Apoliprotein B (107 mg/dl):  This is the protein cap that each LDL particle wears.  By counting these, you get a precise measure of the LDL particles in the bloodstream, a truer indication of your genetic predisposition to heart disease.  These particles may damage your arteries and cause blockages, so it helps to know how many you’ve got.  This is based on the following studies:

"Effects of apolipoprotein and low density lipoprotein receptor gene polymorphisms on lipid metabolism, and the lipid risk factors of coronary artery disease."

Apo B exists in human plasma as two isoforms, apo B-48 and apo B-100. Apo B-100 is the major physiological ligand for the LDL receptor. It is the largest monomeric protein sequenced so far, containing 4536 amino acid residues (Chen et al. 1986, Law et al. 1986). Its gene has been mapped on the short arm of chromosome 2, with an approximate length of 43 kilobases and 29 exons
(Ludwig et al. 1987). The LDL-binding domain of the molecule is proposed to be located between the residues 3129 and 3532 (Knott et al. 1986). Apo B-100 is synthesised in the liver and is required for the assembly of very low density lipoproteins (VLDL). It does not interchange between lipoprotein particles, as do the other lipoproteins, and it is found in IDL and LDL particles after the removal of the apolipoproteins A, E and C (Young 1990).

Apo B-48 is present in chylomicrons and chylomicron remnants and plays an essential role in the intestinal absorption of dietary fats (Kane 1983). Apo B-48 is synthesised in the small intestine. It comprises the N-terminal 48% of apo B-100 and is produced due to posttransscriptional apo B-100 mRNA editing at codon 2153, which creates a stop codon in the intestine instead of a glutamine in the liver (Chen et al. 1987).

Mutations occurring in the apo B gene can alter blood cholesterol levels. Most of the mutations lower blood cholesterol levels due to the production of truncated apo B. The mechanisms by which blood cholesterol is lowered are not yet fully understood. Two mutations in the apo B gene have been associated with elevated blood cholesterol. The apo B-3500 ArgÆGln substitution causes
familial defective hypercholesterolemia (FDB) due to defective binding of LDL to its receptor (Vega & Grundy 1986, Soria et al. 1989). The prevalence of the mutation in the general population in Central Europe is 1/204-1/700 (Innerarity et al.1990, Tybjaerg-Hansen et al. 1990, Schuster et al. 1990). The highest prevalence has been reported from Switzerland (Miserez et al. 1994), and so far apo B -3500 has not been found in Finland (Hämäläinen et al. 1990).  Another mutation in the LDL receptor binding area causing apo B-3531 ArgÆ Cys has been described to cause moderate hypercholesterolemia due to defective binding of LDL to its receptor (Pullinger et al. 1995).

Several restriction fragment length polymorphisms (RFLP) in the Apo B gene have been defined (Humphries & Talmud 1995). The most widely studied of these is the XbaI polymorphism in exon 26, which does not result in an amino acid substitution. In some populations the presence of the XbaI cutting site is associated with hypercholesterolemia in both normolipemic (Berg 1986, Talmud et al. 1987, Aalto-Setälä et al. 1988) and hypercholesterolemic (Leren et al. 1988, Aalto-Setälä et al. 1989) individuals. The absence of the XbaI cutting site was associated with higher triglyceride levels in one study (Deeb et al. 1986). Several studies have failed to reveal any association between the XbaI polymorphism and lipid values (Hegele et al. 1986, Aburatani et al. 1988,              Rajput-Williams et al. 1988, Darnfors et al. 1989, Gajra et al. 1994,) and in one study the association of the presence of the XbaI cutting site with elevated cholesterol and triglyceride levels was only observed in patients with peripheral artery disease (Monsalve et al. 1988).

The EcoRI restriction fragment length polymorphism in exon 29 is associated with an amino acid change Gln Æ Lys4154 . Most studies have revealed no association between the EcoRI polymorphism and cholesterol or triglyceride levels (Ma et al. 1987, Dunning et al. 1988, Jenner et al. 1988, Aburatani et al. 1988, Peacock et al. 1992,). An association between elevated triglycerides and the absence of the EcoRI cutting site has been reported in coronary heart disease patients (Paulweber et al. 1990, Tybjaerg-Hansen et al. 1991) and in healthy males (Paulweber et al. 1990).

The MspI RFLP in exon 26 is associated with an amino acid change Arg Æ Gln3611. The MspI polymorphism is not associated with differences in serum lipid concentrations (Deeb et al. 1986, Hegele et al. 1986, Xu et al. 1989, Genest et al. 1990).

The apo B signal peptide contains a leucine-alanine-leucine insertion/deletion polymorphism affecting the amino acids 14-16 producing signal peptides with 24 or 27 amino acids (Boerwinkle & Chan 1989). The ins allele has been associated with elevated serum triglycerides (Tikkanen & Heliö 1992), low serum cholesterol and apo B (Hansen et al. 1993), and coronary artery disease
(Peacock et al. 1992) in some populations, whereas in others the del allele has been connected with elevated total and LDL cholesterol but not with myocardial infarction (Bohn et al. 1994). No association between the polymorphism and lipids was detected in Asian patients, but the del allele was associated with coronary artery disease (Wu et al. 1994). A strong linkage disequilibrium
between the XbaI and ins/del polymorphisms has been reported (Hansen et al. 1993).
 

RECOMBINANT ADENO-ASSOCIATED VIRUS-MEDIATED GENE DELIVERY OF APOLIPOPROTEIN B mRNA SITE-SPECIFIC RIBOZYME (Abstract No. 430)

Shihua Sun, Talesha Ford, Alan Davis, Ba-Bie Teng

 Research Center for Human Genetics, Institute of Molecular Medicine, University of Texas-Houston, Houston, TX
 Center for Gene Therapy, Baylor College of Medicine, Houston, TX

Abstract

Apolipoprotein B (apoB) plays an obligatory role in the production of triglyceride-rich lipoprotein particles and it is necessary
for the transport of lipids and nutrients in the circulation. However, overproduction of apoB is strongly associated with
premature coronary artery diseases. Patients with familial hypercholesterolemia have markedly elevated plasma levels of
cholesterol and apoB and develop atherosclerosis. To modulate apoB production, we designed a hammerhead ribozyme
targeted at GUA6679Ø of apoB mRNA (designated RB15) to cleave apoB mRNA in vivo. From our previous study, we used
E1-deleted adenovirus vector to deliver RB15 to a dyslipidemia mouse model. The study showed that RB15 cleaved apoB
mRNA efficiently. There was a marked reduction of apoB gene expression and decrease plasma levels of cholesterol,
triglyceride, and human apoB100. Therefore, apoB mRNA-specific hammerhead ribozyme can be used as a potential
therapeutic agent to modulate apoB gene expression and to treat hyperlipidemia.  To have a long-term gene expression, no immune response, and no toxicity in gene therapy, in this study, we sought to construct liver-specific adeno-associated virus (AAV) vector to deliver RB15 to HepG2 cells and animals. RB15 is driven by transthyretin liver-specific promoter (TTR) and a 2773-bp human genomic fragment of hypoxanthine guanine phosphoribosyltransferase (HPRT) was inserted downstream of 5’ ITR of AAV vector (pAAV-TTR-RB15). We produced rAAV-TTR-RB15 by co-transfection of pAAV-TTR-RB15 with helper plasmid pDG in 293 cells, followed by purification using non-ionic iodixanol gradient and by ion exchange with heparin affinity chromatography. The virus titer was 1 x 1012 particles/ml, determined by both real-time PCR and dot-blot hybridization. We characterized the rAAV virial capsid proteins (VP1, VP2 and VP3) by western blotting.

The rAAV-TTR-RB15 (1 x 108 particles) was used to infect HepG2 cells. Total RNA was extracted at days 3 and 7 after infection. Using RNase protection assay the levels of apoB mRNA on day 7 was barely detectable (7.6% compared to that of
non-treated samples). The rAAV-TTR-RB15 (8 x 1010 particles) was used to transduce mouse overexpressing human apoB
gene. Using both PCR and real-time PCR RB15 DNA was detectable in the mouse liver on day 45 after treatment. The RB15
RNA was also detected in mouse liver on day 45 after treatment by RT/PCR. Southern blot analysis show that rAAV-TTR-RB15 was stably transduced into the liver. Using Western blot analysis, the levels of human apoB decreased on days 7, 14, and 28 to 13%, 40%, and 63%, respectively, compared to that of day 0 before treatment. In conclusion, the expressed ribozyme RB15 RNA was active, which decreased apoB production.
 
 

This is about ApoB. (See the whole sequence of lipoprotein interconversions.) Clicking on an *asterisked* word below takes you to that apoprotein. The triglyceride of the other 50% of the IDL is hydrolyzed by another enzyme *hepatic lipase* producing LDL, a lipoprotein that is richer than IDL in cholesterol and its esters. ------------------------------------- See the whole sequence of lipoprotein interconversions or click on the individual steps below again.   Apo B is incorporated into VLDL by hepatocytes, preparing this lipoprotein for transport of triglycerides (TG) and cholesterol from liver to other tissues. The VLDL as it is secreted is in an incomplete *nascent* state.   The conversion of nascent VLDL to its functional form needs the addition of Apo E and Apo C2 donated by *HDL* which in turn acquires them from other lipoproteins to form the "mature" *VLDL* that transports lipids. ×××××   When VLDL encounters lipoprotein lipase *LPL* in tissue capillaries the Apo C2 on the VLDL activates the enzyme, which hydrolyzes much of the triglyceride of the VLDL to produce *IDL*. The latter releases Apo C2 and Apo E which are recycled to HDL and then to VLDL. ×××××   About 1/2 of the resulting IDL, which is poorer than VLDL in triglyceride and relatively richer in cholesterol and its esters, is taken up by the liver by a receptor that recognizes the *Apo B* of the IDL. The *LDL* is taken up by the liver with its Apo B acting as the ligand to the receptor. Another 24% of the LDL is delivered to *other tissues* leaving about 1% of the LDL to be removed from the circulation by scavenger cells such as those found in atheromatous plaques. ×××××   However, the receptors on scavenger cells do not recognize "native" Apo B. Rather, they have a specific affinity for *oxidized* Apo B which is formed when LDL persists for an abnormally long time in the circulation. ×××××   Persistence of LDL in the circulation may result from the excessive VLDL production associated with a high dietary intake of fats, especially those rich in saturated fatty acids. It also occurs in primary and secondary lipidemias in which there is a subnormal uptake of IDL and LDL by the liver and other tissues. ×××××

Fasting Insulin and Apolipoprotein B Levels and Low-Density Lipoprotein Particle Size as Risk Factors for Ischemic Heart Disease

Benoît Lamarche, PhD; André Tchernof, PhD; Pascale Mauriège, PhD; Bernard Cantin, MD; Gilles R. Dagenais, MD; Paul J. Lupien,
MD; Jean-Pierre Després, PhD

Context.  Epidemiological studies have established a relationship between cholesterol and low-density lipoprotein cholesterol (LDL-C)
concentrations and the risk of ischemic heart disease (IHD), but up to half of patients with IHD may have cholesterol levels in the normal range.

Objective.  To assess the ability to predict the risk of IHD using a cluster of nontraditional metabolic risk factors that includes elevated fasting insulin and apolipoprotein B levels as well as small, dense LDL particles.

Design.  Nested case-control study.

Setting.  Cases and controls were identified from the population-based cohort of the Québec Cardiovascular Study, a prospective study conducted in men free of IHD in 1985 and followed up for 5 years.

Participants.  Incident IHD cases were matched with controls selected from among the sample of men who remained IHD free
during follow-up. Matching variables were age, smoking habits, body mass index, and alcohol consumption. The sample included 85
complete pairs of nondiabetic IHD cases and controls.

Main Outcome Measures.  Ability of fasting insulin level, apolipoprotein B level, and LDL particle diameter to predict IHD events, defined as angina, coronary insufficiency, nonfatal myocardial infarction, and coronary death.

Results.  The risk of IHD was significantly increased in men who had elevated fasting plasma insulin and apolipoprotein B levels and
small, dense LDL particles, compared with men who had normal levels for 2 of these 3 risk factors (odds ratio [OR], 5.9; 95%
confidence interval [CI], 2.3-15.4). Multivariate adjustment for LDL-C, triglycerides, and high-density lipoprotein cholesterol (HDL-C) did not attenuate the relationship between the cluster of nontraditional risk factors and IHD (OR, 5.2; 95% CI, 1.7-15.7). On the other hand, the risk of IHD in men having a combination of elevated LDL-C and triglyceride levels and reduced HDL-C levels was no longer significant (OR, 1.4; 95% CI, 0.5-3.5) after multivariate adjustment for fasting plasma insulin level, apolipoprotein B level, and LDL particle size.

Conclusion.  Results from this prospective study suggest that the measurement of fasting plasma insulin level, apolipoprotein B level, and LDL particle size may provide further information on the risk of IHD compared with the information provided by conventional lipid variables.
 

JAMA. 1998;279:1955-1961

OVER THE LAST 30 years, several epidemiological studies have reported a direct relationship between total plasma cholesterol and
low-density lipoprotein cholesterol (LDL-C) concentrations and the risk of coronary artery disease (CAD), and elevated total plasma cholesterol levels are considered by many to be the main cause of coronary atherosclerosis. However, the ability to adequately identify individuals at high risk for the development of CAD solely on the basis of total cholesterol or LDL-C concentration has recently been challenged by evidence suggesting that a considerable proportion of patients with CAD may have cholesterol levels in the normal range (Genest et al reported the proportion to be as high as 50%).  There are also data to suggest that a notable proportion of patients undergoing cholesterol-lowering therapy and who achieve significant LDL-C reduction may still develop CAD. These observations have emphasized the need to find additional markers of risk that would allow a more refined identification of individuals at high risk for CAD.

Clinical data have provided evidence that elevated plasma triglyceride levels and reduced high-density lipoprotein cholesterol (HDL-C)
concentrations may be associated with a considerable increase in CAD risk. Although the independent contribution of plasma
triglycerides to CAD remains controversial, the clinical relevance of elevated triglyceride levels should no longer be overlooked as
hypertriglyceridemia may reflect additional metabolic disturbances highly predictive of CAD risk.  Results from the Helsinki Heart Study and from the Prospective Cardiovascular Münster (PROCAM) study have suggested that hypertriglyceridemia should be considered an important risk factor for CAD, particularly when combined with elevated LDL-C and reduced HDL-C concentrations. This cluster of risk factors may represent the metabolic condition most predictive of CAD risk.

With the 5-year prospective data from the Québec Cardiovascular Study, we have recently reported that elevated fasting plasma insulin levels,11 elevated apolipoprotein B concentrations, and the presence of small, dense LDL particles14 were strongly associated with the development of ischemic heart disease (IHD) in men, independent of established risk factors. Plasma LDL-C, triglyceride, and HDL-C levels were also significant correlates of IHD in the Québec Cardiovascular Study. In the current study, we investigate whether the ability to identify individuals at high risk for the development of IHD could be improved by measuring 3 nontraditional risk factors, namely fasting plasma insulin and apolipoprotein B levels and LDL particle diameter, over and beyond what can be achieved using more traditional lipid risk factors, triglyceride, LDL-C, and HDL-C levels.

METHODS

Study Population and Follow-up

The Québec Cardiovascular Study cohort has been described in detail previously.  In 1973, a random sample of 4637 men aged 35 to 64 years was recruited from 7 suburbs of the Québec metropolitan area for an evaluation of cardiovascular risk factors using the provincial electoral lists. Subsequent evaluations were performed at regular intervals and data collected in 1985 were used as the baseline characteristics for the present prospective analyses. In 1985, 2443 (61%) of the living cohort came to the lipid clinic in a fasting state for their evaluation. Among the 1557 other potential living subjects, 150 (10%) could not be located, 302 (19%) came to the clinic in a nonfasting state, and 1105 (71%) either refused to participate or were evaluated in a nonfasting state at their home by project nurses.  Analyses of data collected in 1973 revealed that the age distribution of the 2443 subjects in 1985 was representative of the original cohort.  At the end of follow-up (September 1, 1990), all subjects were contacted by mail and invited to answer a short standardized questionnaire on smoking habits, medication use, history of cardiovascular disease, and diabetes mellitus. For those who reported such diseases and those who died, hospital charts were reviewed.  Telephone calls were made to subjects who did not answer a second letter and if the call was unsuccessful, another call was made to a close family member. Mortality and morbidity data were obtained in 99% and 96%, respectively, of the subjects of the initial 1973 screening.

Evaluation of Risk Factors

Data on demographic and lifestyle variables as well as medical history and medication were obtained in 1985 through a standardized
questionnaire administered to each subject by trained nurses and further reviewed by a physician. Body weight and height were
recorded. Resting blood pressure was measured after a 5-minute rest in a sitting position. The mean of 2 blood pressure measures taken 5 minutes apart was used in the analyses. Information on personal and family history of IHD and diabetes mellitus, smoking habits, alcohol consumption, and medication use was also obtained. Diabetes mellitus was considered in men who self-reported the disease or who were treated with hypoglycemic agents. Only 2% of men were using hypolipidemic drugs in 1985 (mainly clofibrate and cholestyramine), whereas 8% and 4% of men were using -blockers and diuretics, respectively, on a regular basis at the 1985 screening. Data on drug use at the time of follow-up were not available. Alcohol consumption was computed from the type of beverage (beer, wine, or spirits) consumed in ounces per week and then standardized as an absolute quantity (1 oz of absolute alcohol was equivalent to 22.5 g of alcoholic beverage). Family history of IHD was considered positive if at least 1 parent or 1 sibling had a history of IHD.

Definition of IHD Events

The diagnosis of a first IHD event included typical effort angina, coronary insufficiency, nonfatal myocardial infarction, and coronary
death. All myocardial infarction cases met the criteria previously described,16 namely diagnostic electrocardiographic (ECG) changes alone or 2 of the following criteria: typical chest pain of at least 20 minutes in duration, creatine kinase enzyme level at least twice the upper limit of normal, or characteristic ECG changes. Coronary insufficiency was considered if typical retrosternal chest pain of at least 15 minutes in duration was associated with transient ischemic ECG changes but without significant elevation in levels of creatine kinase. Diagnoses of myocardial infarction and coronary insufficiency were confirmed by hospital charts. All ECG tracings were read by the same cardiologist, who was unaware of the subjects' risk profiles. The diagnosis of effort angina was based on typical symptoms of retrosternal squeezing or pressure-type discomfort occurring on exertion and relieved by rest and/or nitroglycerine. Criteria for the diagnosis of coronary death included confirmation from death certificate or autopsy report confirming the presence of coronary disease without evidence for noncardiac disease that could explain death. Myocardial infarction was considered fatal if death occurred within 4 weeks of the initial event or if it was diagnosed at autopsy.   Deaths related to IHD were confirmed from the Provincial Death Registry. Informed consent was obtained to review relevant hospital files. Autopsies were performed in about one third of deaths. The total IHD event frequency during the 5-year follow-up period was similar in men participating in the study (5.4%) and in nonparticipants (6.5%).

Pairing Procedures

Between 1985 and 1990, 114 of the 2103 men who had no clinical evidence of IHD at baseline had a first IHD event: 50 had a myocardial infarction, 40 had effort angina, 9 had coronary insufficiency, and 15 died of IHD-related causes. Each case subject was matched with a control subject selected from among the remaining 1989 men without IHD during follow-up. Subjects were matched on the basis of age, cigarette smoking, body mass index, and weekly alcohol intake. The mean difference within pairs was 0.6 years, 0.2 kg/m2, and 0.2 oz/wk for age, body mass index, and alcohol intake, respectively. The mean difference within pairs for cigarette smoking was 0.3 cigarettes per day. Subjects who had an IHD event and who were classified as nonsmokers were systematically matched with nonsmoking control-group subjects.

Laboratory Analyses

Fasting lipoprotein lipid and apolipoprotein levels were measured in plasma in 1985 when subjects came to the clinic for evaluation.
Aliquots of fasting plasma were frozen at the time of collection and were later used for the assessment of LDL diameter and fasting
insulin concentrations. Total cholesterol and triglyceride levels were determined on a multianalyzer (Technicon RA-500, Bayer Corp,
Tarrytown, NY) as previously described.  High-density lipoprotein cholesterol was measured in the supernatant fraction after
precipitation of apolipoprotein B–containing lipoproteins with heparin–manganese chloride.  Low-density lipoprotein cholesterol
levels were estimated by the equation of Friedewald et al.  Subjects with triglyceride levels higher than 4.5 mmol/L (399 mg/dL) (n=52) were excluded from the analyses.  Plasma apolipoprotein B levels were measured by the rocket immunoelectrophoresis method of Laurell, as described previously.  Serum standards for the apolipoprotein assay were prepared in the laboratory and calibrated against serum samples from the Centers for Disease Control and Prevention. The standards were lyophilized and stored at-85°C until use. The coefficients of variation for cholesterol, HDL-C, triglyceride, and apolipoprotein B measurements were less than 3%.

Low-density lipoprotein particle diameter was assessed using nondenaturing 2% to 16% polyacrylamide gradient gel electrophoresis
of whole plasma according to Krauss et al and McNamara et al, as described previously.  Plasma samples were applied on gels in a
final concentration of 20% sucrose and 0.25% bromophenol blue. Following a 15-minute pre-run, electrophoresis was performed at 200 V for 12 to 16 hours and at 400 V for 2 to 4 hours. Gels were stained with Sudan black B according to standardized procedures and stored in a solution of 9% acetic acid and 20% methanol until analysis using an optical densitometric image analyzer (BioImage Visage 1101DGEL, Genomic Solutions, Ann Arbor, Mich) coupled with a computer (SPARC Station 2 Sun, Genomic Solutions). Low-density lipoprotein diameter was estimated by comparing the migration distance on the gel of the predominant LDL subspecies for each individual with the migration distance of standards of known diameters. One assay was performed for each subject. Analyses of pooled plasma standards revealed that the assessment of LDL diameter using this method was highly reproducible with a coefficient of variation of less than 3% (A.T., unpublished data, 1996).

Fasting plasma insulin concentrations were measured with a commercial double-antibody radioimmunoassay (human insulin-specific radioimmunoassay method; Linco Research, St Louis, Mo) according to the manufacturer protocol. This assay shows essentially no cross-reactivity with human proinsulin (<0.2%). The coefficient of variation was below 5.5% for both low and high fasting insulin concentrations.

Statistical Analyses

Fasting insulin levels and LDL diameter were measured in 106 and 103 case-control pairs, respectively, but data for both variables were available simultaneously in 100 controls and 102 cases. Men who reported having diabetes mellitus or who were receiving hypoglycemic therapy at the baseline evaluation were excluded (15 cases and 1 control). We therefore had data on 87 IHD cases and 99 controls. After excluding all pairs for which 1 of the 2 subjects had missing data, the study sample included 85 complete pairs of IHD cases and matched controls. Baseline characteristics of subjects who developed IHD during the 5-year follow-up (IHD cases) were compared with the characteristics of those who remained IHD free using paired t tests for means and 2 tests for frequency data.  Variables with a skewed distribution were log-transformed. Correlation analyses were performed using the Pearson and the Spearman coefficients of correlation for parametric and nonparametric variables, respectively.

The median of the control group was used as the cutoff point to identify men with elevated or low levels of each variable of interest
(LDL-C, 3.7 mmol/L [143 mg/dL]; triglycerides, 1.52 mmol/L [135 mg/dL]; apolipoprotein B, 1.1 g/L [110 mg/dL]; fasting insulin, 72
pmol/L [10 µU/mL]; HDL-C, 1.01 mmol/L [39 mg/dL]; LDL particle diameter, 25.82 nm). Thus, by definition, each of these risk factors
was found in 50% of the control subjects. The proportion of cases classified as having 1 or more risk factor based on these arbitrary
cutoff points was compared with that of control subjects. The proportional hazards regression (PHREG) procedure on SAS (SAS
Institute, Cary, NC) for conditional logistic regression analysis was used to estimate the odds ratio (OR) for IHD associated with the
presence of each risk factor, as an isolated condition or combined with others. Odds ratios were adjusted for medication use at baseline (-blockers and/or diuretics), family history, and systolic blood pressure. The potential confounding effects of using -blockers and diuretics were combined because they both yielded similar risk. Thus, medication use (yes or no) and family history (presence or absence) were treated as categoric variables whereas systolic blood pressure was treated as continuous.

RESULTS

Table 1 presents the clinical characteristics of the 85 controls and IHD cases. A higher proportion of case patients was using -blockers and/or diuretics on a regular basis at baseline (17.7% vs 4.7%, P=.007). However, there was no difference between cases and controls in the use of hypolipidemic medication at baseline. As a result of the matching procedure, the frequency of smokers (41%) and the number of cigarettes smoked per day (25 cigarettes per day) were essentially the same in both groups. Systolic blood pressure was also the same in both groups. As expected, there were marked differences in several plasma lipoprotein-lipid parameters as well as in fasting insulin levels at baseline between IHD cases and controls. Triglycerides (18.2%), fasting insulin (18.9%), and apolipoprotein B (15.9%) levels showed the largest case-control differences. Mean plasma HDL-C concentrations and LDL diameter were also significantly different between cases and controls (P=.03). It is important to note that although being tightly matched with IHD cases on the basis of age, body mass index, smoking, and alcohol consumption, the risk profile of control subjects in the current study is very similar to that of the total sample of men who remained free of IHD during follow-up and from which they were selected.

Prevalence of Lipoprotein and Insulin Abnormalities

Because there are currently no reference values for apolipoprotein B and insulin levels and for LDL diameter, and in an attempt to compare the contribution to IHD risk of variables having different scales, lipoprotein-lipid and fasting insulin levels were dichotomized using the median (50th percentile) of the control group. Table 2 presents the prevalence of each of the metabolic abnormalities in IHD cases. Based on these prevalences, ORs for developing IHD during the 5-year follow-up were estimated using conditional logistic regression while taking into consideration the potential confounding effects of systolic blood pressure, medication use, and family history of IHD. Eighty-one percent of cases had elevated fasting insulin concentrations based on these criteria, yielding a 5.5-fold increase in the OR for IHD (95% confidence interval [CI], 2.3-13.6, P<.001) compared with men having insulin levels below the 50th percentile of controls. Elevated plasma triglyceride levels were also associated with a marked increase in the risk of IHD (OR, 3.5; 95% CI, 1.6-7.4; P=.002). Elevated apolipoprotein B and LDL-C levels and small, dense LDL particles were observed in a similar proportion of cases (69.4%, 68.2%, and 69.4%, respectively). These 3 abnormalities were associated with a significant 2.4-fold to 2.7-fold increase in the OR for IHD. Finally, 62.4% of IHD cases had HDL-C levels below the 50th percentile of controls. There was a 60% increase in the risk of IHD associated with reduced HDL-C levels (OR, 1.6), which was not significant after adjustment for confounders (95% CI, 0.85-3.0). This analysis did not take into consideration the fact that cases with 1 abnormality may also have had additional metabolic abnormalities in combination. Nevertheless, results presented in Table 2 suggest that among all variables of interest, elevated fasting plasma insulin concentrations, irrespective of the presence or absence of other lipoprotein abnormalities, were associated with the greatest relative increase in the risk of IHD.

Prevalence of Isolated Abnormalities

The prevalence rates of elevated plasma fasting insulin and apolipoprotein B levels as well as of small, dense LDL in their isolated
form (ie, associated with none of the other 2 abnormalities) were low in both IHD cases and control subjects. Isolated hyperinsulinemia was observed in only 11 (12.9%) of both IHD cases and controls. However, when considering only subjects with elevated fasting insulin levels (42 controls and 69 cases), 11 (15.9%) of 69 hyperinsulinemic IHD cases did not have elevated apolipoprotein B levels or small, dense LDL in combination compared with 11 (26.6%) of 42 controls. Only 2 (2.4%) of 85 IHD cases had isolated elevations in apolipoprotein B levels compared with 9 (10.6%) of 85 controls. Finally, the small, dense LDL phenotype in its isolated form was found in only 5 (5.9%) of 85 IHD cases. In comparison, twice as many controls (11 [12.9%] of 85) had small, dense LDL in isolation. These results suggest that hyperinsulinemia, elevated apolipoprotein B levels, and small, dense LDL particles may be observed more frequently in combination with each other rather than as isolated conditions, and that a smaller proportion of IHD cases may display these abnormalities in their isolated form compared with controls. We therefore tested whether the cluster of these metabolic risk factors may further increase the risk of IHD.

Prevalence of Nontraditional Risk Factors

Figure 1 compares the prevalence rates of the cumulative number of abnormalities in IHD cases and controls. To simplify data
presentation, fasting plasma insulin levels, apolipoprotein B levels, and small, dense LDL particles are referred to as nontraditional risk factors, whereas LDL-C, triglyceride, and HDL-C levels are referred to as traditional risk factors. As shown in Figure 1 (top), only 2 IHD cases (2.4%) had none of the 3 nontraditional metabolic risk factors, compared with 14 controls (16.5%). One of every 5 IHD cases (n =18, 21.2%) had 1 of the nontraditional risk factors in its isolated form, compared with more than a third of controls (n = 31, 36.5%). The proportion of cases that simultaneously had elevated fasting insulin levels, elevated apolipoprotein B levels, and small, dense LDL particles (cumulative number of risk factors, 3) was 2.6-fold greater than that of controls (45.8% vs 17.7%). Consequently, 98% of IHD cases had at least 1 of the nontraditional risk factors compared with 83% of controls. On the other hand, 82% of controls did not have elevated fasting plasma insulin levels, elevated apolipoprotein B levels, and small, dense LDL simultaneously, compared with 54% of IHD cases.

Prevalence of Traditional Risk Factors

A similar analysis was performed using the traditional risk factors (LDL-C, triglycerides, and HDL-C levels) as discriminating variables for the determination of IHD risk (Figure 1, bottom). Although differences in the proportion of cumulative number of traditional risk factors between IHD cases and controls were slightly attenuated compared with differences in the proportion of nontraditional risk factors, a similar pattern of distribution was observed. There was a greater proportion of controls that had relatively low LDL-C and triglyceride levels and high HDL-C levels (number of risk factors, 0) compared with IHD cases (18.8% vs 7.1%), whereas the proportion of IHD cases that had elevated LDL-C and triglyceride levels and low HDL-C concentrations simultaneously (cumulative number of risk factors, 3) was 1.9-fold greater than that of controls (41.2% vs 21.2%).

Risk of Developing IHD During Follow-up

Based on the prevalence of the cumulative number of risk factors presented in Figure 1, the crude OR for developing IHD during the
5-year follow-up was increased 18.2-fold in subjects who had all 3 nontraditional risk factors simultaneously compared with those who had none of the 3 risk factors (results not shown). By comparison, the OR for IHD in subjects with the 3 traditional risk factors
simultaneously was 5.2 (not shown). Multivariate conditional logistic regression analysis was performed to compare the ability to predict IHD using traditional and nontraditional risk factors. The prevalence of IHD cases in subjects with no risk factor (2 and 6 IHD cases for nontraditional and traditional risk factors, respectively) was too small to accurately assess the risk of IHD using this group as a reference. We have therefore performed the multivariate logistic regression analysis by combining subjects with 0 and 1 risk factor only, and by using this group as a reference (OR, 1). As shown in Table 3, subjects that had elevated LDL-C and triglyceride levels and reduced HDL-C concentrations simultaneously (cumulative number of traditional risk factors, 3) showed a 3-fold increase in the risk of IHD (model 1: OR, 3.0; 95% CI, 1.4-6.4; P=.005) compared with men having none or only 1 of these risk factors. This increased risk was no longer significant after multivariate adjustment for fasting insulin and apolipoprotein B levels and LDL particle diameter (model 2: OR, 1.4; 95% CI, 0.5-3.5; P=.50).

The impact of having elevated fasting insulin and apolipoprotein B levels and small, dense LDL particles in combination with each other on the odds of developing IHD was more prominent. The risk of developing IHD was increased almost 6-fold when subjects
simultaneously had elevated fasting insulin and apolipoprotein B levels and small, dense LDL particles (model 3: OR, 5.9; 95% CI,
2.3-15.4; P<.001). This increase in risk was essentially unmodified when LDL-C, triglyceride, and HDL-C levels were included as
cofounders in the multivariate logistic regression model (model 4: OR, 5.2; 95% CI, 1.7-15.7; P=.003).

An analysis was carried out to test the 2-way and 3-way interaction terms as predictors of IHD risk. It was found that none of the 2-way or 3-way interaction terms for continuous variables were significant. However, because of the small sample size, the possibility of a significant interaction among the 3 nontraditional or the 3 traditional risk factors cannot be excluded.

Univariate associations between the traditional and nontraditional risk factors and the variables that were used to match IHD cases to controls were investigated. Plasma triglyceride levels (r=0.15, P=.05) and HDL-C levels (r=-0.17, P=.02) showed significant associations with body mass index. Plasma triglyceride levels also showed a significant but inverse correlation with age (r=-0.23, P=.003) whereas HDL-C levels were positively associated with weekly alcohol consumption (r=0.26, P<.001). Low-density lipoprotein particle size was also a significant correlate of age (r=0.19, P=.01) but the most significant correlation between risk factors and matching variables was observed between plasma fasting insulin concentrations and body mass index (r=0.40, P<.001).

COMMENT

Results of the present prospective study emphasize the potential of plasma fasting insulin and apolipoprotein B levels as well as of small, dense LDL particles as clinically relevant markers of the risk of developing IHD. Our results suggest that this cluster of metabolic abnormalities may even provide more information on IHD risk than the more traditional lipid risk factors, LDL-C, triglycerides, and HDL-C. Indeed, almost 1 (45.8%) of every 2 IHD cases had elevated insulin and apolipoprotein B levels as well as small, dense LDL particles, and this combination of metabolic risk factors resulted in a remarkable 18-fold increase in the risk of IHD.  Adjustment for the more traditional cluster of risk factors through multivariate logistic regression did not attenuate this relationship. These observations have consequential clinical implications, particularly in terms of primary prevention of IHD. They imply that identification of individuals at risk could be substantially improved by measuring fasting plasma insulin and
apolipoprotein B levels and LDL particle diameter. It should be kept in mind that these findings do not in any way lessen the clinical
importance of assessing LDL-C, triglyceride, and HDL-C concentrations. The current study should not be considered an attempt to discredit the well-described and accepted relationship between the so-called lipid triad and the risk of IHD.8-10 It was apparent that an important proportion of IHD cases was characterized by this dyslipidemia compared with controls.

It may be argued that the paired nature of the study population may have had the adverse effect of overmatching for the traditional risk factors, thereby understating their true impact on a randomly selected population.  As expected, there were significant correlations between risk factors and some of the variables used to match IHD cases and controls. Although significant, these correlations were of very low magnitude (with shared variances lower than 7%), with the exception of the relationship between plasma fasting insulin levels and body mass index (with a shared variance of 16%). The paired nature of the study is therefore very unlikely to have biased the estimation of the contribution of the traditional risk factors to IHD risk compared with that of the nontraditional risk factors.

We reported that a very small proportion of IHD cases had no risk factor and that abnormalities in insulin and apolipoprotein B levels and in LDL particle diameter were more frequently observed in combination and not in isolation compared with controls. It is
therefore apparent that the risk of developing IHD is largely dependent on the presence of risk factors that, in most cases, emerge as a cluster of metabolic abnormalities. In this context, arguments have been proposed for why plasma insulin and apolipoprotein B levels and LDL particle size may represent better markers of IHD risk than LDL-C, triglyceride, and HDL-C levels.

Small, Dense LDL and the Risk of IHD

Plasma LDL-C levels are merely measurements of the cholesterol content of a lipoprotein particle that has been described as being very heterogeneous in terms of composition, size, and density. Although the cholesterol content of LDL certainly contributes to its
heterogeneity, we have failed to find a significant association between LDL density or size and LDL-C levels.  Recognition of the
atherogenic potential of small, dense LDL largely came from cross-sectional case-control studies that reported a higher prevalence
of small, dense LDL in patients with IHD compared with healthy controls.24-26 Observations from 3 recent prospective reports provided further support for a critical role of small, dense LDL particles in the etiology of atherosclerosis.14, 27, 28 The greater susceptibility of these particles to oxidation and their reduced affinity for the hepatic LDL receptor have been proposed as potential mechanisms for the increased atherogenic potential of small, dense LDL.

Apolipoprotein B and the Risk of IHD

Apolipoprotein B is the protein moiety of LDL. The clinical interest of this protein lies in the fact that it provides a relatively accurate
estimate of circulating LDL particle numbers. Total plasma apolipoprotein B concentration, as opposed to LDL apolipoprotein B,
also accounts for the number of triglyceride-rich lipoproteins (very low-density lipoprotein and intermediate-density lipoproteins), and
recent data suggest that these 2 lipoprotein subfractions may also play an important role in the etiology of IHD.  Plasma
apolipoprotein B concentration can therefore be considered a crude marker of the number of atherogenic particles in plasma.
Results from the Québec Cardiovascular Study suggest that plasma apolipoprotein B concentration is a strong predictor of IHD risk,
independent of traditional risk factors. It is therefore suggested that apolipoprotein B, as a measure of the number of atherogenic particles in plasma, may yet provide more information than the amount of cholesterol transported by these particles.

Insulin and the Risk of IHD

The concept of insulin resistance as a central component of a potentially atherogenic dyslipidemic state was first introduced in 1988
when it was suggested that a large proportion of individuals resistant to the action of insulin was also characterized by metabolic
disturbances highly predictive of an increased IHD risk.  Using fasting or postglucose insulin levels as crude indices of insulin
resistance, univariate analyses of large cohorts of nondiabetic populations have shown that hyperinsulinemia in the fasting state or
following a glucose load was associated with an increased risk of IHD.  Results from multivariate analyses have, however, yielded
discordant conclusions. We and others have recently reported that elevated plasma insulin levels measured with an antibody showing essentially no cross-reactivity with proinsulin were associated with an increased risk of developing IHD, independent of
other risk factors such as triglyceride, HDL-C, and LDL-C levels.  Nevertheless, whether plasma insulin should or should not be
considered an independent risk factor for the development of IHD remains a matter of considerable debate. It is well accepted, however, that elevated plasma insulin concentrations are most frequently associated with deteriorations in other cardiovascular risk factors.  Hyperinsulinemia and insulin resistance also appear to have direct effects on the arterial wall and contribute to a reduced fibrinolytic potential.  Plasma insulin levels may therefore provide a crude but global description of a number of additional metabolic abnormalities that may, in turn, be associated with an increased risk of IHD, but that may not be adequately assessed by the traditional triad of lipid risk factors. It is important to emphasize that results of the present study apply to nondiabetic men, particularly because patients with type 2 diabetes mellitus were excluded from the analyses. Although inclusion of men with type 2 diabetes mellitus in the study sample essentially had no impact on the results, whether results of the present study can be applied to other populations such as persons with type 2 diabetes mellitus, women, or the elderly population will have to be established more specifically in future studies.

Conclusions

Beyond the mechanisms underlying the atherogenicity of hyperinsulinemia, hyperapobetalipoproteinemia, and small, dense
LDL, and irrespective of whether these mechanisms share common paths, results of the present study suggest that the risk of IHD is increased substantially when these metabolic abnormalities cluster.  The synergistic contribution of the nontraditional cluster of risk factors to IHD risk and the fact that almost 1 of every 2 IHD cases had these abnormalities simultaneously reflect the multifactorial etiology of IHD.  It also emphasizes the importance of defining the risk of IHD based on more than 1 risk factor.

There are a number of critical issues that have to be considered before any decision can be made toward the measurement of these
nontraditional risk factors on a routine basis. Among others, results of this prospective case-control study will have to be confirmed through larger population-based studies, as the relatively low number of IHD cases allowed only a gross assessment of risk. The relatively large CIs associated with the estimated risk in some of the subgroups reflect this phenomenon.  Population reference values such as those used for LDL-C, triglycerides, and HDL-C also will be needed before critical levels of fasting insulin, apolipoprotein B levels, and LDL particle size or density at which a person becomes at greater risk for IHD are identified. Means to achieve effective treatment of the nontraditional risk factors is also a critical issue that deserves a great deal of scrutiny before decisions can be made toward use of these variables in the risk management of IHD. There are data to suggest that LDL particle size can be modulated by changes in plasma triglyceride levels.41 Studies have shown that triglyceride-lowering therapy with fibric acid derivatives can lead to a significant increase in LDL particle size.42, 43 There is also a large body of evidence demonstrating that LDL particle size, apolipoprotein B level, and insulin resistance and/or hyperinsulinemia can be effectively altered by diet and exercise-induced weight loss.44, 45 Thus, the ability to favorably modify the nontraditional risk factors by diet, exercise, and
appropriate pharmacotherapy provides further support for the use of these risk factors in the management of IHD risk. Finally, the
cost-effectiveness of implementing and using new risk factors as a basis for screening and treatment in primary and secondary
prevention of IHD should be established. Irrespective of these important considerations, we hope that these results will help
stimulate research aimed at identifying means that could substantially improve the early diagnosis and treatment of individuals at risk for IHD.

Author/Article Information

From the Lipid Research Center, Laval University Hospital Research Center, Ste-Foy, Québec (Drs Lamarche, Tchernof, Mauriège, Cantin, Lupien, and Després); and the Department of Medicine, University of Montréal, Montréal, Québec (Dr Dagenais).

Reprints: Jean-Pierre Després, PhD, Lipid Research Center, CHUL Research Center, 2705 Laurier Blvd, TR-93, Ste-Foy, Québec GIV 4G2, Canada (e-mail: jean-pierre.despres@crchul.ulaval.ca).

This study was supported in part by the Heart and Stroke Foundation of Canada, the Medical Research Council of Canada, and the Québec Heart Institute Research Foundation. Dr Lamarche is a research scholar of the Medical Research Council of Canada and Dr Tchernof is a recipient of a research fellowship from the Canadian Diabetes Association.

We are grateful to France Gagnon, MSc, and Louise Fleury, MSc, for their important contribution in the data collection and to Paul-Marie Bernard, MSc, biostatistician and professor at the department of Preventive and Social Medicine, Laval University, Ste-Foy, Québec, for his helpful input regarding data analysis. The financial contribution of Fournier Pharma Inc, Montréal, Québec, is also gratefully acknowledged.

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GEN
        The gene length is 43 kb. Exons: 29; introns: 28. Two exons are unusually long: 1.9 kb (the 29th)
        and 7.6 kb (the 26th); the length of the remaining exons vary within the limits of 150-250 bp. The
        mature mRNA, 14.1 kb in length, codes for the protein comprising 4563 amino acid residues."
 FUN
       [1] Circulatory APOB is a ligand for the receptor-mediated transition of very low density
        lipoproteins (VLDL particles) into cells and plays the central role in the transport and metabolism
        of serum cholesterol.
        [2] In addition, APOB, along with glycoprotein Lp(a) (GEM:06q27/APOLPA), participates in
        formation of the complex of serum lipoprotein(a), which plays an important role in atherogenesis
        and pathogenesis of coronary disease."
 MOP
        APOB has two forms: (I) apoB100 (500 kD; 14.1 kb mRNA) is synthesized in liver and participates
        in the packing of VLDL particles, (II) apoB48 (210 kD; 7.5 kb mRNA) is synthesized in intestine
        and participates in formation of chylomicrons (Chen-1987). ApoB-48 represents the amino-terminal
        47% of apoB-100 and that the carboxyl terminus of apoB-48 is in the vicinity of residue 2151 of
        apoB-100 (Innerarity-1987). ApoB-48 contains 2,152 residues compared to 4,535 residues in
        apoB-100 (Higuchi-1988). Both forms are alternative splicing products of the single gene
        (Cladaras-1986)."

Apolipoprotein (apo) E is a 34-kDa protein consisting of 299 amino acids. It is a protein constituent of chylomicrons, very low density lipoproteins and HDL and VLDL remnants (Mahley 1988). On these particles, apo E serves as a ligand for uptake by lipoprotein receptors (Davignon et al. 1988, Mahley 1988,Mahley et al. 1990). Apo E is polymorphic with three common alleles: E2, E3 and E4
(Zannis et al. 1982), which are associated with variations in the blood lipid concentrations. The phenotype E2/2 is associated with type III hyperlipidemia, and E4 is associated with elevated serum total and LDL cholesterol concentrations compared to E2 and E3 (Ehnholm et al. 1986, Utermann 1987, Davignon et al. 1988). The serum concentrations of apo E are higher in individuals with E 3/3 than in individuals with E4, and highest in individuals with E2 (Berglund et al. 1993, Luc et al. 1994).

Apo E polymorphism modifies plasma lipids, at least in Caucasians, partly by affecting the efficiency of cholesterol absorption, so that individuals with E2 absorb less cholesterol than individuals with E4 (Kesäniemi et al. 1987), and individuals with the E4 allele respond better to changes in dietary cholesterol and saturated fatty acids than those without the E4 allele (Lehtimäki et al. 1995). The apo E polymorphism also modifies the metabolism of LDL. Individuals with the apo E phenotype 2/2 catabolise LDL faster than others, and normolipemic apo E 4 homozygotes catabolise LDL at a slower rate than apo E 3 homozygotes (Demant et al. 1991).

The E4 phenotype has been associated with an increased risk of CAD either directly (Kuusi et al. 1989) or via elevated atherogenic lipoprotein levels (Stuyt et al. 1991). The apo E phenotype distribution among CAD patients and myocardial infarction survivors is controversial. Some studies propose a higher frequency of apo E 4 in CAD patients (Nieminen et al. 1992, Wang et al. 1995), or myocardial infarction (AMI) survivors (Cumming and Robertson 1984), whereas others fail to detect any difference (Stuyt et al. 1991, Utermann et al. 1984).