Biosynthesis of a specifically deuterated diunsaturated fatty acid (18:2.DELTA.6,9) for deuterium NMR membrane studies

A unique procedure for the biosynthesis and subsequent isolation of a series of specifically deuteriated cis,cis-octadeca-6,9-dienoic acids has been developed. An auxotroph of Tetrahymena, which lacks Δ9 and Δ12 desaturanse activity, is supplemented with specifically deuteriated oleic acid and converts it to the corresponding deuteriated cis,cis, octadeca-6,9-dienoic acid, 18:2Δ⁶⁹. The deuteriated fatty acid is subsequently isolated by argentation chromatography and HPLC. To demonstrate the utility of the procedure, we describe here the biosynthesis of cis,cis-octadeca-6,9-dienoic acid deuteriated at positions 9 and 10. Gas and thin-layer chromatography of the isolated fatty acid showed that it was greater than 99% pure while ¹³C NMR and mass spectrometry of the O-(trimethylsilyl) derivative confirmed that the 18-carbon fatty acid contains two double bonds located at positions 6 and 9. The yield, from an 11-L culture, was typically 100 mg of which 35% was found to be deuteriated at both the 9- and 10-positions. The deuteriated fatty acid was esterified to 1-hexadecanoyl-sn-glycero-3-phosphocholine, and aqueous, multilamellar dispersions of the lipid were studied by ²H NMR. Each spectrum consists of two overlapping powder patterns and therefore yields two quadrupolar splittings. Over a temperature range from 0 to 40°C, one splitting decreases from 6.6 to 1.8 kHz while the other increases from 4.5 to 5.3 kHz. The magnitudes of the two splittings are equivalent between 10 and 15°C. The values of the splittings, and their response of temperature, differ significantly from those of the corresponding deuteriated oleic acid in microbial membranes [Rance, M., Jeffrey, K.R., Tulloch, A.P., Butler, K W., & Smith, I.C.P. (1980) Biochim. Biophys. Acta 600, 245-262] and in bilayers of 1-hexadecanoyl-2-cis-octadec-9-enoyl-sn-glycero-3-phosphocholine (POPC) [Seelig, J., & Waespe-Šarčevic, N. (1978) Biochemistry 17, 3310-3315]. The results suggests a fatty acyl chain containing two double bonds has physiochemical properties very different from those of the corresponding acyl chain with a single double bond.