Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography–electron impact mass spectrometry

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography–mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.     

Location of double bonds in two unsaturated forms of phytanic acid from Refsum disease as determined by mass spectrometry

A monounsaturated and a triunsaturated form of phytanic acid (3,7,11,15-tetramethylhexacosanoate) were isolated from plasma lipids of a patient with Refsum disease. Both were converted to their methyl esters, oxidized to polyhydroxy acids by treatment with OsO4 and converted to their vicinal trimethylsilyl ethers. These derivatives were analyzed by gas chromatography-mass spectrometry using both electron impact ionization (at 21 and 70 eV) and chemical ionization conditions to obtain clear evidence to establish the structure of the monounsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-15-monoenoic acid and that of the triunsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-6,10,14-trienoic acid. The possible metabolic and dietary sources for these novel fatty acids are discussed.     

Identification by gas chromatography and mass spectrometry of lipids from the rat Harderian gland

Lipids from rat Harderian glands were extracted with ethyl acetate, hydrolysed with base and examined by gas chromatography (GC) and gas chromatography—mass spectrometry (GC—MS) as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS, picolinyl, nicotinate and nicotinylidene derivatives. The latter three derivatives were used to reveal the structures of the alkyl chains of fatty acids, alcohols and glycerol ethers, respectively. Forty-eight compounds were identified, representing about 97% of the total extracted lipids as measured by GC peak areas. The major constituents were fatty acids with chain lengths from 12 to 22 carbon atoms (mainly C₁₈ and C₂₀) and fatty alcohols (C₁₆ to C₂₆) derived from wax esters. Most of these acids and alcohols were unsaturated in the ω-7 position and were accompanied by smaller amounts of the saturated and ω-5 monounsaturated analogues. Glycerol ethers were also identified for the first time in this secretion; the ether chains contained from 14 to 19 carbon atoms (mainly 16) and were straight-chain saturated, unsaturated (ω-5 and ω-7) and branched (iso). The only sterol found was cholesterol amounting to 1.24% of the total extract.     

Properties of lipid bilayer membranes made from lipids containing phytanic acid

Besides the preparation of phytanic acid (3,7,11,15-tetramethylhexadecylic acid) according to the Dumas-Stass reaction, the synthesis of four different lipids containing phytanic acid residues is described. Diphytanoyl phophatidylcholine was systhesized beginning from glycerylphosphorylcholine, whereas the other lipids, diphytanoyl phosphatidylethanolamine, diphytanoyl phosphatidylserine and monophytanoyl glyceride were prepared by total synthesis.Some properties of lipid bilayer membranes made from the lipids containing phytanic acid were investigated. The specific capacity of these membranes was measured. Its value of approximately 400 nF cm^?2 was found to be similar to the value of membranes from lipids with unbranched fatty acid residues. Charge pulse experiments were performed using dipicrylamine as a molecular probe of membrane structure. The results were discussed on the basis of a higher viscosity of the membranes from lipids containing phytanic acid residues compared with unbranched fatty acid residues.     

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.     

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.     

Fatty acid composition of lipids from differentiated tissues and cell cultures of Euonymus europaeus

The fatty acid patterns of Euonymus europaeus callus cultures and cell suspension cultures were analysed at the beginning of stationary growth phase and compared with those from the respective differentiated tissues. The lipid and fatty acid patterns in cell cultures differed remarkably from those in the tissues of the mother plant. No glycerol triacetate was detected in the callus cultures derived from differentiated tissues whereas in seeds this lipid compound amounts to 29%. In addition to fatty acids normally occurring in differentiated tissues, lipids in cultured cells also contained short-chain (C₁₂–C₁₄) as well as very long-chain fatty acids (C₂₀–C₂₄). In tissue culture cells the major fatty acids were found to be saturated, whereas in the mother cells unsaturated fatty acids were predominant. Palmitic acid is the most abundant fatty acid in most of the cultures. Lauric, myristic and palmitic acid amount to 50% in lipids of cell suspension cultures.     

Variation in fatty acid composition of <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (Spirulina) strains

A study of the fatty acid composition was made for 35 Arthrospira strains, concentrating on the most abundant fatty acids, the two polyunsaturated C₁₈ acids, linoleic and γ-linolenic acid, and palmitic acid. When grown at 30 ^∘C and low irradiance (10 μmol photon m⁻² s⁻¹), these three acids together formed 88–92% of total fatty acids. There were considerable differences in the composition of the two polyunsaturated acids. Depending on the strain, linoleic acid formed 13.1–31.5% and γ-linolenic acid formed 12.9–29.4% total fatty acids. In contrast, the range for palmitic acid was narrow: 42.3–47.6% of total fatty acids. Repeat experiments on several strains under defined conditions led to closely similar results for any particular environment, suggesting that fatty acid composition can be used as an aid in differentiating between strains. Five additional strains, which had apparently originated from the same original stock cultures as 3 of the 35 in the main study, but from different culture collections, were also assayed. With four strains the results were similar, irrespective of culture source, but with one strain marked differences occurred, especially in the polyunsaturated C₁₈ fatty acid fraction. These differences were independent of the age of the culture. In addition, straight morphotypes derived during repeat subcultures of four strains; each showed a similar fatty acid composition to that of the helical morphotypes of the same strains. A decrease in temperature from 30 to 20 ^∘C, an increase in irradiance (at 30 ^∘C) from 10 to 70 μmol photon m⁻² s⁻¹ and transfer to dark heterotrophy all favoured an increase in polyunsaturated C₁₈ fatty acids. The highest γ-linolenic acid content of any conditions was found for three strains grown heterotrophically on glucose in the dark at 30 ^∘C. A comparative study of six strains of Spirulina confirmed a previous study showing the absence of γ-linolenic acid in all Spirulina strains, thus permitting the separation of these two genera.     

Determination of plasma fatty acid composition in neonates by gas chromatography

Total fatty acids in plasma of neonates have been analysed as their methyl esters by gas chromatography. They were separated on a capillary column coated with a SP-2380 stationary phase. As little as 100 μl of plasma is used for the analysis. The extraction procedure was performed with dichloromethane—methanol (2:1) and fatty acids were methylated with boron trifluoride—methanol. The quantification of fatty acids is based on an internal standard method. Absolute values (μg fatty acid per 100 μl plasma) are given together with relative values (%). At a signal-to-noise ratio of 3, the detection limits for flame ionisation detection are between 0.08 to 0.51 ng. The high sensitivity and precision permits the effective determination of the fatty acids in neonate plasma.     

IN VIVO STUDIES ON THE METABOLISM OF [15,16-3H]TETRACOSANOIC ACID (LIGNOCERIC ACID) IN ADULT RAT BRAIN

—Adult rats were killed 16 h, 48 h, 6 days and 21 days after intracerebral application of n-[15,16-³H]tetracosanoic acid (lignoceric acid). After incorporation into complex lipids with a strong preference for the ester-bound fatty acids of glycerophospholipids, radioactivity decreased with time. The incorporated activity into the amide-bound fatty acids of sphingolipids was also shown to decrease, with exception of the cerebroside of the hydroxy fatty acid type (cerebron fraction). Only negligible amounts of labelled triglyceride and cholesterol ester could be detected. The fatty acids derived from the complex lipids were analysed by radio gas chromatography. It was revealed that some of the applied labelled lignoceric acid was hydroxylated and incorporated into the cerebron fraction while the rest had their chains shortened. In the latter case all even and odd numbered chain lengths down to C₁₈ and C₁₆ (stearic and palmitic acid) were detected. At this stage, the pool of the degradation products of lignoceric acid is stabilized by the preferred incorporation of fatty acids of these chain lengths into glycerophospholipids. A time-dependent desaturation to oleic acid from stearic acid was observed.     

Changes in the Amount of Complex Lipids in the Seeds and in the Pericarp during the Development of Ivy Fruit (Hedera helix)

By a combination of thin-layer chromatography and gas liquid chromatography, a complete study of the development of the different lipid classes and of their fatty acids, during the development of the fruit of Hedera helix L., the English Ivy, has been achieved. In any part of the fruit observed, at any particular stage, the phospholipids and the neutral lipids are the most abundant lipid classes. They accumulate during the entire process of maturation, whereas significant changes occur in their relative proportions, phospholipids being largely dominant until fruit blackening. The accumulation of fatty acids during maturation is characterized by large amounts of C_18:1 in the neutral lipids, especially in the seed, where petroselinic acid (C_18:1Δ⁶) reaches 86% of the total fatty acids. To a smaller extent, the phospholipids also accumulate and thus have the character of reserve molecules. However, their composition remains more stable, which relates them to the “structural lipids” such as galactolipids that maintain their characteristic fatty acid composition, despite the radical changes occurring in the fatty acid metabolism during fruit ripening.     

Characteristics of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA supramolecular complexes at various developmental stages

Differences in viscoelasticity (η) and molecular mass (M) values, as well as in the fatty acid profile of lipids in DNA supramolecular complexes (SC), isolated from Pseudomonas aurantiaca cultures at the exponential and stationary growth phases, were established for the first time. Typical characteristics of DNA SC from actively growing cells were the following: η = 315 ± 15 dl/g, M_DNA = 39 × 10⁶ Da, C_16:0 > C_18:0 > C_18:1 present as basic fatty acids (FA) in a pool of loosely DNA-bound lipids; the tightly DNA-bound lipid fraction consisted of only two acids C_18:0 > C_16:0. Significantly higher values of viscoelasticity η = 779 ± 8 dl/g and M_DNA = 198 × 10⁶ Da were observed for DNA SC of the stationary phase cells; one more FA, C_14:0, was detected in the loosely bound lipid fraction, while lipids tightly bound to DNA contained mainly C_16:0 > C_18:1 > C_18:0 > C_14:0 FA. The content of saturated FA in the DNA-bound lipids in the stationary phase cells was twice as high than in the exponential phase cells. The fraction of tightly bound lipids from the stationary phase cells contained nine times more unsaturated fatty acids than the fraction from proliferating cells. These differences in FA composition of DNA-bound lipids demonstrate the importance of lipids for the structural organization and functioning of genomic DNA during bacterial culture development.     

Changes in fatty acid composition of lipid classes in developing mustard seed

Maturation of mustard (Sinapis alba) seed proceeds with a sharp decrease in the amounts of palmitic and linoleic acids in the total lipids up to 6 weeks after flowering (WAF). Concomitantly, the concentration of oleic acid increases, reaching a plateau at 4 WAF, which is followed by chain elongation of oleic acid to gadoleic and erucic acids. Compositional changes in constituent fatty acids of individual lipid classes indicate that the very long-chain monounsaturated fatty acids (C₂₀ and C₂₂), as opposed to common long-chain fatty acids (C₁₆ and C₁₈), are metabolized to triacylglycerols mainly by esterification to preformed diacylglycerols and monoacylglycerols, rather than via esterification to glycerol-3-phosphate or lysophosphatidic acids.     

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.     

Simultaneous capillary gas chromatographic profiling of medium- and long-chain fatty acid methyl esters with split injection: Correction for injection-related discrimination by the ‘bracketing’ method

Split injection-related discrimination can be a source of inaccuracy and imprecision in quantitative capillary gas chromatographic profiling methods for compounds with relatively big differences in boiling points, such as the methyl esters of medium- and long-chain fatty acids prepared from biological materials. We systematically investigated a standard containing equal masses of saturated fatty acid methyl esters, with chain lengths from C₅ to C₂₆, under different injection conditions, including injection temperature, sample volume and split ratio. Day-to-day performance was studied under one set of conditions. Normalized peak areas, reciprocal response factors, using either C₁₇ or C₂₃ as an internal standard, and ‘bracketed’ reciprocal response factors (peak area of each analyte divided by half the sum of the peak areas of two adjacent esters were calculated. In all experiments the bracketed reciprocal response factors were found to be closest to unity with the lowest coefficients of variation.     

Temperature-Induced Changes in the Fatty Acid Composition of the Cyanobacterium, Synechocystis PCC6803

Changes in glycerolipid and fatty acid composition with a change in growth temperature were studied in the cyanobacterium, Synechocystis PCC6803. Under isothermal growth conditions, temperature did not significantly affect the composition of the various classes of lipids, but a decrease in temperature altered the degree of unsaturation of C₁₈ acids at the sn-1 position, but not that of C₁₆ acids at the sn-2 position of the glycerol moiety in each class of lipids. When the growth temperature was shifted from 38°C to 22°C, the desaturation of C₁₈ acids, but not that of C₁₆ acids, was stimulated. The desaturation of fatty acids occurred only in the light and was inhibited by chloramphenicol, rifampicin and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, but not by cerulenin, an inhibitor for fatty acid synthesis. These findings suggest that desaturase activities are induced after a shift from a higher to a lower temperature, and that the desaturation of fatty acids is connected with the reactions involved in photosynthetic electron transport.     

Determination of very-long-chain fatty acids in plasma by a simplified gas chromatographic—mass spectrometric procedure

The concentration of very-long-chain fatty acids (VLCFA) (straight chain, more than 22 carbon atoms) in plasma or in cultured fibroblasts is one of the most important diagnostic criteria for the diagnosis of the peroxisomal disorders. A sensitive method for VLCFA assay in plasma, using small sample volume and a simplified procedure, is described. After adequate extraction and derivatization, methyl esters of VLCFA are separated, identificated and quantified by gas chromatography—mass spectrometry (GC—MS). The method is sensitive, reproducible, accurate and relatively simple. GC—MS equipment used for routine organic acid analysis can be used.     

Determination of an antisecretory trimethyl prostaglandin E2 analog in human plasma by combined capillary column gas chromatography—negative chemical ionization mass spectrometry

A method is described for measuring a trimethyl prostaglandin E₂ analog, TM-PGE₂, in human plasma. Trideuterated and monofluorinated analogs of TM-PGE₂ are added to plasma as internal standard and carrier, respectively. The plasma is adjusted to pH 3.0 and is extracted with a mixture of benzene—dichloromethane (9:1). The residue, following removal of the extracting solvent, is reacted consecutively with pentafluorobenzyl bromide and bistrimethylsilyltrifluoroacetamide. The excess derivatizing reagents are removed by evaporation, and an aliquot of the reconstituted residue is analyzed by capillary column gas chromatography using methane as the carrier gas. A quadrupole mass spectrometer is set to monitor in the gas chromatographic effluent the (M − C₇H₂F₅)⁻ fragmention of TM-PGE₂ (m/e 449) and trideuterated TM-PGE₂ (m/e 452) generated by methane negative chemical ionization. Quantitation of unknowns is based on a comparison of the m/e 449 to m/e 452 ion ratio in each unknown to that obtained from the analysis of control plasma spiked with known amounts of TM-PGE₂ and fixed amounts of internal standard and carrier. The sensitivity limit of the assay is approximately 100 pg ml⁻¹, which is equivalent to 1 pg injected. The assay was used to measure the concentration of TM-PGE₂ in the plasma of two subjects following a single 10 μg kg⁻¹ oral dose of the drug.     

Liver fatty acid binding protein expression enhances branched-chain fatty acid metabolism

Although liver fatty acid binding protein (L-FABP) is known to enhance uptake and esterification of straight-chain fatty acids such as palmitic acid and oleic acid, its effects on oxidation and further metabolism of branched-chain fatty acids such as phytanic acid are not completely understood. The present data demonstrate for the first time that expression of L-FABP enhanced initial rate and average maximal oxidation of [2,3-3H] phytanic acid 3.5- and 1.5-fold, respectively. This enhancement was not due to increased [2,3-3H] phytanic acid uptake, which was only slightly stimulated (20%) in L-FABP expressing cells after 30 min. Similarly, L-FABP also enhanced the average maximal oxidation of [9,10-3H] palmitic acid 2.2-fold after incubation for 30 min. However, the stimulation of L-FABP on palmitic acid oxidation nearly paralleled its 3.3-fold enhancement of uptake. To determine effects of metabolism on fatty acid uptake, a non-metabolizable fluorescent saturated fatty acid, BODIPY-C16, was examined by laser scanning confocal microscopy (LSCM). L-FABP expression enhanced uptake of BODIPY-C16 1.7-fold demonstrating that L-FABP enhanced saturated fatty acid uptake independent of metabolism. Finally, L-FABP expression did not significantly alter [2,3-3H] phytanic acid esterification, but increased [9,10-3H] palmitic acid esterification 4.5-fold, primarily into phospholipids (3.7-fold) and neutral lipids (9-fold). In summary, L-FABP expression enhanced branched-chain phytanic acid oxidation much more than either its uptake or esterification. These data demonstrate a potential role for L-FABP in the peroxisomal oxidation of branched-chain fatty acids in intact cells.