G. W. A. Milne

Use of chemical lonization mass spectrometry in analysis of amino acid phenylthiohydantoin derivatives formed during edman degradation of proteins

Abstract A method is described for the identification and quantification of the amino acid phenylthiohydantoins produced during successive steps of the Edman degradation of proteins. The technique employs chemical ionization mass spectrometry and permits rapid identification of all PTH derivatives. Use of this method in the Edman degradation of sperm whale myoglobin is described and compared to gas chromatography.

A major pathway for the mammalian oxidative degradation of phytanic acid.

Abstract A series of branched-chain saturated fatty acids accumulated in tissues of weanling mice fed regular chow supplemented with 2% phytanic acid (3,7,11,15 tetramethylhexadecanoic acid). They were identified by combined gas-liquid chromatography-mass spectrometry as phytanic acid, α-hydroxyphytanic acid, and the 19-, 16- and 14-carbon homologs of phytanic acid. Trace quantities of the 9-carbon homolog have also been detected by gas-liquid chromatography. When the regular chow is supplemented with 2% phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol), Δ 2 -phytenic acid accumulated in liver in addition to the compounds listed above. No evidence was found for the presence of α-ketophytanic acid, or of 15- and 17-carbon intermediates. The latter two would be expected if carbon dioxide fixation to the branch-methyl group were required in phytol or phytanate metabolism, as in the bacterial metabolism of farnesoic acid. Dietary supplementation with 3,7,11-trime thyldodecanoic acid produces accumulation in liver of the fed compound only. Intravenous injection of uniformly 14 C-labeled phytanic acid in mice led to significant incorporation of radioactivity associated on gas-liquid chromatography with 19-16-, 14- and 11-carbon isoprenoid acids. Rat liver in vivo also rapidly converted in high yield uniformly 14 C-labeled phytanic acid or 2,3-dideuterophytanic acid to uniformly 14 C-labeled pristanic acid and 2-deuteropristanic acid, respectively. The oxidation of uniformly 14 C-labeled phytanic acid to 14 CO 2 was not impaired in biotindeficient rats. The proposed main metabolic pathway of phytanic acid in mammals consists of an α-oxidative process leading through α-hydroxyphytanic acid to pristanic acid, followed by a series of β-oxidative steps. The chemical syntheses of a number of isoprenoid acids are described.

Localization of the oxidative defect in phytanic acid degradation in patients with refsum's disease

The rate of oxidation of phytanic acid-U-(14)C to (14)CO(2) in three patients with Refsums disease was less than 5% of that found in normal volunteers. In contrast, the rate of oxidation of alpha-hydroxyphytanic acid-U-(14)C and of pristanic acid-U-(14)C to (14)CO(2), studied in two patients, while somewhat less than that in normal controls, was not grossly impaired. These studies support the conclusion that the defect in phytanic acid oxidation in Refsums disease is located in the first step of phytanic acid degradation, that is, in the alpha oxidation step leading to formation of alpha-hydroxyphytanic acid. The initial rate of disappearance of plasma free fatty acid radioactivity after intravenous injection of phytanic acid-U-(14)C (t(1/2) = 5.9 min) was slower than that seen with pristanic acid-U-(14)C (t(1/2) = 2.7 min) or palmitic acid-1-(14)C (t(1/2) = 2.5 min). There were no differences between patients and normal controls in these initial rates of free fatty acid disappearance for any of the three substrates tested. There was no detectable lipid radioactivity found in the plasma 7 days after the injection of palmitic acid-1-(14)C or pristanic acid-U-(14)C in either patients or controls. After injection of phytanic acid-U-(14)C, however, the two patients showed only a very slow decline in plasma lipid radioactivity (estimated t(1/2) = 35 days), in contrast to the normals who had no detectable radioactivity after 2 days. Incorporation of radioactivity from phytanic acid-U-(14)C into the major lipid ester classes of plasma was studied in one of the patients; triglycerides accounted for by far the largest fraction of the total present between 1 and 4 hr.

Phytenic acid: Identification of five isomers in chemical and biological products of phytol

Abstract 1. 1. Previous studies in the rat indicated that the olefinic acid phytenic acid is an intermediate in the conversion of phytol to phytanic acid. Since phytol is a trans- Δ 2 structure, the phytenic acid product might be expected to retain the same configuration. This study was made to determine which isomers of phytenic acid are formed from phytol by chemical means and by metabolism in the rat. 2. 2. Chemical oxidation of phytol produced virtually only one phytenic acid isomer (trans- Δ 2 ), but dehydrobromination of 3-bromophytanic acid produced 5 isomers. Likewise, the same 5 isomers were found in the intestinal lymph of rats —both conventional and germ-free — that had been fed phytol. Phytenic acid of the lymph was present largely in complex lipids. Saponification caused isomerization of added trans -2-phytenic acid (methyl ester); however, direct transmethylation (with methanol-H 2 SO 4 ) did not cause appreciable isomerization. Using the latter method, all 5 isomers were found in the lymph in differing quantities, which indicates that they were all formed during the absorption of phytol. 3. 3. The individual isomers of phytenic acid were isolated by thin-layer chromatography and gas-liquid chromatography, and were identified by ultraviolet absorption spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectroscopy before and after hydrogenation and oxidative cleavage, respectively. They proved to be the trans - and cis- Δ 2 , the trans - and cis- Δ 3 , and the 3-methylene compounds.