Differential DNA methylation of steatosis and non-alcoholic fatty liver disease in adolescence.

BACKGROUND AND AIMS: Epigenetic modifications are associated with hepatic fat accumulation and non-alcoholic fatty liver disease (NAFLD). However, few epigenetic modifications directly implicated in such processes have been identified during adolescence, a critical developmental window where physiological changes could influence future disease trajectory. To investigate the association between DNA methylation and NAFLD in adolescence, we undertook discovery and validation of novel methylation marks, alongside replication of previously reported marks. APPROACH AND RESULTS: We performed a DNA methylation epigenome-wide association study (EWAS) on DNA from whole blood from 707 Raine Study adolescents phenotyped for steatosis score and NAFLD by ultrasound at age 17. Next, we performed pyrosequencing validation of loci within the most 100 strongly associated differentially methylated CpG sites (dmCpGs) for which ≥ 2 probes per gene remained significant across four statistical models with a nominal p value < 0.007. EWAS identified dmCpGs related to three genes (ANK1, MIR10a, PTPRN2) that met our criteria for pyrosequencing. Of the dmCpGs and surrounding loci that were pyrosequenced (ANK1 n = 6, MIR10a n = 7, PTPRN2 n = 3), three dmCpGs in ANK1 and two in MIR10a were significantly associated with NAFLD in adolescence. After adjustment for waist circumference only dmCpGs in ANK1 remained significant. These ANK1 CpGs were also associated with γ-glutamyl transferase and alanine aminotransferase concentrations. Three of twenty-two differentially methylated dmCpGs previously associated with adult NAFLD were associated with NAFLD in adolescence (all adjusted p < 2.3 × 10-3). CONCLUSIONS: We identified novel DNA methylation loci associated with NAFLD and serum liver biochemistry markers during adolescence, implicating putative dmCpG/gene regulatory pathways and providing insights for future mechanistic studies.

Adiposity associated DNA methylation signatures in adolescents are related to leptin and perinatal factors.

Epigenetics links perinatal influences with later obesity. We identifed differentially methylated CpG (dmCpG) loci measured at 17 years associated with concurrent adiposity measures and examined whether these were associated with hsCRP, adipokines, and early life environmental factors. Genome-wide DNA methylation from 1192 Raine Study participants at 17 years, identified 29 dmCpGs (Bonferroni corrected p < 1.06E-07) associated with body mass index (BMI), 10 with waist circumference (WC) and 9 with subcutaneous fat thickness. DmCpGs within Ras Association (RalGDS/AF-6), Pleckstrin Homology Domains 1 (RAPH1), Musashi RNA-Binding Protein 2 (MSI2), and solute carrier family 25 member 10 (SLC25A10) are associated with both BMI and WC. Validation by pyrosequencing confirmed these associations and showed that MSI2 , SLC25A10 , and RAPH1 methylation was positively associated with serum leptin. These were  also associated with the early environment; MSI2 methylation (ß = 0.81, p = 0.0004) was associated with pregnancy maternal smoking, SLC25A10 (CpG2 ß = 0.12, p = 0.002) with pre- and early pregnancy BMI, and RAPH1 (ß = -1.49, p = 0.036) with gestational weight gain. Adjusting for perinatal factors, methylation of the dmCpGs within MSI2, RAPH1, and SLC25A10 independently predicted BMI, accounting for 24% of variance. MSI2 methylation was additionally associated with BMI over time (17 years old ß = 0.026, p = 0.0025; 20 years old ß = 0.027, p = 0.0029) and between generations (mother ß = 0.044, p = 7.5e-04). Overall findings suggest that DNA methylation in MSI2, RAPH1, and SLC25A10 in blood may be robust markers, mediating through early life factors.

Genetically modified plants are an alternative to oily fish for providing n-3 polyunsaturated fatty acids in the human diet: A summary of the findings of a Biotechnology and Biological Sciences Research Council funded project.

The n-3 polyunsaturated fatty acids (PUFA) present primarily in oily fish, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are important components of cell membranes and that are needed for normal development and cell function. Humans have very limited capacity for EPA and DHA synthesis from α-linolenic acid and so they must be obtained pre-formed from the diet. However, perceived unpalatability of oily fish and fish oil concerns about contamination with environmental pollutants, dietary choices that exclude fish and animal products, and price limit the effectiveness of recommendations for EPA and DHA intakes. Moreover, marine sources of EPA and DHA are diminishing in the face of increasing demands. Therefore, an alternative source of EPA and DHA is needed that is broadly acceptable, can be upscaled and is sustainable. This review discusses these challenges and, using findings from recent nutritional trials, explains how they may be overcome by seed oils from transgenic plants engineered to produce EPA and DHA. Trials in healthy men and women assessed the acute uptake and appearance in blood over 8 hours of EPA and DHA from transgenic Camelina sativa compared to fish oil, and the incorporation of these PUFA into blood lipids after dietary supplementation. The findings showed that postprandial EPA and DHA incorporation into blood lipids and accumulation in plasma lipids after dietary supplementation was as good as that achieved with fish oil. The oil derived from this transgenic plant was well tolerated. This review also discusses the implications for human nutrition, marine ecology and agriculture.

High maternal folic acid intake around conception alters mouse blastocyst lineage allocation and expression of key developmental regulatory genes.

Folate, a cofactor for the supply of one-carbon groups, is required by epigenetic processes to regulate cell lineage determination during development. The intake of folic acid (FA), the synthetic form of folate, has increased significantly over the past decade, but the effects of high periconceptional FA intake on cell lineage determination in the early embryo remains unknown. Here, we investigated the effect of maternal high FA (HFA) intake on blastocyst development and expression of key regulatory genes. C57BL/6 adult female mice were fed either Control diet (1 mg FA) for 4 weeks before conception and during the preimplantation period (Con-Con); Control diet for 4 weeks preconception, followed by HFA (5 mg FA) diet during preimplantation (Con-HFA); or HFA diet for 4 weeks preconception and during preimplantation (HFA-HFA). At E3.5, blastocyst cell number, protein, and mRNA expression were measured. In HFA-HFA blastocysts, trophectoderm cell numbers and expression of CDX2, Oct-4, and Nanog were reduced compared with Con-Con blastocysts; Con-HFA blastocysts showed lower CDX2 and Oct-4 expression than Con-Con blastocysts. These findings suggest periconceptional HFA intake induces changes in key regulators of embryo morphogenesis with potential implications for subsequent development.

Genome-wide methylation analysis identifies differentially methylated CpG loci associated with severe obesity in childhood.

Childhood obesity is a major public health issue. Here we investigated whether differential DNA methylation was associated with childhood obesity. We studied DNA methylation profiles in whole blood from 78 obese children (mean BMI Z-score: 2.6) and 71 age- and sex-matched controls (mean BMI Z-score: 0.1). DNA samples from obese and control groups were pooled and analyzed using the Infinium HumanMethylation450 BeadChip array. Comparison of the methylation profiles between obese and control subjects revealed 129 differentially methylated CpG (DMCpG) loci associated with 80 unique genes that had a greater than 10% difference in methylation (P-value < 0.05). The top pathways enriched among the DMCpGs included developmental processes, immune system regulation, regulation of cell signaling, and small GTPase-mediated signal transduction. The associations between the methylation of selected DMCpGs with childhood obesity were validated using sodium bisulfite pyrosequencing across loci within the FYN, PIWIL4, and TAOK3 genes in individual subjects. Three CpG loci within FYN were hypermethylated in obese individuals (all P < 0.01), while obesity was associated with lower methylation of CpG loci within PIWIL4 (P = 0.003) and TAOK3 (P = 0.001). After building logistic regression models, we determined that a 1% increase in methylation in TAOK3, multiplicatively decreased the odds of being obese by 0.91 (95% CI: 0.86 - 0.97), and an increase of 1% methylation in FYN CpG3, multiplicatively increased the odds of being obese by 1.03 (95% CI: 0.99 - 1.07). In conclusion, these findings provide evidence that childhood obesity is associated with specific DNA methylation changes in whole blood, which may have utility as biomarkers of obesity risk.

Maternal diet as a modifier of offspring epigenetics.

There has been a substantial body of evidence, which has shown that genetic variation is an important determinant of disease risk. However, there is now increasing evidence that alterations in epigenetic processes also play a role in determining susceptibility to disease. Epigenetic processes, which include DNA methylation, histone modifications and non-coding RNAs play a central role in regulating gene expression, determining when and where a gene is expressed as well as the level of gene expression. The epigenome is highly sensitive to a variety of environmental factors, especially in early life. One factor that has been shown consistently to alter the epigenome is maternal diet. This review will focus on how maternal diet can modify the epigenome of the offspring, producing different phenotypes and altered disease susceptibilities.

Changes in rat n-3 and n-6 fatty acid composition during pregnancy are associated with progesterone concentrations and hepatic FADS2 expression.

The mechanisms responsible for changes to long-chain polyunsaturated fatty acid (LC PUFA) status during pregnancy have not been fully elucidated. Tissue samples were collected from virgin and pregnant (day 12 and 20) female rats. LC PUFA status, sex hormone concentrations and hepatic mRNA expression of FADS1, FADS2 and elongase were assessed. Day 20 gestation females had higher plasma and liver docosahexaenoic acid and lower arachidonic acid content than virgin females (P<0.05). There was higher FADS2 mRNA expression during pregnancy (P=0.051). Progesterone and oestradiol concentrations positively correlated with hepatic FADS2 mRNA expression (P=0.043, P=0.004). Progesterone concentration positively correlated with hepatic n-6 docosapentaenoic acid content (P=0.006), and inversely correlated with intermediates in LC PUFA synthesis including n-3 docosapentaenoic acid, γ-linolenic acid and 20:2n-6 (P<0.05). Changes in progesterone and oestradiol during pregnancy may promote the synthesis of LC PUFA via increased FADS2 expression.

Feeding a protein-restricted diet during pregnancy induces altered epigenetic regulation of peroxisomal proliferator-activated receptor-α in the heart of the offspring.

Impaired flexibility in the use of substrates for energy production in the heart is implicated in cardiomyopathy. We investigated the effect of maternal protein restriction during pregnancy in rats on the transcription of key genes in cardiac lipid and carbohydrate metabolism in the offspring. Rats were fed protein-sufficient or protein-restricted (PR) diets during pregnancy. Triacylglycerol concentration in adult (day 105) heart was altered by maternal protein intake contingent on post-weaning fat intake and sex. mRNA expression of peroxisomal proliferator-activated receptor (PPAR)-α and carnitine palmitoyltransferase-1 was increased by the maternal PR diet in adult, but not neonatal, offspring. PPARα promoter methylation was lower in adult and neonatal heart from PR offspring. These findings suggest that prenatal nutrition alters the future transcriptional regulation of cardiac energy metabolism in the offspring through changes in epigenetic regulation of specific genes. However, changes in gene functional changes may not be apparent in early life.

Epigenetic changes in early life and future risk of obesity.

The rapid increase in incidence of obesity over the past two decades cannot be explained solely by genetic and adult lifestyle factors. There is now considerable evidence that the fetal and early postnatal environments also strongly influence the risk of developing obesity in later life. Initially, human studies showed that low birth weight was associated with an increased risk of obesity but increasingly there is evidence that overnutrition in the early life can also increase susceptibility to future obesity. These findings have now been replicated in animal models, which have shown that both maternal under- and overnutrition can induce persistent changes in gene expression and metabolism. The mechanism by which the maternal nutritional environment induces such changes is beginning to be understood and involves the altered epigenetic regulation of specific genes. In this review, we discuss the recent evidence that shows that early-life environment can induce altered epigenetic regulation leading to the induction of an altered phenotype. The demonstration of a role for altered epigenetic regulation of genes in the developmental induction of obesity opens the possibility that interventions, either through nutrition or specific drugs, may modify long-term obesity risk and combat this rapid rise in obesity.

Sex, but not maternal protein or folic acid intake, determines the fatty acid composition of hepatic phospholipids, but not of triacylglycerol, in adult rats.

The aim of the study was to investigate whether the protein and folic acid content of the maternal diet and the sex of the offspring alter the polyunsaturated fatty acid content of hepatic phospholipids and triacylglycerol (TAG). Pregnant rats were fed diets containing 18% or 9% protein with either 1 or 5mg/kg folic acid. Maternal diet did not alter hepatic lipid composition in the adult offspring. Data from each maternal dietary group were combined and reanalysed. The proportion of 18:0, 20:4n-6 and 22:6n-3 in liver phospholipids was higher in females than in males, while hepatic TAG composition did not differ between sexes. Delta5 Desaturase expression was higher in females than in males. Neither Delta5 nor Delta6 desaturase expression was related to polyunsaturated fatty acid concentrations. These results suggest that sex differences in liver phospholipid fatty acid composition may reflect primary differences in the specificity of phospholipid biosynthesis.

The effect of altering the 20:5n-3 and 22:6n-3 content of a meal on the postprandial incorporation of n-3 polyunsaturated fatty acids into plasma triacylglycerol and non-esterified fatty acids in humans.

Previous studies suggest that consuming meals containing large amounts of fish oil is associated with selective postprandial incorporation of 20:5n-3 and 22:6n-3 into plasma non-esterified fatty acids (NEFA). We investigated the effect of consuming meals containing different amounts of 20:5n-3 and 22:6n-3 comparable to dietary habits of western populations on the postprandial incorporation of 18:3n-3, 20:5n-3 and 22:6n-3 into plasma triacylglycerol (TAG) and NEFA over 6h in middle aged subjects. 20:5n-3 incorporation into plasma TAG was greater than 22:6n-3 irrespective of the test meal. Conversely, 22:6n-3 incorporation into plasma NEFA was greater than 20:5n-3, irrespective of the test meal. There was no effect of the amount of 20:5n-3+22:6n-3 in the test meal on the 18:3n-3 incorporation into plasma TAG or NEFA. These findings suggest differential metabolism of 20:5n-3 and 22:6n-3 in the postprandial period when consumed in amounts typical of western dietary habits.

The benefits of oestrogens on postprandial lipid metabolism are lost in post-menopausal women with Type 2 diabetes.

AIMS: Women with Type 2 diabetes appear to lose the protection against cardiovascular disease (CVD) afforded by oestrogens. We examined the effects of oestrogen hormone replacement therapy (HRT) on postprandial clearance of dietary fat in non-diabetic and diabetic post-menopausal women. METHODS: In a cross-sectional study, fasting subjects [HRT+ and HRT- control and diabetic women; Type 2 diabetes (DM) HRT+n = 8, DM HRT-n = 14, control HRT+n = 7, control HRT-n = 11] consumed a meal containing the stable isotope 1,1,1-[13]C-tripalmitin, with blood and breath sampled for 6 and 24 h, respectively, in the postprandial period. RESULTS: In diabetic women, there were no differences between the HRT+ and HRT- groups for any of these parameters. In contrast, in HRT+ compared with HRT- control women, the triglyceride (TG) area under the curve was lower [AUC; HRT+ median (range) 7.7 (4.1, 12.8) mmol/l per 6 h, HRT- 9.7 (3.9, 18.5) mmol/l per 6 h, P < 0.05] and [13]C-palmitic acid in the TG fraction was also lower [HRT+ 23.2 (10.3, 41.3) ng/ml per 6 h, HRT- 47.7 (12.6, 77.2) ng/ml per 6 h, P < 0.05], suggesting the lower postprandial triglyceridaemia associated with HRT in non-diabetic women is because of better chylomicron clearance. CONCLUSIONS: The oestrogen-associated advantage in clearance of dietary lipid we observed in non-diabetic post-menopausal women is not seen in post-menopausal diabetic women. This is likely to promote an atherogenic lipoprotein profile and may contribute to the loss of CVD protection seen in diabetic women.

Metabolism of alpha-linolenic acid in humans.

Alpha-linolenic acid (18:3n-3) is essential in the human diet, probably because it is the substrate for the synthesis of longer-chain, more unsaturated n-3 fatty acids eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) which are required for tissue function. This article reviews the recent literature on 18:3n-3 metabolism in humans, including fatty acid beta-oxidation, recycling of carbon by fatty acid synthesis de novo and conversion to longer-chain polyunsaturated fatty acids (PUFA). In men, stable isotope tracer studies and studies in which volunteers increased their consumption of 18:3n-3 show conversion to 20:5n-3 and 22:5n-3, but limited conversion to 22:6n-3. However, conversion to 18:3n-3 to 20:5n-3 and 22:6n-3 is greater in women compared to men, due possibly to a regulatory effect of oestrogen, while partitioning of 18:3n-3 towards beta-oxidation and carbon recycling was lower than in men. These gender differences may be an important consideration in making dietary recommendations for n-3 PUFA intake.

Effect of meal sequence on postprandial lipid, glucose and insulin responses in young men.

OBJECTIVE: To investigate whether the postprandial changes in plasma triacylglycerol (TAG), nonesterified fatty acids (NEFA), glucose and insulin concentrations in young men were the same if an identical meal was fed at breakfast and lunch, and if the response to lunch was modified by consumption of breakfast. METHODS: In two trials (1 and 2) healthy subjects (age 22+/-1 y, body mass index 22+/-2 kg/m(2)) were fed the same mixed macronutrient meal at breakfast at 08:00 h and lunch at 14:00 h. In the third trial, no breakfast was fed and the overnight fast extended until lunch at 14:00 h. Addition of [1,1,1-(13)C]tripalmitin to one meal in each trial was used to distinguish between endogenous and meal-derived lipids. RESULTS: The postprandial changes in TAG, NEFA and glucose concentrations were similar in trials 1 and 2. The change in plasma total TAG concentration was about two fold less (P<0.05) after lunch compared to breakfast. Postprandial NEFA suppression was the same after breakfast and lunch. Glucose and insulin responses were significantly greater following lunch suggesting decreasing insulin sensitivity during the day. Consumption of breakfast did not alter the postprandial total TAG or NEFA responses after lunch. Measurement of [(13)C]palmitic acid concentration showed that handling of TAG and NEFA from the meal was the same after breakfast and lunch, and was not altered by consumption of breakfast. CONCLUSIONS: Overall, these data suggest that in young, healthy men regulation of plasma TAG from endogenous sources, principally VLDL, but not chylomicrons during the postprandial period leads to differences in the magnitude of lipaemic response when the same meal was consumed at breakfast or at lunch 6 h later.

Conversion of alpha-linolenic acid to palmitic, palmitoleic, stearic and oleic acids in men and women.

The purpose of this study was to determine whether adult humans can recycle carbon from alpha-linolenic acid (18:3n-3) into saturated (SFA) and monounsaturated (MUFA) fatty acids. Six men and six women consumed 700 mg [U-13C]-18:3n-3. Blood was collected over 21 days and breath over 24h. [13C]-labelled SFA and MUFA were detected in plasma phosphatidylcholine (PC) and triacylglycerol (TAG). Total labelled fatty acid incorporation into SFA and MUFA was five- and 25-fold greater in PC than TAG in men and women, respectively. [13C]-16:0 was the major labelled fatty acid in both fractions. Total [13C] incorporation into SFA and MUFA was 20% greater in men than women, and related positively (r(2) = 0.35, P<0.05) to the fractional recovery of labelled 18:3n-3 as 13CO2 on breath. These results suggest that the extent of partitioning towards beta-oxidation and carbon recycling may regulate the availability of 18:3n-3 for conversion to longer-chain fatty acids.

Effect of pregnancy on plasma lipid concentration in Trinidadian women. Result of a pilot study

In Trinidad and Tobago, cardiovascular disease and Type 2 diabetes mellitus are important causes of morbidity and mortality, and birth weight is significantly less than reference standards. Lower birth weight is associated with increased risk of these diseases. Variation in birth weight is due, in part, to deposition of adipose tissue in the foetus during the last trimester at the same time that maternal plasma triacylglycerol (TAG) increases. We conducted a pilot cross-sectional analysis of maternal plasma lipid status and birth weight in healthy, non-pregnant, primigravida Trinidadian women. Non-pregnant and pregnant women, in their second and third trimesters, and at term, were recruited at random from an antenatal clinic. Adult and umbilical cord plasma TAG, non-esterified fatty acids (NEFA) and phosphatidylcholine (PC) concentrations were determined from gas chromatographic analysis of fatty acids. Maternal height, weight, skinfold thickness and infant birth weight were measured. The infants born to Afro-Trinidadian and Indo-Trinidadian women were of low to normal birth weight (medians 3.07 and 3.22 kg, respectively). At term, plasma TAG concentration was approximately two fold (p < 0.05) greater than for non-pregnant women. The increment between 30-34 weeks was 1.5 to 1.9 fold lower than reported in other populations. There was a strong relationship (r = 0.8771, p = 0.019) between maternal and cord plasma TAG and NEFA, but not PC concentrations. There was no significant relationship between maternal TAG concentration at term and birth weight. The result suggests an impaired ability to increase plasma TAG concentrations during late gestation.

Effect of pregnancy on plasma lipid concentration in Trinidadian women. Result of a pilot study.

In Trinidad and Tobago, cardiovascular disease and Type 2 diabetes mellitus are important causes of morbidity and mortality, and birth weight is significantly less than reference standards. Lower birth weight is associated with increased risk of these diseases. Variation in birth weight is due, in part, to deposition of adipose tissue in the foetus during the last trimester at the same time that maternal plasma triacylglycerol (TAG) increases. We conducted a pilot cross-sectional analysis of maternal plasma lipid status and birth weight in healthy, non-pregnant, primigravida Trinidadian women. Non-pregnant and pregnant women, in their second and third trimesters, and at term, were recruited at random from an antenatal clinic. Adult and umbilical cord plasma TAG, non-esterified fatty acids (NEFA) and phosphatidylcholine (PC) concentrations were determined from gas chromatographic analysis of fatty acids. Maternal height, weight, skinfold thickness and infant birth weight were measured. The infants born to Afro-Trinidadian and Indo-Trinidadian women were of low to normal birth weight (medians 3.07 and 3.22 kg, respectively). At term, plasma TAG concentration was approximately two fold (p < 0.05) greater than for non-pregnant women. The increment between 30-34 weeks was 1.5 to 1.9 fold lower than reported in other populations. There was a strong relationship (r = 0.8771, p = 0.019) between maternal and cord plasma TAG and NEFA, but not PC concentrations. There was no significant relationship between maternal TAG concentration at term and birth weight. The result suggests an impaired ability to increase plasma TAG concentrations during late gestation.

Effect of fatty acid supplementation on growth and differentiation of human IMR-32 neuroblastoma cells in vitro.

Polyunsaturated fatty acids play a critical role in the structure and function of the developing nervous system. It has been proposed that fatty acids may effect a variety of biologic processes through the activation of the peroxisome proliferator activated receptors (PPARs)-ligand activated transcription factors. In this report, we demonstrate that fatty acids can inhibit the proliferation of the human neuronal cell line IMR-32. The fatty acids linoleate, alpha-linoleate, arachidonate, docosahexaenoate, and oleate all inhibited [(3)H]thymidine incorporation of IMR-32 cells after 72 h. Fatty acid supplementation also led to the morphologic differentiation of the IMR-32 cells. Linoleate and arachidonate, fatty acids of the n-6 series, induced the most extensive differentiation. Furthermore, the addition of fatty acids to IMR-32 cells led to PPAR activation, suggesting that PPAR activation may be an important event in fatty acid modulation of IMR-32 cell growth. In support of this hypothesis, clofibric acid, a specific ligand of PPARalpha, also inhibited IMR-32 cell proliferation and strongly induced PPAR activation. Together these results suggest that fatty acids may play an important role in the development of neuronal precursor cells and that activation of the PPARs may be one pathway by which fatty acids modulate the growth and differentiation of neuronal precursor cells.

A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction.

Efficient isolation of individual lipid classes is a critical step in the analysis of plasma and lipoprotein fatty acid compositions. Whilst good separations of total lipid extracts are possible by TLC, this method is time consuming and a major rate-limiting step when processing large numbers of specimens. A method for rapid separation of phosphatidylcholine (PC), non-esterified fatty acids (NEFA), cholesterol ester (CE) and triacylglycerol (TAG) from total plasma lipid extracts by solid-phase extraction (SPE) using aminopropyl silica columns has been developed and validated. Following initial separation of polar and neutral lipids, individual classes were isolated by application of solvents with increasing polarity. Recoveries for combined plasma extraction with chloroform-methanol and SPE were (%): PC 74.2 (SD 7.5), NEFA 73.6 (SD 8.3), CE 84.9 (SD 4.9), and TAG 86.8 (SD 4.9), which were significantly greater for TAG and NEFA than by TLC Both GC-flame ionisation detector and GC-MS analysis of fatty acid methyl esters demonstrated that there was no cross-contamination between lipid classes. Measurements of repeatability of fatty acid composition for TAG, PC, CE and NEFA fractions showed similar CV for each fatty acid. The magnitude of the CV appeared to be related inversely to the fractional fatty acid concentration, and was greatest at concentrations of less than 1 g/100 g total fatty acids. There was no evidence of selective elution of individual fatty acid or CE species. In conclusion, this method represents an efficient, rapid alternative to TLC for isolation of these lipid classes from plasma.

Induction of antisense Pax-3 expression leads to the rapid morphological differentiation of neuronal cells and an altered response to the mitogenic growth factor bFGF.

Mutations within the Pax-3 gene lead to a range of developmental abnormalities in both humans and mice. In this report, we have investigated the role that Pax-3 plays in neuronal cell development by specifically downregulating Pax-3 expression within a neuronal cell line. This was achieved by stably transfecting the neuronal cell line ND7 with an expression vector in which antisense Pax-3 RNA was produced under the control of the inducible MMTV promoter. In the stable transfectants, we found that the addition of dexamethasone led to the induction of antisense Pax-3 RNA and a rapid downregulation in endogenous Pax-3 protein expression. The decrease in endogenous Pax-3 protein expression corresponded with a dramatic change in the morphology of the cell: the normally rounded ND7 cells exhibited increased cell to substrate adhesion, extended long neurite processes and expressed genes such as snap-25 that are characteristic of a mature neuron. The morphological differentiation induced by a reduction in Pax-3 expression was followed 24-48 hours later by a cessation in cell proliferation. Interestingly the morphological differentiation and cessation in cell proliferation inducted in the cell lines lacking Pax-3 could be reversed by the addition of the mitogenic growth factor EGF but not by bFGF, whose receptor was downregulated in these cells. These results suggest that the expression of Pax-3 is essential to maintain the undifferentiated phenotype of these immature neuronal cells, and in its absence the cells acquire many of the characteristics of a mature neuronal cell. The slow onset of cell cycle arrest in the cells lacking Pax-3 argues against this transcription factor playing a direct role in the regulation of neuronal cell proliferation.