Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study.

DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process.

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis.

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.

Epididymal maturation of chromatin in spermatozoa from control monkeys (Macaca fascicularis) and those treated with Cetrorelix, a gonadotropin-releasing hormone antagonist.

In order to examine changes in sperm chromatin upon epididymal maturation in the macaque epididymis (Macaca fascicularis), spermatozoa were obtained from six regions of the duct and examined for the state of their chromatin condensation by flow cytometry after staining with acridine orange. To see whether changes were affected by androgens, tissue was obtained from five monkeys treated with the gonadotropin releasing hormone (GnRH) antagonist Cetrorelix. Spermatozoa were recovered from treated and control animals after 16 days (at hemicastration) and another 9 days of treatment. Chromatin condensation of epididymal spermatozoa from controls displayed an increase upon maturation. After 16 days of GnRH-antagonist treatment, spermatozoa in the caput epididymidis displayed greater fluorescence than those from controls, but this was reduced during epididymal transit to values found in the distal epididymal regions of the controls. It is concluded that epididymal chromatin condensation 1) is normal in GnRH-antagonist-treated monkeys as long as sperm are being produced and 2) can compensate for poor testis function so that spermatozoa with normal states of chromatin condensation are found in the distal cauda epididymidis and probably the ejaculate.

Binding of rat and ovine epididymis-specific prealbumins (PES) to rat spermatozoa without effect of heterologous immunization on rat fertility.

The epididymis, under control of testosterone, secretes proteins which bind to the membrane of the spermatozoa during their passage through the lumen. One such class is termed PES (prealbumin epididymal specific). Injection of heterologous oPES (ovine PES) into male rats caused antibody production but failed to induce sterility, unlike results previously obtained when rat PES was injected into male rats. This suggests that only very restricted species-specific epitopes of PES might be useful for causing immunocontraception. Despite this, the sperm binding properties of PES purified from the rat (rat PES) and from the ram (oPES) were shown to be similar. When either rat PES or oPES, conjugated with a fluorescent probe (dimethylamino-fluorescein), was incubated with washed rat spermatozoa originating from the caput, corpus or cauda epididymis, results of flow cytometric analysis showed: (1) the number of spermatozoa bound to isologous or heterologous fluorescent PES, and (2) the binding-affinity of spermatozoa for PES was greater for sperm collected from more distal sites in the epididymis.

Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining.

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.

Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry.

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.

Investigation of epididymal sperm maturation in the golden hamster.

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.

Chromatin condensation in hamster sperm: a flow cytometric investigation.

In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine.

Sperm analysis by flow cytometry using the fluorescent thiol labeling agent monobromobimane.

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.

Neutral lipid transfer and lipolysis convert high molecular weight LDL from cholesterol-fed nonhuman primates towards normal: a molecular analysis.

In cynomolgus monkeys (Macaca fascicularis) fed an atherogenic diet, large, cholesterol ester-rich LDL (Mr greater than 3.5.10(6] are found at the same time that the plasma triacylglycerol levels are low. We studied whether the presence of higher concentrations of triacylglycerol-rich lipoproteins (VLDL) during in vitro incubations would allows depletion from LDL of cholesterol ester and a decreased LDL molecular weight. Three high Mr LDL (Mr = (3.7-4.8).10(6)), rich in cholesterol ester (50 +/- 1.4% by weight), were isolated from three animals by zonal ultracentrifugation, and were then incubated with human VLDL at 37 degrees C for 18 h in lipoprotein-deficient human plasma containing neutral lipid transfer activity. After incubation, modified LDL (M-LDL) was isolated by zonal ultracentrifugation. M-LDL was triacylglycerol-rich (36 +/- 5% by weight) and cholesterol ester-poor (20 +/- 3%), and cholesterol ester had transferred into VLDL. Purified lipoprotein lipase was added to the M-LDL, and triacylglycerol was hydrolyzed. The size of the post-lipolysis M-LDL (Mp-LDL) particles became smaller (mean diameters of 253 A and 228 A for two native LDLs and 215 A and 193 A for Mp-LDL, respectively). Both analytical and zonal ultracentrifugation showed Mp-LDL to be more dense than native LDL. Estimated molecular weights for Mp-LDL were 40%-50% less than that of the original LDL, and fell within the molecular weight range for normal human and monkey LDL. Lipid exchanges, but not apoprotein transfers, were responsible for LDL remodelling, as supported by three separate methods of analysis. Cholesterol ester losses accounted for about two-thirds of the molecular weight decrease. These in vitro results suggest that cholesterol ester enrichment of apoprotein B lipoprotein particles can be reversed by providing adequate levels of VLDL in the presence of neutral lipid transfer processes and lipolytic activity.

Familial lipoprotein lipase deficiency: abnormal lipoproteins and defective metabolism of low density lipoproteins in cultured human skin fibroblasts.

Lipoproteins (chylomicrons + VLDL, VLDL, IDL, LDL and HDL) were separated from the plasma of 2 patients with primary, familial lipoprotein lipase deficiency. Chylomicrons were excessively enriched with cholesteryl esters. VLDL and IDL were of almost normal composition. LDL separated into 2 fractions LDL1 and LDL2, both triglyceride- and protein-rich and cholesteryl ester-poor. LDL2, the main LDL fraction, was denser and smaller than normal LDL. HDL3 was the only HDL population identified and was also triglyceride- and protein-rich and cholesteryl ester-poor. These observations indicate excessive triglyceride and cholesteryl ester transfer between chylomicrons and LDL and HDL. VLDL and its immediate catabolic product, IDL, seem to be spared the effects of the lipid transfer reaction. The biological reactivity of LDL1 and LDL2 was investigated in upregulated cultured human skin fibroblasts. Both exhibited defective specific binding to the LDL receptor and ineffective capacity to down-regulate sterol synthesis. These abnormalities were more pronounced with LDL3. The ineffective downregulation of sterol synthesis is most probably due to both the cholesterol content of the LDLs and their reduced binding to the LDL receptor. The defective binding of the LDLs to the receptor can be attributed to the abnormal composition of the lipoproteins and, to a lesser degree, reduced diameters (only LDL2). It is concluded that abnormal composition of LDL, in particular of lipid moieties, may change the affinity of the moiety of the lipoprotein towards the LDL receptor.

Abnormal regulation of LDL receptor activity and abnormal cellular metabolism of hypertriglyceridaemic low density lipoprotein: normalization with bezafibrate therapy.

The regulation of LDL (B,E) receptor activity and of cellular LDL protein metabolism by hypertriglyceridaemic (HTG) low density lipoprotein before and during hypolipidaemic therapy (with bezafibrate (BZ] were determined in cultured human skin fibroblasts. Defective binding and subnormal capacity to regulate LDL receptor activity was found for HTG-LDL. Binding affinity (Kd) of HTG-LDL to the receptor was 4.97 x 10(-8) M and of N-LDL, 1.74 x 10(-8) M. When assayed with normal 125I-LDL, the capacity of HTG-LDL to down-regulate receptor activity was 46-68% less than N-LDL. Both abnormalities reverted towards normal during treatment. The cellular metabolism of HTG-, BZ- and N-LDL in cells grown for 48 h with the respective lipoproteins was determined. In spite of their defective binding to the receptor, the metabolism of HTG-LDL in the regulated cells was accelerated in comparison to N-LDL, and equal to that of BZ-LDL. That observation is explained by the inefficient ability of HTG-LDL to depress LDL receptor activities in the cells.

Hypolipidemic therapy modulates expression of apolipoprotein B epitopes on low density lipoproteins. Studies in mild to moderate hypertriglyceridemic patients.

Low density lipoproteins (LDL) of untreated moderate to severe hypertriglyceridemic patients (HTG-LDL) are smaller in size and are relatively enriched in triglycerides and proteins compared with normal LDL (N-LDL). HTG-LDL also manifest defective binding to the LDL receptors of normal cultured human fibroblasts. These structural and functional defects are reversible by effective hypolipidemic therapy. The aims of the present study were to confirm the reversibility of the structural and functional defects in mild to moderate hypertriglyceridemic patients and also to test the hypothesis that therapy improved the binding of HTG-LDL to cells by modulating epitopes of apolipoprotein B (apoB-100) on the surfaces of LDL particles. Fasting plasma samples were obtained from five mild to moderate hypertriglyceridemic patients before and 3 weeks after bezafibrate therapy when mean triglyceride levels were 436 and 157 mg/dl (P less than 0.01), respectively. LDL particles were isolated by zonal ultracentrifugation, characterized chemically, and assayed for cell association and proteolytic degradation in-up regulated normal human skin fibroblasts. LDL immunoreactivity was tested in solid phase competitive binding radioimmunoassays (RIA) using three monoclonal antiLDL antibodies (Mab). Mab 464B1B3 and Mab 465B6C3 react against epitopes in the COOH-terminal (T2/K4) fragment of apoB-100. Mab D7.1 reacts with an epitope in the midportion (T3/K3) fragment. Mab 464B1B3 inhibits the binding of LDL to the LDL receptor. Hypolipidemic treatment altered the composition of LDL. Mean LDL triglycerides fell from 9.4 to 5.8% of LDL mass (P less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo conversion of human HDL3 to HDL2 and apoE-rich HDL1 in the rat: effects of lipid transfer protein.

In this study we determined in vivo conversions of human 3H-labeled cholesteryl ester-labeled HDL3 [( 3H]CE-HDL3) in male rats and the effects of partially purified lipid transfer protein on the conversion processes. Zonal centrifugation techniques were used to prepare the [3H]CE-HDL3 and to follow the conversion processes. One hour after the injection, a complete conversion of HDL3 to the HDL2-density species was found. With time, [3H]CE separated with apoE-rich HDL1 and, by 18 hr, 35.9% of plasma radioactivity was associated with the apoE-rich HDL1 lipoprotein fraction. In vitro incubation of [3H]CE-HDL3 in rat plasma reproduced in part the HDL3----HDL2 conversion, but no movement of radioactivity to HDL1 was observed. Injection of the rats with partially purified lipid transfer proteins induced [3H]CE exchange between lipoproteins. The conversion of HDL3 to HDL2, however, was minimally affected. Formation of [3H]CE-HDL1, in contrast, was reduced to about one-half of that found in control animals. It is concluded that in vivo conditions are necessary for conversions of HDL3 (and HDL2) to HDL1, and that lipid transfer reactions delay this process.

Conversion of human plasma high density lipoprotein-2 to high density lipoprotein-3. Roles of neutral lipid exchange and triglyceride lipases.

Cholesterol esters accumulating in human plasma high density lipoproteins (HDL) are important in conversion of HDL3 to larger HDL2. We studied whether mechanisms of removal of cholesterol esters from HDL might be important in a reverse direction, i.e. conversion of HDL2 to HDL3. Native HDL2 or HDL3 is incubated with very low density lipoproteins (VLDL) and lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. After incubation, "modified" (M) VLDL, and HDL2 or HDL3 are reisolated by ultracentrifugation. In modified M-HDL2 or M-HDL3, triglyceride becomes the major core lipid as the triglyceride/cholesterol ester weight ratio increases 8-10-fold relative to native HDL. With only small changes in protein/phospholipid ratios in M-HDLs, the large decrease in cholesterol ester/protein ratios suggest net cholesterol ester loss from HDL. Quantitative recovery analyses prove that the cholesterol esters lost from HDL are transferred to M-VLDL, which is now richer in cholesterol ester and poorer in triglyceride. These substantial exchanges of HDL lipids are not associated by significant transfer of HDL apoproteins but are dependent on neutral lipid transfer factors present in human lipoprotein-poor plasma (d greater than 1.21 g/ml). Similar results are obtained when purified core lipid transfer protein replaces d greater than 1.21 g/ml plasma in these incubations. After depletion of cholesterol ester from HDL, most but not all, exchanged triglyceride can be removed by lipolysis with either hepatic or lipoprotein lipase, resulting in a post-lipolysis HDL2 with an increased triglyceride content relative to normal HDL. With successive incubations with VLDL, and core lipid transfer factors, HDL2 loses more than two-thirds of its cholesterol esters. After lipolysis of acquired triglyceride, HDL2 is remodeled, in both composition and flotation parameters, toward HDL3.

Effect of moderate isocaloric modification of dietary carbohydrate on high-density lipoprotein composition and apolipoprotein A-1 turnover in humans.

Plasma lipid and apolipoprotein (apo) levels were determined in five normolipidemic subjects and five patients with Type IV hypertriglyceridemia (HTG) who were fed for greater than or equal to 6 weeks on two isocaloric diets. The first diet contained carbohydrates (CHO) as 55% of total calories, 29% as fat and 16% as protein. The second diet contained 40% CHO, 45% fat and 15% protein in normolipidemic subjects and 40% CHO, 41% fat and 19% protein in patients with HTG. All diets had a cholesterol content of approximately 400 mg/day and a polyunsaturated/saturated fatty acid ratio of approximately 1:0. Apo A-1 kinetics were measured during the last 2 weeks of each dietary period. The composition and distribution of high-density lipoproteins (HDL), subclasses HDL2 and HDL3, were determined at the end of each dietary term. In the HTG patients, administration of a 40% compared with 55% CHO diet caused a significant decrease of plasma triglyceride levels and an increase of HDL-cholesterol; low-density lipoprotein (LDL) levels increased and very low-density lipoprotein (VLDL) levels decreased (P less than 0.01 and less than 0.07, respectively). Similar quantitative changes of VLDL and HDL levels were found in the normolipidemic subjects. No significant change in plasma levels of apo A-I, A-II and E occurred. Apo A-I kinetic studies revealed decreased synthetic rates and fractional catabolic rates on the low CHO diet. Separation of HDL subfractions by zonal ultracentrifugation in both groups revealed an increase in HDL3-cholesterol ester and protein, and a decrease in HDL2 protein, phospholipid and cholesterol. Our findings indicate that moderate changes in dietary CHO and fat content affect HDL levels, composition and apo A-I metabolism.

Change in very low-, low-, and high-density lipoproteins during lipid lowering (bezafibrate) therapy: studies in type IIA and type IIb hyperlipoproteinaemia.

The effects of lipid lowering therapy (bezafibrate) on plasma lipoproteins was investigated in twelve patients with familial hypercholesterolaemia (type IIA) and eight with familial combined hyperlipidaemia (type IIB). Bezafibrate caused a decrease of plasma cholesterol, plasma triglycerides, plasma apolipoprotein B, VLDL cholesterol and LDL cholesterol and an increase of HDL cholesterol. Post-heparin plasma lipoprotein and hepatic lipase activities increased in both groups (significant only in type IIB). Lipoprotein composition showed the following changes: Increased protein and phospholipids and decreased triglycerides and cholesteryl esters in VLDL. Decreased protein and triglycerides and increased free and esterified cholesterol in LDL. Decreased triglycerides and increased phospholipids in HDL. Cholesteryl ester to protein ratios decreased in VLDL and increased in LDL. The hydrated density of LDL (both groups) and of HDL3 (type IIB) decreased following bezafibrate therapy. These changes were in general similar to those observed in hypertriglyceridaemic patients and could be ascribed, at least in part, to the increase of plasma lipase activities and the decrease of lipid transfer reactions. Comparing the present data with that previously reported, it was found that bezafibrate caused decreased LDL cholesterol in types IIA and IIB but increased levels in type IV. This change was correlated with the initial plasma triglycerides (r = 0.74, P less than 0.0001) and initial plasma LDL cholesterol (r = 0.66, P less than 0.001). It is concluded that varied response of LDL to therapy reflects a complex interaction of metabolic events, including changing rates of VLDL conversion to LDL, lipoprotein compositional changes and effects of therapy on LDL degradation rates.

Defective metabolism of hypertriglyceridemic low density lipoprotein in cultured human skin fibroblasts. Normalization with bezafibrate therapy.

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 less than 0.001). After 6 h preincubation with unlabeled HTG-LDL, the incorporation of [14C]acetate 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 less than 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.

Core modification of human low-density lipoprotein by artificial triacylglycerol emulsion.

To determine whether an apolipoprotein-free artificial triacylglycerol emulsion can substitute for VLDL in studying cholesterol ester-triacylglycerol exchange processes between triacylglycerol-rich lipoproteins and cholesterol ester-rich lipoproteins, we used Intralipid to modify human plasma LDL. Intralipid was incubated with LDL in the presence of lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. Intralipid served as an acceptor for cholesterol ester and as a donor of triacylglycerol, modifying the low-density lipoproteins so that triacylglycerol became the major core lipid in the particle - the contribution of cholesterol ester to LDL mass decreased from 38% to 18%, while that of triacylglycerol increased from 4.9% to 26%. On lipolysis most added LDL triacylglycerol (59-72%) was hydrolyzed, resulting in a smaller particle than the "native' LDL particle with net loss of cholesterol ester. Incubation of LDL with the original Intralipid emulsion resulted in modified LDL with a high relative weight of phospholipid (27.7%). On removal of excess phospholipid from Intralipid and incubation of the resultant "washed' Intralipid with LDL, the relative weight of phospholipid in modified LDL decreased to 20%, which was similar to that observed after incubation of LDL with VLDL. We demonstrate that artificial triacylglycerol emulsion can indeed substitute for VLDL in neutral lipid exchange processes, and further confirm that transfer of core cholesterol ester and triacylglycerol occurs independently of the apolipoproteins present in triacylglycerol-rich lipoproteins and LDL.

Is hypertriglyceridemic very low density lipoprotein a precursor of normal low density lipoprotein?

The precursor-product relationship of very low density (VLDL) and low density lipoproteins (LDL) was studied. VLDL obtained from normal (NTG) and hypertriglyceridemic (HTG) subjects was fractionated by zonal ultracentrifugation and subjected to in vitro lipolysis. The individual subfractions and their isolated lipolysis products, as well as IDL and LDL, were rigorously characterized. A striking difference in the contribution of cholesteryl ester to VLDL is noted. In NTG subfractions, the cholesteryl ester to protein ratio increases with decreasing density (VLDL-I----VLDL-III). This is the expected result of triglyceride loss through lipolysis and cholesteryl ester gain through core-lipid transfer protein action. In HTG subfractions there is an abnormal enrichment of cholesteryl esters that is most marked in VLDL-I and nearly absent in VLDL-III. Thus, the trend of the cholesteryl ester to protein ratios is reversed, being highest in HTG-VLDL-I and lowest in VLDL-III. This is incompatible with the precursor-product relationship described by the VLDL----IDL----LDL cascade. In vitro lipolysis studies support the conclusion that not all HTG-VLDL can be metabolized to LDL. While all NTG subfractions yield products that are LDL-like in size, density, and composition, only HTG-VLDL-III, whose composition is most similar to normal, does so. HTG VLDL-I and VLDL-II products are large and light populations that are highly enriched in cholesteryl ester. We suggest that this abnormal enrichment of HTG-VLDL with cholesteryl ester results from the prolonged action of core-lipid transfer protein on the slowly metabolized VLDL mass. This excess cholesteryl ester load, unaffected by the process of VLDL catabolism, remains entrapped within the abnormal particle. Therefore, lipolysis yields an abnormal, cholesteryl ester-rich product that can never become LDL.