Biochemical Factors

4.1. LIPOPROTEIN LIPASE Plasma lipid and lipoprotein concentrations are important factors linked to CVD risk. In addition to the well-studied dietary factors controlling plasma lipid concentrations, there is growing evidence that genetics plays a factor. Polymorphisms in lipoprotein lipase (LPL) is one gene that has been studied in this regard. The LPL gene is found on chromosome 8p33 and contains 10 exons spanning 30 kb. This gene produces a 475-amino-acid protein that is posttranslationally modified to a signal peptide and the 448-amino-acid mature enzyme that has a molecular weight of about 60 kDa. LPL is anchored to the vascular endothelium and removes lipids from the circulation by hydrolyzing triglycerides present in chylomicrons and very low-density lipopro-tein(VLDL). Familial LPL deficiency causes marked chylomicronemia and premature atherosclerosis, but is relatively rare. There are at least four polymorphic variants of the LPL gene that have a more subtle effect on plasma lipid concentrations and cardiovascular risk. If these polymorphisms are associated with a reduced activity of LPL, they might be genetic risk factors for atherosclerosis.

Two of the most widely studied polymorphisms to lipopro-tein lipase are the restriction fragment length polymorphic sites HindIII and PvuII, located on introns 8 and 6, respectively. The HindIII site results in a T^G substitution and has an allele frequency (presence of the restriction site, H+) of roughly 70%. The PvuII site results in a C^T substitution and has an allele frequency (P+) of about 45%. Given the importance of lipopro-tein lipase to lipid metabolism, attempts have been made to correlate the presence of these polymorphisms to lipid levels. Gerdes et al. showed that subjects with the H+H+ genotype had higher triglyceride (mean = 125 mg/dL) and lower high-density lipoprotein (HDL) cholesterol concentrations (mean = 49 mg/dL) than those with the HH- genotype (110 and 53 mg/dL, respectively) (38). Larson et al. put enrolled subjects on the same low-fat and low-cholesterol, and high-carbohydrate and high-fiber diets prior to lipid testing, and found no difference in HindIII genotypes for triglycerides and HDL cholesterol (39). The total and LDL cholesterol, however, was increased for women in the H+H+ group. Other investigators found similar results for triglycerides for the PvuII polymorphism (158 for P-P- vs 213 mg/dL for P+P+) (40).

The presence of the H+ allele is associated with a higher frequency of coronary disease. When comparing HindIIII polymorphism to the number of disease coronary arteries, the H+H+ genotype had a higher incidence of multivessel disease (OR: 4.4; 95% CI: 1.73-11.33) than the combined H+H- and HH-genotypes among Italians patients surviving AMI (41). Because the H+ allele has the higher prevalence, the presence of the H- variant would be interpreted to offer a protective effect toward CAD. Similar but less dramatic results were obtained for the PvuII polymorphism, where the P+P+ genotype had an odds ratio of 1.73 (95% CI: 1.03-2.89) vs the P'P' genotype (40). Studies examining the risk of particular H and P genotypes with CAD are being initiated. In one study, the H+H+ genotypes was significantly associated with an odds ratio of 2.0 (95% CI: 1.11-3.70) vs the H'H' genotype in 725 patients undergoing angioplasty for ACS and 168 control subjects with normal coronary arteries (42). This same study produced a trend for the P+P+ vs the P-P- genotypes, but results did not reach significance (OR: 1.39; 95% CI: 0.84-2.28).

Another widely studied polymorphism in lipoprotein lipase is the point mutation of a C^G in nucleotide 447 of exon 9, resulting in the substitution of serine for a permature termination codon. The termination polymorphism can be detected with MnFI and has an allele frequency of about 5%. The presence of the stop mutation phenotype (GG) appears to offer a protective effect toward CAD through the lowering of triglyceride and raising the HDL cholesterol concentrations (43). The CAD incidence of the heterozygous (GC) and stop polymorphism (GG) was lower than for the wild-type (CC) phenotype (OR: 0.38; 95% CI: 0.19-0.81) among 189 Japanese subjects. In contrast, a European study of 125 subjects showed no difference in C477G polymorphism between these CAD and control groups (44). Clearly, the number of subjects are too low at this time to make any definitive conclusions about the C477G polymorphism for lipoprotein lipase.

A SNP substitution at nucleotide 9 of exon 2 produces an amino acid change from aspartic acid to asparagine. The allele frequency is rather low at about 1-5% (44). For this reason, this polymorphism has not yet been widely examined in population studies. Another A^G SNP has been identified in nucleotide 291 of exon 6, resulting in the substitution of an asparagine for a serine. The allele frequency of the Asn291Ser substitution is about 2.5%. Preliminary studies have suggested that the heterozygous carriers had increased triglycerides and decreased HDL cholesterol and a predisposition toward ischemic heart disease in women (OR; 1.89; 95% CI: 1.19-3.01) (45).

4.2. PARAOXONASE Paraoxonase (PON) is a 44-kDa calcium-dependent ester hydrolase glycoprotein that is associated with apoA-I and located on the surface of HDL. The enzyme is encoded in the PON1 gene, which is part of a multigene family (PON2 and PON3) with a high degree of sequence homology. Currently, no protein product has been identified for PON2 and PON3 genes. It functions to inhibit the oxidation of LDL by preventing the formation of lipid peroxides and is, therefore, thought to be an important protective factor for atherosclerosis and CAD (46). The gene that is most frequently studied is PON1, where there are two common polymorphisms. A change of a G^A results in an amino acid substitution of a glutamine (Q allele) to an arginine (R) at codon 192. A second polymorphism exists at codon 55, where T^A results in a substitution of a leucine (L allele) for a methionine (M). On the PON2 gene, there is a cysteine^serine substitution at codon 311. The allele frequency for the R and M polymorphism is 30% and 50%, respectively (47). Among Asians, the 192R allele is higher (approx 50%) and the 55L allele is lower (10%) (48). In contrast, among blacks, the 192R is lower (10%) than whites and the 55L allele is higher (73%) (49). These genes exhibit linkage equilibrium (50). The allele frequency for the 331S allele is about 75% (49). Polymorphisms can also be found in the promoter region of PON1, such as a C^T substitution at -108 (51) and a C^G substitution at -907 (52).

The polymorphism of the PON1 gene produces enzymes that have different reactivities toward natural and synthetic substrates. The 192 wild-type allele is more reactive for exogenous nerve gas substrates such as sarin and soman, whereas the R allele is more reactive toward paraoxon and oxon analogs (46). The opposite occurs for the 55 polymorphism, where the wildtype L allele has the highest reactivity toward paraoxone.

There have been several clinical studies that have attempted to correlate the 192 and 55 PON1 polymorphism to cardiac diseases. The data published thus far are contradictory. No association was observed for either polymorphism among Europeans (53,54); however, there have been reports of an association among Asians for the 192R allele (48,55,56). A meta-analysis has suggested that the QR and RR alleles are more prevalent in CHD than controls (OR: 1.44, 95% CI: 1.17-1.77) (57). There have fewer studies conducted for the 311S polymorphism on the PON2 gene. Two studies have suggested a positive correlation with this allele and CAD (58,59).

Because the association between paraoxonase polymorphisms have not produced consistent results, some investigators have suggested that the paraoxonase enzyme activity might be more important than the genotype. In one study, the paraoxonase activity of 417 angiographically proven CAD at 123 nmol/min was substantially lower than the 282 controls at 215 nmol/min (57). In this study, there was no association between PON1 polymorphism and CAD incidence. These authors suggest that there are other factors in addition to the genotype that determine the activity of the final enzyme product.

5. NADH/NADPH OXIDASE

Superoxide (O2^) induces oxidative stress and is an important factor in the development of atherosclerosis and coronary artery disease. O2^ is produced in vascular tissues by NADH/NADPH oxidase. High activity of this enzyme might be equated with increased free-radical production and a higher incidence of CAD. A major component of NADH/NADPH oxidase is the p22phox protein, which is part of a membrane-bound heterodimer and thought to be involved with heme binding. Other subunits of this enzyme include p47phox, p67phox, gp91phox, and Rac-2 proteins. One study showed that the p22phox subunit was upregulated in atherosclerotic coronary arteries vs nonatherosclerotic arteries to suggest a role of this enzyme in the pathophysiology of CAD (60).

A polymorphism exists in the p22phox protein, where a C^T substitution at codon 242 results in the substitution of tyrosine for a histidine at residue 72. There are significant racial differences in the frequency of the 242T allele. Among Caucasians, the frequency is 35% (61), much lower than among Asians at 13% (62). The presence of the 242T allele was associated with significantly reduced vascular NAD(P)H activity of the enzyme (30%) relative to the wild-type (63). One would then presume that the 242T allele would offer some protective effect against the development of CAD.

Studies attempting to correlate the presence of the 242T allele have been discordant. In a study of 402 CAD Japanese patients and controls, the incidence of the 242T (TT and CT) allele was significantly lower than the homozygote 242C wild type (OR: 0.49; 95% CI: 0.28-0.87), suggesting a protective effect for this polymorphism (62). However, different results were observed among Caucasians, where the 242T allele was either not associated with CAD (64) or was associated with progession of atherosclerosis, as measured angiographically by a loss in mean minimum coronary artery lumen diameter (65). One explanation for this apparent discordance is an upregula-tion of antioxidative defenses in Caucasians, despite the production of a defective enzyme.

5.1. ENDOTHELIAL NITRIC OXIDE SYNTHASE Nitric oxide (NO) is an important vasodilating factor that is released together with antioxidants from the endothelium under conditions of laminar arterial blood flow and high shear stress (66). NO also inhibits platelet activation and leukocyte adhesion. Thus, it functions to protect the vessel from the development of atherosclerosis. Under conditions of disturbed blood flow and low shear stress, vasodilators and antioxidant are inhibited, and inflammation and apoptosis are stimulated, making the artery more prone to atherosclerotic lesions. Depressed NO concentrations have been found in early atherosclerosis and overt coronary artery disease. NO is synthesized from l-arginine via a family of nitric oxide synthases (NOS), of which there are at least three isoenzymes: inducible, constitutive neuronal, and constitutive endothelial NOS(eNOS). eNOS is encoded by the NOS3 gene on chromosome 7. Polymorphisms in NOS3 might result in reduced or impaired function of the corresponding synthase.

A well-studied polymorphism occurs at position 894, where a G^T substitution results in a replacement of a glutamine to aspartic acid at position 298, and is thought to be proximal to the active site of the enzyme (67). The frequency for the 298T allele among Caucasians is 30% (67) and about half this amount among Asian (68). The 298T allele is associated with a decreased NO concentration, as measured by its metabolites (NO2- and NO3-) (69). Studies on the association of the 298T allele to CAD have shown an association with CAD (67,68,70); for example, Colombo et al. produced an odds ratio of 2.8 (95% CI: 1.2-6.8) for the 298TT genotype on 315 patients and controls (70). However, like so many other genes, others have contradicted this conclusion (71,72).

A VNTR polymorphism exists in intro 4, where the usual five copies of a 27-bp repeat (allele b) is replaced with four copies (a). The 4a frequency among Caucasians is 15% (73) and 10% among Asians (74). Most studies published to date, however, have failed to find an association with this polymorphism and the presence of CAD among Caucasians (73,75) and Asians (74,76). A less frequent polymorphism in the NOS3 gene is the -786 T^C substitution in the 5' flanking promoter region. There appears to be a linkage between this polymorphism and the 4a VTNR polymorphism. Early studies have shown some association with coronary artery spasm (74) and early coronary artery disease (77).

5.2. APO E GENOTYPES The apoproteins are the protein components of lipoproteins. There are five major apoproteins, labeled A-C, apo E, and apo (a), with subclasses that exist for many of these. Apoproteins function to activate enzymes in lipoprotein metabolism, maintain the structural integrity of the lipoprotein complex, and facilitate the uptake of lipoproteins into cells through surface receptors. Apolipoprotein E is a 34-kDa glycoprotein found in all major lipoproteins except LDL. Apo E plays an important role in the transportation and metabolism of triglycerides, chylomicrons, and VLDL remnants. There are three major isoforms of Apo E, designated E2, E3, and E4, and are encoded by the apo E gene, located on chromosome 19, to produce the e2, e3, and e4 alleles. The e2 contains a cysteine in amino residues 112 and 158, the e3 contains a cysteine and arginine in these positions, and the e4 contains an arginine in both locations. The presence of the e4 genotype is associated with the presence of Alzheimer's disease (78). The frequency of the e2 allele is greatly dependent on the population. In one study of southern Europeans, it was 8%, e3 was about 83%, and e4 was about 9% (79). The frequency of the e4 allele is higher among African blacks.

Studies have shown that individuals with the e2 alleles have the lowest LDL concentration and highest apo E, whereas those with the e4 alleles have the reverse. It is possible that the apo E4 protein binds to the LDL receptor thereby interfering with the clearance of LDL from the circulation. Because LDL is recognized as a risk factor for CAD, studies have been conducted to determine if apo E genotypes are linked. A meta-analysis conducted by Wilson et al. combining the results of 9 published observational studies in men (n = 1971 cases) and 4 studies (n = 181 cases) in women (80). The presence of the e4 allele and presence of coronary heart disease produced an odds ratio of 1.38 (95% CI: 1.22-1.57) and 1.26 (95% CI: 1.13-1.41), respectively. In contrast, Frikke-Schmidt et al. found that the e4 genotype was significantly associated with CHD for men (n = 693 cases) but not women (n = 247 cases) (81).

Polymorphisms in other parts of the apolipoprotein E gene have been identified (i.e., within the promoter region). These include a A^T change at position -491, C^T change at position -427, and a G^T change at -219 (82). In a study of 1245 AMI case and controls, the -219T polymorphism was associated with increased risk for AMI (OR: 1.29; 95% CI: 1.09-1.52). This polymorphism was not linked with the other polymorphisms in the promoter gene or with the e alleles.

5.3. METHYLENE TETRAHYDROFOLATE REDUCTASE Homocysteine is formed from the metabolism of methionine, as shown in Fig. 2. Homocysteine is metabolized back to methionine with vitamins B6 and B12 and folate as cofactors, or transsulfurated to cysteine. High concentrations of homoc-syteine have been implicated as a risk factor for a number of diseases such as peripheral vascular disease, stroke, and CAD. A meta-analysis was conducted examining the relationship of plasma homocysteine and development of CAD (83). Of 14 case-control studies involving over 4700 patients, the odds ratio for development of CAD was 1.7 (95% CI: 1.5-1.9). Clinical trials are being conducted to determine if lowering homocysteine concentrations will result in a reduced risk for CAD. Commercial assays for homocysteine are available and are routinely used in clinical practice. The upper limit of the normal range for homocysteine in plasma is 15 |imol/L. However, a lower concentration that is within the normal range (e.g., 12 |imol/L) might be a more appropriate limit for low or no disease risk.

A major cause of hyperhomocysteinemia is dietary deficiency of vitamins B6 and B12 and folate. As such, the United States has recently introduced folate fortification in grain in an attempt to reduce the prevalence of dietary-induced hyperhomocysteinemia. Folate supplementation is inexpensive and has no side effects. The genetic basis of hyperhomocysteinemia has been known for many years and was the principal basis behind the hypothesis that abnormal homocysteine metabolism led to premature atherothrombotic events. Rare causes of homocystinuria include genetic deficiencies of methionine synthase and cystathionine P-synthase, important enzymes in the degradation of homocys-teine. Pathologic changes within the coronary and cerebral arteries in children with congenital homocystinuria (i.e., intimal damage, hyperplasia of smooth muscles, lipoprotein aggregates, etc.) are identical to that found in adult atherosclerotic plaques.

A severe deficiency in methylenetetrahydrofolate reductase (MTHFR) can also cause hyperhomocysteinemia and homo-cystinuria. MTHFR is a homodimer of 77-kDa subunits and is a key enzyme in the folate cycle that generates 5-methyl-tetrahydrofolate, the substrate for methionine synthase (Fig. 2). The severe MTHFR deficiency is rare with only a few dozen cases described worldwide. At least 18 different point mutations have been demonstrated to produce these severe forms of MTFHR deficiency.

Fig. 2. Metabolic pathway for homocysteine. Methionine from the diet and metabolism of proteins is converted to S-adensylmethionine (S-AM), S-adenosylhomocysteine (S-AH), and then to homocysteine. With 5-methyl-tetrahydrofolate (5-THF) as a substrate, homocysteine is then remethylated back to methionine and tetrahydrofolate by methionine synthase (MS). Using vitamins B6 and B2 as cofactors, respectively, THF remethylated to 5-THF by serine-glycine hydroxymethyltransferase (HMT) and methylenetrahydrofolate reductase (MTHFR), respectively. Polymorphisms in the MTHFR gene is a subject of many clinical investigations. In an alternate pathway, homocysteine is also converted to cystathionine by cystathionine P-synthase (CS) and then to cysteine.

Fig. 2. Metabolic pathway for homocysteine. Methionine from the diet and metabolism of proteins is converted to S-adensylmethionine (S-AM), S-adenosylhomocysteine (S-AH), and then to homocysteine. With 5-methyl-tetrahydrofolate (5-THF) as a substrate, homocysteine is then remethylated back to methionine and tetrahydrofolate by methionine synthase (MS). Using vitamins B6 and B2 as cofactors, respectively, THF remethylated to 5-THF by serine-glycine hydroxymethyltransferase (HMT) and methylenetrahydrofolate reductase (MTHFR), respectively. Polymorphisms in the MTHFR gene is a subject of many clinical investigations. In an alternate pathway, homocysteine is also converted to cystathionine by cystathionine P-synthase (CS) and then to cysteine.

There are five common polymorphisms in MTHFR. The most common and widely studied of these is a C^ T substitution in nucleotide 677 of exon 4. This polymorphism has an allele frequency of about 35% and homozygosity frequencies of 10-15%. The homozygous genotype is associated with <20% of the normal MTHFR activity and thermolability in lymphocyte extracts resulting in low plasma folate concentrations and a mild hyperhomocysteinemia. In the study by Dunn et al., subjects under 50 yr old had a mean homocysteine concentration that varied from 15.8 to 15.9 for the homozygote wild type and heterozygote, which increased to 21.4 |imol/L for the homozygous C677T variant (p < 0.0001) (84).

There have been many studies that have attempted to correlate MTHFR polymorphism to CAD, with conflicting results. A meta-analysis of 10 studies on 5644 patients with CAD produced an odds ratio of 1.30 (95% CI: 1.11-1.52) for CAD and the presence of the 677T variant (3). This association was confirmed on a larger meta-analysis of 40 studies: 11,162 CAD cases and 12,758 controls (OR: 1.15, 95% CI: 1.05-1.28) (85). Among patients with renal failure, the 677T variant was associated with a significantly higher incidence of cardiovascular disease and homocysteine concentrations than patients without such a history (86). No association has been observed for this MTHFR mutation and ischemic stroke (3,87). A major limitation in many of these studies is the failure to consider plasma folate concentrations as a confounding variable. It might be possible that a reduced MTHFR activity can be compensated for by the presence of high plasma folate potentially normalizing the homocysteine concentration. The correlation of MTFHR TT polymorphmism with CAD might be even more significant if the studied population had been folate deficient.

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