Reference values of whole-blood fatty acids by age and sex from European children aged 3-8 years.

OBJECTIVES: To establish reference values for fatty acids (FA) especially for n-3 and n-6 long-chain polyunsaturated FAs (LC PUFA) in whole-blood samples from apparently healthy 3-8-year-old European children. The whole-blood FA composition was analysed and the age- and sex-specific distribution of FA was determined. DESIGN AND SUBJECTS: Blood samples for FA analysis were taken from 2661 children of the IDEFICS (identification and prevention of dietary- and lifestyle-induced health effects in children and infants) study cohort. Children with obesity (n=454) and other diseases that are known to alter the FA composition (n=450) were excluded leaving 1653 participants in the reference population. MEASUREMENTS: The FA composition of whole blood was analysed from blood drops by a rapid, validated gas chromatographic method. RESULTS: Pearson correlation coefficients showed an age-dependent increase of C18:2n-6 and a decrease of C18:1n-9 in a subsample of normal weight boys and girls. Other significant correlations with age were weak and only seen either in boys or in girls, whereas most of the FA did not show any age dependence. For age-dependent n-3 and n-6 PUFA as well as for other FA that are correlated with age (16:0, C18:0 and C18:1n-9) percentiles analysed with the general additive model for location scale and shape are presented. A higher median in boys than in girls was observed for C20:3n-6, C20:4n-6 and C22:4n-6. CONCLUSIONS: Given the reported associations between FA status and health-related outcome, the provision of FA reference ranges may be useful for the interpretation of the FA status of children in epidemiological and clinical studies.

Docosahexaenoic acid for the treatment of fatty liver: randomised controlled trial in children.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in children. We tested whether dietary supplementation with docosahexaenoic acid (DHA) can decrease liver fat content in children with NAFLD. METHODS AND RESULTS: We performed a randomized controlled trial of DHA supplementation (250 mg/day and 500 mg/day) vs. placebo in 60 children with NAFLD (20 children per group). The main outcome was the change in liver fat as detected by ultrasonography after 6, 12, 18 and 24 months of treatment. Secondary outcomes were changes in triglycerides, alanine transaminase (ALT), body mass index (BMI) and homeostasis model assessment of insulin resistance (HOMA). The odds of more severe versus less severe liver steatosis decreased to the same degree at 6 months in children treated with DHA 250 mg/day and DHA 500 mg/day vs. placebo and persisted virtually unmodified for 24 months (OR ≤ 0.02, p ≤ 0.05 for all time points). Triglycerides were lower in the DHA groups than in the placebo group at any time point and ALT was lower in these groups from month 12 onwards. HOMA was lower in the DHA 250 mg group vs. placebo at months 6 and 12. CONCLUSION: DHA supplementation improves liver steatosis in children with NAFLD. Doses of 250 mg/day and 500 mg/day of DHA appear to be equally effective in reducing liver fat content.

The in vitro effects of cigarette smoke on fatty acid metabolism are partially counteracted by simvastatin.

BACKGROUND: Statins enhance the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) from their precursors both in vitro and in vivo. In particular, an increased conversion of linoleic acid (LA) and of alpha-linolenic acid to their derivatives is observed in cultured cells. On the contrary, cigarette smoke (CS) negatively and dose-dependently affects the LC-PUFA production. AIM: To evaluate the effects of CS alone or with simvastatin, on [1-(14)C] LA metabolism in THP-1 cells. RESULTS: CS inhibits LA conversion; after co-incubation, simvastatin nullifies the effects of CS, maintaining LA conversion comparable to controls. However, at the highest CS concentration, simvastatin is unable to counteract the effects of CS. Changes of LA conversion reflect the modulation of desaturase activities by simvastatin and CS. CONCLUSION: CS decreases PUFA conversion and its effects are modulated by the opposite effect of statins. It can be speculated that statin treatments in smoking patients may provide some beneficial effects on PUFA metabolism in addition to lowering cholesterol levels.

Fatty acid composition of plasma, blood cells and whole blood: relevance for the assessment of the fatty acid status in humans.

UNLABELLED: The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of distinct processes: intake, metabolism and peripheral utilization. AIM OF THE STUDY: was to compare the FA profile in plasma, lipoproteins and blood cells with that in whole blood (WB) from healthy volunteers; to assess the quantitative distribution of selected FA in triacylglycerols, cholesteryl esters and phospholipids. Lipid FA profiles are comparable in plasma and lipoproteins but differ from those in blood cells. In WB, the FA profile results from the balanced proportion of FA pools in plasma and cells. The contribution of each lipid class to the total amount of FA differs among blood specimens. Phospholipids of plasma and red blood cell are the major contributors to the FA amount and profile in WB. In conclusion, the FA profile of WB reflects the FA status and WB could be an adequate specimen for the assessment of FA intakes.

Blood fatty acid composition in relation to allergy in children aged 2-9 years: results from the European IDEFICS study.

BACKGROUND/OBJECTIVES: Blood polyunsaturated fatty acids (PUFA) are involved in allergy development, but the etiological role of n-6 and n-3 PUFA is still controversial. A European multicenter study of children (IDEFICS) provided the opportunity to explore the cross-sectional association between fatty acids (FA) and allergy. SUBJECTS/METHODS: Blood FA levels were measured in 2600 children aged 2-9 years and were recorded as the percentage of weight of all FA detected. Logistic regression of allergy status on FA components was adjusted for age, sex, country, body mass index, family history of allergic disease, breast-feeding, and number of siblings. The results were given as odds ratios (OR) for current vs no allergy ever and an increase in FA by 1 s.d. RESULTS: Overall, higher proportions of n-6 PUFA were associated with higher odds of allergy (OR=1.21 (1.05, 1.40)). Monounsaturated FA (MUFA) were associated with reduced risk for allergy (OR=0.75 (0.65, 0.87)), whereas saturated FA did not differ by allergy status. The strongest associations were observed in children <4 years old, with ORs of allergy given as 1.62 (1.15, 2.29) for n-3 PUFA and 0.63 (0.42, 0.95) for MUFA. With regard to individual FA, these associations were independently observed for docosapentaenoic acid (22:5 n-3) and oleic acid (18:1 n-9). CONCLUSIONS: Both PUFA subtypes were positively associated with allergy in an age-dependent manner, whereas MUFA was associated with less allergy. The observation of high proportions of n-3 PUFA in allergic children younger than 4 years might help to understand the nature of early onset of atopic disease.

Whole-blood fatty acids and inflammation in European children: the IDEFICS Study.

BACKGROUND/OBJECTIVES: Fatty acids are hypothesized to influence cardiovascular disease risk because of their effect on inflammation. The aim of this study is to assess the relationship between whole-blood fatty acids (WBFAs) and high-sensitivity C-reactive protein (hs-CRP) in European children. SUBJECTS/METHODS: A total of 1401 subjects (697 boys and 704 girls) aged between 2 and 9 years from the IDEFICS (Identification and prevention of Dietary- and lifestyle-induced health EFfects in Children and infantS) study were measured in this cross-sectional analysis. The sample was divided into three categories of hs-CRP. Associations between WBFA and hs-CRP were assessed by logistic regression models adjusting for body mass index (BMI), country, age, breastfeeding, mother's education and hours of physical activity. RESULTS: Linoleic acid (LA) (P=0.013, 95% confidence interval (CI): 0.822-0.977) and sum of n-6 WBFA (P=0.029, 95% CI: 0.866-0.992) concentrations were associated with lower concentrations of hs-CRP in boys. In girls, a high ratio of eicosapentaenoic acid (EPA)/arachidonic acid (AA) was associated (P=0.018, 95% CI: 0.892-0.989) with lower hs-CRP concentrations. In contrast, sum of blood n-6 highly unsaturated fatty acids (P=0.012, 95% CI: 1.031-1.284), AA (P=0.007, 95% CI: 1.053-1.395) and AA/LA ratio (P=0.005, 95% CI: 1.102-1.703) were associated (P<0.05) with higher concentrations of hs-CRP in girls. CONCLUSIONS: The n-6 WBFAs (sum of n-6 FA and LA) were associated with lower hs-CRP in boys and with higher hs-CRP in girls (AA, sum of n-6 highly unsaturated and AA/LA ratio). More studies are needed to identify the optimal levels of WBFAs to avoid low-grade inflammation in children considering the differences by sex and BMI.

n-6 and n-3 fatty acid accumulation in thp-1 cell phospholipids.

The human monocytic leukemia cell line THP-1 is depleted of the long-chain n-6, AA, when compared to human monocytes. This reflects the low availability of this FA in the growth medium generally used for cultured cells. The effects of AA, as well as EPA, supplementation of THP-1 cells on the incorporation of these FA in cell PL, especially in PC and PE, was investigated. In addition the incorporation of labeled AA in PL from THP-1 cells was compared to that in human monocytes. Measurements were done through HPLC separation of PL, detected by UV absorption and radioactivity, FA analysis by GC and characterization of PC subclasses by FAB-MS. Marked differences were observed in the incorporation of the two FA in cell PL, particularly two PC subclasses, and in the accumulation in individual PL after supplementation of THP-1 cells. Accumulation of AA and EPA in THP-1 cells appeared to be mutually independent. The incorporation of AA was also quite different in THP-1 from that in monocytes. Thus, characterization of the FA content in lipids of cultured cells is an essential requirement for optimal utilization of these cells.

Marine macroalgae analyzed by mass spectrometry are rich sources of polyunsaturated fatty acids.

Algae from cold water (Canada) and warm water (China) were analyzed for their total lipid content, and for their fatty acid (FA) composition and content. The major findings are that FA from Canadian algae are generally richer in polyunsaturated FA (PUFA), with a higher n-3/n-6 FA ratio, and a higher degree of total unsaturation. The 18 C, 4 double bonds FA (18 : 4 stearidonic acid, morotic acid as synonym) was detected in greater amounts in cold water samples. The high levels of total PUFA, and especially of n-3 FA in Canadian algae, suggests their possible utilizations for nutritional purposes.

In vitro differentiation of human monocytes to macrophages results in depletion of antioxidants and increase in n-3 fatty acids levels.

The lipid composition and alpha-tocopherol content of human monocytes were investigated before and after their differentiation to macrophages. The total lipid and protein content per number of cells increased after the differentiation of monocytes by approximately four-fold; a two-fold increase in docosahexaenoic and docosapentaenoic acids and a two-fold decrease in linoleic acid were also noted. As opposed to an initial monocytic vitamin E content of 4.75 pmol/10(6) cells, macrophagic vitamin E levels were undetectable. Changes in vitamin E and fatty acids contents in macrophages, with respect to monocytes, appear to reflect the lipid composition of fetal calf serum, that is low in vitamin E and has a proportionally higher docosahexaenoic acid content than adult human serum.

Formation of 22 and 24 carbon 6-desaturated fatty acids from exogenous deuterated arachidonic acid is activated in THP-1 cells at high substrate concentrations.

Deuterated arachidonic acid (AA, [2H8]20:4 n-6) 1-25 microM, is converted to other fatty acids, as evaluated by gas chromatography-mass spectrometry, in THP-1 cells. The major products, in the 1 to 10 microM range, are 22:4 (elongated) and 20:3 (reduced in 5). At 25 microM, 24:4, 24:5 and 22:5 accumulate, with [2H8]/[2H0] ratios higher than in AA. At high AA concentration preferential conversion to elongated fatty acids with 5 unsaturations, through a 6 desaturase takes place and the 4-desaturated 22:5 appears to be formed through beta-oxidation of 24:5.

Increased thrombogenic potential of human monocyte-derived macrophages spontaneously transformed into foam cells.

This study investigated whether spontaneous lipid enrichment of human macrophages affects their thrombogenic potential as measured by increased production of tissue factor (TF) and plasminogen activation inhibitor types 1 and 2 (PAI-1 and PAI-2). Macrophages were obtained following a 7-day culture period of monocytes, isolated from the same donor, in autologous serum (HS) or in fetal bovine serum (FBS). Those cultured in HS underwent marked lipid accumulation relative to those cultured in FBS that was accompanied by increased production of TF and PAI-1, but not of PAI-2, and decreased production of interleukin-1beta. They also contained more arachidonic and linoleic acid and lower amounts of n-3 polyunsaturated fatty acids, particularly docosahexaenoic acid (22: 6). These data indicate that the transformation of macrophages into foam cells results in an increase in their thrombogenic and antifibrinolytic potential and provide a possible explanation of the thrombotic sequelae frequently consequent on plaque fissuring and disruption.

Persistent impairment of platelet aggregation following cessation of a short-course dietary supplementation of moderate amounts of N-3 fatty acid ethyl esters.

The duration of the effect of a short-course (1-mo twice-daily) supplementation of moderate amounts (2.28 g) of n-3 fatty acid ethyl esters (FA) on platelet lipid composition and aggregation was compared with that of olive oil (3 g/d) supplementation in 14 healthy volunteers. The FA preparation employed contained eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) in a ratio of 1:1.4. A marked rise (p <0.05) in the plasma and platelet content of EPA and DHA, and minimal changes in the content of arachidonic acid (AA) were documented at withdrawal of the n-3 FA supplementation. EPA/AA and DHA/AA ratios in platelet phospholipids showed that the FA accumulation persisted 8-12 wks after stopping the supplementation (p <0.05). The aggregation of platelets in response to collagen or ADP, and thromboxane B2 (TXB2) formation were impaired at withdrawal. The impaired aggregation lasted 8-12 weeks (p always <0.05), whereas TXB2 formation returned to basal values 4 weeks after stopping the n-3 supplementation. No correlation was found between impaired aggregation and TXB2 formation. In contrast, the impaired sensitivity to ADP (p = 0.036) and, to a lesser extent, to collagen (p = 0.068) were related to changes in the intracellular pH (pHi) of the Na+/H+ reverse transport. No changes in platelet composition or function were observed either during or following olive oil supplementation. These results document a long-lasting impairment of platelet sensitivity to ADP and collagen; changes in the pHi values of the Na+/H+ reverse transport, and a simultaneous persistent accumulation of EPA and DHA in platelet phospholipids, after stopping a short-course dietary supplementation of moderate amounts of n-3 fatty acid ethyl esters.

In human monocytes interleukin-1 stimulates a phospholipase C active on phosphatidylcholine and inactive on phosphatidylinositol.

Interleukin-1 (IL-1) can initiate the synthesis of prostaglandins which in turn act as endogenous modulators of IL-1 production. The human monocyte/macrophage synthesizes various eicosanoids through the activation of the cellular phospholipase system. Cell stimulation results in the activation of phospholipase A2 (PLA2) whose major substrate is phosphatidylcholine (PC) and the release of the eicosanoid precursor arachidonic acid (AA) from PC. Another pathway is the stimulation of a phospholipase C (PLC) mainly active on phosphoinositides and the resulting formation of inositol phosphates (IPs) and diacylglycerol (DAG). Phospholipids other than phosphoinositides can also be hydrolysed by PLC to give rise to DAG. Studies have shown that IL-1 does not activate the IP pathway, but it primarily stimulates a PLC linked to phosphatidylethanolamine in cultured rat mesangial cells, and a PLC linked to PC in Jurkart cells. We have stimulated human monocytes with IL-1 and calcium ionophore A23187 and we have observed their effect on the phospholipase system. The results indicate that IL-1 does not activate the formation of IPs in cells labeled with [3H]myo-inositol. In contrast, in cells labeled with [3H]AA, IL-1 causes the formation of DAG associated with the hydrolysis of PC. Moreover, after stimulation with IL-1 there is no accumulation of free AA which would indicate that there has been no activation of PLA2, which occurs instead with A23187 stimulation. These data suggest that, in monocytes, IL-1 does not directly stimulate a PLA2 or a PLC active on phosphatidylinositol; instead it primarily stimulates a PLC active on PC.

Arachidonate release and c-fos expression in various models of hypoxia and hypoxia-hypoglycemia in retinoic acid differentiated neuroblastoma cells.

Hypoxia-hypoglycemia has played an important role in inducing both phospholipase A2 activation and the expression of the early gene c-fos, in the neuroblastoma cell line SK-N-BE, after it has been differentiated by retinoic acid. Under hypoxic-hypoglycemic conditions, arachidonic acid release has found to be significant after 30 min, whereas c-fos expression has required at least 4 h. This model has been obtained by adding glycolytic inhibitor 2-deoxyglucose to the culture and by placing cells in an atmosphere containing 100% N2 for different time periods. This condition has been compared with two different models: NaCN and nitrogen have been used as hypoxic stimuli, without inhibiting the glycolytic pathway, but the same cell cultures have been used. Cell viability and the fall of cellular ATP levels have been evaluated in all the models, in order to monitor and compare the hypoxic cellular damage. Phospholipase A2 activation has been found to be significant in all conditions, even if to a different extent; but only hypoxia combined with the inhibition of the glycolytic pathway, has induced a significant expression of c-fos. It is very difficult to study hypoxic stimuli in 'in vitro' systems. Our study has compared three different models and the one combining gaseous hypoxia and hypoglycemic conditions seems to be very effective in stimulating early events involved in hypoxic phenomena such as phospholipase activation and the expression of the early gene c-fos.

Arachidonic and docosahexaenoic acids differentially affect the expression of fatty acyl-CoA oxidase, protein kinase C and lipid peroxidation in HepG2 cells.

Arachidonic (AA) and docosahexaenoic (DHA) acids (5-20 microM), when supplemented to human hepatoma HepG2 cells, which are depleted in these long-chain polyunsaturated fatty acids in conventional culture conditions, enhance the expression of acyl-CoA oxidase (ACOX), the first enzyme in the peroxisomal beta-oxidation cycle. DHA is effective at lower concentrations (at 5 microM) and to a greater extent (about 60% increment) than AA (about 40%) at 20 microM. Protein kinase C (PKC) appears to be involved in the activity of AA on ACOX, but not in that of DHA, since only the effect of AA is prevented by the PKC inhibitor Staurosporine, and since a remarkable elevation of the PKC activator diacylglycerol occurs only after AA supplementation. AA also induces elevation of lipoperoxides, favoured by the relative vitamin E deficiency occurring in cultured cells, and this effect, which is prevented by supplementation of the vitamin, may contribute to PKC activation.

Regulation of PUFA metabolism: pharmacological and toxicological aspects.

Levels of the long-chain polyunsaturated fatty acids (LCP) of the n-6 and n-3 series in animal plasma and cells are directly or indirectly dependent upon the intakes of either their precursors, the short-chain polyunsaturated fatty acids (SCP), linoleic (LA, 18:2 n-6) and alpha linolenic acid (ALA, 18:3 n-3), respectively, and/or of the preformed products (arachidonic, 20:4 n-6) and eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). We report here that pharmacological agents and cytotoxic compounds significantly affect the production of LCP from SCP in cultured cells. Using labelled substrates and radio HPLC separations, we observed that the potent hypocholesterolemic agent, simvastatin, activates the formation of AA from LA, mainly acting at the delta5 desaturation step, and increases also the mRNA levels, in cultured monocytic cells (THP-1). Elevation of AA occurs also in plasma lipids of hyperlipemic patients treated with statins (but not with fibrates). Conversely, oxysterols (mainly 7-beta-oxysterol), which are detected in circulating lipoproteins of rabbits on a hypercholesterolemic diet, potently inhibit the synthesis of AA from LA in hepatocytic cell lines (Hep-G2). At the same time plasma levels o AA are reduced vs controls, in spite of an identical intake of LA. Finally, on the basis of previous work showing reduced levels of LCP, mainly DHA, in the milk of cigarette-smoking mothers, we have observed that the incubation of human mammary gland cells with sera exposed to cigarette smoke results in marked inhibition of the production of DHA from ALA. The products in smoke responsible for this effect, are being identified through mass spectrometric techniques. In conclusion, pharmacological agents and toxic compounds, such as oxysterols and smoke products affect key steps in the synthesis of the LCP, major bioregulators in mammalian cells.

Manipulation of the fate of long chain polyunsaturated fatty acids in cultured cells.

We have studied the biosynthesis of long chain polyunsaturated fatty acids (LC-PUFA) from their precursors in cultured cells undergoing physiological modifications, or under the influence of lipid-lowering drugs or ethanol. The formation of arachidonic acid (AA, 20:4 n-6) from the percursor linoleic acid (LA, 18:2 n-6) in the neuroblastoma cells SK-N-BE is enhanced at early stages of differentiation, and declines when differentiation is complete, in concomitance with maximal accumulation of AA in cell lipids. In the monocytic cells THP-1, the biosynthesis of LC-PUFA is also enhanced by treatment with the HMGCoA reductase inhibitor simvastatin (S), an effect which is reverted by mevalonate and other intermediates of cholesterol synthesis. Maximal activation of LC-PUFA synthesis by S occurs at concentrations lower than those required for maximal inhibition of cholesterol synthesis. In the hepatoma cells HepG2, ethanol decreases the biosynthesis of LC-PUFA while potentiating the incorporation of acetate into cholesterol. LC-PUFA synthesis appears thus to be modulated in the course of cell differentiation and complex interactions between LC-PUFA and cholesterol synthesis occur, as judged from data obtained through pharmacological manipulations.

Synthesis of long-chain polyunsaturated fatty acids is inhibited in vivo in hypercholesterolemic rabbits and in vitro by oxysterols.

Plasma total lipids, total cholesterol (cholesterol esters and free cholesterol) and oxysterol (mainly 7 beta-hydroxycholesterol (7 beta OH)) concentrations were significantly elevated in New Zealand rabbits fed a 2% cholesterol-containing diet with respect to controls fed the same diet without cholesterol. In addition, linoleic (18:2 n-6) and alpha-linolenic acid (18:3 n-3) plasma concentrations were significantly elevated in hypercholesterolemic rabbits, while concentrations of long-chain n-6 and n-3 derivatives were reduced. Studies in monocytic cell line THP-1 revealed that 7 beta OH markedly inhibited the conversion of 18:2 to 20:4 n-6 and of 18:3 to 22:6 n-3, indicating depression of the desaturation steps; in particular the inhibition was greater for the Delta 5 desaturation step. Furthermore, experiments of Real-Time PCR showed that 5-10 microM 7 beta OH decreased the Delta 5 gene expression. In conclusion, atherogenic oxysterols interfere with the production of long-chain polyunsaturated fatty acids from their precursors both in hypercholesterolemic rabbits and in cultured cells.

Pharmacological modulation of fatty acid desaturation and of cholesterol biosynthesis in THP-1 cells.

In THP-1 cells, simvastatin decreases, in a concentration-dependent manner, cholesterol synthesis and increases linoleic acid (LA) conversion to its long-chain derivatives, in particular to arachidonic acid, activating delta6 and delta5 fatty acid (FA) desaturases. The intermediates in cholesterol synthesis, mevalonate and geranylgeraniol, partially reverse the effects of simvastatin on the LA conversion. The aims of this work were to evaluate: (i) the correlation between cholesterol synthesis and desaturase activity and (ii) the possible involvement of protein isoprenylation in desaturase activity, assessed through pharmacological treatments. THP-1 cells were incubated with [1-14C]LA or with [1-14C]di-homo-gamma-linolenic acid (DHGLA) and treated with simvastatin or with curcumin and nicardipine, inhibitors of desaturases. Curcumin was more active than nicardipine in inhibiting LA and DHGLA conversion: 20 microM curcumin, alone or with simvastatin, totally inhibited delta6 and delta5 desaturation steps; 10 microM nicardipine only partially inhibited the enzymes, being more active on delta5 desaturase. Simvastatin treatment decreased the incorporation of acetate in cholesterol (-93.8%) and cholesterol esters (-70.2%), as expected. Curcumin and nicardipine also decreased cholesterol synthesis and potentiated simvastatin. Finally, the isoprenylation inhibitors (perillic acid and GGTI-286) neither affected the conversion of LA nor inhibited the delta5 desaturase activity. In conclusion, our results indicate that there is no direct relationship between cholesterol synthesis and desaturase activity. In fact, simvastatin decreased cholesterol synthesis and enhanced LA conversion (mainly delta5 desaturation), whereas curcumin and nicardipin decreased delta5 desaturation, with a limited effect on cholesterol synthesis.

Time course and concentration dependence of the incorporation of deuterated/tritiated arachidonic acid and derived fatty acids in THP-1 cell lipids.

We have supplemented THP-1 cells, a human monocytic leukemia cell line, with arachidonic acid (AA), containing [3H8] AA, 1-25 microM, for up to 24 hours, and explored the time and concentration dependent patterns of incorporation in cell lipid classes and subclasses. Twenty-five microM AA consisted of deuterated AA ([2H8] AA), containing also [3H8] AA. Phospholipids (PL) were separated by HPLC with UV and radiodetection, and the fatty acids (FA) methyl esters were analyzed by GC. [2H8] AA pentafluorobenzyl-esters from individual lipid classes were obtained and analyzed by GC-MS. Incorporation of AA in cell lipids increased linearly with increasing concentrations, whereas 22:4 and 22:5 accumulated only at 25 microM AA. Up to 10 microM AA, more than 95% of the FA was incorporated in PL, whereas at 25 microM AA a significant proportion of the exogenous FA was incorporated in triglycerides (TG) and in diacyl phosphatidylcholine (PC). The time-course of AA incorporation showed that the peak was at 3 hours, with minimal incorporation in TG, in the presence of 5 microM, whereas the peak occurred at 6 hours, with about 50 percent incorporation in TG, with 25 microM. The data indicate that the range of AA concentrations and the time course of the incorporation of this FA in cell structural lipids are critical.