Fast Transmethylation of Total Lipids in Dried Blood by Microwave Irradiation and its Application to a Population Study

Abstract

A methodology combining finger-pricked blood sampling, microwave accelerated fatty acid assay, fast gas chromatography data acquisition, and automated data processing was developed, evaluated and applied to a population study. Finger-pricked blood was collected on filter paper previously impregnated with 0.05 mg of the antioxidant butylated hydroxytoluene and air-dried at room temperature. Transmethylation was accelerated by microwave irradiation in an explosion-proof multimode microwave reaction system. The chemical procedure was based on a one-step direct transmethylation procedure catalyzed by acetyl chloride. The short-term stability of PUFA in blood dried on filter paper and stored overnight at room temperature was examined using venous blood. The recoveries ranged from 97–101 % for the categorized fatty acids as well as the ratios of n-6 to n-3 PUFA and the n-3% highly unsaturated fatty acid. Specifically, recoveries were 99, 98, 97, and 97 % for linoleic acid (18:2n-6), arachidonic acid (ARA), α-linolenic acid (ALA), and docosahexaenoic acid (DHA), respectively. The mol% (mean ± SD, 95% confidence interval) of fatty acid composition in subjects from the population study was determined as 36.2±3.8 (35.8, 36.7), 23.2±3.0 (22.8, 23.5), 36.8±3.5 (36.4, 37.2) and 3.79±1.0 (3.68, 3.91) for the saturated, monounsaturated, n-6 and n-3 PUFA, respectively. Individually, the mean mol% (95% CI) was 22.6 (22.3, 22.9) for 18:2n-6, 9.5 (9.3, 9.7) for ARA, 0.51 (0.49, 0.53) for ALA, 0.42 (0.38, 0.47) for eicosapentaenoic acid (EPA), and 1.67 (1.61, 1.73) for DHA. This methodology provides an accelerated yet high-efficiency, chemically safe, and temperature-controlled transmethylation, with diverse laboratory applications including population studies.

Microwave Energy Increases Fatty Acid Methyl Ester Yield in Human Whole Blood Due to Increased Sphingomyelin Transesterification

“…Contrary to our findings with sphingomyelin, at least three previous studies have shown a reduced yield for very-long chain SFA and MUFA with the use of microwave-assisted transesterification both in serum/plasma and whole blood [1,6,16]. Most recently, two studies from one research group using a similar but multi-modal microwave system from the same company found yields of 70-90 % for 20:0, 22:0, 24:0 and 24:1n-9 [6] and 20-70 % for the same fatty acids in an earlier study [16].…”

“…Most recently, two studies from one research group using a similar but multi-modal microwave system from the same company found yields of 70-90 % for 20:0, 22:0, 24:0 and 24:1n-9 [6] and 20-70 % for the same fatty acids in an earlier study [16]. These two studies employed acetyl chloride in methanol (~11 % by volume) as per the method of Lepage and Roy [27], and contrary to what we show in the present study, increasing the reaction time had no effect on fatty acid yield.…”

“…Dried blood spots (DBS) collected by fingertip prick provide fatty acid determinations similar to venous whole blood sampling [1,2], and in recent years have gained in acceptance and been adopted for use in a variety of populations including infants [3,4], the elderly [5] and military personnel [6], among others [2,[7][8][9]. In addition, the % sum of eicosapentaenoic acid (EPA, 20:5n-3) + docosahexaenoic acid (DHA, 22:6n-3) in whole blood can be used as a predictor for risk of sudden cardiac death [10].…”

“…More recently, microwave-assisted direct fatty acid transesterifications have been reported for the conversion of DBS by fingertip prick [1,6,9] and serum [16] lipids into FAME. Although microwave-assisted direct transesterification may fail to fully transesterify triacylglycerols when using 14 % boron trifluoride (BF 3 ) in methanol [1], this may be a limitation of the kitchen-grade microwave utilized in two of the previous studies [1,9].…”

“…Although microwave-assisted direct transesterification may fail to fully transesterify triacylglycerols when using 14 % boron trifluoride (BF 3 ) in methanol [1], this may be a limitation of the kitchen-grade microwave utilized in two of the previous studies [1,9]. This potential limitation may be overcome by systems such as the CEM Discover SP-D single-mode [16] or MARS multi-mode [6,16] microwaves specifically designed for laboratory processes (CEM Corporation, Matthews, North Carolina, USA). Alternative methods to expedite the transesterification process have been adopted using ultrasonic energy [17][18][19][20], with promising results, and microwaveassisted extraction techniques have been implemented previously for the extraction of fats from poultry feeds [21], seeds, meat and bakery products [22], acorns [23] and avocado pulp [24].…”