxidation of cytochrome b. In constras, succinate produced a reduction of all the components of the respiratory chain. The bioenergetic basis of thermogenesis in brown fat is its high respiratory rate. The rapid respiration induced by norepinephrine or fatty acids appears to be characterized by a low yeild of ATP from oxidative phosphorylation and may be controlled by fatty acid mediated release of energy coupling, possibly by an indirect mechanism. One of nine papers to be published from the Symposium “Brown Adipose Tissue,” presented at the AOCS-AACC Joint Meeting, Washington, D. C., March 1968. This work was done during the tenure of an Established Investigatorship from the American Heart Association.     

Changes in the structure of soybean triglycerides during maturation

Soybeans of the Hawkeye variety were picked at eleven periods from 30 to 111 days after flowering and extracted with chloroform-methanol. The triglyceride fraction of five pickings, selected 35 to 91 days after flowering (when synthesis of lipid was most active), were isolated by silicic acid thin layer chromatography (TLC) and species composition determined using argentation TLC and lipase hydrolysis. The triglyceride content of the total lipid increased from 6.5% at 30 days after flowering to 85% in the mature bean (111 days). The major changes in fatty acid composition of the triglycerides occurred during the first 52 days after flowering. During this period linolenic acid decreased from 34.2% to 11.7%, the percentages of linoleic and oleic acids increased, stearic remained fairly constant and palmitic decreased slightly. Large quantitative changes occurred in the molecular species of the triglycerides of the bean during maturation; some triglycerides containing linolenic acid could not be detected approximately 66 days after flowering. Although changes occurred in the percentage and amount of each triglyceride species, the positional distribution of fatty acids remained virtually unchanged throughout maturation. Linolenic acid was distributed fairly uniformly between the β-position and the α-positions, linoleate favored esterification in the β-position, and oleate the α-positions. Most of the stearic and palmitic acids were esterified in the α-positions. The consistency of the positional arrangement of the fatty acids indicated that the mode of glyceride synthesis was established very early during maturation and molecular species composition was controlled by the fatty acids available for synthesis.