Carboxylic acid esters react with hydroxylamine in alkaline solution to form hydroxamic acids. These acids produce a red to violet reaction with ferric chloride which has been utilized by Feigl (1949) for the microchemical detection of carboxylic acid derivatives. Lipmann and Tuttle (1945) developed a method for the quantitative assay of acylphosphates by allowing hydroxylamine to react with these substances in aqueous solutions at pH 6.0. Hestrin (1949) adapted the reaction for the estimation of short-chain esters by employing alkaline aqueous reagents. Under these conditions esters of higher fatty acids did not react. In order to achieve a reaction of neutral fats and other natural lipids with these reagents, Bauer and Hirsch (1949) employed strictly anhydrous conditions and developed a method for the determination of the total esterified fatty acids in blood serum. Since almost all the fatty acids in blood serum are in esterified form, this method gives results identical with those testing total fatty acids in blood serum.Although the method of Bauer and Hirsch is simpler than most other available methods for blood fat determination, it still involves several time-consuming steps. The blood extracts have to be evaporated to dryness at 600 C. and the evaporation has to be repeated, with the strict exclusion of water.In connexion with work on the uptake of fat from blood serum by tissues in vitro, a convenient and fairly accurate method was required for blood fat estimation. It was learnt by personal information from Oren and Hestrin that higher fatty acid esters react at room temperature with hydroxylamine in alkaline solutions in aqueous alcohol. The presence of alcohol makes the reaction possible and also keeps the long-chain *Part of a
The isolation and purification of palmitoyl-CoA synthetase from rat liver microsomes is described. Several methods suitable for enzyme assay are described. The general properties and kinetic parameters of the purified enzyme were determined and are discussed in relationship to microsomal fatty acid activation.
Up to 60% of i.v. injected doses of 1-C14 palmitic acid was found in the liver 15 minutes after injection, two thirds in the form of triglycerides and the rest as phospholipids. Almost no radioactive UFA could be demonstrated in the liver or in blood plasma. Thereafter, the activity in the triglyceride fraction fell rapidly, only 50% remaining after 1 hour. At the same time the phospholipid activity increased. With 1-C14 linoleic acid a similar pattern was found, except that the percentage retained in the liver was lower and maximum retention was found after 5 minutes. Fifteen minutes after injection of 1-C14 palmitate most of the radioactive glycerides in the liver were found in the microsomes and mitochondria, with very little in the floating fat. Equilibration between these cytoplasmatic particles occurred only after 2 hours. Perfusion of the liver in situ with blood or blood substitutes, after various periods following 1-C14 palmitic acid injection, caused a release of about 5–20% of the liver triglycerides and 0.5–2% of the liver phospholipids per hour.
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