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Apolipoprotein B (apoB) metabolism was investi- gated in 20 men with plasma triglyceride 0.66-2.40 mmol/l and plasma cholesterol 3.95-6.95 mmol/l. Kinetics of VLDL 1 (S f 60-400), VLDL 2 (S f 20-60), IDL (S f 12-20), and LDL (S f 0-12) apoB were analyzed using a trideuterated leucine tracer and a multicompartmental model which al- lowed input into each fraction. VLDL 1 apoB production varied widely (from 5.4 to 26.6 mg/kg/d) as did VLDL 2 apoB production (from 0.18 to 8.4 mg/kg/d) but the two were not correlated. IDL plus LDL apoB direct production accounted for up to half of total apoB production and was inversely related to plasma triglyceride ( r 5 2 0.54, P 5 0.009). Percent of direct apoB production into the IDL/ LDL density range ( r 5 0.50, P , 0.02) was positively re- lated to the LDL apoB fractional catabolic rate (FCR). Plasma triglyceride in these subjects was determined princi- pally by VLDL 1 and VLDL 2 apoB fractional transfer rates (FTR), i.e., lipolysis. IDL apoB concentration was regulated mainly by the IDL to LDL FTR ( r 5 2 0.71, P , 0.0001). LDL apoB concentration correlated with VLDL 2 apoB pro- duction ( r 5 0.48, P 5 0.018) and the LDL FCR ( r 5 2 0.77, P , 0.001) but not with VLDL 1 , IDL, or LDL apoB produc- tion. Subjects with predominantly small, dense LDL (pat- tern B) had lower VLDL 1 and VLDL 2 apoB FTRs, higher VLDL 2 apoB production, and a lower LDL apoB FCR than those with large LDL (pattern A). Thus, the metabolic conditions that favored appearance of small, dense LDL were diminished lipolysis of VLDL, resulting in a raised plasma triglyceride above the putative threshold of 1.5 mmol/l, and a prolonged residence time for LDL. This lat- ter condition presumably permitted sufficient time for the processes of lipid exchange and lipolysis to generate small LDL particles. —Packard, C. J., T. Demant, J. P. Stewart, D. Bedford, M. J. Caslake, G. Schwertfeger, A. Bedynek, J. Shepherd, and D. Seidel. Apolipoprotein B metabolism and the distribution of VLDL and LDL subfractions. J. Lipid Res. 2000. 41: 305-317.

The St. Thomas' mixed hyperlipidemic (SMHL) rabbit (previously St. Thomas' Hospital rabbit) is a putative model of familial combined hyperlipidemia (FCH). When fed a low (0.08%) cholesterol diet, it exhibits elevations in both plasma cholesterol and triglyceride compared to New Zealand White (NZW) controls. To determine the mecha- nism for this hyperlipidemia we studied the secretion of apolipoprotein B (apoB)-containing lipoproteins from per- fused livers of both young and mature rabbits. During a 3-h perfusion we measured the total cholesterol and triglycer- ide content of the medium and the cholesterol, triglyceride, and apoB content of very low density lipoprotein (VLDL) 1 (S f 60-400), VLDL 2 (S f 20-60), intermediate (S f 12-20), and low (S f 0-12) density lipoproteins (IDL, LDL). Lipo- protein concentrations increased linearly throughout the perfusion period. The rate of cholesterol output was 3-fold higher (459 vs. 137 ng/g liver/min, P 5 0.003) in SMHL versus NZW rabbits whilst that of triglyceride was similar (841 vs. 662 ng/g liver/min, NS). VLDL 1 cholesterol output was elevated 2-fold (232 vs. 123 ng/g liver/min, P , 0.05) and VLDL 2 1 IDL 1 LDL cholesterol output, 4.5-fold (106 vs. 23 ng/g liver/min, P , 0.005) in SMHL versus NZW rab- bits. ApoB output in VLDL1 was 38 ng/g liver per min in SMHL and 14 ng/g liver per min in NZW (NS). In SMHL VLDL 2 1 IDL 1 LDL apoB was increased 9-fold at 53 ver- sus 6 ng/g liver per min in NZW ( P , 0.001). We con- clude that the SMHL rabbit overproduces apoB-containing lipoproteins particularly in the VLDL 2 1 IDL 1 LDL frac- tion, a characteristic consistent with its use as a model of FCH. —Ardern, H. A., G. M. Benson, K. E. Suckling, M. J. Caslake, J. Shepherd, and C. J. Packard. Apolipoprotein B overproduction by the perfused liver of the St. Thomas' mixed hyperlipidemic (SMHL) rabbit. J. Lipid Res. 1999. 40: 2234-2243.

A multicompartmental model has been devised to explain apolipoprotein B (apoB) kinetics in very low density lipoprotein subfractions (VLDL1 Sf 60-400 and VLDL2 Sf 20-60), intermediate density (IDL Sf 12-20) and low density lipoproteins (LDL Sf 0-12). Normal and hyperlipemic subjects were given tracer doses of 131I-labeled VLDL1 and 125I-labeled VLDL2 and the metabolism of apoB in VLDL1, VLDL2, IDL, and LDL was followed over a period of 13 days. VLDL1 apoB and VLDL2 apoB clearance curves had an initial shoulder, a rapid decay, and a 'tail' of slowly metabolized lipoprotein. ApoB derived from VLDL1 appeared in IDL over 10-50 h and exhibited bi-exponential decay that was attributed to the presence of two metabolically distinct species. A further compartment was required to explain the observation that a substantial proportion of apoB from VLDL2 appeared and disappeared from the IDL density range faster than apoB derived from VLDL1 delipidation. Both of the more rapidly removed IDL species gave rise to LDL apoB that was also modeled as a heterogeneous entity with two plasma compartments. The final model, which has much in common with previous versions (M. Berman et al. 1978. J. Lipid Res. 19: 38-56), a multi-step delipidation pathway and slowly metabolized remnant compartments in VLDL, incorporates parallel delipidation routes in VLDL2, IDL, and LDL. These parallel pathways linked kinetic heterogeneity in VLDL with that in IDL and LDL.

An analysis of apolipoprotein (apo) B turnovers conducted in subjects with moderate hypercholesterolemia was performed to discover relationships that may exist between apoB kinetic parameters and plasma lipid and lipoprotein levels. A group of 21 subjects with plasma cholesterol in the range 250-300 mg/dl and triglyceride < 265 mg/dl were injected with tracers of 131I-labeled very low density lipoprotein 1 (VLDL1, Sf 60-400) and 125I-labeled VLDL2 (Sf 20-60) prepared by cumulative flotation ultracentrifugation. The metabolism of apoB in these fractions was followed through intermediate density (IDL, Sf 12-20) to low density (LDL, Sf 0-12) lipoprotein. The most consistent feature giving rise to the higher apoB levels that occurred in VLDL2, IDL, and LDL in the hypercholesterolemic group was increased input of VLDL2 (787 +/- 607 (SD) mg/day vs. 349 +/- 213 in normals, P < 0.01). VLDL1 apoB input was variably affected and not significantly different from normal. However, the plasma residence time of this subfraction was increased (0.15 +/- 0.07 days vs. 0.08 +/- 0.03 days in normals, (P < 0.001) due to a decreased fractional rate of direct catabolism. Fractional transfer rates (FTR) down the delipidation cascade and other fractional rates of direct catabolism were, overall, not significantly different from normal. The plasma residence time of VLDL2 apoB and LDL apoB was similar in hypercholesterolemic and normal subjects, while that of IDL apoB was slightly increased. Variation in LDL apoB mass within the hypercholesterolemic group correlated with VLDL1 apoB input (r = 0.58, P = 0.006), the fractional rate of transfer from IDL to LDL (r = 0.61, P = 0.003), and direct LDL input (r = 0.64, P = 0.002). The proportion of LDL apoB mass derived by direct, i.e., VLDL-independent input, varied from 5 to 50% and was inversely correlated with plasma triglyceride (r = -0.53, P = 0.014) and positively with HDL2 (r = 0.66, P = 0.002). In addition, the amount of direct LDL input was related to the amount of VLDL1 removed by direct catabolism (r = 0.53, P = 0.013). The analysis indicated that moderate hypercholesterolemia arose principally from overproduction of small VLDL, while variation in VLDL1 input and the IDL to LDL conversion rate (presumably hepatic lipase-mediated) modulated the extent of the elevation in LDL apoB.

This study was designed to investigate the response of Type III hyperlipoproteinemic subjects to bezafibrate therapy. The metabolism of apolipoprotein B was examined in four lipoprotein subclasses of Sf 60-400 (large very low density lipoprotein (VLDL)), Sf 20-60 (small VLDL), Sf 12-20 (intermediate density lipoprotein (IDL)), and Sf 0-12 (low density lipoprotein (LDL)) before and during bezafibrate therapy. Treatment reduced the plasma concentration of VLDL and raised high density lipoprotein (HDL) cholesterol. There was no net change in LDL cholesterol or its associated apolipoprotein B. The decrease in plasma VLDL derived mainly from an inhibition of synthesis of both large and small subfractions which reduced the number of particles in the circulation without normalizing their lipid composition. Catabolism of the larger VLDL also increased, presumably as a result of lipoprotein lipase activation. Although the plasma concentration of LDL was unchanged, both its synthesis and catabolism were perturbed. Its fractional catabolic rate fell by 50%, but the impact that this would have had on its steady state level in the circulation was apparently blunted by a decrease in its synthesis from Sf 12-20 IDL. In the control phase of the study, most IDL apolipoprotein B was converted to LDL. Bezafibrate therapy channelled this material towards direct catabolism.

Two methods are compared for measuring the kinetic parameters of apolipoprotein A-I and A-II metabolism in human plasma. In the first, high density lipoprotein apoproteins were radioiodinated in situ in the lipoprotein particle (endogenous apoprotein labeling) while in the second, individually labeled apolipoprotein A-I or A-II was incorporated into the particle by in vitro incubation (exogenous apoprotein labeling). The catabolic clearance rate of exogenously labeled apolipoprotein A-I was consistently faster than that of endogenous apolipoprotein A-I. Conversely, endogenously and exogenously labeled apolipoprotein A-II were catabolized at identical rates. The fractional plasma clearance rates of endogenous apolipoproteins A-I and A-II were the same.

This study examined the effects of dietary saturated and polyunsaturated fat on the chemical composition and metabolism of low density lipoproteins (LDL) in eight normal male subjects. The influence of these diets on fecal sterol excretion was also measured in four of the subjects. When compared with the saturated fat diet, the polyunsaturated diet lowered both plasma cholesterol polyunsaturated diet lowered both plasma cholesterol (23%, P less than 0.001) and triglyceride (14%, P less than 0.001) levels. Sixty-seven percent of the reduction in the former lipid resulted from a fall in LDL cholesterol (23%, P less than 0.001), although very low density (VLDL) and high density lipoprotein (HDL) cholesterol levels also fell (by 27% and 20% of their respective control value). These changes were accompanied by significant alterations in LDL composition. Specifically, during polyunsaturated fat feeding, the relative percentage cholesterol in the LDL fraction fell while that of phospholipid rose. There was no change in the percentage protein or triglyceride. The fatty acid components of LDL triglyceride, cholesteryl esters, and phospholipid were also affected by dietary fat saturation level. Overall, polyunsaturated fat feeding produced an enrichment in linoleate with reciprocal changes in palmitate, stearate, and oleate which affected triglycerides more than cholesteryl esters and phospholipids. The above changes in LDL composition were associated with alterations in the metabolism of LDL apoprotein (apoLDL). The polyunsaturated deit lowered plasma apoLDL by 13% (P less than 0.05). This resulted from an increase in the fractional catabolic rate of LDL (whether determined by plasma decay curve analysis (P less than 0.05) or urine/plasma radioactivity ratios (P less than 0.001) without significant alteration of its corporeal distribution or synthetic rate. The polyunsaturated fat diet did not cause a consistent change in fecal neutral or acidic steroid excretion. We conclude that the hypocholesterolemic action of polyunsaturated fat diets is effected by multiple mechanisms whose expression may vary from patient to patient.

Binding of human low density lipoproteins (LDL) to their specific receptor on cultured cells can be inhibited by treatment with 1,2-cyclohexanedione which blocks a number of functionally significant arginyl residues on the apolipoprotein. We have used this observation to examine the role of the receptor pathway in LDL catabolism in man. The plasma clearance rates of 125I-LDL and 131I-cyclohexanedione-treated LDL were measured in four normal and four heterozygous familial hypercholesterolemic subjects. Chemical modification of the lipoprotein significantly reduced its fractional clearance rate and permitted calculation of receptor-mediated and receptor independent catabolism in both groups. The normal subjects cleared 11% of their plasma LDL pool (corresponding to 3.0 mg/kg per day) by a receptor-independent path. In tared daily by these pathways, respectively. Because the mean apoLDL pool size in the group was increased 3-fold over normal, this gave absolute clearance rates for the apoprotein of 2.5 mg/kg per day via the receptors and 12.8 mg/kg per day by the nonreceptor pathway. We conclude that the specific LDL receptor mechanism operates in vivo and probably accounts for 33% and 16% of overall LDL catabolism in normal and heterozygous familial hypercholesterolemic subjects, respectively.

The utility of 1,2 cyclohexanedione-modified low density lipoprotein (CHD-LDL) as a marker for the measurement of receptor independent LDL catabolism has been assessed by examining its metabolic properties in cultured human fibroblasts and in rabbits. Cell culture studies showed that the inhibition of high affinity membrane receptor binding produced by the modification could be partially reversed by prolonged incubation of the CHD-LDL at 37 degrees C. Pre-exposure of the complex to alkaline pH (pH 10.5) prevented this and yielded a product that was apparently stable. Despite its regained ability to bind to the fibroblast receptor, 125I-labeled CHD-LDL incubated at 37 degrees C for 24 hr either in vivo or in vitro was removed from rabbit plasma in the same manner as freshly prepared 131I-labeled CHD-LDL and as 131I-labeled CHD-LDL that had been treated at pH 10.5. However, its plasma clearance was significantly faster than that of reductively methylated LDL. We believe that this may result from differential catabolism of these modified lipoproteins rather than from susceptibility of the CHD-LDL to receptor-directed catabolism.

This study examines the effects of probucol (1 g/ day) on the plasma concentration, composition, and metabo- lism of low and high density lipoproteins (LDL and HDL) in eleven hyperlipidemic subjects, (seven Type I1 and four Type IV). The drug lowered plasma cholesterol in the Type I1 pa- tients by 11% (P < 0.02) without affecting triglyceride. Both LDL and HDL cholesterol levels fell by 6% and 2696, respec- tively. The small reduction in the former was not associated with a change in the composition of the lipoprotein nor with a measurable alteration in the level of circulating apoLDL. Kinetic studies revealed that probucol had no consistent effect on either the synthesis or catabolism of apoLDL. However, probucol did exert a potent influence on HDL, lowering the level of this lipoprotein in both the Type I1 and Type IV patients despite the fact that total plasma cholesterol in the latter group was unchanged by treatment. The fall in HDL mass largely affected the HDLs subfraction; HDLp, which was initially low in our subjects, did not show a consistent response to therapy. Not all of the constituents in HDL were equally affected by the drug. Specifically, the fall in total plasma apoA levels (which derived from significant reductions in the rates of synthesis of apoproteins A-I and A-11) was less than that of HDL cholesterol. Direct measurement of the composition of the lipoprotein confirmed that during therapy it carried less cholesterol per unit protein. The significance of these obser- vations in relation to the prophylaxis of ischemic heart disease is not yet clear, but it seems prudent at present to use probucol selectively in subjects who show a substantial hypocholester- olemic response that derives primarily from a reduction in circulating LDL.-Atmeh, R. F., J. M. Stewart, D. E. Boag, C. J. Packard, A. R. Lorimer, and J. Shepherd. The hypo- lipidemic action of probucol: a study of its effects on high and low density 1ipoproteins.J. Lipid Res. 1983. 24: 588-595.

This study was designed to identify a method for the measurement of human high density lipoprotein subfraction (HDL2 and HDL3) metabolism. Apolipoproteins A-I, A-II, and C, the major HDL apoproteins, were radioiodinated and incorporated individually into HDL2 and HDL3 in vitro. Using a double label technique, the turnover of apoA-I in HDL2 and HDL3 was measured simultaneously in a normal male. The apoprotein exchanged rapidly between the two subfractions, evidenced by equilibration of their apoA-I specific activity. Radiolabeled apoA-II, incorporated into the subfractions, showed a similar exchange in vitro. Incubation of 131I-labeled very low density lipoproteins (VLDL) with HDL or its subfractions resulted in transfer of C proteins from VLDL to the HDL moiety. The extent of transfer was dependent on the HDL subfraction present; 50% of the VLDL apoC was transferred to HDL3, while the transfer to total HDL and HDL2 was 69% and 78%, respectively. ApoC also exchanged between HDL2 and HDL3, again showing a preference for the former and suggesting a primary metabolic relationship between VLDL and HDL2. Overall, the study indicates that apoA-I, apoA-II, and the C proteins exist in equilibrium between HDL2 and HDL3. This phenomenon precludes their use as probes for HDL subfraction metabolism in humans.

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein.

Hepatic lipase deficiency produces significant distortion in the plasma lipoprotein profile. Particles with reduced electrophoretic mobility appear in very low density lipoprotein (VLDL). Intermediate density lipoprotein (IDL) increases markedly in the circulation and plasma low density lipoprotein (LDL) levels fall. At the same time there is a mass redistribution within the high density lipoprotein (HDL) spectrum leading to dominance in the less dense HDL2 subfraction. The present study examines apolipoprotein B turnover in a patient with hepatic lipase deficiency. The metabolism of large and small very low density lipoproteins was determined in four control subjects and compared to the pattern seen in the patient. Absence of the enzyme did not affect the rate at which large very low density lipoproteins were converted to smaller particles within this density interval (i.e., of VLDL). However, subsequent transfer of small very low density lipoproteins to intermediate density particles was retarded by 50%, explaining the abnormal accumulation of VLDL in the patient's plasma. Despite this, intermediate density particles accumulated to a level 2.4-times normal because their subsequent conversion to low density lipoprotein has been almost totally inhibited. Consequently, the plasma concentration of low density lipoprotein was only 10% of normal. On the basis of these observations, hepatic lipase appears to be essential for the conversion of small very low density and intermediate density particles to low density lipoproteins. The pathways of direct plasma catabolism of these species were not affected by the enzyme defect. In vitro studies were performed by adding purified hepatic lipase to the patient's plasma.(ABSTRACT TRUNCATED AT 250 WORDS)