Fatty acids bound to alpha-fetoprotein and albumin during rat development

The time-course levels and composition of the fatty acids bound to rat alpha-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n-3], either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8-10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n-3]. These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.     

Plasma binding of oestradiol in progesterone-treated rats

Progesterone treatment of female rats causes an increase in body weight possibly via suppression of oestradiol secretion. This study was carried out to investigate the effect of progesterone on the non-protein bound and hence presumably biologically active fraction of oestradiol. Oestradiol binding to plasma proteins was studied in female Wistar rats during the oestrous cycle and after 12 days of treatment with progesterone (5 mg/day). There was no change in either the unbound fraction of oestradiol or plasma albumin concentrations during the oestrous cycle. Plasma oestradiol concentrations in progesterone-treated rats were similar to those seen during dioestrus, as were the degree of oestrogen binding and the plasma albumin concentrations. Although it was not feasible to calculate unbound concentrations, these results suggest that the increased body weight seen in progesterone-treated rats, and also during pregnancy, may be a result of suppression of unbound oestradiol concentrations to levels similar to those occurring during dioestrus.     

Utilization of free fatty acids complexed to human plasma lipoproteins by mammalian cell suspensions

The purpose of this study was to determine whether lipoprotein-bound free fatty acid could be utilized by isolated mammalian cells. Ehrlich ascites tumor cells were incubated in vitro with radioactive free fatty acids that were bound to human plasma lipoproteins. Under these conditions, lipoprotein-bound free fatty acids were readily taken up by the cells. After 2 min of incubation with free fatty acids bound to low density lipoproteins, most of the radioactivity that was associated with the cells was in the form of free fatty acids. As the incubation continued, increasing amounts of radioactivity were incorporated into CO(2) and cell lipids, particularly phospholipids. Most of the free fatty acid uptake was the result of fatty acid transfer from low density lipoproteins to the cell, not from irreversible incorporation of the intact free fatty acid-low density lipoprotein complex. Fatty acid uptake increased as the ratio of free fatty acid to low density lipoprotein was raised. When albumin was added to the medium, free fatty acid uptake decreased. A large percentage of the newly incorporated cellular radioactivity was released into the medium if the cells were exposed subsequently to a solution containing albumin. Most of the released radioactivity was in the form of free fatty acid. The results with this experimental model suggest that lipoprotein-bound free fatty acid, like albumin-bound free fatty acid, is readily available for uptake by isolated cells. The mechanism of free fatty acid utilization by the Ehrlich cell is similar when either low density lipoprotein or serum albumin serves as the fatty acid carrier.     

Recombinant Human Albumin in Cell Culture: Evaluation of Growth-Promoting Potential for NRK and SCC-9 Cells In Vitro

Serum-derived albumin has for a long time been used in cell culture media, but the exact role of albumin and/or impurities bound to albumin has not been precisely defined. In this study, recombinant human albumin was evaluated for its growth-promoting activity on two cell lines, NRK and SCC-9. For NRK cells, the recombinant human albumin was found to exert an inhibitory effect. The fact that fatty acid free HSA was also inhibitory while HSA fraction V was stimulatory suggested a role for fatty acids or some other bound moieties in growth stimulation by HSA fraction V. Addition of oleic acid, cholesterol, phosphatidylcholine, phosphatidylserine or a combination of these lipids, however, did not significantly improve the growth stimulating activity of either fatty acid free HSA or the recombinant human albumin. For SCC-9 cells, both recombinant human albumin and fatty acid free HSA showed slight stimulation (although they were not as active as HSA fraction V), suggesting that in some cell systems, the albumin molecule per se may promote cell growth and survival. This revised version was published online in July 2006 with corrections to the Cover Date.     

The displacement of albumin bound bilirubin by free fatty acids in vivo

Variations in bilirubinaemia in response to alterations of the free fatty acid level was studied in conscious and unstressed Gunn rats. When only one molecule of bilirubin is bound to albumin (without bilirubin overload), no displacement of bilirubin is observed, even if the protein binds as much as 6 molecules of free fatty acids. After overloading with exogenous unconjugated bilirubin, the second site of fixation of bilirubin on albumin is partly occupied; in this situation, a displacement is observed, but only when more than 3.5 molecules of free fatty acids are simultaneously bound to the protein. In vivo, free fatty acids do not spontaneously reach such levels as those responsable for the observed displacement of bilirubin. In the ranges of bilirubin and free fatty acids concentrations likely to be encountered clinically, free fatty acids might not represent an effective agent of displacement for bilirubin, as it is commonly thought.     

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.     

Role of very low density lipoproteins in the energy metabolism of the rat

The role of very low density lipoproteins (VLDL) in the energy metabolism of conscious, 24-hr fasted rats was studied. VLDL labeled with [2-3H]glycerol and [1-14C]palmitate were infused into the rats, along with [1-13C]palmitate bound to albumin and d-8-glycerol, and various metabolic factors were assessed. The rates of appearance in plasma of fatty acids in VLDL and albumin-bound free fatty acids (FFA) were about equal, on a molar basis, and only a small fraction of the FFA flux was derived from VLDL. The rate of direct oxidation of the fatty acids from VLDL was 4.4 +/- 0.9 mumol of FA/kg X min, as compared with the value of 4.0 +/- 0.42 mumol of FA/kg X min for plasma FFA. Four percent of the plasma glycerol flux was derived from VLDL. Thus, the direct oxidation of fatty acids in VLDL played an important role in the energy metabolism of the rats, accounting for a percentage of the total CO2 production that was equal to the amount that arose from the oxidation of plasma FFA. The oxidation of VLDL-fatty acids did not involve prior entry of the fatty acids into the plasma FFA pool to any significant extent.     

Validation and use of centrifugal ultrafiltration-dialysis in the measurement of percent free oestradiol in serum

The validity of the centrifugal ultrafiltration-dialysis method in the measurement of the percentage of oestradiol and testosterone which is not bound to protein (free) in human serum was assessed. In an initial validation, the values of % free steroid obtained for oestradiol and testosterone in protein-free ultrafiltrates of human serum were higher than expected. Filter paper discs used in the system were shown to adsorb [3H]oestradiol from protein-free solution. Their replacement with glass-fibre discs, which did not adsorb oestradiol, decreased but did not eliminate the discrepancy between observed and expected results for % free oestradiol in ultrafiltrates. The use of polyethylene rather than polypropylene vials then reduced the discrepancy to insignificant levels. The estimates for % free oestradiol in serum were reduced by these modifications.     

Peripheral oestrogen metabolism in postmenopausal women with or without breast cancer: the role of dietary lipids and growth factors

Studies we have carried out have revealed significant differences in oestrogen production and metabolism between normal women and postmenopausal women with breast cancer. The free, biologically available fraction of oestradiol is elevated in plasma from women with breast cancer and we have found that metabolic clearance rates and production rates of oestradiol are also increased. In vitro studies have suggested that lipids can influence the distribution of sex steroids in plasma and we have therefore examined the effect of dietary lipids on the distribution of sex steroids in plasma in vivo. Consumption of a meal with a high saturated fat content or the oral or i.v. administration of "Intralipid", a stabilised emulsion of soya bean oil that is high in unsaturated free fatty acids, had little effect on the available fractions of oestradiol in plasma. However, results from a preliminary study suggest that long-term changes in dietary fat intake can alter the distribution of steroids in plasma. It is concluded that dietary lipids may influence the availability of sex steroids to tissues. Such a mechanism could account for the significant correlation that has been found between dietary fat consumption and the incidence of breast cancer on a world-wide basis.     

Serum albumin stimulates serotonin uptake into human blood platelets

Active uptake of serotonin (5-hydroxytryptamine, 5-HT) into blood platelets from healthy donors exhibited a lower Vmax value in buffer media than in plasma. Also in plasma ultrafiltrate Vmax was reduced, but it returned to the level measured in plasma upon addition of human serum albumin (40 milligrams) containing fatty acids. Fatty-acid-free albumin was even more stimulatory and when added to platelets in a phosphate-buffered medium, it increased Vmax beyond the value observed in plasma. Km values calculated on the basis of unbound 5-HT were not affected by the media except for a slight decrease in ultrafiltrate as compared to plasma. The fraction of 5-HT (0.5 mumol/l) bound to 40 milligrams albumin was 17% with the preparation containing fatty acids and 22% with fatty-acid-free albumin, while total plasma proteins dissolved in phosphate buffer bound 24%. The uptake of 1 mumol/l 5-HT was enhanced by both albumin preparations already at 1 milligram and near-maximal effects occurred at 10 milligrams.     

Seasonal variation of neutral lipids in Pinus sylvestris L. sapwood and heartwood

Summary The lipid levels and fatty acid composition of three fractions (free fatty acids, triacylglycerols and sterol/triterpenoid esters) extracted from the sapwood and heartwood of three stems of Pinus sylvestris were determined to investigate both seasonal changes in sapwood and possible metabolic changes related to heartwood formation. Seasonal changes were observed only in the amount of the free fatty acids in the sapwood: the level of free fatty acids was greatest at the beginning and end of the growing season. In the January and March samples the amount of the free fatty acid fraction in the sapwood was very small. The amount of the other fractions remained at the same level throughout the study. Marked seasonal changes in the fatty acid composition occurred only in the free fatty acid fraction of the sapwood: the saturation grade increased during the winter.     

Free fatty acids: a possible regulator of the available oestradiol fractions in plasma

Consumption of dietary fats has been linked to the high incidence of breast cancer found in Western women. In vitro studies we have carried out show that unsaturated free fatty acids can increase the biologically available oestradiol fractions in plasma. It is possible therefore that the increased risk for breast cancer associated with a diet high in fats may be related to an elevation in the biologically available oestradiol fractions in plasma.     

Influence of albumin and non-esterified fatty acids on serum prostacyclin binding in pregnancy

We investigated the etiology of the previously documented decrease in serum prostocyclin binding during pregnancy. Addition of albumin to the serum of pregnant women failed to raise binding to non-pregnant levels. Pregnancy serum bound significantly more prostacyclin following the removal of non-esterified fatty acids and the addition of fatty acid free albumin resulted in a rise in binding to non-pregnant levels. We conclude that serum protein prostacyclin binding is affected by both albumin concentration and non-esterified fatty acids.     

Fatty acids increase the circulating levels of oxidative stress factors in mice with diet-induced obesity via redox changes of albumin

Plasma concentrations of free fatty acids are increased in metabolic syndrome, and the increased fatty acids may cause cellular damage via the induction of oxidative stress. The present study was designed to determine whether the increase in fatty acids can modify the free sulfhydryl group in position 34 of albumin (Cys34) and enhance the redox-cycling activity of the copper-albumin complex in high-fat diet-induced obese mice. The mice were fed with commercial normal diet or high-fat diet and water ad libitum for 3 months. The high-fat diet-fed mice developed obesity, hyperlipemia, and hyperglycemia. The plasma fatty acid/albumin ratio also significantly increased in high-fat diet-fed mice. The increased fatty acid/albumin ratio was associated with conformational changes in albumin and the oxidation of sulfhydryl groups. Moreover, an ascorbic acid radical, an index of redox-cycling activity of the copper-albumin complex, was detected only in the plasma from obese mice, whereas the plasma concentrations of ascorbic acid were not altered. Plasma thiobarbituric acid reactive substances were significantly increased in the high-fat diet group. These results indicate that the increased plasma fatty acids in the high-fat diet group resulted in the activated redox cycling of the copper-albumin complex and excessive lipid peroxidation.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Role of acetate in the reduction of plasma free fatty acids produced by ethanol in man

To investigate the mechanism by which ethanol lowers plasma free fatty acids, we tested the ability of two products of alcohol metabolism, acetate and lactate, to lower free fatty acids in man. Sodium acetate was given orally to five healthy fasting volunteers and caused a significant fall in plasma free fatty acids. After amounts of ethanol and acetate that produced similar reductions in free fatty acids, plasma acetate increased 3- to 4-fold within 20 min. In each of three subjects the fall of free fatty acids observed after acetate ingestion occurred at plasma acetate levels less than or equal to those reached after ethanol. In all studies plasma glucose remained stable. Oral administration of sodium lactate to another volunteer in amounts sufficient to raise plasma lactate concentrations to a level similar to that found after ethanol administration failed to lower plasma free fatty acids. Thus acetate, a metabolite of ethanol, reduces plasma free fatty acids at plasma acetate levels comparable to those resulting from ethanol metabolism, which suggests that the lowering of plasma free fatty acids produced by ethanol is mediated, at least in part, by acetate.     

Free amino acids mimic the anabolic but not the proliferative effect of albumin in OK proximal tubular cells

When plasma proteins leak from circulation into the renal tubular lumen in the proteinuric renal diseases, nephrotoxicity of filtered albumin (and/or molecules bound to it) may be important in the subsequent development of tubulo-interstitial damage which contributes to the progression of the disease. When cultured opossum kidney (OK) proximal tubular cells were exposed to bovine serum albumin for 3 days in vitro, increased cell division ([3H]-thymidine incorporation) and cellular hypertrophy (increased protein/DNA ratio) were observed. Both effects were halved if defatted albumin was used. A trivial explanation for the growth responses is that free fatty acids carried on the albumin, and amino acids generated by intracellular degradation of the albumin, are exerting a non-specific growth effect as metabolic fuels which are oxidized to generate ATP. However, the water-soluble free fatty acid octanoate (1 mmol l(-1)) had no significant effect on protein/DNA ratio and a very variable stimulatory effect on [3H]-thymidine incorporation, whereas an essential amino acid mixture or 1 mmol/l(-1) l-Ala or l-Phe only increased the protein/DNA ratio. Furthermore no carnitine was added to the culture medium. This absence would have impaired mitochondrial transport (and hence oxidation) of long-chain free fatty acids derived from the albumin. l-Phe is also a poor substrate for mitochondrial oxidation in kidney. It is therefore concluded that the growth effects of albumin in OK proximal tubular cells are specific effects of the albumin protein and of the free fatty acids and amino acids derived from it, and not a non-specific effect on metabolic fuel supply.     

EFFECT OF FATTY ACIDS ON RAT BRAIN PARTICULATE HEXOKINASE

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo , is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.     

Nonsteroidal substances that affect serum free testosterone

Several steroid hormones affect free testosterone (FT) levels in blood by competing with testosterone for binding sites on testosterone-binding globulin (TeBG). However, the effect of endogenous nonsteroidal substances in serum has not been reported. Some of these potential modifiers of FT were studied using equilibrium dialysis. Nonesterified fatty acids at 0.9 mM elevated FT approx 10% at pH 7.4. Investigation of the curvilinear relationship of percent FT (pFT) vs pH showed that pH-dependent changes of testosterone binding to albumin were responsible for a small linear increase in pFT with decreasing pH. The greater portion of the curvilinear increase of pFT with decreasing pH was due to fatty acids competing with testosterone for TeBG binding sites. Ketone bodies significantly affected FT (7.5% elevation) only at levels found in diabetic ketoacidosis. Sodium ions improved binding 11% when 7 mM was compared to 157 mM sodium, but physiological changes in sodium would result in only +/- 1% changes in FT. Very low levels (0.03 mM) of calcium may be essential for normal testosterone binding to TeBG since 1.0 mM EGTA raised FT by 75%. This study shows that dialysis at 37 degrees C should not be performed overnight, that thimerosol should not be used as a preservative, and that the dialysis buffer should contain physiological concentrations of sodium and calcium.     

Diet and serum sex hormones in healthy men

The possible effect of dietary fat content and the ratio of polyunsaturated to saturated fatty acids (P/S-ratio) on serum sex hormones was studied in 30 healthy male volunteers. The customary diet of the subjects, which supplied 40% of energy as fat (mainly from animal sources, P/S-ratio 0.15) was replaced for a 6 weeks period by a practically isocaloric experimental diet containing significantly less fat (25% of energy) with a higher P/S-ratio (1.22) and other environmental factors were stabilized. Serum testosterone and 4-androstenedione decreased from 22.7 +/- 1.1 nmol/l to 19.3 +/- 1.2 nmol/l, (SEM, P less than 0.001) and from 4.6 +/- 0.2 nmol/l to 4.3 +/- 0.2 nmol/l (SEM, P less than 0.01), respectively. These changes were paralleled by a reduction in serum free (non-protein bound) testosterone (P less than 0.01) suggesting a possible change in biological activity. During the low fat period a significant negative correlation between serum prolactin and androgens was observed. All the changes in androgen levels were reversible. With the exception of a small but non-significant decrease in serum estradiol-17 beta, the other hormone parameters were practically unaffected by the dietary manipulation. Our results indicate that in men a decrease in dietary fat content and an increase in the degree of unsaturation of fatty acids reduces the serum concentrations of androstenedione, testosterone and free testosterone. The mechanism and importance of this phenomenon is discussed in the light of epidemiological and experimental data.