In vivo conversion of linoleic acid to arachidonic acid in human adults

Human adults are shown to be capable of conversion of linoleic acid (LA, 18:2 n-6) to arachidonic acid (AA, 20:4 n-6) in vivo. It is confirmed that they can also convert alpha-linolenic acid (LNA, 18:3 n-3) to eicosapentaenoic acid (EPA, 20:5 n-3) and to docosahexaenoic acid (DHA, 22:6 n-3) in vivo. The time course and the maximal response for these processes during the first week after a single dose of the 18-carbon precursor is described. A stable-isotope method in which the protons of the C17 and C18 carbons are substituted with deuterium atoms is used in order to provide for a safe method for the study of human metabolism. High sensitivity and selectivity of detection is assured with negative ion, gas chromatography/mass spectrometry analysis. It is clear that human adults on an ad lib diet carry out EFA metabolism in vivo.     

Mitochondrial and peroxisomal oxidation of arachidonic and eicosapentaenoic acid studied in isolated liver cells

The partitioning between peroxisomal and mitochondrial beta-oxidation of [1-14C]eicosapentaenoic acid (20:5(n-3] and [1-14C]arachidonic acid (20:4(n-6)) was studied. In hepatocytes from fasted rats approximately 70% of the fatty acid substrate was oxidized with oleic, linoleic, eicosapentaenoic and docosahexaenoic (22:6(n-3)) acid, even more with adrenic (22:4(n-6)) and less with arachidonic acid. When the mitochondrial oxidation was suppressed by fructose refeeding and by (+)-decanoylcarnitine, the fatty acid oxidation in per cent of that in cells from fasted rats was with 18:1(n-9) 7%, 18:2(n-6) 8%, 20:4(n-6) 12%, 20:5(n-3) 20%, 22:4(n-6) 57% and for 22:6(n-3) 29%. The fraction of 14C recovered in palmitate and other newly synthesized fatty acids after fructose refeeding decreased in the order 22:4(n-6) greater than 22:6(n-3) greater than 20:5(n-3) greater than 20:4(n-6) and was very small with 18:1(n-9) and 18:2(n-6). In cells from both fed and fructose-refed animals 20:5(n-3) was efficiently elongated to 22:5(n-3) and 22:6(n-3). 20:5(n-3) and 20:4(n-6) were not elongated after fasting. The phospholipid incorporation with [1-14C]20:5(n-3) decreased during prolonged incubations while it remained stable with [1-14C]arachidonic acid. The results suggest that peroxisomes contribute more to the oxidation of 20:5(n-3) than with 20:4(n-6) although both substrates are probably oxidized mainly in the mitochondria.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

Role of lysosomal acid ceramidase in the metabolism of ceramide in human skin fibroblasts

A 2.8-fold accumulation of ceramide was demonstrated in cultured skin ftbroblasts from a patient with Farber's disease, an inborn error of metabolism in which acid ceramidase activity is deficient. To investigate the role of acid ceramidase in the metabolism of ceramide in fibroblasts, we have investigated the lysosomal degradation of ceramide that was taken up by fibroblasts from an exogenous lipid suspension. Fluorescent 4-nitrobenz-2-oxa-1,3-diazole-7-aminododecanoyl-sphingosine (NBD-ceramide) from an exogenous ceramide suspension was incorporated into the intracellular structures of fibroblasts at 37 °C. Study of the cellular uptake of exogenous [³H]oleylsphingosine showed that the rate of ceramide accumulation was nearly identical in Farber's disease and normal fibroblasts. The deficiency of acid ceramidase in Farber's fibroblasts resulted in the decrease of cellular degradation and uptake of ceramide and the increase of retention time of ceramide in these diseased cells. Studies of subcellular fractionation of these fibroblasts showed that the accumulated ceramide was located in the lysosomal fraction. As a result, the density of the lysosomal fraction of Farber's fibroblasts was found to be less than that of controls. These results suggest the defect of cellular metabolism in this inherited disease is located within the lysosome.     

Phytanic acid alpha-oxidation in human cultured skin fibroblasts.

We studied the oxidation of [1-14C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts. The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.     

Transcellular conversion of endogenous arachidonic acid to lipoxins in mixed human platelet-granulocyte suspensions

Incubation of mixed human platelet/granulocyte suspensions with ionophore A23187 led to a platelet dependent formation of several lipoxin isomers from endogenous substrate. The major metabolite coeluted with authentic lipoxin A4 (5(S), 6(R), 15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) in several HPLC-systems and showed an identical UV-spectrum. Furthermore, a similar profile of lipoxins was formed in pure platelet suspensions incubated with exogenous leukotriene A4 (5(S) -5, 6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid). The conversion of exogenous leukotriene A4 to lipoxin A4 was markedly increased in the presence of ionophore A23187.     

Characterization of acid phosphatase isoenzymes in human skin fibroblasts.

The multiple acid phosphatase isoenzymes of cultivated skin fibroblasts were investigated in an effort to further clarify the basis of the observed molecular heterogeneity. Treatment with neuraminidase did not alter the migration of isoenzymes present prior to treatment with neuraminidase did not alter the migration of isoenzymes present prior to treatment, but additional isoenzymes were detectable after treatment. Variation of enzymes, permitting prediction of net charge and molecular size differences. Isoelectric focusing between pH 5.0 and pH 8.0 demonstrated three isoenzymes of acid phosphatase in the total cell homogenate and in the lysosomal fraction, two of which appeared to have similar isoelectric points.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.     

Tumor necrosis factor is cytotoxic to human fibroblasts in the presence of exogenous arachidonic acid

Recombinant human tumor necrosis factor (TNF) stimulated the growth of confluent human fibroblasts (FS-4) in serum-free culture medium. However, TNF had a cytotoxic effect upon the growth of FS-4 cells in combination with arachidonic acid. When arachidonic acid was added to culture medium in the absence of TNF, however, it had no effect on the cell growth. Arachidonic acid inhibited the TNF-induced cell growth in a dose-dependent manner: it reversed the TNF-stimulated growth to the control level at a concentration of 10 microM and was cytotoxic to TNF-treated FS-4 cells at higher concentrations. This cytotoxicity of TNF was not observed in FS-4 cells treated with palmitic acid. Indomethacin, a cyclooxygenase inhibitor, decreased the cytotoxic effect that TNF exerted in the presence of arachidonic acid. These results suggest that TNF becomes cytotoxic to FS-4 cells when arachidonic acid present in the culture medium is converted to prostaglandins.     

Linoleic acid kinetics and conversion to arachidonic acid in the pregnant and fetal baboon.

Linoleic acid plasma kinetics in pregnant baboons and its conversion to long chain polyunsaturates (LCP) in fetal organs is characterized over a 29-day period using stable isotope tracers. Pregnant baboons consumed an LCP-free diet and received [U-13C]linoleic acid (18:2*) in their third trimester of gestation. In maternal plasma, 18:2* dropped to near baseline by 14 days post-dose, while labeled arachidonic acid (20:4*) plateaued at 10 days at about 70% of total labeled fatty acids. After 2;-5 days, total tracer fatty acids decreased in visceral organs, but increased in the fetal brain. Maximal fetal incorporation of 18:2* was 1;-2 days post-dose; thereafter it dropped while 20:4* increased reciprocally. Labeled 20:4 replaced 18:2* in neural tissues by 5 days post-dose. In liver, kidney, and lung, 20:4* became dominant by 12 days, but in heart the crossover was >29 days. Fetal brain 20:4* plateaued by 21 days at 0. 025% of dose, while fetal liver 20:4* was constant from 1 to 29 days at 0.006% of dose. Under these dietary conditions we estimate that the fetus derives about 50% its 20:4 requirement from conversion of dietary 18:2, with the balance from maternal stores, and conclude that 1) fetal organs accumulate 18:2 within a day of a maternal dose and convert much of it to 20:4 within weeks, 2) modest dietary 18:2 levels may support fetal brain requirements for 20:4, and 3) the brain retains n;-6 fatty acids uniquely compared with major visceral organs.     

Factors influencing the latency of the peroxisomal enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) in permeabilized human skin fibroblasts.

In selectively permeabilized fibroblasts suspended in a medium mimicking the composition of the cytosol the peroxisomal enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) was found to exhibit about 80% latency (Wolvetang, E.J., Tager, J.M. and Wanders, R.J.A. (1990) Biochem. Biophys. Res. Commun. 1035, 6-11). We investigated which components of the cytosol mimicking medium are important for latency of DHAP-AT and unmasking of latent DHAP-AT activity by ATP. We show that the latency of DHAP-AT is critically dependent upon the presence of reduced glutathione in the medium and that the in vivo prevailing GSH/GSSG ratio is sufficient to maintain DHAP-AT latency. Although thiol-groups in the peroxisomal membrane seem to be essential for the integrity of peroxisomes in selectively permeabilized fibroblasts no latency of DHAP-AT is observed in buffered sucrose media or in cell homogenates, irrespective of the presence of GSH in the medium used. We suggest that during homogenization irreversible damage is inflicted upon the peroxisomal membrane and/or that more factors than at present investigated are involved in maintaining peroxisomal integrity. Furthermore, we demonstrate that cations play a role in the stimulatory effect of ATP on latent DHAP-AT activity while a proton gradient is not directly involved in the stimulatory effect of ATP on latent DHAP-AT activity.     

Hepatic peroxisomal and mitochondrial fatty acid oxidation in the riboflavin-deficient rat.

The effects of riboflavin deficiency on hepatic peroxisomal and mitochondrial palmitoyl-CoA oxidation were examined in weanling Wistar-strain male rats. The specific activities of peroxisomal catalase and palmitoyl-CoA-dependent NAD+ reduction were not affected by up to 10 weeks of riboflavin deficiency. In contrast, the specific activity of mitochondrial carnitine-dependent palmitoyl-CoA oxidation was depressed by 75% at 10 weeks of deficiency. The amount of peroxisomal protein per g of liver was not affected by riboflavin deficiency, whereas, expressed per liver, both riboflavin-deficient and pair-fed controls showed decreased peroxisomal protein compared with controls fed ad libitum. Hepatic mitochondria, but not peroxisomes, were sensitive to riboflavin deficiency.     

Role of cytoskeletal elements in the retractile activity of human skin fibroblasts

Giant axonal neuropathy skin fibroblasts, which are characterized by a selective and partial disorganization of vimentin filaments [1] exhibited, when compared with normal skin fibroblasts, less fibrin clot retractile (FCR) activity and spreading within the fibrin clot both during active growth and resting stage. Skin fibroblasts derived from patients affected with adenomatosis of the colon and rectum, which display a disorganized actin network [2], exhibited reduced FCR activity and spreading within the fibrin clot only during resting stage. FCR inhibition was also obtained by treating the cells with colcemid, cytochalasin B (CB) and dihydrocytochalasin B. The data suggest that FCR activity is under the control of different cytoskeletal structures. For the first time, a direct involvement of intermediate-sized filaments could be demonstrated in the interaction between fibroblasts and an organic substratum.     

Histiocytic conversion of human adult skin fibroblasts by the Snyder-Theilen feline sarcoma virus

Oncogenes are apparently involved in the transformation/neodifferentiation of human cells. We now report a novel example of transformation/neodifferentiation: the specific conversion of cultured human adult skin fibroblasts (HSF) to histiocytes (tissue macrophages (TM)) by the Snyder-Theilen feline sarcoma virus (ST: (FeSV)). The de novo conversion of cultured HSF was demonstrated in a large fraction of ST:FeSV (FeLV)-transformed foci in the presence of dexamethasone (DX). Identification of tissue macrophages in ST:FeSV(FeLV)-infected HSF cultures was established by light-and transmission electron microscopy, histochemistry, immunohistochemistry, adherence-reattachment, latex particle uptake, and secretion of bioactive interleukin-1. The ST:FeSV gene and glucocorticosteroids, or other naturally occurring hormones, may play a role in morphogenetic processes within cells from a variety of normal and diseased tissues in situ, including induction of non-bone marrow, mesenchyme-derived, tissue macrophages.     

Insulin-induced insulin resistance of alpha-aminoisobutyric acid transport in cultured human skin fibroblasts.

Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin.     

Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

The chain length of [3H]hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of [3H]glucosamine was investigated. [3H]Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.     

Intracellular oxidation by human glioma cell populations: effect of arachidonic acid

Arachidonic acid (AA) and Gamma linolenic acid have been shown to limit glioma cell growth, stimulate apoptosis and lipid peroxidation. However, brain tumours are characterised by cellular heterogeneity and responding cell populations have not been identified. Brain tumour samples from patients were disaggregated. In cell preparations from 7 gliomas, reactive oxygen species (ROS), morphology and plasma membrane integrity were monitored +/-18-36 microM AA for 15-120 min using flow cytometry. Basal oxidative activity related to cell size/morphology, small granular cells showed lower activity. AA stimulation of ROS formation depended on cell size/morphology. Large, less granular cells showed greater AA stimulation. In 17 gliomas, GFAP immunofluorescence was demonstrated in larger cell populations. The large GFAP positive cell population with low side scatter was the highest responding cell population, suggesting selective tumour cell sensitivity to AA induced ROS formation. ROS may have a role in AA induced cell death and anti-tumour activity of AA in glioma.