Differences in apolipoproteins and low-density lipoprotein subfractions in postmenopausal women on and off estrogen therapy: results from the Framingham Offspring Study

The use of estrogens by postmenopausal women has been associated with reduced risk of coronary artery disease (CAD) in some studies, possibly due to favorable effects of estrogens on plasma lipoproteins. In order to examine such effects, we studied 180 postmenopausal women from the Framingham Offspring Study, selected by type of menopause (natural or oophorectopic) and estrogen use. We determined fasting plasma total cholesterol, triglyceride, very-low-density lipoprotein (VLDL) cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and apolipoprotein (apo) A-l and B concentrations, as well as LDL particle size (LDL 1 to LDL 6). Apo A-l levels were significantly (P less than .005) higher, and diastolic blood pressure and glucose levels were significantly (P less than .05) lower in postmenopausal women taking estrogen regardless of type of menopause. HDL cholesterol levels were also higher in women taking oral estrogens, but differences were significant only for the oophorectomized group (P less than .02). Total cholesterol, VLDL cholesterol, and LDL cholesterol levels were significantly lower (P less than .01) in women with natural menopause who were taking estrogens than in women with natural menopause not taking this medication. No significant differences between estrogen users and nonusers were found with regard to triglyceride levels or LDL particle score, in either the natural menopause or oophorectomy groups. These data indicate that estrogen use in postmenopausal women is associated with significantly elevated plasma apo A-l levels and decreased LDL cholesterol concentrations.     

Platelet secreted lipoprotein-like particle is taken up by the macrophage scavenger receptor and enhances cellular cholesterol accumulation

Enhanced macrophage cholesterol accumulation is associated with foam cell formation in the atherosclerotic lesion. Since platelet activation plays an important role in atherogenesis, we questioned whether products released from activated platelets could affect macrophage cholesterol metabolism. The addition of platelet-conditioned medium (PCM, obtained from collagen activated platelets) to a J-774 macrophage cell line, enhanced cellular cholesteryl ester content by 32%. The cholesterol esterification rate was also increased by 29%. Pre-loading the macrophages with cholesterol by incubation with acetyl-LDL, resulted in a further elevation of 48% in PCM-mediated cholesterol esterification. Possible mechanisms for the enhanced cholesterol esterification by J-774 macrophages following incubation with PCM include increased cholesterol influx and/or decreased cholesterol efflux (These cells were recently shown not to synthesize cholesterol). However, both increased uptake of PCM cholesterol by the macrophages as well as increased cellular cholesterol efflux (by 22%) were noted. The enhancement of cholesterol esterification by PCM was competitively inhibited by fucoidin and polyinosinic acid, implicating PCM binding to the scavenger receptor. This was further evidenced by the observations that apolipoprotein E which reduces cellular uptake via the scavenger receptor but not via the LDL receptor, also inhibited the effect of PCM, whereas IgG C-7, the LDL receptor antibody, did not alter the effect of PCM. Lysosomal involvement in the cellular processing of PCM was observed since PCM activity was inhibited by the lysosomal inhibitor, chloroquine. Partial purification of PCM by gel filtration revealed that the cholesterol component was associated with both phospholipids and proteins in a lipoprotein-like particle. Delipidation of PCM resulted in its inactivation but both heat treatment and tryptic digestion of PCM, revealed that the protein (and not only the cholesterol) component was also essential for the effect of PCM on cellular cholesterol esterification. Furthermore, PCM prepared from platelets of a patient with Gray Platelet Syndrome that lack platelet alfa granules (which contain platelet specific proteins), failed to enhance cholesterol esterification. These results demonstrate that lipoprotein-like particles released during platelet activation can interact with the macrophage scavenger receptor thus leading to enhanced cellular cholesterol accumulation.     

Effect of simvastatin on receptor-dependent low density lipoprotein catabolism in normocholesterolemic human volunteers

Simvastatin, an inhibitor of HMG-CoA reductase was given to 7 normolipidemic healthy volunteers for 1 month at a dose of 20 mg/day. Measurements of turnover of low density lipoprotein apolipoprotein B (LDL-apo B) were determined before and after drug treatment using intravenous injection of 125I-labeled LDL and 131I-labeled cyclohexanedione-treated LDL to quantify the receptor pathway. In addition to a 13% increase in HDL cholesterol and apolipoprotein A-I concentrations, plasma cholesterol was reduced by 20%, LDL-cholesterol by 32%, and apolipoprotein B by 23%. Assuming a heterogeneous pool of LDL, the new model presented in the companion paper was built to calculate the contribution of the receptor-dependent and the receptor-independent pathways and the corresponding fractional catabolic rates. Simvastatin did not modify constantly the synthetic rate of LDL-apo B but increased the fractional catabolic rate of the receptor-dependent pathway and the contribution of this pathway in the catabolism. The fall in LDL plasma levels observed in normocholesterolemic subjects can be then entirely explained by an enhanced fractional removal of LDL from the circulation by the receptor route.     

Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.

Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.     

Short-term egg yolk feeding in humans. Increase in apolipoprotein B and low density lipoprotein cholesterol.

In animal studies, hypercholesterolemia induced by cholesterol feeding results in the plasma cholesterol being transported by lipoproteins of lower densities. Little information is available for humans. To determine the specific lipoprotein responses to dietary cholesterol challenge in humans, four volunteer subjects ingested a liquid formula diet containing 5000 mg of egg yolk cholesterol per day for 30 days and the changes in their lipoprotein fractions were examined. The high dietary cholesterol (above the range of normal diet) was associated with marked increases in apolipoprotein B and low density lipoprotein (LDL) cholesterol levels. An elevated cholesterol : triglyceride ratio in the LDL fraction indicated that the diet altered both LDL level and composition. High density lipoprotein cholesterol and apolipoprotein AI increased slightly. Very low and intermediate density lipoprotein cholesterol and apolipoprotein E levels did not increase during the diet. Thus, high dietary cholesterol was associated with major changes in LDL level and composition, but only minor changes in the other lipoprotein fractions and suggested only minor accumulation of remnant particles.     

Accumulation and loss of cholesterol esters in monkey arterial smooth muscle cells exposed to normal and hyperlipemic serum lipoproteins.

The effects of high low and very low density lipoprotein fractions from normal or hyperlipemic rhesus monkey serum on the accumulation or removal of cholesterol esters from rhesus monkey smooth muscle cells in tissue culture were determined. Serum or serum lipoproteins were labeled with [14C] free cholesterol and adjusted to the same free cholesterol level in the incubation medium. Of the two normal lipoproteins examined, the LDL fraction caused more esterification than the HDL. Cells incubated in hyperlipemic serum showed a 2-fold stimulation in esterification as compared to cells in normal serum. This was contributed by hyperlipemic VLDL and LDL and led to a concomitant increase in cellular cholesterol ester content. Both hyperlipemic LDL and HDL stimulated esterification when compared to their normal counterparts. Cholesterol ester removal was examined by incubating the serum or lipoprotein fractions with cells enriched in cholesterol ester through a prior exposure to hyperlipemic serum. The cells incubated in normal or hyperlipemic HDL or lipoprotein-deficient serum had the lowest cholesterol ester content. Thus, the lipoprotein fractions which caused the lowest levels of cholesterol esterification were also the most efficient in the removal of cellular cholesterol esters.     

Platelet secretory products enhance LDL receptor activity and inhibit scavenger receptor activity in human monocyte derived macrophages

Macrophage cholesterol accumulation is an early event in atherogenesis. Platelet secretory products have the potential to affect macrophage cholesterol accumulation through their effect on cellular lipoprotein uptake via the low density lipoprotein (LDL) or the scavenger receptor pathways. Preincubation of human monocyte-derived macrophages (HMDM) for 16 hours at 37 degrees C with serotonin, ADP, fibrinogen, fibronectin and platelet-derived growth factor (PDGF), followed by washout of these substances, significantly enhanced LDL uptake by 25% to 75%, whereas acetyl LDL (AcLDL) degradation (AcLDL is taken up by the scavenger receptor), was substantially reduced by 40% to 60% (except for ADP). The effect of serotonin (0 to 75 mumol/L) on macrophage interaction with lipoproteins was further analyzed and revealed a dose-dependent effect on both stimulation of macrophage LDL degradation and cholesterol esterification by up to 2.5 times, as well as an inhibition of the cellular uptake of AcLDL by up to 1.5 times. Analysis of the regulatory effect of serotonin on macrophage lipoprotein uptake revealed that the main effect of serotonin on the uptake of both lipoproteins was to change the affinity of the lipoproteins toward their specific receptor without a significant effect on the number of binding sites. The next questions addressed are whether substances that are known to be secreted by activated platelets can also modify LDL, and whether this modification can alter the interaction between LDL and macrophages. LDL treated with all of the studied substances demonstrated significantly enhanced cellular degradation compared with untreated LDL. The data thus demonstrate that substances such as those released from activated platelets can selectively affect macrophage LDL and scavenger receptor activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Management of lipoprotein-X accumulation in severe cholestasis by semi-selective LDL-apheresis

Liver disorders characterized by prolonged bile stasis are often associated with the accumulation of an abnormal lipoprotein, lipoprotein-X (LP-X), in plasma. LP-X is separated in the low-density lipoprotein (LDL) density range, but lacks apolipoprotein B and does not interact with the LDL receptor; LP-X can cause hyperlipidemia, cutaneous xanthomas, and worsening of arterial disease. We report the case of a patient with severe cholestasis, markedly elevated plasma cholesterol levels (26.8 to 31.5 mmol/L), mainly due to a massive accumulation of LP-X in plasma, and diffuse xanthomas. To reduce the elevated cholesterol levels, the patient was given extracorporeal treatment aimed at removing atherogenic lipoprotein (LDL-apheresis). LDL-apheresis was performed at weekly or bi-weekly intervals, either by a semi-selective technique using filters with a defined pore diameter (double filtration, DF) or by a more selective technique using dextran-sulfate-cellulose (DSC) columns able to bind LDL. The semi-selective DF technique proved more effective than DSC, removing 48% of total cholesterol (compared to 30% with DSC), and lowering cholesterol levels to 11.1 mmol/L in 6 weeks. DF removed both LDL and LP-X from plasma, whereas DSC selectively decreased the LDL content. The reduction of plasma cholesterol levels was associated with a complete regression of the xanthomas, supporting DF apheresis as a first-choice treatment for patients with massive LP-X accumulation due to cholestasis.     

Apolipoprotein B gene mutations affecting cholesterol levels.

In the past 5 years, many different mutations in the apolipoprotein (apo) B gene have been described that affect plasma cholesterol levels. More than 20 different mutations in the apoB gene have been shown to cause familial hypobetalipoproteinaemia, a condition characterized by abnormally low plasma concentrations of apoB and LDL cholesterol. Almost all of the mutations are nonsense or frameshift mutations that interfere with the translation of a full-length apoB100 molecule. Many, but not all, of these apoB gene mutations result in the synthesis of a truncated species of apoB that can be detected within the plasma lipoproteins. Familial hypobetalipoproteinaemia heterozygotes are almost always asymptomatic and have LDL cholesterol levels about one-quarter to one-third of those of unaffected family members. Several homozygotes and compound heterozygotes for familial hypobetalipoproteinaemia have been described. In these individuals, the LDL cholesterol levels are extremely low, usually less than 5 or 10 mg dl-1, and the clinical phenotype is variable, ranging from completely asymptomatic to severe problems related to intestinal fat malabsorption. One missense mutation in the apoB gene (an Arg----Gln substitution at apoB amino acid 3500) is associated with very poor binding of apoB100 to the cellular LDL receptor. This syndrome has been designated familial defective apolipoprotein B (FDB). The amino-acid substitution at residue 3500 delays the clearance of LDL from the plasma and results in hypercholesterolaemia. In some Western populations, the frequency of FDB heterozygotes appears to be as high as 1 in 500 individuals.     

LDL concentration is correlated with the removal from the plasma of a chylomicron-like emulsion in subjects with coronary artery disease

In animal model studies, the uptake of chylomicron remnants after entering in the space of Disse occurs mainly by low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP). In subjects, the relative importance of each one of these receptors for the clearance of chylomicron remnants is not fully understood. In our study, LDL cholesterol and apolipoprotein (apo) B were correlated to the plasma kinetics of a chylomicron-like emulsion in 77 subjects (11 women, mean age 58 +/- 12 years) with coronary artery disease (CAD). Their total cholesterol was 227 +/- 25 mg/dl, triglyceride 159 +/- 25 mg/dl, LDL cholesterol 148 +/- 27 mg/dl, HDL cholesterol 40 +/- 9 mg/dl, apo A1 1.80 +/- 0.53 g/l and apo B 1.65 +/- 0.48 g/l. The emulsion was double-labeled with 3H-triolein and 14C-cholesteryl oleate and injected intravenously after 12-h fasting. The decay curves of the radioisotopes were determined from blood samples collected at predetermined intervals during 60 min. A negative correlation between FCR of the emulsion cholesterol esters and LDL cholesterol and apo B plasma concentrations was found (r=-0.4, P=0.005 and r=-0.3, P=0.01, respectively) whereas FCR of the emulsion triglycerides did not correlate with any of the plasma lipids or apolipoprotein parameters. Concluding, in patients with CAD, LDL catabolic pathway significantly influences the removal from plasma of chylomicron remnants.     

Lipid-protein particles secreted from activated platelets reduce macrophage uptake of low density lipoprotein.

Cellular uptake of low density lipoprotein (LDL) was reduced by 30-40% in macrophages that were preincubated with platelet conditioned medium (PCM) obtained from activated platelets. LDL mediated cholesterol accumulation and cholesterol esterification in macrophages were substantially inhibited by macrophages preincubation with PCM. This inhibitory effect was found to be dose dependent, and resulted from a reduction in the number of LDL receptors (decrement of 35% in "apparent Vmax"). The active component in PCM was present only in medium obtained from activated platelets and was found to be of a molecular weight higher than 25,000 dalton. It comprised of both protein and cholesterol but upon PCM delipidation only the lipid fraction demonstrated the inhibitory effect on macrophage uptake of LDL. Specific uptake of the PCM lipoprotein-like particle via the scavenger receptor on macrophages was found to be essential for the expression of LDL receptor reduced activity. Furthermore, LDL mediated cholesterol esterification was not inhibited by PCM in U937 macrophages, a cell line that lacks the scavenger receptors. It is concluded that activated platelets secrete a lipoprotein-like particle which is recognized by the macrophage scavenger receptor. Subsequent to PCM-macrophage interaction, cellular LDL uptake was reduced. This effect could be attributed to the PCM lipid constituents.     

Synergistic upregulation of low-density lipoprotein receptor activity by tamoxifen and lovastatin

OBJECTIVE: To study the mechanism involved in the cholesterol-lowering activity of tamoxifen, an estrogen receptor (ER) modulator widely used in breast cancer therapy. METHODS AND RESULTS: We used MOLT-4 cells, which do not express estrogen receptors and require important amounts of cholesterol for proliferation. We firstly confirmed that tamoxifen reduced cholesterol biosynthesis by inhibiting sterol Delta(8,7)-isomerase and Delta(24)-reductase activities, which resulted in the accumulation of zymosterol. In cells incubated in the presence of low-density lipoprotein (LDL) (120 microg cholesterol/ml), tamoxifen stimulated LDL receptor activity and expression in a dose-dependent manner, as determined by 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-labeled LDL uptake, LDL receptor expression on the cell surface and LDL receptor mRNA levels. Furthermore, tamoxifen, but not lovastatin, inhibited the egress of LDL-derived cholesterol from lysosomes, as ascertained by filipin staining in both MOLT-4 and HepG2 cells. When studied in combination, especially at relatively high LDL concentrations in the medium, tamoxifen and lovastatin stimulated LDL receptor activity synergistically, which is attributed to the different mechanism of action these drugs exhibit. CONCLUSIONS: The present study demonstrates the stimulation of the LDL receptor by tamoxifen. These results explain the long-known hypolipidemic effect of tamoxifen and support its use, or that of other intracellular cholesterol trafficking inhibitors, in combination with statins for the reduction of plasma LDL cholesterol levels.     

Cholesterol flux in cholesterol ester-loaded macrophages in an in vitro perfusion system

Cholesterol ester-loaded J774 macrophages attached to microcarrier beads were perfused or incubated with lipoproteins in vitro. Cholesterol influx was reduced by decreasing LDL cholesterol, efflux was promoted by increasing HDL cholesterol or by adding apolipoprotein A-I/phosphatidylcholine complexes to the perfusate or incubation medium. Addition of sera obtained from patients after LDL apheresis or plasma exchange resulted in much smaller increments in cell cholesterol than pretreatment sera, due to decreased influx, but efflux was unchanged despite the reduction in HDL cholesterol by plasma exchange. These data suggest that extracorporeal cholesterol removal promotes mobilization of intracellular cholesterol ester mainly by reducing cholesterol influx.     

Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice with attenuated expression of scavenger receptor B1

Scavenger receptor BI (SR-BI) is a multiligand cell-surface receptor that plays a central role in high density lipoprotein homeostasis in rodents. To investigate a role for SR-BI in atherosclerosis, mice with attenuated SR-BI expression were crossed with low density lipoprotein (LDL) receptor-deficient mice. Compound-homozygous mutants showed increased plasma cholesterol, surprisingly due primarily to increased LDL cholesterol and apolipoprotein B levels. LDL turnover studies showed that this resulted from increased LDL cholesterol production rather than decreased LDL catabolism. Atherosclerotic lesion size was significantly increased in male compound-mutant mice relative to LDL receptor-deficient controls (93 427+/-16 079 versus 34 448+/-5 331 microm(2), respectively; P=0.003). The proatherogenic effect of attenuated SR-BI expression may in part be due to increased LDL cholesterol levels. These findings suggest that upregulation of the receptor could have therapeutic potential for the treatment of atherosclerosis.     

Autosomal recessive hypercholesterolemia in three sisters with phenotypic homozygous familial hypercholesterolemia: diagnostic and therapeutic procedures

Familial hypercholesterolemia is an autosomal-dominant inherited disorder caused by mutations in the low-density lipoprotein (LDL) receptor gene. The homozygous form is characterized by high-serum LDL cholesterol concentrations, xanthoma formation and premature atherosclerosis. Recently, another molecular defect that also results in severely elevated LDL cholesterol levels was identified: autosomal recessive hypercholesterolemia. This inherited disorder is caused by a mutation in a putative LDL receptor adaptor protein. In our lipid clinic, three sisters with phenotypic homozygous hypercholesterolemia were recently diagnosed as having autosomal recessive hypercholesterolemia. They presented in 1990 with massive tuberous xanthomas at the knees, thighs, elbows and buttocks. LDL receptor and apolipoprotein B gene defects were excluded through mutation analysis. From 1992 onward they underwent LDL-apheresis on a weekly basis. To date the clinical outcome is very satisfying with no evidence of coronary heart disease or aortic valve lesions and almost complete regression of xanthomatosis.     

Receptor-mediated endocytosis: insights from the lipoprotein receptor system.

The low density lipoprotein (LDL) receptor system coordinates the metabolism of cholesterol, an essential component of the plasma membrane of all mammalian cells. Study of this system has led to an enhanced understanding of the cellular basis of cholesterol homeostasis. It has also brought into focus an important mechanism of metabolic regulation--the process of receptor-mediated endocytosis. In this article, we first describe the receptor-mediated endocytosis of LDL, a sequence of events in which receptor binding and internalization are coupled in specialized regions of the plasma membrane called coated pits. Second, we trace the cellular functions of the cholesterol derived from internalized LDL. Third, genetic evidence is presented to indicate that both the binding and internalization of LDL are mediated by a single receptor molecule that contains two active sites, one mediating binding and the other internalization. Finally, the characteristics of the LDL receptor system are used to suggest models for receptor systems in general.     

Plasma lipoprotein(a) concentration in familial hypercholesterolemic patients without coronary artery disease.

Familial Hypercholesterolemia (FH) is a condition characterized by markedly elevated blood cholesterol, low-density lipoproteins (LDL), and apolipoprotein B-100 (apo B). The molecular basis of this monogenic disease is the defective functioning of the cellular receptor for LDL that recognizes apo B. Lipoprotein(a) [Lp(a)] is a circulating lipoprotein that is structurally related to LDL, as it also contains apo B. To assess the impact of the LDL receptor deficiency on the plasma Lp(a) concentration, we measured Lp(a) in 28 FH patients and in 31 unaffected relatives. Because elevation of Lp(a) concentration in plasma of patients with coronary artery disease (CAD) appears to occur independently from plasma cholesterol levels, to avoid potentially confounding problems, members of the families chosen had no history for the disease. Whereas apo B clearly showed a bimodality of distribution by being significantly higher in the FH patients (166 +/- 38 mg/dL) than in the unaffected relatives (92 +/- 18 mg/dL), Lp(a) concentration did not differ in the two groups of patients (30 +/- 24 mg/dL in the FH patients v 31 +/- 23 in the normolipidemic relatives). Similar results were obtained when only siblings were further considered. We conclude that although Lp(a) is closely related to LDL structurally, its level in plasma is not significantly affected by the LDL receptor activity.     

Differences in [14C]glycerol utilization in normal and familial hypercholesterolemic fibroblasts

It is known that cultured fibroblasts from familial hypercholesterolemia (FH) patients lack the normal cell receptor for low density lipoprotein (LDL) and that the absence of receptor-mediated transport of LDL cholesterol into these cells results in increased cellular synthesis of cholesterol. After 20 h perincubation in lipid-free medium, cultured FH fibroblasts incorporated significantly greater amounts of [14C]glycerol into cellular lipids than did normal fibroblasts. Relative to the control medium which contained only bovine serum albumin (BSA), preincubation with 5% fetal bovine serum or 50 micrograms LDL/ml decreased [14C]glycerol incorporation by both cell types. FH cells utilized more [14C]glycerol for phospholipid synthesis and less for triglyceride synthesis than normal cells. This study indicates that LDL may be important in the transport of glycerides, as well as cholesterol, to cells.     

Comparisons of apolipoprotein B levels estimated by immunoassay, nuclear magnetic resonance, vertical auto profile, and non-high-density lipoprotein cholesterol in subjects with hypertriglyceridemia (SAFARI Trial)

Low-density lipoprotein (LDL) cholesterol and triglyceride-rich lipoproteins constitute non-high-density lipoprotein (non-HDL) cholesterol. These are atherogenic lipoproteins and non-HDL cholesterol is a secondary target of treatment beyond LDL cholesterol in patients with hypertriglyceridemia. Some investigators favor total apolipoprotein B over non-HDL cholesterol as the secondary target of treatment. This is based on publications suggesting that total apolipoprotein B is more predictive of cardiovascular events than non-HDL cholesterol. Several methods are available for estimating total apolipoprotein B. This study compared total apolipoprotein estimated by immunonephelometric assay (INA), vertical auto profile (VAP), nuclear magnetic resonance (NMR), and non-HDL cholesterol levels in patients with hypertriglyceridemia from the previously reported Simvastatin plus Fenofibrate for Combined Hyperlipidemia (SAFARI) trial. Total apolipoprotein B levels were found to be highest by INA, intermediate by NMR and non-HDL cholesterol, and lowest by VAP. Concordance for non-HDL cholesterol levels among the INA, VAP, and NMR methods was better than that for total apolipoprotein B levels; the correlation between non-HDL cholesterol and apolipoprotein B by INA was strongest (0.929). In patients with a low triglyceride/HDL cholesterol ratio (<3.5), total apolipoprotein B determined by INA was higher than that estimated from non-HDL cholesterol levels, whereas in patients with a high triglyceride/HDL C ratio (≥3.5), apolipoprotein B predicted using non-HDL cholesterol was in better agreement with INA-determined apolipoprotein B levels. Similar trends were observed with VAP using equations specific for LDL particle size. In conclusion, more work is needed to improve agreement of apolipoprotein B measurements among methods employed clinically. Non-HDL cholesterol is also useful to predict total apolipoprotein B and some improvement may be attained by taking into account the ratio of triglyceride/HDL cholesterol as a measurement of LDL particle size.