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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1074-1081.)
© 1997 American Heart Association, Inc.

Articles

Urbanization Elicits a More Atherogenic Lipoprotein Profile in Carriers of the Apolipoprotein A-IV-2 Allele Than in A-IV-1 Homozygotes

Hannia Campos; José López-Miranda; Carmen Rodríguez; Marta Albajar; Ernst J. Schaefer; ; José M. Ordovás

From the Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University (J.L.-M., C.R., M.A., E.J.S., J.M.O.), and the Department of Nutrition, Harvard School of Public Health (H.C.), Boston, Mass.

Correspondence to Hannia Campos, PhD, Department of Nutrition, Room 353A, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115. E-mail hphac{at}gauss.bwh.harvard.edu

	Abstract

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Abstract Coronary heart disease (CHD) is increasing in developing countries, particularly in urban areas. The impact of urbanization and apolipoprotein (apo) A-IV genetic polymorphism on plasma lipoproteins was studied in 222 men and 236 women from rural and urban Costa Rica. The apoA-IV allele frequencies were 0.937 for apoA-IV-1 and 0.062 for apoA-IV-2. Significant interactions between the apoA-IV polymorphism and area of residence (rural versus urban) were detected for HDL cholesterol (P=.003), apoA-I (P=.05), LDL particle size (P=.01), and LDL/HDL cholesterol ratio (P=.005). Urban compared with rural carriers of the apoA-IV-2 allele had significantly lower plasma HDL cholesterol (0.95 versus 1.17 mmol/L) and apoA-I (980 versus 1140 mg/L), a significantly higher LDL/HDL cholesterol ratio (3.35 versus 2.39), and significantly smaller LDL particles (258 versus 263 Å). In contrast, no significant rural-urban differences for these parameters were found in apoA-IV-1 homozygotes. Regardless of their apoA-IV phenotype, urban residents consumed more saturated fat (P=.02) and smoked more cigarettes per day (P=.03) than rural residents. A significant interaction between saturated fat intake and apoA-IV phenotype was found for HDL cholesterol (P<.0003) and LDL/HDL cholesterol ratio (P<.003). Increased saturated fat intake (13.6% versus 8.6% of calories) was significantly associated with 6% higher HDL cholesterol and no change (0.7%) in LDL/HDL cholesterol ratio in apoA-IV-1 homozygotes and with 19% lower HDL cholesterol and 37% higher LDL/HDL cholesterol ratio among carriers of the apoA-IV-2 allele. Smokers (1 cigarette per day) had significantly lower HDL cholesterol (P<.005) and apoA-I (P<.01) concentrations than nonsmokers (<1 cigarette per day), particularly among carriers of the apoA-IV-2 allele (-19% and -13%) compared with apoA-IV-1 (-4% for both). After taking these lifestyle characteristics into account, the areas of residence by phenotype interactions for plasma lipoprotein concentrations were no longer statistically significant. Lifestyles associated with an urban environment, such as increased smoking and saturated fat intake, elicit a more adverse plasma lipoprotein profile among Costa Rican carriers of the apoA-IV-2 allele than in apoA-IV-1 homozygotes. Therefore, under the conditions studied, persons with the apoA-IV-2 allele may be more susceptible to CHD.

Key Words: gene polymorphism • LDL particle size • dietary intake • smoking • cholesterol

	Introduction

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As developing countries control communicable diseases and promote urban, industrial, and commercial expansion, they encounter new health problems.¹ For example, CHD and its risk factors have been increasing progressively in Costa Rica, and CHD is now the major cause of death,^{2 3} with comparable mortality rates to those in the United States and Europe.³ Plasma LDL cholesterol concentrations are low in developing countries such as Costa Rica, where dietary intakes are low in fat and high in carbohydrate compared with industrialized countries.^{2 4} In contrast, Costa Ricans have high concentrations of plasma triglycerides, low concentrations of HDL cholesterol, and a predominance of small LDL particles.^{2 4} Since this lipoprotein phenotype is associated with CHD,^{5 6} lipids other than LDL cholesterol concentrations may be more important indicators of CHD in Costa Rica, as well as in other nonindustrialized societies. Genetic and environmental factors such as diet, smoking, and obesity are involved in determining the concentrations of plasma lipoproteins.⁷ Therefore, studies on gene-environment interactions become of particular interest in the context of urbanization, where environmental exposure varies over a fixed genetic substrate.

Intravascular lipoprotein metabolism is regulated by a family of proteins called apolipoproteins.⁸ One of these, apoA-IV, is a 46-kD protein synthesized in the small intestine.⁹ It has been proposed that apoA-IV plays an important role in the metabolism of triglyceride-rich lipoproteins and HDL. Several studies show that apoA-IV mediates the activation of lipoprotein lipase,¹⁰ thereby promoting triglyceride-rich lipoprotein clearance and HDL formation, and activates LCAT,^{11 12} an essential step in reverse cholesterol transport.¹³ Genetic variants of apoA-IV have been identified, with the major allele in all populations being apoA-IV-1 (gene frequency 0.88 to 0.99).^{14 15 16 17 18 19 20} A common variant, apoA-IV-2, results from the substitution of histidine for glutamine at position 360.²¹ The apoA-IV-2 allele frequency varies worldwide from being completely absent in Japan to 0.11 in Iceland.^{14 15 16 17 18 19 20} In some population studies,^{17 18} the apoA-IV-2 allele has been associated with higher HDL cholesterol concentrations, but others have found no association.^{19 20} In dietary intervention trials, the apoA-IV-2 allele modulates the effect of dietary fat and cholesterol on plasma HDL and LDL cholesterol concentrations.^{22 23} Furthermore, obese NIDDM carriers of the apoA-IV-2 allele are at increased risk of myocardial infarction compared with obese NIDDM apoA-IV-1 homozygotes.²⁴

Data are scarce on the prevalence of CHD risk factors in subjects living in different environments within developing countries. Urban compared with rural residents of Costa Rica have reduced physical activity and increased obesity, cigarette smoking, and dietary fat intake.^{2 4} These factors were associated with a more atherogenic plasma lipoprotein profile, defined by increased concentrations of plasma triglyceride and LDL cholesterol and decreased HDL cholesterol.^{2 4} It is likely that the change in lifestyle that accompanies urbanization in developing countries is associated with the observed increase in CHD, particularly among subjects with genetic susceptibility. We have tested the hypothesis that the apoA-IV genetic polymorphism is associated with the effect of environment (rural versus urban) on the plasma lipoprotein profile.

	Methods

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Study Population
This study was carried out in the rural and urban areas of the canton (county) of Puriscal, Costa Rica, which comprises about 800 km². The population is predominantly mestizo (mixture of white and Amerindian ethnic groups). While data are scarce, anthropological research suggests that racial mixing in Costa Rica took place before the 19th century, when a comparatively homogeneous Hispanic-American society was formed.²⁵ Stratified random sampling was performed in the rural and urban areas to obtain a similar number of participants in each group, as previously described.^{4 26} This process was achieved using the Costa Rican Census and Statistics Bureau maps and population information for the definition of area of residence. The urban area was defined as the canton's capital, Santiago, which has a population of 8000 people living in 918 households. The rural area included 150 small localities with <500 inhabitants each, distributed in 3415 identified households. Eligible subjects were men and nonpregnant women aged 20 to 65 years, from 260 randomly selected households.

Data and Sample Collection
Data collection was coordinated from the fieldwork station in Puriscal at the Institute of Health Research (INISA), University of Costa Rica, as previously described.^{4 26} Trained field workers visited participants in their households for recruitment, where subjects signed an informed consent form. Subsequent appointments were made for data collection, consisting of a health questionnaire including sociodemographic characteristics, smoking status, and medication use. Dietary assessment was carried out using a food-frequency questionnaire previously validated in this population.⁴ Anthropometric measurements including height and weight were taken and a fasting blood sample was collected, always in the morning. Blood tubes were immediately stored at 4°C and centrifuged at 2500 rpm for 20 minutes at 4°C to isolate and aliquot plasma. HDL supernatants were obtained after precipitation of apoB-containing lipoproteins with dextran sulfate–Mg²⁺. All plasma aliquots were then stored at -70°C until they were transported to the USDA Human Nutrition Research Center on Aging at Tufts University in Boston, Mass, for the determination of total triglyceride; total, LDL, and HDL cholesterol; apoA-I; apoB; LDL particle size; and apoA-IV phenotype.

Laboratory Analyses
Plasma cholesterol, triglyceride, and HDL cholesterol concentrations were measured using an Abbott Diagnostics ABA-200 bichromatic analyzer and Abbott A-Gent enzymatic reagents. LDL cholesterol was estimated for all subjects by using the equation of Friedewald et al.²⁷ Total cholesterol, triglyceride, and HDL cholesterol assays were standardized through the Centers for Disease Control Lipid Standardization Program. Plasma apoB and apoA-I quantification was performed with noncompetitive enzyme-linked immunosorbent assay. Standards used for these assays were calibrated with purified protein standards assayed by amino acid analysis. LDL particle size was determined by 2% to 16% gradient gel electrophoresis as previously described.⁵ For comparison with previous studies on LDL particle size in whites,^{5 6} we classified the population into two LDL size groups. The group with small LDL (pattern B) was defined by the predominance of LDL particles with diameter <260 Å and large LDL (pattern A) by the predominance of LDL particles 260 Å.^{5 6} ApoA-IV phenotyping was performed by isoelectric focusing of whole plasma followed by immunoblotting¹⁷ with a specific anti-human apoA-IV antiserum. The frequencies of the apoA-IV alleles were estimated by the gene-counting method. The apoA-IV-1 phenotype included 406 subjects homozygous for the apoA-IV-1 allele, whereas the apoA-IV-2 phenotype included 47 apoA-IV-1/2 heterozygous and 5 apoA-IV-2/2 homozygous subjects. One subject with an apoA-IV-1/3 phenotype was identified.

Statistical Analyses
Statistical analyses were performed with the Statistical Analysis Systems software (SAS, Inc). All variables were examined for outliers and sample distribution using the univariate/plot procedure. Logarithmic transformations were used for statistical analyses of plasma triglyceride concentrations because of the skewed distribution. Unadjusted rural-urban comparisons for continuous variables including age, BMI (calculated as weight in kilograms divided by height in meters squared), plasma lipoproteins, and LDL particle size (Å) were carried out using two-tailed t tests. Pearson correlation coefficients were calculated to test the association between age, BMI, LDL particle size, and plasma lipoproteins. Differences in distribution of categorical variables, including medication use (yes versus no), smoking status (nonsmokers <1 versus smokers 1 cigarette per day), LDL size group (pattern A versus B), area of residence (rural versus urban), and apoA-IV phenotype (A-IV-1 versus A-IV-2), were tested using the Mantel-Haenszel ² statistic. Mean plasma lipoprotein concentrations in the heterozygous and homozygous carriers of the apoA-IV-2 allele were similar and therefore combined as one group for analyses. The general linear model procedure with the least-square-means option was used for the analyses of covariance. Age, sex, and BMI were included as covariates in all our models because of their association with plasma lipoprotein concentrations, LDL particle size, dietary intake, and smoking status.^{2 4 26 28 29} The probability values of the main effects and interaction terms were used for hypothesis testing. The multiple t test analyses carried out with the least-squares option are presented to describe the between-group comparisons. It is possible that due to multiple t tests, some of the between-group comparison results are due to chance. The first analysis of covariance model was used to test the effect of area of residence, apoA-IV phenotype, and area by phenotype interactions on various parameters, including plasma lipoproteins, LDL particle size, dietary intake, and cigarette smoking. The second set of covariance analyses tested whether rural-urban differences and/or area by phenotype interactions in plasma lipoproteins could be explained by differences in smoking status or dietary fat intake and the interactions of smoking by phenotype and dietary fat by phenotype. Dietary intake class variables (low versus high) were created by dividing the population into two groups using the median level for each dietary fat variable. Total, saturated, monounsaturated, and polyunsaturated dietary fat, as a percent of caloric intake, and dietary cholesterol, expressed in milligrams per 1000 kcal, were examined. Each class category (smoking or dietary composition) and its interaction by phenotype was tested individually in a model that also included area, phenotype, and interaction area by phenotype, age, sex, BMI, and caloric intake. Caloric intake was always included in the models testing the effects of fat intake because of the known association between dietary composition and total caloric intake.²⁸ When smoking or dietary intake were significantly associated with plasma lipoproteins, they were simultaneously included in the model. For example, smoking status was included in the model testing for the effect of saturated fat on LDL cholesterol, HDL cholesterol, and LDL/HDL cholesterol ratio, and saturated fat intake was included in the model testing for the effect of smoking status on HDL cholesterol and apoA-I. Since the area by phenotype interaction was not statistically significant when environmental factors were adjusted for, this interaction was not included in the final models. All unadjusted data are presented as arithmetic mean±SD and adjusted data as mean±SEM, except data on triglyceride concentrations, which are presented as geometric mean±SD or SEM.

	Results

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Participation rates were very similar in the rural and urban areas in women (95%, n=123 and 92%, n=120, respectively) and men (85%, n=111 in both areas). Enough plasma sample volume for apoA-IV phenotyping was not available in 6 women; therefore, the final sample size in women was 237. The one female subject with an apoA-IV-1/3 phenotype and subjects taking hormones (n=39), which are medications known to affect plasma lipoproteins (n=40), or both (n=2) were not included in the analyses in which plasma lipoprotein concentrations were examined. Proportionally, there were more apoA-IV-2 carriers on medication (15.4%, n=8) than apoA-IV-1 (8.4%, n=34), but the distribution was not statistically significant (P<.09).

ApoA-IV Allele Frequencies and Rural-Urban Differences in Plasma Lipoproteins
Table 1 shows the apoA-IV allele frequencies in Costa Rica and other selected populations. The frequency of the apoA-IV-2 allele was lower in the urban than the rural area, but the differences were not statistically significant (P=.2). The apoA-IV-2 allele frequency in Costa Rica (0.062) was lower than in white populations, higher than in blacks, Amerindians, or Japanese, and very similar to Mexican Americans. The unadjusted subject characteristics by area of residence are shown in Table 2. Urban area of residence was significantly (P<.05) associated with increased BMI, cigarettes smoked per day among smokers, plasma triglyceride, total cholesterol, LDL cholesterol, apoB, and the LDL/HDL cholesterol ratio. The percent of current smokers was higher in the urban (32.4%) than the rural (25.5%) area but not statistically significant (P=.14). No significant rural-urban differences were found for age, HDL cholesterol, or LDL particle size. The adjusted plasma lipoprotein concentrations by area of residence and apoA-IV phenotype are shown in Table 3. After adjustments for age, sex, and BMI were made, subjects living in the urban area still had significantly (P<.05) higher plasma triglyceride, total cholesterol, LDL cholesterol, apoB, and LDL/HDL cholesterol ratio. In addition, urban subjects exhibited significantly lower HDL cholesterol (P=.01) and apoA-I (P=.02) concentrations than rural subjects.

View this table: [in this window] [in a new window]   Table 1. ApoA-IV Allele Frequencies in Costa Rica and Other Selected Populations

View this table: [in this window] [in a new window]   Table 2. Unadjusted Subject Characteristics by Area of Residence

View this table: [in this window] [in a new window]   Table 3. Plasma Lipoprotein Concentrations by ApoA-IV Phenotype in Rural and Urban Puriscal, Costa Rica

Impact of ApoA-IV Polymorphism and Area of Residence on Plasma Lipoprotein Concentrations and LDL Size
While the effect of the apoA-IV phenotype was not significant for any plasma lipoprotein parameter studied, significant interactions between the apoA-IV polymorphism and area of residence were detected for HDL cholesterol (P=.003), apoA-I (P=.05), and LDL/HDL cholesterol ratio (P=.005) (Table 3). Compared with rural apoA-IV-2 carriers, urban residents carrying the apoA-IV-2 allele had lower HDL cholesterol (0.95 versus 1.17 mmol/L; P=.004) and apoA-I (980 versus 1140 mg/L; P=.02) and a higher LDL/HDL cholesterol ratio (3.35 versus 2.39; P=.0006). However, no significant rural-urban differences for these parameters were found in subjects with the apoA-IV-1 phenotype. Urban carriers of the apoA-IV-2 allele also had significantly (P<.05) lower HDL cholesterol and apoA-I and a higher LDL/HDL cholesterol ratio than apoA-IV-1 homozygotes, whether rural or urban. While no significant interaction was found for triglyceride, total cholesterol, LDL cholesterol, and apoB, the rural-urban differences in these parameters were greater in the apoA-IV-2 carriers than in apoA-IV-1 homozygotes. To further investigate these gene-environment interactions on biochemical markers of CHD, we determined the predominance of small LDL particle size, a common plasma lipoprotein phenotype recently associated with increased CHD.⁶ While this trait was not significantly associated with area of residence or apoA-IV phenotype, we found a significant gene-environment interaction (P=.01) in which urban residents carrying the apoA-IV-2 allele have significantly smaller LDL particles than rural residents. Urban residents with the apoA-IV-I phenotype had decreased (P=.05) predominance of small LDL pattern B (35%) compared with rural residents (46%). In contrast, urban apoA-IV-2 carriers had increased (P=.06) predominance of small LDL (60%) compared with rural carriers (29%). Small LDL particle size was significantly (P=.0001) associated with decreased HDL cholesterol (r=.56), increased triglyceride (r=.65), and an increased LDL/HDL cholesterol ratio (r=.30).

Impact of ApoA-IV Polymorphism and Lifestyle Characteristics on Plasma Lipoproteins
To test whether phenotype by area differences in plasma lipoproteins could be explained by lifestyle differences between phenotypes, we examined dietary intake and smoking status by area of residence, apoA-IV polymorphism, and area by polymorphism interactions. Table 4 shows the average daily nutrient intake and smoking habits by apoA-IV phenotype and area of residence adjusted for age, sex, and BMI. No significant phenotype effect or phenotype-environment interaction was detected for any of these environmental variables. Regardless of their apoA-IV phenotype, urban residents consumed significantly (P<.02) more protein, total fat, saturated fat, and monounsaturated fat and less carbohydrate than rural residents. A higher prevalence of smokers was found in the urban (32%) than the rural area (25%), and smokers in the urban area smoked more cigarettes per day compared with rural (15.6 versus 9.1 cigarettes per day, P=.03). Among the lifestyle characteristics studied, saturated fat intake and smoking status explained the area effect and/or area by phenotype interactions on LDL cholesterol, HDL cholesterol, apoA-I, and the LDL/HDL cholesterol ratio. No significant phenotype by dietary fat or smoking status interactions were detected for triglyceride, total cholesterol, or apoB concentrations. Fig 1 shows the effect of saturated fat intake on LDL cholesterol and HDL cholesterol concentrations. Increased saturated fat intake (13.6% versus 8.6% of calories) was significantly (P<.05) associated with higher LDL cholesterol, particularly among carriers of the apoA-IV-2 allele (3.02±0.12 versus 2.67±0.12 mmol/L, 13% difference), compared with apoA-IV-1 homozygotes (2.95±0.06 versus 2.73±0.07 mmol/L, 8% difference), but no significant interaction was detected (P=.6). In contrast, a significant interaction (P=.0003) between saturated fat intake and apoA-IV phenotype was detected for HDL cholesterol. Compared with a low saturated fat dietary intake, increased saturated fat was significantly associated with 6% higher HDL cholesterol concentrations (1.12±0.02 versus 1.06±0.02 mmol/L, P=.03) in apoA-IV-1 homozygotes and with 19% lower HDL cholesterol (0.97±0.05 versus 1.20±0.06 mmol/L, P=.003) among carriers of the apoA-IV-2 allele. As a result of these individual effects of saturated fat intake on LDL and HDL cholesterol concentrations, a significant apoA-IV by saturated fat–intake interaction was detected for the LDL/HDL cholesterol ratio (P=.003). Carriers of the apoA-IV-2 allele on a high saturated fat diet had significantly higher (P<.01) LDL/HDL cholesterol ratio (3.20±0.18) than carriers in the low saturated fat–diet group (2.33±0.20) and apoA-IV-1 homozygotes, whether on a low or high saturated fat diet (2.71±0.07 and 2.73±0.07, respectively). Saturated fat intake was not associated with the LDL/HDL cholesterol ratio in apoA-IV-1 homozygotes, since both LDL and HDL cholesterol concentrations were proportionally increased on the high saturated fat diet. On average, 8.6% of calories was consumed as saturated fat in the low compared with 13.6% in the high saturated fat group. Subjects in the high saturated fat–intake group also reported decreased carbohydrate intake (percent of calories) and increased intakes of total, monounsaturated, and polyunsaturated fat and of cholesterol. The effects of these other fats and their interactions with apoA-IV phenotype were not independently associated with plasma lipoprotein concentrations in the multiple regression models described in the "Methods" section. The effects of dietary intake and smoking status on LDL particle size were less striking. Compared with low total fat intake (<28.7% of calories), increased total fat intake (28.7% of calories) was associated with larger LDL particles in apoA-IV-1 homozygotes (260 versus 262 Å, P=.0006), and with smaller LDL particles in carriers of the apoA-IV-2 allele (262 versus 260 Å). The total fat intake by phenotype interaction was not statistically significant (P=.06), and the interaction of area by phenotype was still statistically significant (P=.01) after adjusting for dietary fat. No significant interaction between saturated fat intake and apoA-IV phenotype was found for LDL size.

View this table: [in this window] [in a new window]   Table 4. Average Daily Nutrient Intake and Smoking Habits by ApoA-IV Phenotype in Rural and Urban Puriscal, Costa Rica

View larger version (37K): [in this window] [in a new window]   Figure 1. Effect of a higher saturated fat diet on LDL and HDL cholesterol concentrations by apo A-IV phenotype. Bar graph shows the percent difference in LDL cholesterol and HDL cholesterol concentrations between low vs high saturated fat intake (mean±SD, 8.6±1.8% vs 13.6±1.9% calories) consumed by A-IV-1 vs A-IV-2 residents from Puriscal, Costa Rica. Low and high saturated fat intake groups were defined as below and above the median saturated fat intake level in the studied population (11% of calories). The values for saturated fat and the saturated fat group by phenotype interaction were P=.03 and P=.6 for LDL cholesterol and P=.04 and P=.0003 for HDL cholesterol. The model also included the class variables area of residence, phenotype, and smoking status and the covariates age, gender, BMI, and caloric intake. *P<.05, P<.005 for between group comparisons of low vs high saturated fat.

The effects of smoking on HDL cholesterol and apoA-I concentration are shown in Fig 2. Compared with nonsmokers, smokers (mean=12.6 cigarettes per day) had significantly lower HDL cholesterol (P<.005) and apoA-I concentrations (P<.01), particularly among carriers of the apoA-IV-2 allele (0.93±0.07 versus 1.15±0.05 mmol/L, -19%; and 969±62 versus 1120±39 mg/L, -13%, respectively) compared with apoA-IV-1 (1.07±0.03 versus 1.12±0.02 mmol/L, -4%; and 1087± 21 versus 1133±13 mg/L, -4%, respectively). The interaction between smoking and phenotype was not statistically significant for HDL cholesterol (P=.06) or apoA-I (P=.15) or any of the other lipoprotein parameters (P>.20).

View larger version (34K): [in this window] [in a new window]   Figure 2. Effect of cigarette smoking on HDL cholesterol and apo A-I concentrations by apo A-IV phenotype. Bar graph shows the percent difference in HDL cholesterol and apo A-I concentrations between nonsmokers (<1 cigarette per day) vs smokers (1 cigarettes per day) (mean±SD cigarette per day among smokers, 12.6±10), A-IV-1 vs A-IV-2 residents form Puriscal, Costa Rica. The values for smoking status and the smoking by phenotype interaction were P=.005 and P=.06 for HDL cholesterol and P=.01 and P=.1 for apo A-I cholesterol concentrations. The model also included the class variables area of residence, phenotype, and saturated fat intake and the covariates age, gender, BMI, and caloric intake. *P<.05, P<.01 for between group comparisons of smokers vs nonsmokers.

	Discussion

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We have shown that urban residents carrying the apoA-IV-2 allele had significantly lower HDL cholesterol and apoA-I concentrations and higher LDL/HDL cholesterol ratio than rural apoA-IV-2 carriers. A predominance of small LDL particle size was found among urban apoA-IV-2 carriers compared with rural. The urban apoA-IV-2 carriers also had adverse concentrations of these lipoprotein measurements compared with apoA-IV-1 homozygotes, whether rural or urban. In contrast, area of residence did not have a significant effect on these lipoproteins among apoA-IV-1 homozygotes. These data demonstrate an interaction between apoA-IV and environment that is significant for HDL cholesterol, apoA-I, LDL/HDL cholesterol ratio, and LDL particle size. Other lipoprotein parameters, such as triglyceride, total cholesterol, LDL cholesterol, and apoB, were significantly higher in urban than in rural subjects. For these latter parameters, the rural-urban difference was greater in the apoA-IV-2 carriers than in apoA-IV-1 homozygotes, although not significantly. Regardless of their apoA-IV phenotype, urban residents consumed more saturated fat and smoked more cigarettes per day than rural residents. These two unfavorable urban characteristics were identified as significant determinants of the lower HDL cholesterol and apoA-I concentrations and the higher LDL/HDL cholesterol ratio among urban carriers of the apoA-IV-2 allele. The finding of low HDL cholesterol concentrations, increased LDL/HDL cholesterol ratio, and predominance of small LDL particle size in urban apoA-IV-2 carriers indicates that the apoA-IV-2 allele may cause increased susceptibility to CHD in the presence of unfavorable environmental risk factors such as increased smoking and saturated fat intake.

The frequency of the major apoA-IV allele (apoA-IV-1) was 0.937 in Costa Rica, whereas the most common variant (apoA-IV-2) had a frequency of 0.062. The frequency of the apoA-IV-2 allele in our population was lower than in whites, in whom this allele is commonly found,^{15 17 20} but higher than in Amerindian populations, in which the apoA-IV-2 allele is completely lacking.¹⁴ Our results were very similar to those found in Mexican Americans¹⁹ and support strong admixture between white and Amerindian ethnic groups in Costa Rica. These findings are expected, as European settlement took place before the 19th century in this population.²⁵

Our study suggests that the apoA-IV-2 allele lowers HDL cholesterol and apoA-I concentrations when carriers are exposed to an urban environment characterized by increased smoking and saturated fat intake. Previous population studies have been inconsistent regarding the effect of the apoA-IV-2 allele on HDL cholesterol concentrations. Two studies by Menzel et al^{17 18} in European populations reported that carriers of the apoA-IV-2 allele have significantly higher HDL cholesterol concentrations than apoA-IV-1 homozygotes, but their analyses did not take into account the potentially confounding effects of BMI and other environmental factors on HDL cholesterol concentrations. A more comprehensive study by Ehnholm et al²⁰ pooled data from five different regions of Europe and found no overall correlation between the apoA-IV-2 allele and HDL cholesterol concentrations in young subjects between 18 and 26 years of age. However, this study showed variable regional effects of the apoA-IV-2 allele. Carriers of the apoA-IV-2 allele living in industrialized cities of Finland and Great Britain, where CHD mortality is high, exhibited 11% lower HDL cholesterol concentrations than carriers living in Southern European Mediterranean cities, where CHD mortality is low. In contrast, no difference (1%) in HDL cholesterol concentrations was found among apoA-IV-1 homozygotes living in these areas. Some environmental factors that could be responsible for this effect in Finland and Great Britain, as well as in urban Costa Rica, are smoking, saturated fat intake, obesity, and reduced physical activity.^{2 4} Our data indicate that carriers of the apoA-IV-2 allele may be more susceptible than apoA-IV-1 homozygotes to the lowering effects of smoking on HDL cholesterol and apoA-I.³⁰ The apoA-IV-2 allele may also modulate the effect of dietary fat on plasma LDL and HDL cholesterol concentrations.^{22 23} Consistent with these observations in dietary intervention trials, we found a significant interaction between saturated fat intake (8.6% versus 13.6% of calories) and the apoA-IV phenotype on HDL cholesterol and the LDL/HDL cholesterol ratio. Increased saturated fat intake in our study resulted in a 0.22-mmol/L increase in LDL cholesterol, a 0.06-mmol/L increase in HDL cholesterol, and no significant difference in the LDL/ HDL cholesterol ratio among apoA-IV-1 homozygotes. In contrast, high saturated fat intake was associated with a 0.35-mmol/L increase in LDL cholesterol, a 0.23-mmol/L decrease in HDL cholesterol, and 37% increase in the LDL/HDL cholesterol ratio among carriers of the apoA-IV-2 allele. Using the Mensink and Katan³¹ equations derived from dietary intervention trials to predict the expected change in plasma lipoproteins induced by changes in dietary fat intake, one would expect that a 5% increase in saturated fat (as observed in our study) would raise LDL cholesterol 0.17 mmol/L and HDL cholesterol 0.06 mmol/L, with no significant change in the LDL/HDL cholesterol ratio. The expected differences in lipoprotein concentrations predicted using these equations were very similar to the observed effects of saturated fat on LDL and HDL cholesterol concentrations in apoA-IV-1 homozygotes in our study but very different from the observed results in carriers of the apoA-IV-2 allele. These observations underscore the importance of genetic background and environmental factors in determining the effect of apoA-IV on HDL metabolism and CHD risk. Rewers et al²⁴ have shown that in obese patients with NIDDM, carriers of the apoA-IV-2 allele are at increased risk of myocardial infarction compared with apoA-IV-1 homozygotes.

One unique finding in the Costa Rican population was the differential effect of the apoA-IV polymorphism on LDL particle size in the context of an urban compared with a rural lifestyle. The prevalence of subjects with a predominance of small LDL particles was 60% in urban compared with 29% in rural apoA-IV-2 carriers. In contrast, the prevalence of this trait among apoA-IV-1 homozygotes was 35% in the urban compared with 46% in the rural area. The predominance of small LDL is associated with CHD risk^{5 6} and is produced by obesity and sedentary habits.⁶ Preliminary evidence suggests linkage of the small-LDL phenotype to the region of the apoA-I/C-III/A-IV gene cluster on chromosome 11.³² As urban residents from Costa Rica are more obese and sedentary than rural residents,^{2 4} our current data suggest that apoA-IV is involved in the response of LDL to these environmental factors, producing an adverse effect on particle size among apoA-IV-2 carriers. The expression of small LDL is also influenced by low fat, high carbohydrate diets.³³ Compared with populations in the United States,³⁴ the prevalence of small LDL is higher in Costa Rica in both men (52 versus 33) and women (30 versus 10). This difference is probably determined in part by the consumption of diets lower in total fat among Costa Ricans.⁴ Interestingly, urban carriers of the apoA-IV-2 allele were more likely to express the small LDL phenotype despite higher dietary fat intake. This association is probably influenced by the lower HDL cholesterol concentrations found among carriers of the apoA-IV-2 allele on high saturated fat diets and the strong association between small LDL particle size and reduced HDL cholesterol concentrations.^{5 6}

An intriguing metabolic explanation for the link between apoA-IV-2, low HDL cholesterol, small LDL, and CHD is through LCAT, an essential enzyme that esterifies HDL cholesterol and acts early in the antiatherogenic process of reverse cholesterol trans-port.¹³ Plasma lipoproteins containing apoA-IV have the highest LCAT activity per microgram of protein.³⁵ A high LCAT activity could indicate that apoA-IV plays an important role in the process of reverse cholesterol transport despite the low plasma concentration of apoA-IV compared with apoA-I.⁸ The Gln₃₆₀His substitution that characterizes the apoA-IV-2 variant is localized in the amphipathic helix, which may be involved in lipid binding and LCAT activation.³⁶ This structural difference could be responsible for the decreased ability of apoA-IV-2 to activate LCAT compared with apoA-IV-1.¹² Transgenic animal models have shown that low expression of LCAT reduces HDL.³⁷ Cigarette smoking inhibits LCAT activity and lowers HDL.³⁰ Therefore, carriers of the apoA-IV-2 allele may also be more susceptible to the effects of smoking on LCAT activity and HL metabolism, since the effect of smoking on HDL cholesterol was greater in apoA-IV-2 carriers (-19%) than in apoA-IV-1 homozygotes (-4%). On the basis of our findings, we conclude that apoA-IV-2 carriers develop a more adverse plasma lipoprotein profile when exposed to an urban lifestyle characterized by increased smoking and saturated fat intake, as opposed to a rural environment, where smoking and diets high in saturated fat are less prevalent. Thus, the apoA-IV-2 allele may contribute to increased susceptibility to CHD in populations experiencing urbanization.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BMI = body mass index CHD = coronary heart disease LCAT = lecithin:cholesterol acyltransferase NIDDM = non–insulin-dependent diabetes mellitus

	Acknowledgments

 
This work was supported by grant HL49086 from the National Institutes of Health and contract 53-3K06-5-10 from the USDA Department of Agriculture Research Service. Drs López-Miranda, Rodríguez, and Albajar were supported by fellowships from the Spanish Ministries of Health and Education, Madrid, Spain.

Received March 1, 1996; accepted August 2, 1996.

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