A. Kuksis

Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase

Abstract A hyperlipoproteinemic patient was investigated for lipoprotein abnormalities as a result of an abnormally high proportion (30%) of triglyceride in low density lipoprotein (LDL), the presence of β-VLDL as well as a reduced post-heparin lipolytic activity. Analysis of the component lipase activities in the proband and his brother by selective inactivation with protamine or by affinity chromatography revealed that lipoprotein lipase was present in normal concentrations while the triglyceride lipase characteristic of that released from liver was reduced to 5% of normal levels. Although evidence was obtained to indicate inhibition, in vitro, of post-heparin lipolytic activity of a normal subject, analysis of the patients post-heparin plasma revealed that pre-β-VLDL was catabolized while β-VLDL- and TG-enriched LDL were poor substrates. Very low density lipoprotein (VLDL) was markedly elevated in the proband and his brother and possessed pre-β- and β-mobility on agarose gel electrophoresis. Apolipoproteins A-I, A-II and D were present in normal concentrations while apolipoproteins B, C-II, C-III and E were elevated 2–3-fold over normal concentrations. There was no deficiency of apolipoprotein E-III in VLDL which is usually characteristic of the accumulation of β-migrating VLDL in primary Type III hyperlipoproteinemia. Both LDL and high density lipoprotein (HDL) were enriched in triglyceride and phosphatidyl choline at the expense of cholesterol ester and sphingomyelin. Mass analysis and analytic ultracentrifugation indicated that HDL was elevated in these subjects in contrast to a tendency for decreased HDL in most hypertriglyceridemic subjects. It is proposed that the presence of LDL and HDL 2 , which are relatively enriched in triglyceride and phosphatidyl choline, are a consequence of a deficiency of hepatic lipase. The accumulation of β-VLDL may also be associated with this enzyme deficiency or possibly result from a lipoprotein lipase which may be less active in the presence of lipoprotein components accumulating as a result of the hepatic lipase deficiency.

TOTAL PARENTERAL NUTRITION AT HOME: STUDIES IN PATIENTS SURVIVING 4 MONTHS TO 5 YEARS

Total parenteral nutrition at home has been delivered to 12 patients surviving for periods ranging from 4 months to 5 years. The route used is a silicone rubber catheter introduced into the superior vena cava. The mean survival of uncomplicated catheters is 15.8 months, but in 4 patients it is in excess of 28 months. The delivery system is a simple pneumatic infusor set, portable and safe to use, and not requiring complicated electronics. All patients have maintained ideal body weight and have been socially rehabilitated, with two exceptions. Detailed metabolic studies are presented and the data include routine biochemical analyses, liver function tests, liver biopsy results, blood aminograms, the plasma phospholipid fatty acid pattern, the plasma lipoprotein pattern, and blood vitamin and trace element profiles. Based on these findings a nutrient program is recommended and discussed.

Apolipoprotein A-I promotes the formation of phosphatidylcholine core aldehydes that are hydrolyzed by paraoxonase (PON-1) during high density lipoprotein oxidation with a peroxynitrite donor.

High density lipoprotein (HDL) is rich in polyunsaturated phospholipids that are sensitive to oxidation. However, the effect of apolipoprotein A-I and paraoxonase-1 (PON-1) on phosphatidylcholine oxidation products has not been identified. We subjected native HDL, trypsinized HDL, and HDL lipid suspensions to oxidation by the peroxynitrite donor, 3-morpholinosydnonimine. HDL had a basal level of phosphatidylcholine mono- and di-hydroperoxides that increased to a greater extent in HDL, compared with either trypsinized HDL or HDL lipid alone. Phosphatidylcholine core aldehydes, which were present in small amounts, increased 10-fold during oxidation of native HDL, compared with trypsinized HDL (p = 0.004), and 4-fold compared with HDL lipid suspensions (p = 0.0021). In addition, the content of lysophosphatidylcholine increased 300% during oxidation of native HDL, but only 80 and 25%, respectively, during oxidation of trypsinized HDL and HDL lipid suspensions. Phosphatidylcholine isoprostanes accumulated in comparable amounts during the oxidation of all three preparations. Incubation of apolipoprotein A-I with 1-palmitoyl-2-linoleoyl glycerophosphocholine proteoliposomes in the presence of 3-morpholinosydnonimine or apoAI with phosphatidylcholine hydroperoxides resulted in a significant increase in phosphatidylcholine core aldehydes with no formation of lysophosphatidylcholine. We propose that apolipoprotein A-I catalyzes a one-electron oxidation of alkoxyl radicals. Purified PON-1 hydrolyzed phosphatidylcholine core aldehydes to lysophosphatidylcholine. We conclude that, upon HDL oxidation with peroxynitrite, apolipoprotein AI increases the formation of phosphatidylcholine core aldehydes that are subsequently hydrolyzed by PON1.

Molecular species of glycerophospholipids and sphingomyelins of human erythrocytes: Improved method of analysis

This study reports the application of modern methods of molecular species analysis in determination of the structure of both major and minor glycerophospholipids and sphingomyelins of human erythrocytes. Individual phospholipid classes were resolved from total lipid extracts by thin-layer chromatography. Diradylglycerols were released by phospholipase C and converted into trimethylsilyl ethers, which were resolved into the alkenylacyl, alkylacyl and diacylglycerol subclasses by normal phase high performance liquid chromatography. Molecular species of diradylglycerols and ceramides were quantitated according to carbon and double bond number by gas liquid chromatography using a fused silica capillary column wall-coated with bonded RTx-2330. The molecular species of ceramides were determined by GC/MS. The diradyl glycerophosphocholines contained 93.0% diacyl, 4.6% alkylacyl and 2.5% alkenylacyl, white the diradyl glycerophosphoethanolamines were made up of 48.8% diacyl, 47.8% alkenylacyl and 3.4% alkylacyl subclasses. Analysis of the molecular species showed that the long chain polyunsaturated acids were mainly combined with C16 in all diradyl GPC subclasses and in diacyl GPE, while in the alkylacyl and alkenylacyl GPE and in diacyl glycerophosphoinositol and diacyl glycerophosphoserine they were combined mainly with C18 saturated fatty chains. In addition to the C16 and C18 alkyl and alkenyl, the ether fractions also contained significant proportions of C20, C22 and C24 chains. The molecular species of the ceramide moieties of the SPH were made up largely of mono- and diunsaturated species. Over 200 molecular species were identified and quantitated in a representative sample of human red blood cells.

Molecular Species of Glycerophospholipids and Sphingomyelins of Human Plasma: Comparison to Red Blood Cells

In addition to diacyl glycerophosphocholine and sphingomyelin, human plasma also contains small amounts of other glycerophospholipids, which may have special metabolic function. The structure and origin of these minor plasma lipids has not been determined. Knowledge of the detailed composition of the phospholipids of red blood cells (Myheret al., Lipids 24, 1989) permits evaluation of one of the possible sources. This study reports the detailed analyses of plasma glycerophospholipids made in parallel to those of the erythrocyte lipids obtained from the same blood using HPLC and GLC methods. The proportions of the major phospholipid classes in the plasma and erythrocytes were similar to published values, including the essential absence of diradyl glycerophosphoserine from plasma. Plasma diradyl glycerophosphocholine contained 93.0% diacyl, 3.4% alkylkacyl and 3.6% alkenylacyl, whereas the diradyl glycerophosphoethanolamine consisted of 71.8% alkenylacyl, 19.9% diacyl and 8.3% alkylacyl subclasses. The diradyl glycerophosphoinositol was 100% diacyl. The content of the minor subclasses of plasma diradyl glycerophosphocholine is similar to that of the red cells, but the ether content of the diradyl glycerophosphoethanolamine is higher in plasma than in cells. The lipid ether subclasses of plasma glycerophospholipids also contained a higher proportion of the C20, C22 and C24 alkyl and alkenyl chains than those of the cells. Furthermore, the C16 and C18-containing species in diradyl glycerophosphoethanolamine subclasses varied with the nature of the polyunsaturated acid, whereas in diradyl glycerophosphocholine subclasses the polyunsaturated acids were combined with the C16 and C18 acids in equal proportions. The significant differences in the molecular species of glycerophospholipids and sphingomyelin between plasma and red cells would appear to limit any direct transfer or equilibration of their lipid components.

Determination of the complete structure of natural lecithins

A method is described for the separation, identification, and quantitative estimation of the individual molecular species occurring in natural lecithin mixtures. Purified lecithin preparations are converted into diglyceride acetates by enzymic dephosphorylation and acetylation. The diglyceride acetates are separated on the basis of the degree of unsaturation and the molecular geometry by means of chromatography on thin layers of silica gel which are impregnated with silver nitrate. The various acetates thus resolved are separately recovered from the plates and diluted with tridecanoin internal standard; the quantitative distribution of the molecular weights is determined by gas chromatography.Suitable aliquots of the saturated and unsaturated diglyceride acetates are further analyzed for over-all and for positional distribution of fatty acids. The identity and proportions of the various lecithins are deduced by integration and normalization of all the experimental data. Where doubt exists, specific diglyceride acetates are isolated by preparative gas chromatography, and their fatty acid composition is determined. The method is illustrated with data obtained for the mixed lecithins of egg yolk. The general approach is applicable to the determination of the structure of other phospholipids of comparable complexity.

Triglyceride structure of milk fats.

The enantiomeric nature of the triglycerides of bovine milk fat was reinvestigated by determining the stereospecific distribution of fatty acids in rearranged butterfat, following partial hydrolysis with pancreatic lipase, and in certain molecular distillates of native butterfat, following Grignard degradation. The results with rearranged butterfat confirmed the validity of pancreatic lipase hydrolysis as a means of generating representative diglycerides from milk fat triglycerides. The Grignard degradation and lipolysis gave identical distributions for fatty acids when included as part of the assay system in the stereospecific analysis. Characteristically, butyric acid and the other short chain acids occupied the 3 position in the native butterfat, while in the rearranged oil they were distributed more or less randomly. Gas chromatographic analysis of the short chain glycerides on polyester columns allowed an effective resolution of butyryl, caproyl and caprylyl glycerides of identical numbers of total acyl carbons and double bonds. The method was especially well suited for resolution of the 2,3-diglycerides, which were recovered either as the more polar fraction from thin layer chromatography of the X-1,2-diacylglycerols, or by acetolysis of the residual phenolphosphatides resulting from phospholipase A digestion. It was shown that butyric acid in the 3 position was preferentially paired with myristic, palmitic and oleic acid in the 2 position, and palmitic and oleic acid in the 1 position, which was also characteristic of the other short chain acids.

Role of luminal lecithin in intestinal fat absorption.

The effects of biliary lecithin on fat absorption were studied in 1 day bile fistula rats fed micellar solutions of bile salt, monoglyceride and radioactive free fatty acids. By electron microscopy and measurement of uptake of radioactivity into liver and adipose tissue, it was shown that in the absence of bile lecithin there was significant impairment of fat release from mucosa. Fat clearance was effected by the feeding of phosphatidyl choline or choline, but not phosphatidyl ethanolamine, inositol or cholesterol. In the absence of luminal choline there was a decrease in incorporation of radioactive leucine into mucosal protein. It is concluded that biliary and dietary lecithin or choline play an important role in triglyceride transport out of intestinal mucosa, by providing surfactant lecithin for the chylomicron envelope and by supporting mucosal protein biosynthesis.

General strategies in chromatographic analysis of lipids

Lipid extracts of natural sources contain a large number of lipid classes and molecular species. Completely reproducible samples are obtained only with great care and skill. Analytical methods other than chromatography and/or mass spectrometry are of little use for resolution and identification of lipid molecules even in simple mixtures. The analytical information desired governs the selection of the chromatographic and mass spectrometric method, which determine the sample preparation and derivative needed. Usually a combination of chromatographic methods is necessary to identify specific species of lipids. The recent development of soft ionization techniques, that are readily interfaced with mass spectrometers, have greatly simplified the sample preparation and have largely eliminated the need for derivatization. Because these techniques require expensive equipment and dedicated operators, the methods selected must be consistent with the true analytical needs and the available resources. Although personal preference cannot be eliminated entirely, the general strategies outlined below should help to reduce the number of possibilities facing a lipid analyst to a few practical choices.

A comparative study of the lipids of globule membrane and fat core and of the milk serum of cows

Nine samples of fresh raw cows milk were separated into fat globules and milk serum by centrifugation. After destabilization by freezing and thawing, the milk fat globules were resolved into membranes and fat cores. The lipid composition of these structures was compared to that of the surrounding milk serum. Of the total milk fat, 95–98% was in the fat cores, 0.5–1% in the globule membranes and the rest (1.5–4%) in the milk serum. The fat cores contained 88–93% triglyceride, 5.2–9.8% diglyceride, 1.5–7.3% free fatty acid and 0.2–0.4% cholesterol, but no phospholipid. The lipids of the membrane contained 21–44% phospholipid, made up of about equal proportions of phosphatidyl ethanolamine, phosphatidyl choline, and sphingomyelin. The other lipids of the membrane (56–79%) consisted of 83–88% triglyceride, 5.1–10.7% diglyceride, 1–5.1% free fatty acid and 0.4–1.9% cholesterol. The milk serum contained 30–45% phospholipid divided about equally among phosphatidyl ethanolamine, phosphatidyl choline and sphingomyelin. The rest (55–70%) of the milk serum lipids was made up of 71–83% triglycerides, 4.3–10.1% diglycerides, 8.7–15.7% free fatty acids, and 1.2–8.4% cholesterol. Corresponding phospholipid classes of milk serum and globule membranes had identical fatty acid compositions. The triglycerides and diglycerides of the globule membranes possessed increased proportions of palmitic and stearic acids in comparison to the glycerides of the fat cores.