Because the association of hypertriglyceridemia and premature atherosclerosis is not due to the direct effects of the triglyceride molecule itself, we studied the effects of increased plasma triglyceride-rich lipoproteins on the composition and structure of low density lipoprotein (LDL) and high density lipoprotein (HDL). We found profound changes in the core and surface domains of both lipoproteins with increasing triglyceridemia. Core cholesterol esters were progressively depleted and replaced by triglyceride molecules. Highly significant negative correlations were found between cholesterol ester/protein ratios (r = cause of accelerated coronary artery disease in familial hypercholesterolemia. 5 The increased risk for coronary artery disease observed in hypertriglyceridemia, however, is poorly understood and probably does not reflect the direct effects of the triglyceride molecule itself. Recently, Hulley and associates 6 have suggested that the low HDL cholesterol levels found in hypertriglyceridemic patients contribute significantly to this phenomen. Such patients also have low LDL cholesterol levels and high VLDL cholesterol and triglyceride levels. The metabolic pathways responsible for these observations have not been previously elucidated.-In this paper we suggest that these abnormalities may merely reflect the state of hypertriglyceridemia. Specifically, we propose that the replacement of cholesterol ester in HDL and LDL by triglycerides contributes to the low levels of cholesterol in these lipoproteins. In recent in vitro experiments, 7 " 9 we showed that cholesterol ester-triglyceride bidirectional transfer processes, originally observed by Nichols and Smith, 10 play a major role in determining the structure and composition of LDL and HDL. We

A s bstract. The effects of triglyceridemia on plasma lipoproteins were investigated in 16 hypertriglyceridemic (HTG) subjects (222-2,500 mg/dl) before and after the initiation ofbezafibrate therapy. Bezafibrate caused a mean reduction of 56% in plasma triglyceride and increased the levels of lipoprotein and hepatic triglyceride lipases by 260 and 213%, respectively. The natures of very low density lipoprotein (VLDL), isolated at plasma density and of low and high density lipoprotein (LDL and HDL), separated by zonal ultracentrifugation, were determined. HTG-LDL appears as multiple fractions whereas HTG-HDL is seen predominantly as HDL3.HTG-VLDL is relatively poor in apoproteins and triglycerides but enriched in free and esterified cholesterol. HTG-LDL (main fraction) is depleted offree and esterified cholesterol but enriched in apoprotein and triglyceride. It is also denser and smaller than normal. HTG-HDL3 is denser than N-HDL3 and demonstrates compositional abnormalities similar to those of HTG-LDL. With the reduction of the VLDL mass, all abnormalities revert towards normal. This is accompanied by an increase in LDL-apoprotein B and cholesterol levels, which indicates an increased conversion of VLDL to LDL.Significant correlations between plasma triglyceride and the degree of all abnormalities are shown. The data obtained during treatment corroborate these relationships.The observations support the concept that most abnormalities reflect the degree of triglyceridemia.Address reprint requests to Dr. Eisenberg, Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital.Received for publication 12 December 1983 and in revised form 4 April 1984.We suggest that plasma core-lipid transfer protein(s) is an effector ofthe abnormal cholesteryl ester distribution. Its prolonged action on increasingly large and slowly metabolized VLDL populations would entail a correspondingly excessive transfer of cholesteryl ester to VLDL and of triglyceride to LDL and HDL. It is calculated that, in moderate HTG, LDL and HDL contain only 50% of the normal cholesterol load. It is suggested that cholesteryl ester redistribution in HTG might be important in regulating metabolic events.

Inasmuch as caput epididymal and even testicular spermatozoa are now being used to generate pregnancies by direct injection into the oocyte, differences in the chromatin of spermatozoa from proximal and distal locations in the epididymis were studied. Acridine Orange staining was used to investigate chromatin structure in human spermatozoa which had left the testis and were undergoing maturation in the epididymis. Measurement of green and red fluorescence intensities of human spermatozoa by flow cytometry demonstrated a decrease in binding of Acridine Orange to DNA as the spermatozoa traversed the epididymis. Using spermatozoa from the cauda epididymis as the standard, the percentages of spermatozoa from the efferent duct, proximal corpus epididymis, midcorpus epididymis, distal corpus epididymis, proximal cauda epididymis and distal cauda epididymis that had matured with regard to chromatin condensation were 28 +/- 5, 39 +/- 3, 49 +/- 5, 64 +/- 5, 69 +/- 6 and 74 +/- 4% respectively. It may be concluded that eggs fertilized by ejaculated spermatozoa receive a more highly condensed form of chromatin than that received by eggs inseminated with proximal epididymal or testicular spermatozoa.

The metabolism of hypertriglyceridemic low density lipoprotein (HTG-LDL) was investigated in upregulated cultured human skin fibroblasts. Low density lipoprotein (LDL) was isolated by zonal centrifugation from the plasma of seven HTG subjects, before and 2 wk after the initiation of bezafibrate (BZ) therapy. HTG-LDL is a cholesterol-poor, triglyceride-rich lipoprotein of smaller diameter than BZ-LDL or normal LDL (N-LDL). Binding, cell association, and proteolytic degradation of HTG-LDL were compared with that of BZ-LDL and N-LDL and were found to be significantly lower by a paired t test analysis (P < 0.001). After 6 h preincubation with unlabeled HTG-LDL, the incorporation of [14Clacetate to sterols was significantly higher than with BZ-LDL or N-LDL (577±43.7; 330±41.5; 262±47, mean±SE, picomoles sterols per milligram cell protein per 2 h, respectively; P < 0.001 by paired t test).To determine the effectiveness of HTG-LDL and BZ-LDL on the down-regulation of LDL receptor activity, up-regulated cells were incubated for 48 h with HTG-LDL and BZ-LDL. LDL receptor activity was significantly higher after preincubation with HTG-LDL compared with BZ-LDL, and the rates of sterol synthesis were similarly increased. These results demonstrate that HTG-LDL does not down-regulate the LDL receptor activity as efficiently as BZ-LDL and that its cholesterol content is not enough to adequately suppress cellular sterol synthesis.Significant correlation between LDL composition and cholesterol synthesis by cultured cells was found with all LDL preparations over a wide range of cholesteryl ester to protein ratio (0.8-2.2). This correlation indicates that the compositional and structural abnormalities of HTG-LDL, and especially the low cholesterol content of the lipoprotein, alter LDL metabolism and cellular cholesterol formation.

The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

Plasma lipids, lipoprotein and apolipoprotein levels were determined in seven women and seven men with moderate obesity before, during 7 weeks of continuous weight loss (10.4% to 9.6% of body weight, 1000 kCal/day diet), and after 3 months at a stable, reduced weight. Plasma triglyceride levels decreased by 30.4% in men and by 39.4% in women (p < 0.0001) after 1 week of caloric restriction and remained at this level throughout the study period. The plasma cholesterol decreased by 19.0% in men (p < 0.001) and by 10.9% in women (p < 0.01) in the period of active weight loss, but returned to prediet values after stabilization at a leaner body mass. Similar changes were observed in LDL cholesterol levels. No change in high density lipoprotein (HDL) cholesterol levels occurred during active weight reduction, but after 3 months at a reduced weight, a significant increase in HDL cholesterol was evident, and the ratio of HDL cholesterol to plasma cholesterol increased over prediet values (p < 0.001, women). Separation of HDL subpopulations by zonal ultracentrifugation before and after weight reduction revealed that HDL 2 increased slightly in men and decreased slightly in women. In both genders, HDL 3 tended to decrease after weight reduction. Plasma levels of apolipoprotein A-l decreased during active weight loss, but this was significant only in women (p < 0.05). After 3 months of reduced weight, plasma apo A-l increased to prediet levels. No significant changes in plasma apo A-ll or apo E were noted. Our results indicate that in moderately obese, but otherwise healthy, subjects weight reduction achieved by caloric restriction does not affect HDL composition or subpopulation distribution significantly. Moreover, maintenance of a leaner body mass has a beneficial long-term effect by increasing HDL cholesterol and decreasing plasma triglycerides. (Arteriosclerosis 4:115-123, March/April 1984)

The fluorescent labeling agent monobromobimane (mBBr) was used to label thiols and disulfides (after reduction of sperm disulfides by dithiothreitol) in intact spermatozoa. Bimane-labeled sperm of several mammalian species were analyzed by flow cytometry (FCM) and examined by fluorescent microscopy. FCM analysis showed sperm thiol oxidation to disulfides during epididymal maturation. FCM of labeled mature spermatozoa showed differences among species in the sperm thiol content. Heterogeneity in thiol content of sperm within individual samples was also observed. In addition, FCM patterns showed heterogeneity among and within samples in the content of disulfides and their resistance to reduction. FCM analysis reflected the microscopic appearance of the labeled spermatozoa. FCM analysis of bimane-labeled spermatozoa offers a convenient method for the study of sperm thiol-disulfide status and permits detection of sperm subpopulations within an individual sample. FCM analysis of mBBr-labeled spermatozoa may serve as a test to evaluate sperm quality.

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