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.

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.

Different patterns characterize Omega 6 and Omega 3 long chain polyunsaturated fatty acid levels in blood from Italian infants, children, adults and elderly.

Long chain polyunsaturated fatty acids (LC-PUFA), especially the Omega 3, modulate key functions in the body. Their circulating levels are representative of their "status", and may vary at different ages. We have compared the FA status in Italian subjects from neonates to adulthood, assessed through FA analysis of blood drops from fingertips. Data from four cohorts of Italian subjects (total number 1835), have been pooled in four age-groups: neonates (4 days, n=81), children (2-9 years, n=728), adults (40-59 years, n=434) and elderly (60-79 years, n=592). LC-PUFA of both series (Omega 3 and 6) are higher in the blood of neonates than at subsequent ages, reflecting the efficient transfer of these FA from mothers to the fetus. In contrast, the lowest levels of Omega 3 PUFA, especially of DHA, are found in children, probably reflecting inadequate dietary intakes, with possible consequences on the health status at subsequent ages.

A randomized placebo-controlled study on high-dose oral algal docosahexaenoic acid supplementation in children with cystic fibrosis.

Low plasma concentrations of docosahexaenoic acid (DHA) are reported in unsupplemented cystic fibrosis (CF) patients. Forty-one CF patients aged from 6 to 12 years were randomized to receive high-dose DHA (100 mg/kg/day in the first month and 1g per day thereafter through a 12-month supplementation) or placebo (germ oil). Primary outcome was percentage change in plasma AA:DHA ratio. Secondary outcomes were changes in the number of pulmonary exacerbations compared to previous year, lung function, BMI, skinfold thicknesses, and body composition assessed by DXA and in serum concentrations of C-reactive protein, cytokines and vitamin (α-tocopherol and retinol). Compared to the control group plasma AA:DHA ratio decreased in the intervention group after 6 months (median percentage changes: -73% in the intervention group vs. -10% in the control group, P=0.001). No differences were detected between groups for secondary outcomes. Despite a decrease of the AA/DHA ratio, DHA supplementation for one year did not induce any significant biochemical and clinical improvement in CF patients.

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.

Early retinol-binding protein levels are associated with growth changes in infants born to diabetic mothers.

BACKGROUND: Biochemical predictors of infants' growth changes are not available. OBJECTIVES: We tested whether retinol-binding protein (RBP), docosahexaenoic acid and insulin (I) measured within 72 h from birth are associated with growth changes in infants born to mothers with gestational diabetes mellitus (GDM). METHODS: Fifty-six children, 32 born to diabetic mothers treated with insulin (GDM-I) and 24 born to diabetic mothers treated with diet (GDM-D), were evaluated at 0, 1, 3, 6 and 12 months of life. RESULTS: At multivariable regression performed using generalized estimating equations, early RBP levels and maternal body mass index were associated to average weight changes and early RBP and insulin levels to average length changes, respectively. There was no difference between GDM-I and GDM-D infants. CONCLUSIONS: This exploratory study suggests that early RBP levels may be a predictor of growth changes.

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.

Maternal smoking habits are associated with differences in infants' long-chain polyunsaturated fatty acids in whole blood: a case-control study.

OBJECTIVE: To study the effects of maternal smoking on the status of long-chain polyunsaturated fatty acids (LCPUFA) in infants' whole-blood lipids. DESIGN: A case-control matched study planned on the basis of preliminary observations. SETTING: Maternity ward. PATIENTS: A total of 159 healthy, term, breastfed infants with weight appropriate for gestational age, subdivided (53 per group) into those born to non-smokers (reference), smokers (> or = 5 cigarettes per day) who either stopped within the first trimester of pregnancy (early smokers) or who continued througout pregnancy (late smokers). INTERVENTIONS: The fatty acid profile of 4-day-old infants was determined on whole blood. RESULTS: Higher levels of linoleic (LA) and alpha-linolenic acid (ALA) and lower levels of the metabolic products di-homo-gammalinolenic (DHGLA) and arachidonic (AA), of the n-6 series, and docosahexaenoic acid (DHA), of the n-3 series, were found in infants born to late smokers compared with the reference group. The DHGLA/LA and AA/DHGLA ratios in the n-6 series and DHA/ALA in the n-3 series, which are indices of the metabolic processes in LCPUFA synthesis, were lower in infants born to smokers compared with those born to non-smokers. Infants born to early smokers showed n-6 PUFA levels and ratios similar to references and n-3 parameters closer to those born to late smokers. No dietary differences were found among the three groups of mothers. All the independent associations with smoking persisted after adjustment for maternal covariates. Pre-pregnancy body weight, which is lower in late smokers compared with non-smokers, independently correlated with LCPUFA levels in both series. CONCLUSIONS: Maternal smoking is associated with a reduction in LCPUFA pools in infants, which might have structural and functional consequences.

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.

Marine macroalgae as sources of polyunsaturated fatty acids.

Algae from cold water (Canada) and warm water (China) were analysed for the total lipid content, and for their fatty acid (FA) composition and content. The major findings are that fatty acids (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 C 18:4 FA (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 possible utilization for nutritional purposes.

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.

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.

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.

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.

Statins enhance arachidonic acid synthesis in hypercholesterolemic patients.

BACKGROUND AND AIM: It has been shown that statins enhance arachidonic acid biosynthesis from linoleic acid in vitro, and there is also evidence that statin-treated patients have high plasma and cellular arachidonic acid levels. The aim of this study was to investigate the possible effects of statins on the desaturating steps of arachidonic acid biosynthesis in patients. METHODS AND RESULTS: Plasma polyunsaturated fatty acids (percentages and concentrations), total cholesterol, LDL-cholesterol and triglycerides were measured in three groups of hypercholesterolemic patients before and after treatment with statins or fibrates for about three years, or before and after four months on a low-fat diet. As expected, there was a significant reduction in plasma total cholesterol (-23%) and LDL-cholesterol (-27%) levels in the statin group, and a significant reduction in triglycerides (-29%) and some decrease in total cholesterol (-12%) in the fibrate group; no change was observed in the control group. The selective increase in plasma arachidonic acid levels (in terms of absolute concentration and as a percentage of total fatty acids) was of the same order as the reduction in cholesterol, and the selective increase in the product/precursor ratios for delta 5 desaturation in the statin group indicated that this key step in arachidonic acid synthesis is affected. CONCLUSIONS: Our data show that an increase in arachidonic acid synthesis due to enhanced delta 5 desaturation is a major effect of statin treatment, and has the same order of magnitude as the reduction in plasma cholesterol levels.

Very low intakes of N-3 fatty acids incorporated into bovine milk reduce plasma triacylglycerol and increase HDL-cholesterol concentrations in healthy subjects.

Eight normolipidaemic volunteers, habitual partial skim milk drinkers and non-eaters of fish during the study, were given 500 ml day(-1) of partial skim milk for 1 month; they were then switched to 500 ml day(-1) of a novel commercially available milk preparation, supplying 400 mg of N-3 fatty acids-of which 300 mg were EPA+DHA-and 15 mg vitamin E, for 6 weeks. No changes in plasma lipid parameters were observed after the first run-in month; at 3 and 6 weeks on the N-3-rich milk, marked increments of plasma EPA (44 and 31%, respectively) and DHA (13 and 31%, respectively) were observed. Triacylglycerol (TG) concentrations decreased by 19% and high-density lipoprotein (HDL) concentrations increased by 19% at 6 weeks; plasma vitamin E rose by 21% while the susceptibility of plasma to oxidation was unaffected. Correlations were found between plasma EPA or DHA and TG, cholesterol, and HDL. In conclusion, the intake of a milk preparation providing low amounts of EPA+DHA to healthy individuals led to marked increases of N-3 fatty acids and vitamin E in plasma and in associated favourable changes in HDL and TG.

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.

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.