Manford D. Morris

Improved graft patency associated with altered platelet function induced by marine fatty acids in dogs

Female mongrel dogs fed a marine fish diet rich in long-chain polyenoic fatty acids had improved patency of small-diameter arterial prosthetic grafts as compared to controls. Also, in vivo platelet function as measured by bleeding times was significantly prolonged. Eicosapentaenoic acid, not found in the serum of control animals, was present in relatively high concentrations in both the serum and a platelet-rich fraction of the marine oil-fed group. Eicosapentaenoic acid, unlike arachidonic acid, does not induce platelet aggregation and this phenomonon may account for the altered platelet function demonstrated in our animals and hence the improved graft patency. These data lend further support to the role of platelets in determining the patency of vascular grafts.

Human low density lipoprotein structure: correlations with serum lipoprotein concentrations

Human low density lipoproteins (LDL) were isolated and purified from individuals having widely differing serum lipid concentrations. Very low density lipoproteins (VLDL) and high density lipoproteins (HDL) were also isolated and quantitated. HDL2 and HDL3 were separated by flotation velocity in the analytical ultracentrifuge and their relative weight percent determined. The mean density of LDL from 41 individuals was determined by flotation velocity at two different solvent densities. The mean density of LDL was directly proportional to the triglyceride (r=0.65) and VLDL (r=0.50) concentrations and inversely proportional to the HDL (r=−0.55) and HDL2 (r=−0.74) concentrations (all significant at P<0.001). The mean molecular weight of LDL from 42 individuals was determined by flotation equilibrium centrifugation. The mean molecular weight of LDL was directly proportional to the HDL (r=0.49) and HDL2 (r=0.48) concentrations and inversely proportional to the serum triglyceride (r=−0.60) and VLDL (r=−0.48) concentrations (all significant at P<0.005 except triglyceride—P<0.001). The molecular weight of LDL was inversely proportional to its density, and thus inversely proportional to its protein/lipid ratio which was confirmed by composition measurements. The density and molecular weight of LDL had no relationship to the concentration of LDL (r=0.04 and 0.03).

The ultracentrifugal heterogeneity of serum low density lipoproteins in normal humans.

Abstract Studies of pure human low density lipoproteins (LDL) in the analytical ultracentrifuge revealed three groups of normolipidemic individuals of which 19 41 (46%) had heterogeneous LDL. The most common finding in individuals with heterogeneous LDL, 16 19 individuals, was the presence of a major fast LDL component, group Fs. The three other individuals with heterogeneous LDL had a major slow LDL component, group fS. Fifty-four percent of individuals ( 22 41 ) had homogeneous LDL, group S, in which the S f ,1.2,KBr of 39.8 closely agreed with the major LDL component of the fS group, S f = 40.7. In group Fs, the major LDL component was S f of 45.5 and was highly correlated with low serum triglyceride content, mean triglycerides concentration of 48 vs 91 mg/dl in group fS and S. Neither age nor sex appeared to be related to the occurrence of heterogeneous LDL, except as it is correlated with serum triglyceride values.

Quantitative determination of low density lipoprotein oxidation by FTIR and chemometric analysis

This study was conducted to develop a quantitative FTIR spectroscopy method to measure LDL lipid oxidation products and determine the effect of oxidation on LDL lipid and protein. In vitro LDL oxidation at 37°C for 1 h produced a range of conjugated diene (CD) (0.14–0.26 mM/mg protein) and carbonyl contents (0.9–3.8 μg/g protein) that were used to produce calibration sets. Spectra were collected from the calibration set and partial least squares regression was used to develop calibration models from spectral regions 4000-650, 3750-3000, 1720-1500, and 1180-935 cm−1 to predict CD and carbonyl contents. The optimal models were selected based on their standard error of prediction (SEP), and the selected models were performance-tested with an additional set of LDL spectra. The best models for CD prediction were derived from spectral regions 4000-650 and 1180-935 cm−1 with the lowest SEP of 0.013 and 0.013 mM/mg protein, respectively. The peaks at 1745 (cholesterol and TAG ester C=O stretch), 1710 (carbonyl C-O stretch), and 1621 cm−1 (peptide C=O stretch) positively correlated with LDL oxidation. FTIR and chemometrics revealed protein conformation changes during LDL oxidation and provided a simple technique that has potential for rapidly observing structural changes in human LDL during oxidation and for measuring primary and secondary oxidation products.

Characterization of the serum low-density lipoproteins of normal and two rhesus monkeys with spontaneous hyperbeta-lipoproteinemia

Abstract Cholesterol distribution among the serum lipoproteins was determined and the LDL characterized for normal Rhesus monkeys and two male Type IIb Rhesus monkeys. In fasted normal monkeys, lipoprotein analyses showed no chylomicrons and only small amounts of VLDL. While about 40% of the serum cholesterol was found in the LDL, the majority was in the HDL. The genetically altered monkeys carried much more of the serum cholesterol in the VLDL and markedly less in the HDL. A comparison of the LDL 2 of normal to that of the Type IIb Rhesus showed no major differences. Cholesterol was the major component of the LDL 2 (% by wt) and triglyceride was present in a relatively minor amount. Phospholipid and protein were found in a 1:1 ratio and the cholesterol-phospholipid ratio was about 1.3. About 70% of the phospholipid was lecithin and sphingomyelin made up 11%. The fatty acid composition of each lipid component was unique. Cholesteryl esters were primarily linoleic acid, triglycerides had about 40% oleic acid, and phospholipids had about 60% palmitic and stearic acid (30% of each). When normal and Type IIb LDL 2 were compared, no major differences were found in phospholipids or in the fatty acid compositions. The amino acid composition of the protein moiety of the LDL 2 was determined. Similar results were found for both the normal and the type IIb monkeys. Likewise, a similar molecular weight was found for the intact LDL 2 of both groups. The weight average molecular weight was 3.3 million as determined by sedimentation equilibrium. It was concluded that the LDL 2 of Rhesus monkeys with spontaneous hyperbeta-lipoproteinemia was similar to the LDL 2 of normal Rhesus monkeys. Furthermore, since Rhesus monkey LDL is similar to the human LDL, these genetically altered monkeys may serve as a useful experimental model in which to study human Type IIb hyperlipoproteinemia.

The distribution of serum high density lipoprotein subfractions in non-human primates.

The ultracentrifugal flotation patterns in 1.2 g/ml solvent and ultracentrifugal gradient distribution of high density lipoproteins (HDL) from the primates-human, apes and monkeys-were determined, with emphasis on the gorilla species of apes and rhesus monkeys. Diets for non-human primates were commercial chow, which is low in cholesterol. Molecular weights and protein, cholesterol, phospholipid and triglyceride compositions of various density fractions were determined on human, gorilla and rhesus HDL. The HDL2/HDL3 ratio was determined from the two peaks observed upon flotation in high salt in the analytical ultracentrifuge. The HDL2 of all three species of apes-gorillas (Gorilla gorilla), chimpanzees (Pan troglodytes) and orangutans (Pongo pygmaeus)—was always greater than HDL3, while that of all six species of Old World monkeys-Rhesus (Macaca mulatta), sooty mangabeys (Cercocebus atys), cynomolgus (Macaca fascicularis), stumptails, (Macaca arctoides) patas (Erythrocebus patas) and African greens (Cercopithecus aethiops)—was less. In addition, the HDL3 concentration in five gorillas was about 15 mg/dl as cholesterol while the HDL2 concentration was 92 mg/dl, much lower and higher, respectively, than humans. HDL2 of gorillas was similar in density and molecular weight to that of humans. The distribution of densities in gorilla HDL was predominantly in HDL2, while rhesus HDL usually, but not always, was unimodal, having a density distribution similar in heterogeneity to human HDL3, but somewhat less dense (peaking at 1.109 vs 1.129 g/ml). The molecular weight of rhesus HDL was about the same as human HDL3 in all three density subfractions and at the peak density. Likewise, the chemical compositions were similar for the subfractions 1.10–1.125 and>1.125 g/ml for rhesus HDL and human HDL3. Consequently most but not all chow-fed rhesus HDL was very similar to human HDL3, but lighter in density.

The effect of cholesterol feeding on primate serum lipoproteins. I. Low density lipoprotein characterization from rhesus monkeys with high serum cholesterol.

Abstract The effect of dietary cholesterol on the serum low density lipoproteins (LDL 2 ) of the rhesus monkey ( Macaca mulatta ) was studied by comparison of monkeys on a standard commercial monkey chow diet to those on a high cholesterol, high fat diet. The serum lipoproteins were fractionated by ultracentrifugation. Monkeys ingesting the control diet had 2% of the serum cholesterol in VLDL and an equal amount in the LDL 1 (1.006–1.019 g/ml) fraction. Upon ingesting the high cholesterol, high fat diet, the percentage in the VLDL and the LDL 1 fractions increased to 34–40% of the total. There was little change in the percentage of the serum cholesterol in the LDL 2 fraction except the absolute amount was higher in the cholesterol-fed animals. A 50% reduction in the percentage of serum cholesterol in the HDL was found upon cholesterol feeding (60% for controls vs 30% for cholesterol fed). A comparison of the LDL 2 composition of the control and cholesterolfed monkeys showed that the cholesteryl esters increased while protein and triglyceride decreased. The cholesterol/phospholipid ratio increased from 1.34 to 1.69 after cholesterol feeding. Cholesterol feeding had little effect on the phospholipid composition other than a twofold increase in phosphatidylethanolamine of LDL 2 . The percentage of unsaturated fatty acids in LDL 2 increased in the triglycerides, decreased in the phospholipids, and were unchanged in the cholesteryl esters after cholesterol-fat feeding. However, individual changes in unsaturated fatty acids seemed unrelated. In the LDL 2 phospholipid, arachidonic acid rose eightfold to 23% of the total while other unsaturated fatty acids showed little change. In the LDL 2 triglycerides, linoleic acid fell 73%, oleic 37%, and palmitoleic doubled upon cholesterol feeding. These changes were not a reflection of the dietary fatty acid composition except that four times as much fat was available to the cholesterol-fed animals. The amino acid composition of the apo-LDL 2 was not affected by cholesterol feeding. Determination of the molecular weights on the native LDL 2 showed that cholesterol feeding resulted in an increase of about 12% (3.1 to 3.5 million).

A new serum lipoprotein found in many rhesus monkeys

Abstract A new serum lipoprotein was found in about 10 out of 30 rhesus monkeys ( Macaca mulatta ) which contained 28% by weight of protein, 42% total cholesterol, 22% phospholipid, and 8% triglyceride. This is in contrast to LDL (which the ten monkeys also contained) which had 24% protein, 46% total cholesterol, 24% phospholipid and 7% triglyceride. An Sf, 1.063 in KBr of 3.0 to 3.7 and molecular weight of 3.5 – 3.7 million were observed compared to means of 8.1 and 3.0 million for normal rhesus LDL. The lower Sf was caused by its higher density. This new lipoprotein was most easily demonstrated and isolated by preparative ultracentrifugation of all serum lipoproteins at density 1.22 g/ml, followed by 6% agarose gel filtration at 6°. The new lipoprotein appeared as a distinct peak eluting before LDL.

Effects of cholesterol feeding on primate serum lipoproteins. III. The change in high density lipoprotein components.

Sooty mangabey (Cercocebus atys) monkeys had a lower serum HDL cholesterol concentration than any other Old World monkey species reported. In addition, they had a higher serum Lp(a) concentration than other species. The mangabeys were fed a cholesterol-fat diet for 5 weeks. HDL2 and HDL3 amounts were determined from the two peaks apparent upon analytical ultracentrifugation. In the first 1-3 weeks, 13 of the 14 mangabeys increased 30% (mean) in total HDL, this increase occurring only in the HDL2 fraction. After 5 weeks, HDL and HDL2 decreased markedly. During the cholesterol feeding, HDL3 continually decreased in flotation rate, indicating it was either smaller and/or denser. HDL2 and HDL3 separated well on molecular sieving agarose columns during the diet period, whereas a single symmetrical elution peak was found for chow-fed HDL. Thus on a cholesterol-fat diet, HDL2 and HDL3 increased in difference in molecular size.

Effects of cholesterol feeding on primate serum lipoproteins: II. Low density lipoprotein characterization from rhesus monkeys with a moderate rise in serum cholesterol

Abstract Rhesus monkeys ( Macaca mulatta ) were fed diets containing added cholesterol and fat for 2 months during which time their serum cholesterol rose to 308 ± 75 mg/dl. The LDL were isolated by agarose gel filtration of the serum lipoproteins floating at a density of 1.22 g/ml. Upon cholesterol-fat feeding, the LDL increased in amount and heterogeneity, rose in S f , 1.063, KBr from 8.1 to 10.8 (major peak) and S f 14–17 (minor peak), increased in average molecular weight from 3.1 to 3.6 million (major component), decreased in elution volume (indicating a higher molecular weight), decreased in protein (24 to 18.5%) and triglyceride (6.5 to 2.5%) content, and increased in cholesterol content from 46.5 to 55%. Fractions of the cholesterol-fed LDL were studied on the ascending, peak, and descending limbs of the agarose LDL peak. The fractions across the cholesterol-fed LDL peak were similar in composition, even though the S f 14–17 component was in much greater amount on the ascending side than on the descending side (pure S f 10–11 component). This indicated that in cholesterol feeding which produced a moderate serum cholesterol rise, the LDL size increased to a limiting molecular weight of about 3.6 million and a larger species formed, having an S f around 14–17, but with a similar composition. These results are in agreement with results obtained in studies of human LDL only in the increased amount of LDL. Human LDL has not been observed to change composition to that extent, nor alter in its molecular size or heterogeneity.