The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation

The fatty acid compositions of the lipids and the lipid peroxide concentrations and rates of lipid peroxidation were determined in suspensions of liver endoplasmic reticulum isolated from rats fed on synthetic diets in which the fatty acid composition had been varied but the remaining constituents (protein, carbohydrate, vitamins and minerals) kept constant. Stock diet and synthetic diets containing no fat, 10% corn oil, herring oil, coconut oil or lard were used. The fatty acid composition of the liver endoplasmic reticulum lipid was markedly dependent on the fatty acid composition of the dietary lipid. Feeding a herring-oil diet caused incorporation of 8.7% eicosapentaenoic acid (C_20:5) and 17% docosahexaenoic acid (C_22:6), but only 5.1% linoleic acid (C_18:2) and 6.4% arachidonic acid (C_20:4), feeding a corn-oil diet caused incorporation of 25.1% C_18:2, 17.8% C_20:4 and 2.5% C_22:6 fatty acids, and feeding a lard diet caused incorporation of 10.3% C_18:2, 13.5% C_20:4 and 4.3% C_22:6 fatty acids into the liver endoplasmic-reticulum lipids. Phenobarbitone injection (100mg/kg) decreased the incorporation of C_20:4 and C_22:6 fatty acids into the liver endoplasmic reticulum of rats fed on a lard, corn-oil or herring-oil diet. Microsomal lipid peroxide concentrations and rates of peroxidation in the presence of ascorbate depended on the nature and quantity of the polyunsaturated fatty acids in the diet. The lipid peroxide content was 1.82±0.30nmol of malonaldehyde/mg of protein and the rate of peroxidation was 0.60±0.08nmol of malonaldehyde/min per mg of protein after feeding a fat-free diet, and the values were increased to 20.80nmol of malonaldehyde/mg of protein and 3.73nmol of malonaldehyde/min per mg of protein after feeding a 10% herring-oil diet in which polyunsaturated fatty acids formed 24% of the total fatty acids. Addition of α-tocopherol to the diets (120mg/kg of diet) caused a very large decrease in the lipid peroxide concentration and rate of lipid peroxidation in the endoplasmic reticulum, but addition of the synthetic anti-oxidant 2,6-di-t-butyl-4-methylphenol to the diet (100mg/kg of diet) was ineffective. Treatment of the animals with phenobarbitone (1mg/ml of drinking water) caused a sharp fall in the rate of lipid peroxidation. It is concluded that the polyunsaturated fatty acid composition of the diet regulates the fatty acid composition of the liver endoplasmic reticulum, and this in turn is an important factor controlling the rate and extent of lipid peroxidation in vitro and possibly in vivo.     

Lipid and Fatty Acid Composition in the Flesh of an Edible Marine Fish: Amadi (Coilia reynaldi)

Amadi is a small sized edible marine fish species (Coilia reynaldi) under the order-Clupeiformes. It is important for principal lipids and in particular for highly unsaturated fatty acids which have potential biomedical benefits. Among the lipid classes, phospholipids were found to be the most predominant constituents than the glycolipid and neutral lipid in Amadi. Twenty six fatty acids were quantified by open tube gas–liquid chromatography. Dominant fatty acids in this fish are Palmitic acid (C_16:0), Stearic acid (C_18:0), Oleic acid (C_18:1n?9), Myristic acid (C_14:0), Palmitoleic acid (C_16:1), Docosahexanoic acid (C_22:6n?3), Pentadecanoic acid (C_15:0), and Eicosatetraenoic acid (C_20:4n?3). Fatty acid deficiency in fish species is indicated by the presence of C_20:3n?9 acid. It is absent in this fish.The content of DHA and EPA are maximum in amount in neutral lipid than other lipid classes.     

Mitochondria as a site of C4 acid decarboxylation in C4-pathway photosynthesis.

One group of C₄, species utilize a NAD-malic enzyme to decarboxylate C₄ acids. This enzyme, together with a major isoenzyme of aspartate aminotransferase and a NAD-malate dehydrogenase, is localized in the mitochondria of the bundle sheath cells and the following pathway for C₄, acid decarboxylation has been proposed: aspartate → oxaloacetate → malate → CO₂ + pyruvate. The present study reports that mitochondria isolated from the bundle sheath cells of one of these species, Atriplex spongiosa, are capable of decarboxylating C₄, acids at rates between 5 and 8 μmol/min/mg chlorophyll. For maximum decarboxylating activities, these particles required aspartate, 2-oxoglutarate and phosphate as well as malate; in the absence of any one of these compounds, activity was reduced to 0.3–0.8 μmol/min/mg chlorophyll. Rates for C₄ acid decarboxylation were much greater than the respiratory activities of these particles, including the capacity to form citrate or to oxidize malate, succinate, pyruvate or 2-oxoglutarate (0.03–0.6 μmol/min/mg chlorophyll). A comparison of mitochondria prepared from leaves of various C₄, and C₃, species showed that only the mitochondria from the bundle sheath cells of plants with high NAD-malic enzyme have capacities for rapid C₄ acid decarboxylation. The effects of a variety of experimental conditions on C₄ acid decarboxylating activities are also reported. The role of these mitochondria in C₄ photosynthesis is discussed.     

Effects of sodium chloride concentration on phospholipid fatty acid composition of yeasts differing in osmotolerance

The effect of growth medium NaCl concentration on the fatty acid composition of phospholipids of 3 strains of Saccharomyces cerevisiae and 6 osmotolerant yeast strains was examined. The S. cerevisiae strains were characterized by a high content of palmitoleic (C_16:1) acid and by having no polyunsaturated C₁₈ acids, whereas the osmotolerant strains had a low content of C_16:1 and a high proportion of polyenoic C₁₈ acids. An increase of the NaCl concentration from 0% to 8% resulted in a decrease of the cellular phospholipid content on a dry-weight basis, for all strains but one of the osmotolerant strains. For the S. cerevisiae strains increased salinity produced a slight decrease of the proportion of C₁₆ fatty acids with a concomitant increase of C₁₈ acids, whereas the osmotolerant strains showed an increase of the relative content of oleic acid (C_18:1) at the expense of the proportion of polyenoic C₁₈ acids.     

Elongation of mitochondrial fatty acids during brain development

Abstract— Elongation of mitochondrial fatty acids was studied in whole brain samples from rats before, during and after the period of myelination. The mitochondria were isolated by centrifugation in a discontinuous sucrose gradient and incubated under N₂ in a medium containing NADH, NADPH, ATP and acetyl-[1-¹⁴C]coenzyme A. Fatty acids were extracted, methylated and analysed by gas-liquid chromatography. A distinct pattern emerged in which brain mitochondria from rats undergoing myelination synthesized longer chain fatty acids preferentially, particularly C_22:4. Mitochondria from brains of mature rats synthesized shorter chain fatty acids preferentially, mainly C_18:0 and C_20:4. We suggest that eicosamonoenoic acid (C_22:1) is a precursor in vivo of nervonic acid (C_24:1).     

The effect of carnitine on the oxidation of saturated fatty acids by pea cotyledon mitochondria

Summary Carnitine was found to stimulate fatty acid oxidation by pea (Pisum sativum L.) cotyledon mitochondria. The stimulation was at a maximum for long chain (C_16:0) and short chain (C_4:0 and C_6:0) fatty acids. Evidence was also provided which indicated that mid-chain (C_10:0 and C_12:0) fatty acid oxidation by mitochondria was stimulated by carnitine. It is postulated that carnitine acts by facilitating transport of these species of fatty acids across the mitochondrial membranes to intramitochondrial -oxidation sites.Abbreviations ADP adenosine-5¹-diphosphate - ATP adenosine-5¹-triphosphate - BSA bovine serum albumin - CoA coenzyme A - EDTA ethylenediamine tetra-acetate - RCR respiratory control ratio     

The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria

The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.     

Fatty acid and lipid analysis of the house cricket,Acheta domesticus

The fatty acid content and composition of the house cricket Acheta domesticus have been investigated in entire insects at different developmental stages and in selected organs of male and female adults. We have also determined the fatty acid composition of the various lipid classes within extracts of the organs of adult female insects. Fatty acids were analysed by capillary gas chromatography or mass spectrometry as their methyl esters (FAMEs) after direct transesterification of insect material or separated lipid classes.The major esterified fatty acids in all extracts were palmitate (C_16:0), stearate (C_18:0), oleate (C_18:1) and linoleate (C_18:2). Levels of esterified fatty acid varied considerably between organs but the fatty acid compositions showed only small variations. The levels of polyunsaturated fatty acids of the C₁₈ series were considerably higher in phospholipid fractions than in other lipid classes. Triacylglycerols formed the major lipid class in ovaries, fat-body and newly-laid eggs, whereas diacylglycerols and phospholipid predominate in the haemolymph. Triacylglycerols, phospholipids, diacylglycerols and free fatty acids were all found in significant amounts in the gut tissue.     

Variations in the Lipid Compositions and in the Lipid Molecular Species of Lipomyces starkeyi Cultivated in the Glucose Sufficient and the Glucose Deficient Media

The lipid compositions, fatty acid compositions, positional distributions of fatty acids in glycerides, and molecular species of phospholipids of L. starkeyi, cultured in the glucose sufficient and the glucose deficient media were compared.

Under the glucose sufficient condition, the triglyceride content increased, accompanied by the remarkable increase of C_16:0–C_18:1–C_18:0. The phospholipid content also increased with the variations of the compositions of molecular species in phosphatidylethanolamine and phosphatidylcholine.

Under the glucose deficient condition, the triglyceride content remarkably decreased, especially in C_18:1–C_18:1–C_18:1. The compositions of phospholipid molecular species were considerably different from those of the glucose sufficient condition.     

Protein deficiency and age related alterations in rat peritoneal macrophage lipids

The effects of dietary protein restriction and age on the thioglycollate elicited peritoneal macrophage lipid constituents were studied. Impact of subtle changes in lipid components on macrophage functions have been assessed. Lipid profiles of macrophages recovered from rats fed 20 and 4% protein diets and stock diet fed rats (0 and 3 wk) were comparable qualitatively. Quantitative analysis however revealed significant decrease in phospholipids (30–40%) and consequent elevation of cholesterol/phospholipid molar ratios in the protein depleted and young rats (0 wk), compared to the protein fed groups. The protein deficient and the young rats also exhibited accumulation of certain neutral lipids and reduction in triglycerides. Analysis of fatty acid methyl esters of macrophage phospholipids revealed the predominance of long chain polyunsaturated fatty acids even when oleic (C_18:1) and linoleic (C_18:2) formed the bulk of unsaturated fatty acids in the diet. However, the long chain poly unsaturated fatty acid content, particularly the docosahexaenoic acid (C_22:6n-3) was greatly reduced in the protein depleted and 0 wk rats. Observed changes in the long chain polyunsaturated fatty acids of macrophage phospholipids may be of physiological significance as they modulate the immunological functions of the cell.     

Identification of the alga known as <Emphasis Type="Italic">Nannochloropsis</Emphasis> Z-1 isolated from a prawn farm in Hainan,China as <Emphasis Type="Italic">Chlorella</Emphasis>

An alga known as “Nannochloropsis”, isolated from a prawn farm in Hainan, China, has been critically investigated and identified as Chlorella, a member of the Chlorophyceae based on fatty acid composition, ultrastructure, and 18S rDNA. Cells of this alga were spherical, measured by 1–6 μm in diameter and were enclosed in thin walls of approximately 0.04 μm thickness. They contained several small mitochondria, two to three thylakoids and had no vacuoles. There were many pyrenoids in the algal cells and their thylakoid lamellae were sparse and not translucent. Many lipid droplets were present in the cytoplasm. The total lipid content of this alga was 3% per gram dry weight and its major fatty acids were C_16:0, C_18:0, C_18:1, C_18:2, C_18:3 and C_20:0. Eicosapentaenoic acid (C_20:5, EPA) was not detected. The length of its 18S rDNA sequence was 1,712 bp. 18S rDNA sequence analyses indicated that this alga was a species of Chlorella.     

The activity of sphingomyelin cycle enzymes and concentration of sphingomyelin degradation products in rat liver in dynamics of acute toxic hepatitis

Activity of key enzymes of the sphingomyelin cycle and the content of its components (sphingomyelin, ceramide and sphingosine-1-phosphate) have been studied in livers of rats in dynamics of acute toxic hepatitis induced by subcutaneous administration of oil solution of CCl₄. Sphingomyelinase activity significantly increased already on early terms and remained increased over the whole period of observation. Ceramidase activity insignificantly differed from the control level. The levels of sphingomyelin and sphingosine-1-phosphate did not undergo marked changes while ceramide content significantly increased. The balance between liver content of ceramide (proapoptotic) and sphingosine-1-phosphate (the antiapoptotic factor) was shifted towards ceramide over the whole observation period. In sphingomyelin molecules there was a significant decrease in the content of the fatty acids C_18:1 and C_22:2, while in ceramide molecules and sphingosine-1-phosphate only the fatty acid C_22:2 changed. In spite of a significant decrease in the content of some unsaturated fatty acids, calculated unsaturation coefficients of the fatty acid component of the sphingomyelin cycle metabolites insignificantly differed from control. Taking into consideration literature data our results suggest involvement of ceramide-mediated apoptosis in the pathogenesis of acute toxic hepatitis. Elimination of damaged hepatocytes permits realization of repair processes and promotes optimization of cellular community in the liver.     

The distribution of fatty acids including petroselinic and tariric acids in the fruit and seed oils of the Pittosporaceae, Araliaceae, Umbelliferae, Simarubaceae and Rutaceae

The fatty acid composition of the fruit oils or seed oils of Pittosporaceae (eight genera, 10 species), Araliaceae (two species), Simarubaceae (three species), and of one umbelliferous and one rutaceous species were determined by gas chromatography, argentation TLC and ozonolysis. In the Pittosporaceae, in which the major C₁₈ fatty acid of all species was either oleic acid (18:1, 9c) or linoleic acid (18:2, 9c, 12c), large amounts of C₂₀ and C₂₂ fatty acids seem to occur regularly. Petroselinic (18:1, 6c) and tariric (18:1, 6a) acids were absent. However, petroselinic acid was the major fatty acid in the Araliaceae and Umbelliferae. In these two families only small amounts of C₂₀ and C₂₂ acids were detected and tariric acid was absent. The Rutales contained relatively high amounts of trans-octadecenoic acids (18:1, 9t). Tariric acid was the major fatty acid in the two species of Picramnia (Simarubraceae), which also contained small amounts of petroselinic acid. The major fatty acids in Ailanthus glandulosa (Simarubaceae) and Phellodendron amurense (Rutaceae) were linoleic or linolenic acid (18:3, 9c, 12c, 15c); these species contained neither tariric nor petroselinic acid and the levels of C₂₀ and C₂₂ fatty acids were low. The appearance of schizogenous resin canals and polyacetylenes and the absence of iridoids and petroselinic acid allows the Pittosporaceae to be separated from the Rutales and Araliales and to be placed in an independent order, the Pittosporales. Arguments for a rather close relationship of the Pittosporales to the Araliales and Cornales (including the Escalloniaceae) are presented.     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

Acute administration of 3,5-diiodo-L-thyronine to hypothyroid rats stimulates bioenergetic parameters in liver mitochondria

The role of 3,5-diiodo-L-thyronine (T₂), initially considered only a 3,3′,5-triiodo-L-thyronine (T₃) catabolite, in the bioenergetic metabolism is of growing interest. In this study we investigated the acute effects (within 1 h) of T₂ administration to hypothyroid rats on liver mitochondria fatty acid uptake and β-oxidation rate, mitochondrial efficiency (by measuring proton leak) and mitochondrial oxidative damage (by determining H₂O₂ release). Fatty acid uptake into mitochondria was measured assaying carnitine palmitoyl transferase (CPT) I and II activities, and fatty acid β-oxidation using palmitoyl-CoA as a respiratory substrate. Mitochondrial fatty acid pattern was defined by gas-liquid chromatography. In hypothyroid + T₂ vs hypothyroid rats we observed a raise in the serum level of nonesterified fatty acids (NEFA), in the mitochondrial CPT system activity and in the fatty acid β-oxidation rate. A parallel increase in the respiratory chain activity, mainly from succinate, occurs. When fatty acids are chelated by bovine serum albumin, a T₂-induced increase in both state 3 and state 4 respiration is observed, while, when fatty acids are present, mitochondrial uncoupling occurs together with increased proton leak, responsible for mitochondrial thermogenesis. T₂ administration decreases mitochondrial oxidative stress as determined by lower H₂O₂ production. We conclude that in rat liver mitochondria T₂ acutely enhances the rate of fatty acid β-oxidation, and the activity of the downstream respiratory chain. The T₂-induced increase in proton leak may contribute to mitochondrial thermogenesis and to the reduction of oxidative stress. Our results strengthen the previously reported ability of T₂ to reduce adiposity, dyslipidemia and to prevent liver steatosis.     

Transformation of sterols byMycobacterium vaccae: effect of lecithin on the permeability of cell envelopes to sterols

An enhancement of Β-sitosterol transformation to androstendione byMycobacterium vaccae observed in medium containing egg-yolk lecithin, was associated with the incorporation of a considerable amount of lecithin into the cell envelope lipids. By GC/MS measurements, fatty acids ranging from 14 to 22 carbon atoms were identified in the lipids removed from the cells by organic solvents. Octadecenoic (18:1), 2-methyl-octadecenoic (2-Me 18:1), and hexadecanoic (16:0) acids were the major components of the lipid preparation obtained from both the control cells, and the cells grown in lecithin-containing medium. However, in the fatty acid pattern of the latter a distinct increase in the C_18:1 component, concomitant with the decrease in the 2-Me 18:1 fatty acid was demonstrated. The C₁₆ fatty acid fraction also showed a higher content of methyl-branched components in the control cell preparation. The enrichment in unsaturated fatty acids increases fluidity of lipids, whereas the decrease in methyl-branched fatty acids may affect the conformation of the surface lipidic components, which may result in enhanced sterol penetration through the cell wall barrier in the presence of lecithin.     

Structure and composition of hydrocarbons and fatty acids from a marine blue-green alga,Synechococcus sp.

The major hydrocarbons of Synechococcus sp., a marine blue-green alga isolated from the Gulf of Mexico, were identified as 1-nonadecene and 1,14-nonadecadiene. The content of 1-nonadecene increased with culture age from about 0.5 mg/g dry weight in young cells to about 2.3 mg/g dry weight in old cells, while the content of 1,14-nonadecadiene remained constant with culture age at about 1 mg/g dry weight. Both [1-¹⁴C]acetate and [2-¹⁴C]acetate were incorporated to equal extents into the hydrocarbons. [1-¹⁴C]Stearate was incorporated into the hydrocarbons, but [³H]arachidate was not. The fatty acids of Synechococcus sp. were typical of blue-green algae, consisting of C_16:0, C_16:1, C_16:1, C_16:0, C_18:0, C_18:1, C_18:2 and C_18:3 fatty acids were detected. The hexadecenoic acid was shown to be 9-C_16:1 while the octadecenoic acid was a mixture of 93% 11-C_18:1 and 7% 9-C_18:1. The fatty acid content increased during the first 4 days of growth and then decreased slightly.     

CHANGES OF FATTY ACIDS IN LARVAE OF THE WHEAT BLOSSOM MIDGE,SITODIPLOSIS MOSELLANA (GEHIN) (DIPTERA: CECIDOMYIIDAE)*

Abstract The changes of fatty acids in larvae of the wheat blossom midge, Sitodiplosis mosellana (Gehin) at different periods were examined by gas choromatography. There were 10–16 kinds of fatty acids, of which the predominant ingredients were palmitic (C_16:0), oleic (C_18:1) and linoleic (C_18:2) acids which were more than 95% in total fatty acids, stearic acid (C_18:0) about 2%‐3.5% and any of the others was less than 1%. The fatty acid compositions increased from mid‐May, when larvae of the wheat blossom midge left the wheat‐ears and fallen on the ground, to April of next year before pupating and emerging. No arachidic acid (C_20.0) was discovered in over‐summering, over‐wintering as well as inactive over‐wintered larvae. The content of saturated fatty acids in over‐summering, overwintering as well as inactive over‐wintered larvae were less than those of in active over‐wintered larvae and wheat‐ear larvae. Therefore, changes of the arachidic acid and the proportions of saturated fatty acids/unsaturated fatty acids could be used as one of the biochemical criteria to determine the active state and the degree of diapause in larvae of the wheat blossom midge.     

Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C_12:0), N-myristoyl- (C_14:0) and N-palmitoyl- (C_16:0) sphingosine (C_18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C_18:0) substrate. The activity towards N-linolenoylsphingosine (C_18:3/C_18:1), N-linoleoylsphingosine (C_18:2/C_18:1) and N-oleoylsphingosine (C_18:1/C_18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C_18:0/C_18:1). The activity towards N-stearoyldihydrosphingosine (C_18:0/C_18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C_6:0/C_18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher K_m values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.