Enrichment of brain docosahexaenoic acid (DHA) is highly dependent upon the molecular carrier of dietary DHA: lysophosphatidylcholine is more efficient than either phosphatidylcholine or triacylglycerol.

Docosahexaenoic acid (DHA) is highly concentrated in the brain, and its deficiency is associated with several neurological disorders including Alzheimer's disease. However, the currently used supplements do not appreciably enrich brain DHA, although they enrich most other tissues. We tested the hypothesis that the ability of the dietary carrier to augment brain DHA depends upon the generation of DHA-lysophosphatidylcholine (LPC), the preferred carrier of DHA across the blood brain barrier. We compared the efficacy of DHA-triacylglycerol (TAG), di-DHA phosphatidylcholine (PC) and DHA-LPC to enrich brain DHA following their gavage to normal rats for 30 days, all at a dose of 10 mg DHA/day. The results show that DHA from TAG, which is released as free DHA or monoacylglycerol during digestion and is absorbed as TAG in chylomicrons, was incorporated preferentially into adipose tissue and heart but not into brain. In contrast, LPC-DHA increased brain DHA by up to 100% but had no effect on adipose tissue. Di-DHA PC, which generates both free DHA and LPC-DHA during the digestion, enriched DHA in brain, as well as in heart and liver. Brain-derived neurotrophic factor was increased by di-DHA PC and DHA-LPC, but not by TAG-DHA, showing that enrichment of brain DHA correlated with its functional effect. We conclude that dietary DHA from TAG or from natural PC (sn-2 position) is not suitable for brain enrichment, whereas DHA from LPC (at either sn-1 or sn-2 position) or from sn-1 position of PC efficiently enriches the brain and is functionally effective.

Metabolism of uniformly labeled 13C-eicosapentaenoic acid and 13C-arachidonic acid in young and old men.

Background: Plasma eicosapentaenoic acid (EPA) and arachidonic acid (AA) concentrations increase with age.Objective: The aim of this study was to evaluate EPA and AA metabolism in young and old men by using uniformly labeled carbon-13 (13C) fatty acids.Design: Six young (∼25 y old) and 6 old (∼75 y old) healthy men were recruited. Each participant consumed a single oral dose of 35 mg 13C-EPA and its metabolism was followed in the course of 14 d in the plasma and 28 d in the breath. After the washout period of ≥28 d, the same participants consumed a single oral dose of 50 mg 13C-AA and its metabolism was followed for 28 d in plasma and breath.Results: There was a time × age interaction for 13C-EPA (Ptime × age = 0.008), and the shape of the postprandial curves was different between young and old men. The 13C-EPA plasma half-life was ∼2 d for both young and old men (P = 0.485). The percentage dose recovered of 13C-EPA per hour as 13CO2 and the cumulative ß-oxidation of 13C-EPA did not differ between young and old men. At 7 d, however, old men had a >2.2-fold higher plasma 13C-DHA concentration synthesized from 13C-EPA compared with young men (Page = 0.03). 13C-AA metabolism was not different between young and old men. The 13C-AA plasma half-life was ∼4.4 d in both young and old participants (P = 0.589).Conclusions: The metabolism of 13C-AA was not modified by age, whereas 13C-EPA metabolism was slightly but significantly different in old compared with young men. The higher plasma 13C-DHA seen in old men may be a result of slower plasma DHA clearance with age. This trial was registered at clinicaltrials.gov as NCT02957188.

Dietary supplementation of arachidonic acid increases arachidonic acid and lipoxin A4 contents in colon, but does not affect severity or prostaglandin E2 content in murine colitis model.

BACKGROUND: Arachidonic acid (ARA) is an essential fatty acid and a major constituent of biomembranes. It is converted into various lipid mediators, such as prostaglandin E2 (PGE2) and lipoxin A4 (LXA4). The effects of dietary ARA on colon maintenance are unclear because PGE2 has both mucosal protective and proinflammatory effects, and LXA4 has an anti-inflammatory role. Our objective is to clarify the effects of dietary ARA on an experimental murine colitis model. METHODS: C57BL/6 mice were fed three types of ARA diet (0.075%, 0.15% or 0.305% ARA in diet), DHA diet (0.315% DHA) or control diet for 6 weeks, and were then administered dextran sodium sulphate (DSS) for 7 days to induce colitis. We evaluated colitis severity, fatty acid and lipid mediator contents in colonic tissue, and the expression of genes related to lipid mediator formation. RESULTS: ARA composition of colon phospholipids was significantly elevated in an ARA dose-dependent manner. ARA, as well as DHA, did not affect colitis severity (body weight loss, colon shortening, diarrhea and hemoccult phenomena) and histological features. PGE2 contents in the colon were unchanged by dietary ARA, while LXA4 contents increased in an ARA dose-dependent manner. Gene expression of cyclooxygenase (COX)-1 and COX-2 was unchanged, while that of 12/15-lipoxgenase (LOX) was significantly increased by dietary ARA. ARA composition did not correlate with neither colon length nor PGE2 contents, but significantly correlated with LXA4 content. CONCLUSION: These results suggest that dietary ARA increases ARA and LXA4 contents in colon, but that it has no effect on severity and PGE2 content in a DSS-induced murine colitis model.

Supplementation of arachidonic acid-enriched oil increases arachidonic acid contents in plasma phospholipids, but does not increase their metabolites and clinical parameters in Japanese healthy elderly individuals: a randomized controlled study.

BACKGROUND: The importance of arachidonic acid (ARA) among the elderly has recently gained increased attention. The effects of ARA supplementation in the elderly are not fully understood, although ARA is considered to be associated with various diseases. We investigate whether ARA supplementation to Japanese elderly subjects affects clinical parameters involved in cardiovascular, inflammatory, and allergic diseases. We also examine the levels of ARA metabolites such as prostanoids during intervention. METHODS: We conducted a randomized, double-blind and placebo-controlled parallel group intervention trial. ARA-enriched oil (240 or 720 mg ARA per day) or placebo was administered to Japanese healthy men and women aged 55-70 years for 4 weeks followed by a 4-week washout period. The fatty acid contents of plasma phospholipids, clinical parameters, and ARA metabolites were determined at baseline, 2, 4, and 8 weeks. RESULTS: The ARA content in plasma phospholipids in the ARA-administrated groups increased dose-dependently and was almost the same at 2 weeks and at 4 weeks. The elevated ARA content decreased to nearly baseline during a 4-week washout period. During the supplementation and washout periods, no changes were observed in eicosapentaenoic acid and docosahexaenoic acid contents. There were no changes in clinical blood parameters related to cardiovascular, inflammatory and allergic diseases. ARA supplementation did not alter the level of ARA metabolites such as urinary 11-dehydro thromboxane B2, 2,3-dinor-6-keto prostaglandin (PG) F1α and 9,15-dioxo-11α-hydroxy-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid (tetranor-PGEM), and plasma PGE2 and lipoxin A4. ARA in plasma phospholipids was not correlated with ARA metabolite levels in the blood or urine. CONCLUSION: These results indicate that ARA supplementation, even at a relatively high dose, does not increase ARA metabolites, and suggest that it does not induce cardiovascular, inflammatory or allergic diseases in Japanese elderly individuals.

Simultaneous quantification of total eicosapentaenoic acid, docosahexaenoic acid and arachidonic acid in plasma by high-performance liquid chromatography-tandem mass spectrometry.

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC-tandem mass spectrometry (HPLC-MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid-liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r(2) > 0.997, n = 18). Based on matrix addition, accuracy deviation was <15%, except for AA at 10 mg/L (30-90%), whereas precision was <8% for all fatty acids studied. The method was applied to the measurement of these omega-3 fatty acids in a fish oil supplement study with healthy volunteers. Healthy males (n = 4) were administered a supplement containing 465 mg EPA and 375 mg DHA per capsule (Omacor®). A dose of two capsules was given daily over a 4 week period. Pre-treatment concentrations varied between subjects for EPA (17-68 mg/L), DHA (36-63 mg/L) and AA (121-248 mg/L). During the dosing period EPA increased 460-480% from the baseline concentration, while DHA increased 150-160%. The EPA-AA ratio increased from 0.07-0.56 to 0.3-3.1 after 4 weeks of dosing. In conclusion, the method described could be suitable for monitoring EPA, DHA and AA in clinical studies that may aid in achieving optimal concentrations of these fatty acids in patients who could be at risk of sudden cardiac death.

Low-dose arachidonic acid intake increases erythrocytes and plasma arachidonic acid in young women.

Arachidonic acid (ARA) is considered to be a minor contributor to the diet. Previous reports regarding the effect of ARA supplementation on the composition of long-chain polyunsaturated fatty acids (LCPUFA) in the blood of humans are extremely limited. In the present study, we conducted a crossover double-blind, placebo-control study. Twenty-three young Japanese women consumed one capsule containing triacylglycerol enriched with 80 mg ARA, equivalent to the amount in one egg, daily for 3 weeks. Blood samples were drawn before and after treatment periods, and the compositions of the LCPUFA in blood lipid fractions were measured. The supplementation of ARA increased the composition of ARA, but did not decrease the composition of n-3LCPUFA in erythrocyte phospholipids and plasma phospholipids, esterified cholesterol, and triacylglycerol. We found that dietary ARA increased the ARA level in all lipid fractions of the blood, even at a very low dose.

Differential tissue dose responses of (n-3) and (n-6) PUFA in neonatal piglets fed docosahexaenoate and arachidonoate.

Docosahexaenoic acid (DHA) and arachidonic acid (ARA) are commonly added to infant formula worldwide; however, dietary concentrations needed to obtain optimal tissue levels have not been established. Hence, we studied tissue responses in piglets fed various doses of DHA and ARA. Doses were 0, 1, 2, and 5 times those used in U.S. infant formulas and DHA/ARA in Diet 0, Diet 1, Diet 2, and Diet 5 were 0, 4.1/8.1, 8.1/16.2, and 20.3/40.6 mg/100 kJ formula, respectively. Supplementation of dietary DHA and ARA increased DHA in brain, retina, liver, adipose tissue, plasma, and erythrocyte by 1.1- to 25.8-fold of Diet 0 (P-trend < 0.01). Tissue ARA (1.1- to 6.0-fold of Diet 0) responded to dietary ARA in liver, adipose tissue, plasma, and erythrocytes (P-trend < 0.05); brain and retina ARA was, however, unresponsive to dietary DHA and ARA. Plasma and erythrocyte DHA were positively associated with DHA in neural (brain and retina) and visceral (liver and adipose) tissues (r(2) = 0.11-0.56; P < 0.001-P = 0.042). Plasma and erythrocyte ARA did not correlate with neural ARA. Only plasma ARA was associated with liver ARA (r(2) = 0.222; P = 0.02) and adipose ARA (r(2) = 0.867; P < 0.001) and erythrocyte ARA correlated with adipose ARA (r(2) = 0.470; P < 0.001). We conclude that dietary DHA supplementation affords an effective strategy for enhancing tissue DHA, ARA in visceral but not neural tissues is sensitive to dietary ARA, and erythrocyte and plasma DHA can be used as proxies for tissue DHA, although blood-borne ARA is not an indicator of neural ARA.

Antioxidant modulation of oxidant-stimulated uptake and release of arachidonic acid in eicosapentaenoic acid-supplemented human lymphoma U937 cells.

Omega-3 polyunsaturated fatty acids (PUFA) are increasingly finding use as treatments for a variety of medical conditions. PUFA supplementation can, however, result in increased oxidative stress causing elevated turnover rate of membrane phospholipids, impairment of membrane integrity and increased formation of inflammatory mediators. The aim of this study was to determine which antioxidant compounds were most effective in ameliorating the stimulation of phospholipid turnover by oxidative stress. U937 cells were supplemented with eicosapentaenoic acid and either ascorbic acid, alpha-tocopherol, beta-carotene or astaxanthin prior to being challenged with oxidant. Although all antioxidants were found to be effective in decreasing oxidant-stimulated peroxide formation, only alpha-tocopherol significantly decreased oxidant-stimulated release of 3H-labeled arachidonic acid (AA), while ascorbic acid markedly increased release. All antioxidants except alpha-tocopherol decreased oxidant-stimulated 3H-AA uptake. Our data suggest that antioxidants are not equally effective in combating the effects of oxidative stress upon membrane phospholipid turnover, and that optimal protection will require mixtures of antioxidants.

Dose response of bone mass to dietary arachidonic acid in piglets fed cow milk-based formula.

BACKGROUND: The addition of arachidonic acid (AA) and docosahexaenoic acid (DHA) to infant formula was recently approved in North America. In piglets, dietary AA is linked to elevations in bone mass. OBJECTIVE: The objective was to investigate the effects of varied amounts of dietary AA on bone modeling and bone mass with the use of the piglet model for infant nutrition. DESIGN: Male piglets (n = 32) were randomly assigned to receive 1 of 4 formulas supplemented with AA (0.30%, 0.45%, 0.60%, or 0.75% of fat) plus DHA (0.1% of fat) from days 5 to 20 of life. Measurements included biomarkers of bone modeling, fatty acid status, and whole-body and femur bone mineral content; bone area was measured by dual-energy X-ray absorptiometry. Differences among groups were detected with two-factor analysis of variance. Regression analyses were used to determine factors responsible for bone mineral content after dietary AA was accounted for. RESULTS: Proportions of AA in plasma, liver, and adipose were modified by the dietary treatments, but bone modeling was not affected. Liver AA was positively related to plasma insulin-like growth factor 1 and calcitriol and urinary N-telopeptide. Whole-body bone mineral content was elevated in the piglets fed 0.60% and 0.75% AA and was best predicted by dietary AA and bone resorption. CONCLUSIONS: This study confirms that dietary AA alters bone mass and clarifies the best amount of AA to add to the diet of pigs born at term. Because the amount of dietary DHA was held constant, whether other amounts of DHA are related to bone mass requires investigation.

The influence of prematurity and long chain polyunsaturate supplementation in 4-week adjusted age baboon neonate brain and related tissues.

Clinical studies show that docosahexaenoic acid (DHA) and arachidonic acid (ARA) supplemented formula improve visual function in preterm infants, however improved fatty acid status is known only for plasma and red blood cells (RBC) since target organs cannot be sampled from humans. Baboons were randomized to one of four groups: Term breast-fed (B); Term formula-fed (T-); Preterm formula-fed (P-); and Preterm DHA/ARA-supplemented formula-fed (P+). The P+ contained 0.61 +/- 0.03% DHA and 1.21 +/- 0.09% ARA, and breast milk had 0.68 +/- 0.22% and 0.62 +/- 0.12% as DHA and ARA, respectively. The B and P+ groups had significantly higher DHA concentration in all tissues than T- and P-. The P- group showed dramatically lower DHA content of 35%, 27%, 66%, and 75% in the brain, retina, liver, and plasma, respectively, compared with B. Supplementation prevented declines in DHA levels in the retina, and liver, and attenuated the decline in brain, plasma and RBC of preterm animals. In contrast, ARA was not significantly lower compared with B in any group in any tissue but was significantly elevated in liver and brain. RBC and plasma DHA were correlated with DHA in tissues; RBC/plasma ARA were uncorrelated with tissue ARA. We conclude that 1) DHA drops precipitously in term and preterm primates consuming formula without long chain polyunsaturates, while 22:5n-6 concentration rises; 2) tissue ARA levels are insensitive to dietary LCP supplementation or prematurity, 3) plasma and RBC levels of ARA are uncorrelated with total ARA levels; 4) DHA levels are correlated with group effects and are uncorrelated within groups.

Effects of tanshinone I isolated from Salvia miltiorrhiza bunge on arachidonic acid metabolism and in vivo inflammatory responses.

Arachidonic acid (AA) mainly released from the cell membrane by phospholipase A(2) (PLA(2)) is converted to eicosanoids by the action of cyclooxygenase (COX) and lipoxygenase (LO). In order to find the specific inhibitors of AA metabolism especially PLA(2) and COX-2, 300 plant extracts were evaluated for their inhibitory activity on PGD(2) production from cytokine-induced mouse bone marrow-derived mast cells in vitro. From this screening procedure, the methanol extract of Salvia miltiorrhiza was found to inhibit PGD(2) production and the ethyl acetate subfraction gave the strongest inhibition of five subfractions tested. From this ethyl acetate subfraction, an activity-guided isolation finally gave tanshinone I as an active principle. This investigation deals with the effects of tanshinone I on AA metabolism from lipopolysaccharide (LPS)-induced RAW 264.7 cells and in vivo antiinflammatory activity. Tanshinone I inhibited PGE(2) formation from LPS-induced RAW macrophages (IC(50) = 38 microM). However, this compound did not affect COX-2 activity or COX-2 expression. Tanshinone I was found to be an inhibitor of type IIA human recombinant sPLA(2)(IC(50) = 11 microM) and rabbit recombinant cPLA(2) (IC(50) = 82 microM). In addition, tanshinone I showed in vivo antiinflammatory activity in rat carrageenan-induced paw oedema and adjuvant-induced arthritis.

Effect of caffeic acid on apical transporters' dysfunction of renal proximal tubule cells under oxidative stress in vitro.

The protective effect of caffeic acid (CA) against oxidative stress-induced inhibition of proximal tubule apical transporter was investigated. In the present study, 10 (-4) M H2O2 did not affect cell viability regardless of incubation time. However, it decreased apical transporters' activity such as Na (+)/glucose cotransporter, Na (+)/Pi cotransporter, and Na (+)/H(+) antiporter in the proximal tubule cells. CA (>10(-6) M) prevented H2O2-induced inhibition of apical transporters. Thus, we investigated its action mechanism. CA also prevented H2O2-induced lipid peroxides formation, arachidonic acid (AA) release, and Ca(2+) uptake. In conclusion, CA, in part, prevented H2O2-induced inhibition of apical transporter activity via decrease of AA release and Ca(2+) uptake in primary cultured renal proximal tubule cells.

Perinatal supply and metabolism of long-chain polyunsaturated fatty acids: importance for the early development of the nervous system.

The long-chain polyunsaturated fatty acids, arachidonic (AA) and docosahexaenoic acid (DHA), are essential structural lipid components of biomembranes. During pregnancy, long-chain polyunsaturated fatty acids (LC-PUFA) are preferentially transferred from mother to fetus across the placenta. This placental transfer is mediated by specific fatty acid binding and transfer proteins. After birth, preterm and full-term babies are capable of converting linoleic and alpha-linolenic acids into AA and DHA, respectively, as demonstrated by studies using stable isotopes, but the activity of this endogenous LC-PUFA synthesis is very low. Breast milk provides preformed LC-PUFA, and breast-fed infants have higher LC-PUFA levels in plasma and tissue phospholipids than infants fed conventional formulas. Supplementation of formulas with different sources of LC-PUFA can normalize LC-PUFA status in the recipient infants relative to reference groups fed human milk. Some, but not all, randomized, double-masked placebo-controlled clinical trials in preterm and healthy full-term infants demonstrated benefits of formula supplementation with DHA and AA for development of visual acuity up to 1 year of age and of complex neural and cognitive functions. From the available data, we conclude that LC-PUFA are conditionally essential substrates during early life that are related to the quality of growth and development. Therefore, a dietary supply during pregnancy, lactation, and early childhood that avoids the occurrence of LC-PUFA depletion is desirable, as was recently recommended by an expert consensus workshop of the Child Health Foundation.

Fatty acid absorption in preterms on formulas with and without long-chain polyunsaturated fatty acids and in terms on formulas without these added.

BACKGROUND/OBJECTIVE: Long chain polyunsaturated fatty acids have beneficial effects in preterm neurophysiological development and are semi-essential. Their levels and variation in plasma and red cells in term and preterms are better known than their intestinal absorption. In this paper the absorption of supplemented arachidonic acid (AA) and docosahexaenoic acid (DHA) is evaluated in a preterm group. DESIGN: Four newborn randomized groups were studied. Group T comprised 11 terms on regular formula. Preterms: group P (n=9) was on a classic preterm formula. INTERVENTION: groups PA (n=9) and PB (n=13) were on the same formula but PB contained AA and DHA in similar proportion to breast milk. At 20 days a 3 day metabolic balance was taken for Ca, P(i), Mg, total fat and individual fatty acids (C8-C24, saturated unsaturated). RESULTS: Calcium absorption was (mean+/-s.d.) 51+/-13% in terms. In preterms it was respectively 45+/-18, 38+/-11 and 37+/-21%. Total fat absorption was 92.0+/-8.0% in terms, and from 95.0+/-2.0 to 91.0+/-8.0% in preterms. Absorption of 8:0, 10:0 and 12:0 showed a very high and constant rate despite significant intake differences (715-33 mg/kg/day). Linoleic acid and alpha-linolenic acid were absorbed in the three groups at around 94% regardless of a greater LA intake in group P. Details of absorption (mg/kg/day) were: for AA, intake 17+/-7, fecal excretion 5+/-4, net retention 12+/-5 (75.0+/-18%); for DHA, intake 10+/-3, fecal excretion 3+/-2, net retention 6+/-4 (62.3+/-30%). CONCLUSION: Intestinal absorption of fatty acids is high and is comparable in terms and preterms as regards the studied acids. Longer acids were less well absorbed. The supplemented amounts of AA and DHA were less well absorbed and probably not impairing calcium absorption. SPONSORSHIP: University of Alicante, University of Miguel Hernández.

Fatty acid induced glioma cell growth is mediated by the acyl-CoA synthetase 5 gene located on chromosome 10q25.1-q25.2, a region frequently deleted in malignant gliomas.

Acyl-CoA synthetase (ACS) ligates fatty acid and CoA to produce acyl-CoA, an essential molecule in fatty acid metabolism and cell proliferation. ACS5 is a recently characterized ACS isozyme highly expressed in proliferating 3T3-L1 cells. Molecular characterization of the human ACS5 gene revealed that the gene is located on chromosome 10q25.1-q25.2, spans approximately 46 kb, comprises 21 exons and 22 introns, and encodes a 683 amino acid protein. Two major ACS5 transcripts of 2.5- and 3.7-kb are distributed in a wide range of tissues with the highest expression in uterus and spleen. Markedly increased levels of ACS5 transcripts were detected in a glioma line, A172 cells, and primary gliomas of grade IV malignancy, while ACS5 expression was found to be low in normal brain. Immunohistochemical analysis also revealed strong immunostaining with an anti-ACS5 antibody in glioblastomas. U87MG glioma cells infected with an adenovirus encoding ACS5 displayed induced cell growth on exposure to palmitate. Consistent with the induction of cell growth, the virus infected cells displayed induced uptake of palmitate. These results demonstrate a novel fatty acid-induced glioma cell growth mediated by ACS5.

Incorporation of polyunsaturated fatty acids into CT-26, a transplantable murine colonic adenocarcinoma.

Previous studies in our laboratory have shown that marine oils, with high levels of eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acids (DHA, 22:6n-3), inhibit the growth of CT-26, a murine colon carcinoma cell line, when implanted into the colons of male BALB/c mice. An in vitro model was developed to study the incorporation of polyunsaturated fatty acids (PUFA) into CT-26 cells in culture. PUFA-induced changes in the phospholipid fatty acid composition and the affinity with which different fatty acids enter the various phospholipid species and subspecies were examined. We found that supplementation of cultured CT-26 cells with either 50 microM linoleic acid (LIN, 18:2n-6), arachidonic acid (AA, 20:4n-6), EPA, or DHA significantly alters the fatty acid composition of CT-26 cells. Incorporation of these fatty acids resulted in decreased levels of monounsaturated fatty acids, while EPA and DHA also resulted in lower levels of AA. While significant elongation of both AA and EPA occurred, LIN remained relatively unmodified. Incorporation of radiolabeled fatty acids into different phospholipid species varied significantly. LIN was incorporated predominantly into phosphatidylcholine and had a much lower affinity for the ethanolamine phospholipids. DHA had a higher affinity for plasmenylethanolamine (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) than the other fatty acids, while EPA had the highest affinity for phosphatidylethanol-amine (1,2-diacyl-sn-glycero-3-phosphoethanolamine). These results demonstrate that, in vitro, significant differences are seen between the various PUFA in CT-26 cells with respect to metabolism and distribution, and these may help to explain differences observed with respect to their effects on tumor growth and metastasis in the transplantable model.

Different contribution of phospholipid and triacylglycerol metabolism to esterification of free intracellular arachidonate: a study on SK-N-BE(2) human neuroblastoma cells.

When SK-N-BE(2) human neuroblastoma cells were exposed for 1h to growth medium supplemented with [14C]arachidonic acid (AA) at final concentrations ranging from 1 microM to 100 microM, an amount of this fatty acid was uptaken ranging form a 2% to a 120% of that present in cells at steady state. As more [14C]AA was uptaken by cells, a larger fraction was progressively incorporated into triacylglycerols (TAG) in comparison to phospholipids (PL), with minor amounts remaining in a free form. By gas chromatographic analysis it was estimated that TAG from cells grown in ordinary medium contained about 2 nmoles AA per mg protein, but, after 1 h exposure to medium supplemented with 100 microM AA (label-free) this value rose to about 28 nmoles/mg protein; furthermore, as estimated on the basis of total fatty acid content, TAG mass was increased by a 16%. Cell exposure to medium enriched with 100 microM AA did not cause PL mass changes, whereas AA content was significantly increased only in phosphatidylcholine. Medium enrichment with 100 microM AA dramatically enhanced [3H]glycerol incorporation into TAG, as assessed after 1 h cell pulse, with minor but significant changes observed also for phosphatidylinositol and phosphatidylethanolamine, but not for phosphatidylcholine. In the light of these data, the contribution of PL and TAG to the removal of free intracellular AA is discussed.

Docosahexaenoic and arachidonic acid absorption in preterm infants fed LCP-free or LCP-supplemented formula in comparison to infants fed fortified breast milk.

The absorption of long-chain polyunsaturated fatty acids (LCP) with particular respect to docosahexaenoic (DHA) and arachidonic acid (AA) has been studied in 39 very-low-birth-weight infants appropriate for gestational age after a 10-day feeding period. The infants were fed either a LCP-supplemented formula (n = 11), or a LCP-free formula (n = 11) or breast milk fortified with protein and carbohydrates to have similar protein and energy intakes as in the formula-fed infants (n = 17). Total fat content and fatty acid profile were measured in the human milk, the two formulas, and in the stool samples. After a 10-day feeding period, the fecal excretions of total fat, DHA and AA were measured during a 3-day balance period. The total fat apparent absorption rates were similar in all groups (84.1, 82.1 and 80.6% of intake, respectively). The DHA and AA intakes were significantly (p < 0.01) higher in the group fed the fortified breast milk than in the group fed the LCP-supplemented formula (DHA: 75.5 +/- 12.4 vs. 50.2 +/- 4.2 mg/72 h; AA: 45.5 +/- 5.8 vs. 30.2 +/- 2.7 mg/72 h). There was a tendency for lower apparent absorption rates for both LCPs studied in the group fed fortified breast milk when compared to the group fed LCP-supplemented formula (AA: 70.6 +/- 10.9 vs. 73.0 +/- 8.7% of intake, DHA: 69.0 +/- 10.6 vs. 74.2 +/- 9.5% of intakes, but the differences were not significant. As consequence of the different intakes, the net absorption of the two studied LCP fatty acids were significantly (p < 0.01) higher in the breast milk group than in the group fed the LCP-supplemented formula (DHA: 52.6 +/- 6.1 vs. 36.8 +/- 4.5 mg/72 h; AA: 31.4 +/- 3.1 vs. 22.4 +/- 2.3 mg/72 h). The data demonstrate that DHA and AA are absorbed from the studied LCP-supplemented formula at least as effectively as from human milk. The net absorption of these LCP depend on the amount of dietary intake, and seems to be influenced by the dietary LCP source.

Biological effects of dietary arachidonic acid. Introduction.

The intent of this symposium is to assemble current knowledge of the role of arachidonic acid (AA) in the diet to provide a conceptual and mechanistic framework for future research. The principal focus is on the varied biological effects of dietary AA, including opposing effects of n-3 and n-6 polyunsaturated fatty acids (PUFA); regulation of n-6 PUFA metabolism, eicosanoid synthesis and gene expression; the importance of AA in infant nutrition and the contemporary Western diet in general; and the effects of AA on tumor promotion. Through its myriad actions and remarkably ubiquitous presence in cells, AA can be argued to affect every cell of the body. Although the varied molecular events associated with the metabolism of AA have been subjects of intense investigation, the ability of AA in the diet to alter AA levels in cellular membranes is poorly described and is thus the focus of this symposium.

Swelling, acidosis, and irreversible damage of glial cells from exposure to arachidonic acid in vitro.

Swelling and damage of C6 glioma cells and of primary cultured astrocytes were analyzed in vitro during incubation with arachidonic acid (AA; 20:4). The cells were suspended in a physiological medium supplemented with AA at concentrations of 0.001-1.0 mM. Cell swelling was quantified by flow cytometry with hydrodynamic focusing. Flow cytometry was also utilized for assessment of cell viability by exclusion of the fluorescent dye propidium iodide and for measurement of the intracellular pH (pHi) by 2',7'-bis-(2-carboxyethyl)-5(and -6)carboxy-fluorescein. Administration of AA caused an immediate dose-dependent swelling of C6 glioma cells, even at a concentration of 0.01 mM. At this level cell volume increased within 20 min to 105.0% of control, at 0.1 mM to 111.0%, while at 1.0 mM to 123.7%. Following a phase of rapid cell volume increase, swelling leveled off during the subsequent observation period of 70 min. Viability of the C6 glioma cells was 90% under control conditions. It remained unchanged after raising AA concentrations to 0.1 mM. At 0.5 mM, however, cell viability fell to 72.8%, and at 1.0 mM to 32.7%. pHi of the glioma cells was 7.3 under control conditions. In parallel with the early swelling phase, AA led to a dose-dependent decrease of the intracellular pH and an elevated lactate production of the cells. During incubation with 0.1 mM AA, pHi decreased to 7.06 after 5 min, but recovered to normal subsequently. In addition, swelling-inducing properties of linoleic (18:2) or stearic (18:0) acid were analyzed for evaluation of the specificity of glial swelling induced by AA. Whereas stearic acid (0.1 mM) failed to induce a swelling response, linoleic acid (0.1 mM) was found to be effective. The volume increase of the glial cells, however, was only half of that found during exposure to AA at the same concentration. Further, glial swelling from AA or linoleic acid was completely inhibited by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Finally, omission of Na+ ions in the suspension medium with replacement by choline led also to inhibition of the cell volume increase by AA. Experiments using astrocytes from primary culture confirmed the swelling-inducing properties of AA at a quantitative level, whereas vulnerability of the cells to AA was increased. The present results demonstrate an important role of AA in cytotoxic swelling and irreversible damage of glial cells at concentrations that occur in vivo in cerebral ischemia or trauma.(ABSTRACT TRUNCATED AT 250 WORDS)