Girls Are Affected by Lipoprotein Lipase, but Not Apolipoprotein. CIII Polymorphism ... lipid and lipoprotein concentrations in Vietnamese girls. ..... Frohlich JJ.
Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Plasma Triglyceride and HDL-Cholesterol Concentrations in Vietnamese Girls Are Affected by Lipoprotein Lipase, but Not Apolipoprotein CIII Polymorphism Nghiem Nguyet Thu,* Ta Thi Tuyet Mai,y Reiko Ohmori,**1 Masatoshi Kuroki,z Nguyen Van Chuyen,yy Nguyen Thi Kim Hung,y Masanobu Kawakami,z and Kazuo Kondo* * Institute of Environmental Science and Human Life, Ochanomizu University, Tokyo 112-8610, Japan; y Center for Child Nutrition, Hochiminh, Vietnam; ** First Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan; zOmiya Medical Center, Jichi Medical School, Saitama 330-8503, Japan; and yyDepartment of Food and Nutrition, Japan Women’s University, Tokyo 112-8681, Japan ABSTRACT Lipoprotein lipase (LPL) and apolipoprotein CIII (apoCIII) play an important role in HDL-cholesterol and triglyceride metabolism. This study examined the effects of LPL S447X and apoCIII SstI polymorphisms on the plasma lipid and lipoprotein concentrations in Vietnamese girls. Three hundred and fifty-one Vietnamese girls, from 7 to 9 y of age, were randomly selected from the urban and rural areas of Ho Chi Minh City, Vietnam. The presence of LPL S447X and apoCIII polymorphism was determined using the Invader assay, and the plasma lipid concentrations were measured by an enzymatic method. Dietary intakes and anthropometrics were assessed for each child. The frequencies of LPL 447X (11.9%) and apoCIII S2 (32.6%) polymorphisms in the Vietnamese were similar to those of other Asian populations. The frequency of LPL 447X was the same as that in Caucasians but the frequency of apoCIII S2 was ;3.8 times that in Caucasians. Overall, LPL 447X polymorphism was associated with a higher HDL-cholesterol concentration (7.4%, P ¼ 0.007) and a lower triglyceride concentration (13.6%, P ¼ 0.04) than LPL 447S. The apoCIII S2 polymorphism was not associated with an increase in the plasma triglyceride concentration in Vietnamese girls with a low fat intake. However, due to the high frequency of the apoCIII SstI polymorphism and the increasingly westernized diet in Vietnam, attention should be paid to the interaction of genotype with the Vietnamese diet. J. Nutr. 136: 1488–1492, 2006. KEY WORDS: lipoprotein lipase S447X apolipoprotein CIII SstI HDL-cholesterol triglyceride Vietnamese
The plasma concentrations of triglyceride (TG)2 and HDLcholesterol (HDL-C) have been considered independent risk factors for coronary artery disease (CAD) (1–3). Several prospective epidemiological studies have shown an inverse relation between the risk of CAD and the plasma HDL-C concentration (4–6). The association of common gene variants within the candidate genes regulating TG and HDL metabolism might thus be an important factor in CAD. Lipoprotein lipase (LPL) is a multifunctional protein that hydrolyzes core TG from circulating chylomicron and VLDL (7). During this process, surface free cholesterol and phospholipids are transferred to HDL particles, thereby increasing the concentration of HDL-C (8). The S447X mutation in exon 9, which truncates the LPL protein by 2 amino acids (Ser-Gly), is common in the general 1
population (9). This polymorphism was found to relate to high HDL-C, low TG concentrations, and to help prevent premature CAD (10). Apolipoprotein CIII (apoCIII), which is a major component of TG-rich lipoproteins (chylomicron and VLDL), has been shown in vitro to be an inhibitor of LPL (11,12). The first reported polymorphism in the apoCIII gene was the Sstl polymorphism in the 39 untranslated region of the gene (13). This polymorphism is caused by a C!G change at position 3238. An SstI polymorphism in the 39 untranslated region of the apoCIII gene was also reported to be associated with the higher plasma TG concentration and CAD in a number of studies (14–16), but not in others (17). Therefore, depending on the ethnicgeographical origin of the studied population, it appears that additional genetic or environmental factors play a role in the potential associations of these polymorphisms. An increasing intake of energy and fat has contributed to the increasing mortality and morbidity due to CAD in Vietnam (18,19). In addition to dietary changes, whether Vietnamese people have any genetic risk factors for atherosclerosis, such as
To whom correspondence should be addressed. E-mail: rhirano@ndmc.
ac.jp. 2 Abbreviations used: ApoCIII, apolipoprotein CIII; CAD, coronary artery disease; HDL-C, HDL-cholesterol; LDL-C, LDL-cholesterol; LPL, lipoprotein lipase; RDA, recommended dietary allowances; TG, triglyceride.
0022-3166/06 $8.00 Ó 2006 American Society for Nutrition. Manuscript received 16 December 2005. Initial review completed 29 December 2005. Revision accepted 1 March 2006. 1488
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high plasma TG and low HDL-C concentrations, or a protective gene against atherosclerosis, has yet to be elucidated. The aim of this study was to determine whether LPL S447X and apoCIII SstI polymorphisms exist, and to determine whether they affect plasma lipid and lipoprotein concentrations in 2 groups of Vietnamese girls with different dietary intakes due to living in urban or rural areas.
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developed by Dr. Yukio Yoshimura, Shikoku University, Japan (21). Excel Eiyokun data were obtained from the Nutritive Composition Table of Vietnamese Food (22), an official database used for calculating nutrient intake that was developed by the National Institute of Nutrition in Vietnam. The Vietnamese standard recommended dietary allowances (RDA) was used to estimate the nutrient intake status (23). LPL S447X and apoCIII SstI polymorphism genotyping. Genomic DNA was isolated from 2 mL of whole blood obtained from the subjects using a commercially available kit (QIA amp DNA Mini Blood Kit, QIAGEN). We used the Invader assay to screen for the presence of LPL S447X and apoCIII SstI polymorphisms. The Invader assay combines structure-specific cleavage enzymes and a universal fluorescent resonance energy transfer system, with details described elsewhere (24,25). Statistical analyses. The allele frequency of LPL S477X and apoCIII SstI were estimated by gene counting. A chi-square test was used to examine the Hardy-Weinberg equilibrium (26) for the presence of LPL S447X and apoCIII SstI polymorphisms. Continuous variables were expressed as means 6 SD, and 2-way ANOVA was used to test the effects of genotype, environment, and their interaction on the variables measured. Statistical procedures were performed using the StatView statistical program 5.0 (SAS Institute). Differences were considered significant at P , 0.05.
MATERIALS AND METHODS Study subjects. A cross-sectional study was conducted as part of a evaluation of a nutritional anemia program for 351 school girls, aged 7–9 y, in Ho Chi Minh City, Vietnam. A rural or an urban area is classified according to the main-income occupations of households as issued by the Ministry of Labor and Welfare in Vietnam. The subjects were randomly selected from 2 schools in an urban area known as the First district, where the majority of people are traders, and from 3 schools in a rural area, the Binh Chanh district, where most people are farmers or fishermen. All the girls in each group (rural or urban) were therefore homogeneous and mono-ethnic (Kinh, the major ethnic group in Vietnam). In the rural group, 1 child was found to have urethrorectalosma disease. In the urban group, 1 child had suffered urinary tract infections and another had a history of hepatitis. Three school girls were excluded from the study due to these diseases, and 193 rural and 155 urban school girls participated. The research protocol was approved by the Research and Ethical Review Board of the Ho Chi Minh Child Nutrition Center. Informed consent to participate in the study was obtained from the parents of the subjects. Laboratory analyses. Fasting blood samples were obtained in EDTA-coated vacutainer tubes during clinical examinations of the girls. The samples were stored at 2808C until analysis. The plasma total cholesterol, LDL-cholesterol (LDL-C), HDL-C, and TG concentrations were determined by standard laboratory methods (Daiichi Pure Chemicals) using a medical autoanalyzer (BioMajesty, JCABM2250, JEOL). Anthropometric and dietary intake measurements. Anthropometric measurements, including the body weight, height, and left midarm circumference, were taken according to a standardized protocol, by nutritionists who worked at the Ho Chi Minh Center for Child Nutrition (20). The BMI (kg/m2) was calculated from the baseline measurements of body weight and height. The body fat was measured using a bioelectric impedance method on a body fat analyzer TBF-511 (Tanita). Dietary intake was measured by a single dietitian-administered 24-h recall/child during 3 consecutive days, using food models of food portion sizes, photographs, a common set of household measures, and a food-weighing scale to facilitate the estimate of portion size. The parents were requested to recall what their children consumed during the previous day. Three consecutive days were chosen, excluding weekends and special events. All dietitians worked at the Ho Chi Minh Center for Child Nutrition in Vietnam. The nutrient intake was calculated using a Vietnam Eiyokun software package, version 08.E,
RESULTS The LPL S447X polymorphism and plasma lipid concentrations. The frequency of LPL 447XX carriers was 2.0% and that of LPL 447SX carriers was 19.7%, resulting in an LPL 447X allele frequency of 11.9% (Table 1). This frequency was in Hardy-Weinberg equilibrium. The frequency of the LPL S447X polymorphism between urban and rural girls did not differ. The energy intake of urban girls was higher than that of rural girls (P , 0.0001). The energy intake of urban girls (;7.2 MJ/ d) was nearly adequate to the Vietnamese RDA (7.5 MJ/d), whereas that of rural girls (;5.0 MJ/d) did not reach the Vietnamese RDA (Table 2). The percentage of energy intake from fat was ;22% in urban girls but only 16% in rural girls (P , 0.0001). Fat intake of the rural girls was much lower than the Vietnamese RDA (20–25% of total energy intake). The nutritional status and anthropometric variables did not differ among the LPL genotype subgroups for either urban or rural girls. Plasma TG concentrations differed among LPL 447SS allele carriers and LPL 447SX/XX allele carriers in both urban and rural groups (P 5 0.04, Table 3). Neither environment nor the interaction between environment and genotype affected plasma triglycerides. The plasma HDL-C concentration was
TABLE 1 Genotype and allele frequencies of LPL S447X and apoCIII SstI in Vietnamese girls1 Genotype frequency LPL S447X Vietnamese girls, n
SS
SX
Allele frequency ApoCIII SstI
XX
S1S1
S1S2
LPL S447X S2S2
S allele
X allele
n (%) Total, 348 Rural, 193 Urban, 155 1
272 (78.3) 148 (76.7) 124 (80.0)
69 (19.7) 43 (22.3) 26 (16.8)
7 (2.0) 2 (1.0) 5 (3.2)
163 (46.9) 92 (47.7) 71 (45.8)
Frequencies are in Hardy-Weinberg equilibrium chi-square, P . 0.05.
ApoCIII Sstl S1 allele
S2 allele
% 143 (41.1) 79 (40.9) 64 (41.3)
42 (12.0) 22 (11.4) 20 (12.9)
88.1 87.8 88.4
11.9 12.2 11.6
67.4 68.1 66.5
32.6 31.9 33.5
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TABLE 2 Nutrient intakes and anthropometric variables of urban and rural Vietnamese girls according to LPL S447X polymorphism1 Urban, n LPL 447SS, 124
Rural, n
LPL 447SX/XX, 31
LPL 447SS, 148
LPL 447SX/XX, 45
Urban vs. rural P-value2
Energy intake, MJ/d Protein intake, % energy Fat intake, % energy Carbohydrate intake, % energy BMI, kg/m2 Body fat, % Left mid arm circumference, cm 1 2
7.2 15.8 22.2 59.8 15.8 15.0 17.9
6 6 6 6 6 6 6
1.4 2.1 4.2 4.6 2.4 5.8 2.2
7.6 16.3 23.1 58.0 15.9 15.5 18.2
6 6 6 6 6 6 6
1.5 2.7 4.8 7.1 2.5 6.3 2.7
5.1 14.4 15.4 70.2 13.9 10.5 15.9
6 6 6 6 6 6 6
1.3 2.1 5.7 6.5 1.1 2.7 1.2
5.0 14.5 16.2 69.3 14.1 11.0 16.1
6 6 6 6 6 6 6
1.2 2.0 5.2 6.2 1.4 3.6 1.5
,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
Values are means 6 SD. P-values are from 2-way ANOVA; the effects of genotype and the environment by genotype interaction were NS, P . 0.05.
higher in LPL 447 SX/XX allele carriers than in LPL 447SS allele carriers in both urban and rural groups (P 5 0.007). The environment also affected the plasma HDL-C concentration (P , 0.0001). Overall, the plasma HDL-C concentration was 7.4% higher, and the plasma TG concentration was 13.6% lower, in LPL 447X than in LPL 447S allele carriers. Plasma total cholesterol and LDL-C concentrations were not affected by the LPL S447X polymorphism in either urban or rural girls. The apoCIII SstI polymorphism and plasma lipid concentrations. The frequency of the apoCIII S2 allele was 32.6% in Vietnamese girls (Table 1) and did not differ from the expected frequency according to the Hardy-Weinberg equilibrium. There was no difference in frequency of apoCIII SstI polymorphism between urban and rural girls. Although the energy, protein, and fat intakes by urban girls were greater than those in rural girls (P , 0.0001), intakes did not differ among apoCIII SstI genotype subgroups of rural or urban girls (Table 4). Similarly, the BMI, body fat, and left mid-arm circumference in urban girls was greater than those in rural girls (P , 0.0001), but these anthropometric variables did not differ between the apoCIII SstI genotype subgroups in either rural or urban girls. The plasma lipid concentrations were not affected by either the environment or the apoCIII SstI polymorphism (data not shown).
apoCIII play an important role in the metabolism of TG rich lipoproteins and HDL. The present study showed that LPL 447X was related to a significant increase in the plasma HDL-C concentration and a significant decrease in the plasma TG concentration, whereas the apoCIII SstI S2 allele, with its high frequency, was not related to an increase in the plasma TG concentration in Vietnamese girls with a regular low fat intake. The frequency of the LPL 447X allele mutation ranged from 0.085 to 0.11 in all populations (10). Compared with other populations, the frequency of this polymorphism in Vietnamese girls is similar to that in Japanese (27) and Chinese (28) in Asia and to that in Caucasians (29,30). The LPL S447X polymorphism is different from the other known LPL polymorphisms because it does not cause an increase in the plasma TG concentration (10). Our results were compatible with those of other studies of adult Caucasian populations that showed the LPL 447X polymorphism to be related to a protective lipid profile for CAD (9,10,29,30). Even in rural Vietnamese girls who have low energy and low fat intakes and low plasma lipid concentrations, LPL 447X still affected the plasma lipid concentrations. The effect of the LPL 447X allele on the HDL-C and TG concentrations therefore seems to be independent of any difference in the nutritional status of Vietnamese girls compared with Caucasians. This finding was inconsistent with an American cohort study (the Bogalusa study) (31), which investigated 829 subjects aged 5– 18 y at baseline and followed them for a mean of 18.8 y. The Bogalusa study showed that LPL 447X allele carriers have higher plasma HDL-C and lower TG concentrations than noncarriers in adulthood, but not in childhood. This discrepancy may be due to the fact that the age of the subjects in the Bogalusa study
DISCUSSION Multiple environmental factors affect plasma lipid and lipoprotein concentrations in the general population. LPL and
TABLE 3 Plasma lipid concentrations of urban and rural Vietnamese girls according to LPL S447X polymorhism1 Urban, n LPL 447SS, 124
Rural, n
LPL 447SX/XX, 31
LPL 447SS, 148
LPL 447SX/XX, 45
1 2
4.46 2.58 1.24 1.29
6 6 6 6
0.7 0.6 0.3 0.8
4.53 2.65 1.31 1.07
6 6 6 6
0.9 0.7 0.7 0.5
SS vs. SX/XX
P-value2
mmol/L Total cholesterol LDL-cholesterol HDL-cholesterol Triglyceride
Urban vs. rural
3.68 2.10 0.95 1.22
6 6 6 6
0.6 0.5 0.2 0.5
3.71 2.09 1.06 1.08
6 6 6 6
0.6 0.6 0.3 0.5
,0.0001 ,0.0001 ,0.0001 NS
Values are means 6 SD. P-values are from 2-way ANOVA; the effects of genotype and the environment by genotype interactions were NS, P . 0.05.
NS NS 0.007 0.04
LPL S447X AND APOCIII POLYMORPHISM IN VIETNAMESE
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TABLE 4 The nutritional status of urban and rural Vietnamese girls according to apoCIII SstI polymorphism1 Urban, n S1S1, 71
Rural, n
S1S2/S2S2, 84
S1S1, 92
S1S2/S2S2, 101
P-value2
mmol/L Energy intake, MJ/d Protein intake, % energy Fat intake, % energy Carbohydrate intake, % energy BMI, kg/m2 Body fat, % Left mid-arm circumference, cm 1 2
7.2 15.9 22.2 59.3 16.0 15.0 17.8
6 6 6 6 6 6 6
1.4 2.4 4.2 5.0 2.7 6.3 2.3
7.4 15.8 22.3 59.6 15.7 15.3 18.1
6 6 6 6 6 6 6
1.5 2.1 4.6 5.3 2.1 5.6 2.3
Urban vs. rural
5.1 14.4 15.8 69.7 14.2 11.1 16.1
6 6 6 6 6 6 6
1.3 2.3 5.2 5.9 1.3 2.8 1.3
5.1 14.4 15.4 70.2 13.8 10.2 15.9
6 6 6 6 6 6 6
1.2 1.9 6.0 6.9 1.1 3.0 1.3
,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
Values are means 6 SD. P-values are from 2-way ANOVA; the effects of genotype and the environment by genotype interaction were NS, P . 0.05.
ranged from 5 to 18 y, whereas those in our study ranged from 7 to 9 y. We used female children at the age ranging rrom 7–9 y to eliminate any confounding effects of age, sex, smoking, and medications on the relation between genes and the plasma lipid concentrations. The SstI site (3238C . G) in the 39 untranslated region of the apoCIII gene was first described in 1983 (13), and its association with hypertriglyceridemia has been confirmed in a majority of studies as well as in a review (32). However, no relation between apoCIII SstI polymorphisms and plasma TG concentration was found in Vietnamese girls. The frequency of the apoCIII SstI S2 allele in the Vietnamese was similar to other Asians, such as Japanese (27), Chinese (33), and Koreans (34). In contrast, this frequency was ;3.8 times that found in Caucasians, according to the Framingham Offspring Study (35). ApoCIII SstI polymorphism has been associated with a high plasma apoCIII and hypertriglyceridemia in several, but not all studies. Our study showed that apoCIII SstI polymorphism was not related to high plasma TG concentration. To date, studies on SstI polymorphism have mostly been carried out in adults. There was one previous study on 503 healthy Italian children (aged 11–13 y) that showed that children with an S1S2 genotype have a 12.7% higher plasma TG concentration than their S1S1 peers (16). The Framingham study was carried out on 2485 healthy Caucasians (mean age ;52 y) and showed the TG concentration to be insignificantly higher in men carriers of the S2 allele (35). Another Italian study on 800 patients showed S2 carriers to have significantly higher plasma TG and apoCIII concentrations (36). A recent study of 2267 Japanese adults showed a higher TG concentration in S2 allele carriers, but the change of age- and sex-adjusted means was not significant (27). Other studies of adult Taiwanese-Chinese (mean age ;55 y) and adult Koreans (mean age ;40 y) did not mention the relation between apoCIII polymorphism and the plasma TG concentration but it did show the frequency of the apoCIII S2 allele to be higher in subjects with hypertriglycemia than in controls (42.5 vs. 30% in Taiwanese and 36.5 vs. 24.5% in Koreans, respectively) (33,34). Discrepancies among results reported in the literature could be due to gene–environment interactions, such as those described for dietary fat consumption (37). So far, the effects of apoCIII SstI polymorphism on the plasma lipid and lipoprotein concentrations and the relation between apoCIII SstI polymorphism and hypertriglyceridemia were investigated in other populations with profiles of both a high dietary intake and high plasma lipid and lipoprotein concentrations, such as in Caucasians (35–37) and Japanese
(27), and were also investigated in a case-control design in subjects with high TG concentrations (33,34). To our knowledge, our findings indicate, for the first time, that apoCIII SstI polymorphism does not effect the TG concentration in girls with low nutritional status and low plasma lipid concentrations. We hypothesized that the stronger effect of apoCIII on plasma TG concentrations that was reported in other populations might be the result of an interaction between the gene and the westernized environment, a factor not present in the majority of our study subjects. The frequency of the S2 allele in Asians was 3.8 times the frequency found in Caucasians. The role of apoCIII polymorphism in hypertriglycemia should therefore be paid close attention to in Vietnamese people due to the trend of a high-energy diet pattern. In conclusion, LPL S447X polymorphism influenced the plasma HDL-C and TG concentrations, whereas no effect of apoCIII SstI polymorphism on the TG concentration was observed in Vietnamese girls with a low fat intake. However, due to the high frequency of this polymorphism and the increasingly westernized diet in Vietnam, its role in hyperlipidemia and CAD still needs further investigation. ACKNOWLEDGMENT We are indebted to Dr. M. Nagano for his technical assistance in genotyping.
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