Standardization and validation of assay of selected omega-3 and omega-6 fatty acids from phospholipid fraction of red cell membrane using gas chromatography with flame ionization detector.

BACKGROUND: Docosahexaenoic acid (DHA) is an important structural component of human brain and retina. Evidence exists linking nutritional status of pregnant mothers and cognitive functions of their born infants. The DHANI (Maternal DHA Supplementation and Offspring Neurodevelopment in India) trial was implemented to evaluate the effect of maternal supplementation with DHA during pregnancy and for 6 months following delivery on motor and mental development of infants at 1 and 12 months. We describe here the standardization and validation of an assay for measurement of selected omega-3 and omega-6 fatty acids from the phospholipid fraction of red blood cells to assess their status in mothers at baseline, delivery and 6 months post-delivery and for infants in cord blood and at 1 and 12 months of age. The validated method has been used for the analysis of samples for DHANI. METHODS: Lipids were extracted from a pool of red blood cells, separated using thin layer chromatography. The phospholipid fraction was esterified, and fatty acids were separated by gas chromatography using a flame ionization detector. RESULT: The method accuracy for DHA was between 97 - 98% and between 91 - 95% for arachidonic acid (AA) at three different concentrations. The intra-assay and inter-assay coefficient of variation for the fatty acids ranged from 1.19 to 5.7% and 0.78 to 13.0% respectively. Intraclass correlation (ICC), as a measure of reproducibility, ranged between 0.689 and 0.996. A good linearity was observed for all the fatty acids between concentrations of 0.2-4 µg/ml. CONCLUSION: The standardized and validated method is suitable for implementation in large epidemiological studies for evaluation of fatty acids and in nutritional trials for assessment of fatty acid content of various lipid classes.

Use of dried blood for measurement of trans fatty acids.

BACKGROUND: Fatty acid measurements especially trans fatty acid has gained interest in recent times. Among the various available biomarkers, adipose tissue is considered to be the best for the long term dietary intake but the invasive nature of tissue aspiration reduces its utility. Phlebotomy is a much less invasive method of sample collection when a large number of participants are involved in the study and therefore is an alternative, most suitable for large population based studies. In the present study fatty acid (with special emphasis on trans fatty acid) extraction from blood spotted and dried on filter paper was carried out to simplify the sample collection procedure and transportation. METHODS: Blood samples were collected from 19 healthy volunteers. The blood was spotted (30 spots of 10 microl each) on filter paper, dried at room temperature and stored at 4 degrees C in zip-lock poly bags. For comparison whole blood stored at -70 degrees C was simultaneously analyzed. RESULTS: A good agreement was seen between trans fatty acid values obtained in dried blood and whole blood as evident from the pearson correlation coefficients ('r' for monounsaturated (trans) 0.70 and for polyunsaturated (trans) 0.692 respectively). The intraclass correlation coefficient for monounsaturated trans was 0.805 and for polyunsarurated trans was 0.776. CONCLUSION: Dried blood spots can be used for trans fatty acid analysis.

Association of trans fatty acids with lipids and other cardiovascular risk factors in an Indian industrial population.

OBJECTIVE: Trans-fat, an invariable component of industrial fat is considered as one of the major dietary factors associated with CVD. Although the use of trans-fat is completely banned in some of the high-income countries where the CVD epidemic is declining, it is widely used in LMIC. We aimed to investigate the association of trans fatty acid in serum with risk markers of CVD in an industrial population in India. Participants were randomly selected from a study conducted in an industrial setting among employees and their family members. Information related to their demographic profile, anthropometric measurements, oil intake were recorded. Fasting samples were collected and stored at - 80 °C for analysis. Their lipid profile and hs CRP were measured and fatty acids analyzed using gas chromatography (GC) with flame ionization detector (FID). RESULTS: Complete data was available for 176 participants. Among trans fatty acids, mono trans fatty acid was significant predictor of serum triglycerides [Unadjusted ß (95% CI) 22.9 (2.6, 43.2); Adjusted ß (95% CI) 20.4 (3.5, 37.3)]. None of the other trans fatty acids either individually or in group correlated with any of the biochemical markers studied.