Chain Free Fatty Acids Research Articles

Influence of chain length and degree of unsaturation on plasma free fatty acid uptake by the perfused rat liver

1. 1. The fractional uptake of free fatty acids by perfused liver is inversely related to the chain length and directly related to the number of double bonds in the molecule. 2. 2. The affinity of the liver for long chain free fatty acids is myristic > palmitoleic > linoleic > oleic ≅ palmitic > stearic. Partial in vitro extraction by charcoal of fatty acids bound to albumin shows similar behaviour supporting the view that the first step in the uptake of fatty acids by the cell is a non-selective, physical adsorption. Therefore, it is suggested that the rate of fractional uptake of the individual free fatty acids depends less on the selectivity of absorption to the cellular membrane than on the relative affinity of the individual fatty acids for the binding sites on the albumin molecule. 3. 3. The uptake of the total exchangeable pool of plasma free fatty acids by the liver can be measured rather accurately by using oleic or palmitic acid as tracer. 4. 4. The difference in the uptake of individual fatty acids could explain the finding that the proportion of stearic acid in the plasma is greater than in adipose tissue. At the same time, it can explain the relative variations (increase of stearic acid and decrease of oleic acid) observed when there is a fall in the plasma free fatty acids concentration.

Quantitative preparation and gas chromatography of short and medium chain fatty acid benzyl esters (C 1 -C 12 ).

Abstract The benzyl esters of short and medium chain fatty acids (C1—C12) were prepared with N,N′-dicyclohexyl-O-benzylisourea or with phenyldiazomethane. The extent of estertification was checked by using 14C-labeled fatty acids. The fatty acid benzyl esters were separated gas chromatographically on both SE-30 and EGSS-X columns. For quantification, the relative response for equal weights of fatty acids was determined. The mild esterification with phenyldiazomethane allows this method to be used for the assay of short and medium chain free fatty acids in biological materials.

A method for determination of the total dissolved free fatty-acid content of sea water

A new method is proposed for the determination of total dissolved free fatty acids (FFA) in sea water. After preliminary extraction by chloroform, a copper complex is formed which is then extracted by a chloroform-heptane solution. Copper concentration is measured by atomic absorption spectrophotometry. The method is particularly adapted to the determination of long chain FFA (longer than C10) and dissolved organic matter usually present in sea water gives no interference. At the 30 μ/l. level the standard deviation is 1 μ/l. for seven determinations.

Medium Chain Fatty Acid Binding to Human Plasma Albumin

Abstract The binding of hexanoic, octanoic, and decanoic acids to defatted human plasma albumin was measured by equilibrium dialysis at 37° in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The results were analyzed in terms of multiple stepwise equilibria. For each of the albumin binding sites, the magnitude of the equilibrium constants increased as the chain length of the acid increased: decanoate g octanoate g hexanoate. The first six equilibrium constants ranged from 1.5 x 104 to 4.7 x 10 for hexanoate, from 3.4 x 104 to 1.2 x 103 for octanoate, and from 105 to 3.4 x 103 for decanoate. In each case, the equilibrium constants occurred in a generally descending order, suggesting that major cooperative binding effects do not occur over the physiologically important range of fatty acid-albumin molar ratios. The equilibrium constants calculated for each of the three acids could not be grouped in a common way in terms of classes of binding sites, indicating that a single, uniform class-site binding model cannot be applied to these medium chain fatty acids. Octanoate binding was relatively insensitive to pH changes over the range of 6.0 to 8.2. Decanoate binding also was similar at pH 6.5 and 7.4. A decrease in octanoate binding occurred when the albumin was acetylated or when the medium contained 6 m urea. Octanoate binding also was decreased when either palmitate or oleate was added to albumin, suggesting that medium chain fatty acid transport may be influenced by changes in the plasma long chain free fatty acid concentration.

Metabolism of Free Fatty Acids in Isolated Liver Cells: FACTORS AFFECTING THE PARTITION BETWEEN ESTERIFICATION AND OXIDATION

Abstract The structural and metabolic integrity of isolated rat liver cells was verified by the high percentage of trypan blue exclusion, a low degree of lactate dehydrogenase release into the medium, a constant rate of gluconeogenesis from l(+)-lactate, and increased oxygen consumption following the addition of 2,4-dinitrophenol. Palmitic acid, incubated in an albumin-bound form with isolated liver cells, was esterified to form phospholipids, triglycerides, diglycerides, and cholesterol esters and was oxidized to CO2 and ketone bodies. In liver cells from fed rats, the major portion of palmitate was esterified, an intermediate quantity was oxidized to ketone bodies, and a smaller amount was oxidized to CO2. The partition of palmitic acid between esterification and ketogenesis was inversed by fasting, whereas oxidation to CO2 and the total rate of palmitate utilization were unaltered. Greater esterification of [14C]palmitate in cells from fed rats was not a result of carbon recycling via chain elongation or de novo synthesis. Liver cells from fasted rats derived more energy from fatty acid oxidation than cells from fed rats. The results indicate that citric acid cycle flux and endogenous lipolysis were unaffected by fasting. These observations signify that altered partition of free fatty acids between the pathways of oxidation and esterification in the liver is a major causative factor in the increased ketogenesis in the fasting state. An increase in [1-14C]palmitate concentration augmented palmitate uptake, ketogenesis, and esterification, whereas 14CO2 production was only slightly affected. The estimated citric acid cycle flux and the acetoacetate to β-hydroxybutyrate ratio were diminished. Increased ketogenesis in response to sequential elevation of the palmitate concentration could not be accounted for by diminished citric acid cycle flux and therefore resulted from increased β oxidation. Ketone body specific activity approached a constant value at v of 3 to 4. Results indicate intracellular mixing of free fatty acids derived from endogenous lipolysis and from the medium, prior to β oxidation. Phospholipid was the predominant esterification product at low concentrations of added palmitate, but, as phospholipid formation approached saturation, a sigmoid increase in diglyceride and triglyceride formation occurred. Fructose and glycerol each decreased ketogenesis from added palmitate. Fructose, glycerol, and, to a lesser extent, glucose increased palmitate esterification in liver cells isolated from fasting rats. This effect was characterized by increased conversion of added palmitate to diglycerides, triglycerides, and phospholipids and decreased conversion to cholesterol esters. These substrates did not alter the rate of fatty acid uptake. Fructose, glycerol, and, to a lesser extent, glucose elevated 14CO2 production from [1-14C]palmitate. At higher fructose concentrations the elevated 14CO2 production was reversed. Results suggest that substrates which enter glycolysis beyond fructose 1,6-diphosphate decrease ketogenesis by competition with fatty acid oxidation and enhance esterification by the resulting increased availability of long chain free fatty acids and by a separate preferential stimulation of glycerolipid formation. Results indicate that decreased availability of non-fatty acid substrates and, thereby, decreased competitive oxidation of these substrates is a participating causative factor in the increased oxidation, and the decreased esterification, of long chain fatty acids in the liver in the fasting state.

Effect of glycerol deprivation on the phospholipid metabolism of a glycerol auxotroph of Staphylococcus aureus.

A study of the effects of glycerol deprivation on the content and metabolism of the phospholipids of a glycerol auxotroph of Staphylococcus aureus showed that (i) there was an increase in the proportions of lysylphosphatidylglycerol (LPB) and a concomitant decrease in the proportion of phosphatidylglycerol. The total phospholipid content per sample and the proportion of cardiolipin did not change, but the phosphatidic acid increased transiently and then fell to pretreatment levels. (ii) The loss of (32)P from the lipids during the chase in a pulse-chase experiment was essentially the same in phosphatidylglycerol, cardiolipin, and phosphatidic acid during glycerol deprivation or growth in the presence of glycerol. LPG lost half the radioactivity in slightly more than two doubling times when grown with glycerol. In the absence of glycerol, (32)P accumulated in LPG for about 20 min and then stopped, after which time there was no apparent turnover. (iii) During glycerol deprivation, the initial (32)P incorporation decreased sixfold compared to that of the control with glycerol. The initial incorporation into LPG decreased only 2.5-fold, whereas that of PG decreased 45-fold. (iv) During glycerol deprivation, the free fatty acid content increased from 1.2 to 12.5% of the total extractable fatty acids and then slowly decreased. The increase was largely iso- and anti-iso-branched 21-carbon-atom fatty acids. In glycerol-supplemented cultures, the major fatty acids were branched 14- to 18-carbon fatty acids. The decrease in longer chain free fatty acids after 60 min represented their esterification into lipids. (v) During glycerol deprivation ribonucleic acid synthesis and cell growth continued for 40 min and protein synthesis continued for 90 min. Then synthesis and growth stopped. (vi) After the addition of glycerol to glycerol-deprived cells, (32)P and (14)C-glycerol were incorporated into the phospholipids without lag; ribonucleic acid, protein synthesis, and cell growth began after a 5- to 10-min lag at the pretreatment rate. The initial rate of lipid synthesis after the addition of glycerol was three times greater than the growth rate. This rapid rate continued for about 25 min until the lipid content and proportions of LPG and phosphatidylglycerol were restored.

魚類筋肉脂質の冷凍貯蔵中の変化-III

The development of rancidity in fish flesh during storage at -20°C effected the subsequent decrease of protein soluble in 5% NaCl (pH 7.0). Actomyosin (1.6-2.0mg/ml), a protein affecting the qualities of fish flesh, was reacted at pH 7.2 and ionic strength 0.5 with short chain free fatty acids or aliphatic aldehydes (3 μM/mg protein), the oxidation products of unsaturated lipids. The most reactive acid and aldehyde at -20°C were caproic acid and propanal respectively. In the presence of phospholipids (more than about 20% of total protein) insolubilization of actomyosin was prevented in caproic acid reaction medium but not in propanal one. Neutral lipids alone showed no effects on actomyosin insolubilization, while in the presence of phospholipids (over 20% of lipids) preventive effects were observed in caproic acid reaction medium. The differences in preventive effects of phospholipids on actomyosin-caproic acid and propanal systems seemed to have originated from differentiae of caproic acid and propanal reaction mechanisms with actomyosin.

Inhibition of Gluconeogenic Enzymes by Free Fatty Acids and Palmitoyl Coenzyme A

The effects of free fatty acids on rat liver phosphoenolpyruvate carboxykinase (EC 4.1.1.32), pyruvate carboxylase (EC 6.4.1.1), and glucose 6-phosphatase (EC 3.1.3.9) were determined to evaluate the suggestion that free fatty acid inhibition of key glycolytic enzymes constitutes a specific inhibitory effect. Activities of all of these gluconeogenic enzymes were found to be markedly inhibited by long chain free fatty acids as well as by palmitoyl coenzyme A. The manner of the inhibition of gluconeogenic enzymes was indistinguishable from that reported for the glycolytic enzymes. It is therefore concluded that such effects in vitro of free fatty acids or long chain acyl-CoA esters on glycolytic and gluconeogenic enzymes may not be relevent for the regulation of metabolism in vivo.

Binding of long chain fatty acids to beta-lactoglobulin.

Abstractβ‐lactoglobulin (BLG), a bovine milk protein that is available commercially in crystalline form, binds long chain free fatty acids (FFA). The binding data were analyzed with a model containing one primary FFA binding site and a large number of weak secondary binding sites. At 37C and pH 7.4, the apparent association constant for binding of FFA to the primary site was of the order of 105 M−1 and that for binding to the secondary sites was approximately 103 M−1. The strength of binding was: palmitate > stearate > oleate > laurate. The affinity of BLG for palmitate increased as the pH of the incubation medium was raised from 6.5 to 8.7 and decreased as the ionic strength of the medium was raised. Palmitate binding was decreased in the presence of 6 M urea and when the protein either was exposed to elevated temperature or was acetylated prior to incubation. BLG took up methyl palmitate, cetyl alcohol, hexadecane and cholesterol to a lesser extent than FFA. Binding of FFA to BLG was associated with a small increase in the intensity of the fluorescent emission of the protein at 333 mμ. BLG can serve as an FFA acceptor or carrier in biological experiments. FFA released from adipose tissue during in vitro incubation was taken up by BLG. Net transfer of fatty acid to the incubation medium ceased when the molar ratio of FFA to BLG exceeded 1.1.14C‐1‐Palmitate bound to BLG was taken up by Ehrlich ascites tumor cells in vitro. At a given palmitate‐protein molar ratio, much more labeled fatty acid was taken up by these cells from media containing BLG than from those containing bovine albumin, apparently because FFA is bound less firmly to BLG than to albumin.

Regulation of hepatic carbohydrate metabolism by FFA and acetyl-CoA: sequential feedback inhibition.

AbstractIn recent years experiments in slices, perfused liver and in the whole animal demonstrated that liver carbohydrate metabolism can be controlled by free fatty acids (FFA). Our investigations suggest that FFA and acetyl‐CoA might provide regulation of the rate and direction of opposing pathways of hepatic glycolysis and gluconeogenesis by controlling the activity of key enzyme systems. Long and short chain FFA were observed to selectively inhibit the key enzymes of glucose catabolism, glucokinase, hexokinase, phosphofructokinase and pyruvate kinase. The long chain FFA were inhibitors two magnitudes stronger than octanoate. The FFA were also able to inhibit lactate production in a fortified supernatant fluid system. Acetyl‐CoA inhibited hepatic glucokinase and pyruvate kinase but did not affect liver hexokinase, phosphofructokinase, lactate dehydrogenase, glucose‐6‐phosphatase or fructose‐1, 6‐diphosphatase. The inhibition of glucokinase and pyruvate kinase was dependent on the dose and preincubation time with acetyl‐CoA. The acetyl‐CoA is the end product of the degradation of FFA which in turn is an end product of glucose catabolism; therefore, the inhibition of the three key glycolytic enzymes by FFA and the subsequent reinforcement of the inhibition of glucokinase and pyruvate kinase may be called sequential feedback inhibition. The regulatory role of phosphoenol‐pyruvate, NADH, ATP, alanine and oxaloacetate in the control of hepatic carbohydrate metabolism is discussed.

A modified copper reagent for the colorimetric determination of free fatty acids

A colorimetric assay for measuring long chain (C > 10) free fatty acids in plasma is described. The basic assay procedure of R. J. Schimmel and E. Knobil ((1970) Amer. J. Physiol.218, 1540–1547) was modified in three ways: 1) by not buffering the cobalt-triethanolamine reagent, 2) by replacing potassium sulfate with lithium sulfate, and 3) by making a single reagent of chloroform, heptane, and methanol. In the absence of the buffer system, the soap complex was chemically more stable. A loss of sensitivity incurred by omitting the buffer system was corrected by using a salt solution of 20% (w/w) Na2SO4/10% (w/w) Li2SO4 instead of saturated Na2SO4/K2SO4. The number of pipettings was reduced by making a single reagent of chloroform, heptane, and methanol. Because of improved assay stability, large numbers of samples (90) can be processed simultaneously. The method is adequately linear over the range of 100–1250 μequiv/liter. The standard deviation is independent of the mean over the range of 250–1000 μequiv/liter and is about 40 μequiv/liter (n = 15).

Effects of guanidine derivatives on mitochondrial function

Long chain free fatty acids interfere with the inhibitory action of phenethylbiguanide and related compounds on mitochondrial respiration in vitro. This interference depends on binding of fatty acids to mitochondria and diminishes with decreasing chain length. Reversal of guanidine-derivative inhibition by fatty acids differs from that caused by dinitrophenol in that the effect of fatty acid is achieved without alteration in coupling or respiratory control. The binding of phenethylbiguanide to mitochondria is inhibited by both fatty acid and dinitrophenol. Serum albumin potentiates the inhibitory potency of guanidine derivatives, probably by removing endogenous mitochondrial free fatty acids.

Quantitative recovery of short chain free fatty acids after gas chromatography

LipidsVolume 1, Issue 3 p. 230-231 Short Communication Quantitative recovery of short chain free fatty acids after gas chromatography O. K. Reiss, O. K. Reiss Department of Biochemistry and the Webb-Waring Institute for Medical Research, University of Colorado Medical Center, Denver, ColoradoSearch for more papers by this authorJ. G. Warren, J. G. Warren Department of Biochemistry and the Webb-Waring Institute for Medical Research, University of Colorado Medical Center, Denver, ColoradoSearch for more papers by this authorJoyce K. Newman, Joyce K. Newman Department of Biochemistry and the Webb-Waring Institute for Medical Research, University of Colorado Medical Center, Denver, ColoradoSearch for more papers by this author O. K. Reiss, O. K. Reiss Department of Biochemistry and the Webb-Waring Institute for Medical Research, University of Colorado Medical Center, Denver, ColoradoSearch for more papers by this authorJ. G. Warren, J. G. Warren Department of Biochemistry and the Webb-Waring Institute for Medical Research, University of Colorado Medical Center, Denver, ColoradoSearch for more papers by this authorJoyce K. Newman, Joyce K. Newman Department of Biochemistry and the Webb-Waring Institute for Medical Research, University of Colorado Medical Center, Denver, ColoradoSearch for more papers by this author First published: 01 May 1966 https://doi.org/10.1007/BF02531878Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume1, Issue3May 1966Pages 230-231 RelatedInformation

Rapid and Sensitive Assay for Milk Lipase1,2

An improved micromethod has been developed for measuring lipase activity in milk by the pH stat technique. The silica-gel procedure measures only 16.8% of the lipase activity observed in milk by the pH stat method. The major factors contributing to this apparent low lipase activity by the silica-gel method were a) incomplete titration of the long chain free fatty acids; b) lack of proportionate response of milk lipase activity to increased concentrations of milk in the assay system; and c) variations in the pH of the lipase assay mixture during the silica-gel incubation period. Compared to the silica-gel method, the pH stat assay had greater sensitivity, accuracy, and rapidity and allowed measurement of the initial reaction velocity.During storage at 4C the lipolytic activity of fresh raw milk was constant up to 24 hr, after which it decreased. Homogenization of raw milk does not appear to affect lipase activity per se.It appears to be a substrate activating phenomenon, in that there is a direct relationship between homogenization pressure and susceptibility of milk fat to lipolysis.