BWECKENR~GE, W. C., MAMI, L., AND KUKSIS, A. Triglyceride structure of human milk fat. Can. J. Biochem. 47, 761-7669 (11969).The triglycerides of the fat globules of human milk were resolved by thin-layer chromatography on silica gel impregnated with silver nitrate. The chemical structure of the glycerides was determined by gas chromatography of the glycerides and fatty acids and by a stereospecific analysis of total milk fat sampk. The major triglycerides of human milk fat contained 48-54 acyl carbons which were made up sf Ci2-Ffa acids of various degrees of unsaturation. The fully saturated triglywrides (6-873 contained mainly SPB-glycerol 1-stearate 2-palmitate esterified at position 3 with Cae-Cas acids. The monoensic triglycerides (25-2'6" %) were comprised mainly of sn-glycerol 1-oleate Zpalmitate esterified at position 3 t s Ci2-Cas acids, as well as their rammates. The dienes (2433%) were made up largely of sn-glycerol 1-stearate 2,3=dioleate and %-palmitate 83-diokate. The trienes (18-20%) contained mostly sn-glycerol 8,2,3-triokate and 1-sleate Zpalmitate 3-linoleate. The tetraenes (7-21 %) were identified as mainly S P B -~I~C~~O I 1 ,Zdioleate 3-linoleate and I -palmitate 2,3-dilholeate. Arachidsnic acid was found in the polyene fraction (6 %) in the Zpodtion, while the linolenic acid was preferentially associated with position 3 of the tetraenes.

Glycosylation is a major pathway for posttranslational modification of tissue protein and begins with nonenzymatic addition of carbohydrate to the primary amino groups. Excessive glycation of tissue protein has been implicated in the pathogenesis of diabetes and ageing. While glycation of aminophospholipids has also been postulated, glycated aminophospholipids have not been isolated. Using normal phase HPLC with on-line electrospray mass spectrometry we found glycated ethanolamine phospholipids to make up 10-16% of the total phosphatidylethanolamine (PE) of the red blood cells and plasma of the diabetic subjects. The corresponding values for glycated PE of control subjects were 1-2%.Key words: Glucosylated aminophospholipid; Glucose; Phosphatidylethanolamine; Phosphatidylserine; Electrospray; Thin-layer chromatography; Liquid chromatography, mass spectrometry; Normal phase HPLC bovine brain phosphatidylinositol (PI) and sphingomyelin (SPH) were obtained from Sigma Chemical Co., St. Louis, MO. All chemicals were of reagent grade quality, while the solvents were of chromatographic purity and were obtained from local suppliers. The purity of the reference compounds was ascertained by thin-layer chromatography (TLC) [3,7]. Isolation of phospholipids from bloodBlood was obtained from six diabetic patients and six non-diabetic donors. The diabetics were selected for elevated blood glucose levels indicated by their content of glycosylated hemoglobin (9-15%). EDTA blood was centrifuged (2300xg for 10 min) in a swinging bucket rotor to separate the plasma from the red cells. The cells were washed three times with five volumes of phosphate buffered saline (150 mM NaC1, 50 mM sodium phosphate, pH 8.0) and centrifuged (2300xg for 10 min). The red blood cell phospholipids were extracted according to Rose and Oaklander [8]. The plasma phospholipids were extracted with chloroform-methanol 2:1 modified from Folch et al. [9]. Glucosylated PE could be stored at -20°C in neutral chloroform methanol for several days without decomposition. The Schiff base dissociated in dilute acetic acid.

Selected elution factors were determined for model oxotriacylglycerols as an aid in identification of the peroxidation products of natural triacylglycerols by reverse-phase high-performance liquid chromatography (HPLC) with electrospray mass spectrometry (LC/ES/MS). For this purpose synthetic triacylglycerols of known structure were converted to hydroperoxides, hydroxides, epoxides, and core aldehydes and their dinitrophenylhydrazones by published procedures. The oxotriacylglycerols were resolved by normal-phase thin-layer chromatography and reverse-phase HPLC, and the identities of the oxotriacylglycerols confirmed by LC/ES/MS. Elution factors of oxotriacylglycerols were determined in relation to a homologous series of saturated triacylglycerols, ranging from 24 to 54 acyl carbons, and analyzed by reverse-phase HPLC, using a gradient of 20-80% isopropanol in methanol as eluting solvent and an evaporative light-scattering detector. It was shown that the elution times varied with the nature of the functional group and its regiolocation in the triacylglycerol molecule. A total of 31 incremental elution factors were calculated from chromatography of 33 oxygenated and nonoxygenated triacylglycerol species, ranging in carbon number from 36 to 54 and in double-bond number from 0 to 6.

Synthetic cholesteryl 5-oxovalerate and 9-oxononanoate were used as reference standards for the isolation and identification of cholesteryl ester core aldehydes from tert-butyl hydroperoxide/Fe++ oxidation of synthetic and natural cholesteryl esters. The core aldehydes were recovered from the peroxidation products by thin-layer chromatography as the free aldehydes or the 2,4-dinitrophenylhydrazones and were identified, respectively, by gas-liquid chromatography (GLC) and by GLC combined with mass spectrometry (GC/MS) or by reverse-phase high-performance liquid chromatography (HPLC) and by HPLC with MS (LC/MS). The core aldehydes produced by peroxidation of cholesteryl linoleate were identified as mainly 9-oxononanoates of cholesterol and oxycholesterols, with smaller amounts of the 8-oxooctenoates, 10-oxodecenoates, 11-oxoundecenoates and 12-oxododecenoates. Peroxidation of cholesteryl arachidonate yielded 5-oxovalerates of cholesterol and the oxycholesterols as the main products with smaller amounts of the 4-oxobutyrates, 6-oxohexenoates, 7-oxoheptenoates, 8-oxooctenoates, 9-oxononenoates, 9-oxononadienoates and 10-oxodecadienotes. The oxycholesterols resulting from the peroxidation of the steroid ring were identified as mainly 7-keto-, 7 alpha-hydroxy- and 7 beta-hydroxy-cholesterols and 5 alpha,6 alpha- and 5 beta,6 beta-epoxy-cholestanols. Cholesteryl palmitate and oleate did not yield core aldehydes in the present peroxidation system. In these esters, the sterol and linoleic acid moieties appeared to be oxygenated at about the same rate, while the arachidonic acid moiety reacted more rapidly than did the sterol moiety.

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