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* Centro de Investigacion de Alimentos y Desarrollo (CIAD), Hermosillo, Mexico;
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269; and
** Columbia University, New York, NY 10032
2To whom correspondence should be addressed. E-mail: maria-luz.fernandez{at}uconn.edu.
| ABSTRACT |
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KEY WORDS: chronic disease Mexican children LDL phenotype saturated fat plasma triglycerides
The etiology of chronic disease is multifactorial and comprises modifiable and nonmodifiable risk factors. Among the nonmodifiable risk factors, heredity and sex are relevant, whereas the modifiable risk factors include modifications in diet, increases in aerobic exercise, and weight loss. There is a high prevalence of coronary heart disease (CHD),3 insulin resistance, and type II diabetes in the northern part of Mexico (1). It is not clear whether biomarkers for chronic disease are already identifiable in childhood or whether lifestyle plays the dominant role in the prevalence of chronic disease in the adult population. Studies aimed at understanding the contribution of modifiable risk factors for chronic disease, as well as those that are genetically based, are needed for this population.
In studies conducted with young male adults in this area of Mexico (2,3), we previously observed an increased prevalence of low plasma concentrations of HDL, high triglycerides (TG), high blood pressure, and BMI > 26 kg/m2, all factors indicative of the metabolic syndrome (4) and the potential for both CHD and type II diabetes. In addition, evaluation of risk factors in this region indicated that subjects from a low socioeconomic background not only exhibited these parameters of increased risk for CHD, but also consumed very high-fat diets (5). Based on these data, we examined children from the same geographical area to evaluate the potential presence at a young age of these biomarkers for chronic disease.
We recruited children of low socioeconomic background to determine dietary intake, level of physical activity (as measured by number of steps taken per day recorded with a pedometer), blood pressure, plasma lipids and apolipoproteins (apos), plasma glucose, and insulin. Our hypothesis was that both lifestyle and genetic components would affect the potential risk for CHD or type II diabetes in children from this region.
| MATERIALS AND METHODS |
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Subjects and dietary assessment. The experimental protocol was approved by the University of Connecticut Institutional Review Board and by the Review Board of CIAD. Parents of children participating in the study attended informational meetings and signed the consent form. Fifty-four children (25 boys and 29 girls) 8 to 12 y old were recruited from the Mauricio Kelly school located in one of the poorest neighborhoods of the city of Hermosillo, Mexico, to assess the presence of biomarkers for chronic disease in this pediatric population. Three-day weighed food records (6) were used to evaluate dietary intake of macronutrients and dietary cholesterol. Both the children and the individual in charge of preparing meals at home were provided with food scales (Ohaus CS 2000) and a chart to record the subjects daily intake. Parents were instructed how to keep the records and researchers in the study worked very closely with children to document their food intake during the day. Diet intake was analyzed using the ESHA Food Processor Program (ESHA, Food Processor, 7.20, ESHA Research Editor, 1998). Regional foods that were not included in the database were analyzed for individual components and added to the database. These foods included snacks, organ meats, and candies, which were usually consumed by the children.
Two fasting (12-h) blood samples were collected, on different days, from each subject into tubes containing 0.15 g/100 g EDTA to determine plasma lipids, plasma glucose, insulin, leptin, LDL peak size, and plasma apos. Plasma was separated by centrifugation at 1500 x g for 20 min at 4°C and placed into vials containing PMSF (0.05 g/100 g), sodium azide (0.01 g/100 g), and aprotinin (0.01 g/100 g).
Systolic and diastolic blood pressure were measured in the right arm with the participant seated and following a 5-min rest using an Omron manual blood pressure Hem 185 Hem 18.
Plasma lipids and apos. Total cholesterol was determined by enzymatic methods using Roche Diagnostics standards and kits (7). HDL cholesterol (HDL-C) was measured in the supernatant after precipitation of apo Bcontaining lipoproteins (8). LDL cholesterol (LDL-C) was determined using Friedewalds equation (9). TGs were determined using Roche Diagnostics kits, which adjust for free glycerol (10). Apo B concentrations were measured by an immunoturbidimetric method and turbidity was determined at 340 nm (11). Apo C-III (12) and apo E (13) were measured on an Hitachi Autoanalyzer 740 utilizing kits from Wako.
Plasma glucose and insulin.
Plasma glucose was determined enzymatically (14). Briefly, 3 mL of working solution was added to 0.20 mL of sample and mixed, transferred to cuvettes, incubated at 37°C for 5 min, and then read at 505 nm on a DU-640 UV spectrophotometer (Beckman Coulter). Insulin was measured in plasma using a radioimmunoassay kit that utilizes the double-antibody/polyethylene glycol technique as previously described (15). In addition, the homeostasis model assessment (HOMA) (16) was used to calculate insulin resistance (IR) according to the following equation: IR (HOMA IR) = fasting insulin (µU/mL) x fasting glucose (mmol/L) ÷ 22.5. The HOMA model has been shown to be a reliable method of measuring insulin resistance in various populations when other more invasive methods are not feasible (16). Based on the equation, children were classified as having IR if the calculated value was
3.8 (17).
LDL size determination.
The Lipoprint LDL system (Quantimetrix) was used to identify the size of LDL using a nongradient high-resolution polyacrylamide gel electrophoresis system. Briefly, 25 µL of plasma was added to precast polyacrylamide gel tubes and overlaid with 200 µL of loading gel. Tubes were then photopolymerized for
30 min and placed into the electrophoresis chamber. Electrophoresis buffer (Tris-hydroxymethyl aminomethane 66.1 g/100 g, boric acid 33.9 g/100 g, pH 8.28.6) was added to the top and bottom portions of the chamber. The gel was run for
60 min at 36 mV or until the HDL fraction was
1 cm from the end of the gel. Gels were allowed to sit for 30 min and scanned with a densitometer. LDL size was determined using software supplied by the manufacturer.
Statistical analysis. Students independent t test was used to compare boys and girls. Differences with P < 0.05 were considered significant.
| RESULTS |
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Intake of total fat (39% of total energy) and saturated fat (12% of total energy) was higher than NCEP recommendations (Table 3). In contrast, carbohydrate intake was low (49% of total energy) compared to the average intake of 55% in the United States. The average intake of dietary fiber was high, due mostly to the consumption of tortillas and beans. The high level of dietary cholesterol (395 mg/d) reflects the high amount of animal (saturated) fat consumed by the children (Table 3).
| DISCUSSION |
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In our previous studies with adult sedentary males from this same region, we observed extremely low HDL levels (ranging from 0.54 to 1.15 mmol/L) and high fasting plasma TG (mean 2.61 ± 0.85 mmol/L) (2,3), with 43% of these subjects having plasma LDL-C levels higher than 3.09 mmol/L. These biomarkers can be linked to the high prevalence of CHD and type II diabetes in this part of Mexico (1). In addition, when the diet from these subjects was analyzed using 3-d dietary records, there was a high consumption of saturated fat, mainly derived from meat products (3). Further, Mexican subjects from a low socioeconomic group also exhibited the biomarkers for CHD and insulin resistance and presented the same dietary pattern of high consumption of saturated fat (5). All these studies in adults plus our current study in children clearly indicate that interventions targeted at modifying dietary intakes and increasing physical activity are seriously needed in this population.
Biomarkers of chronic disease. Children from this study had 3 identifiable risk factors, which could be associated with the increased risk for insulin resistance and CHD later in life, mainly high plasma TG, low HDL-C concentrations, and the predominance of small dense LDL or phenotype B. Identifying risk factors for the metabolic syndrome and CHD in young populations is essential for disease management and for the reduction of overall prevalence of these diseases. In addition, about 39% of children were classified as either overweight or obese according to CDC standards (18). We also measured the waist circumference in these children and, according to published values for Mexican children (22) and those from Cyprus (23), we found the same pattern as for BMI. The positive correlations detected between diastolic and systolic blood pressure with BMI were also important, suggesting that body weight may be associated with other parameters of heart disease and obesity in the population under study.
The elevated levels of plasma TG and the low levels of HDL-C present in this group of children represent a major concern in terms of biomarkers for chronic disease because both independently constitute a risk factor for CHD. In addition, there was a high prevalence of small dense LDL among these children and they also had elevated concentrations of plasma insulin. Furthermore, 11% of the population was classified as having insulin resistance.
Elevated plasma TG concentrations are correlated with increased risk for CHD. Apo C-III is known to inhibit TG hydrolysis by lipoprotein lipase (24), decreasing the removal of TG from VLDL or chylomicrons. Similar to our study, others have shown that plasma apo C-III concentrations correlate with TG levels (25) and are known to be increased in hypertriglyceridemic individuals (12). Plasma apo C-III levels were very high in children in the present study compared to values reported for adults (20,21), indicating that both risk factors (high apo C-III and high TG) are present in this population.
Low levels of HDL are better predictors for CHD than total or LDL-C (26). In agreement with the importance of HDL-C levels for CHD risk, the recommendations of the NCEP Adult Treatment Panel III report include an increased cutoff point for risk levels of HDL-C for both men (>1.03 mmol/L) and women (>1.16 mmol/L) (4). The low HDL-C concentrations observed in children in this study constitute an independent risk factor for CHD and may be a major reason for the prevalence of CHD in older populations.
In addition to the observed dyslipidemias in this population, the high prevalence of phenotype B contributes to the risk factors for chronic disease. Pattern B is associated with increased concentrations of TG, apo C-III, and apo B and decreased HDL (27) and is associated with a 3-fold increase in CHD risk (28). Pattern B results in LDL particles with decreased affinity to hepatic LDL receptors, extended residence time in circulation, increased migration into endothelial cells, increased propensity for oxidation, a stimulatory effect on macrophages, and enhanced coagulant activity (29). Small dense LDL is also a prominent feature associated with insulin resistance. Individuals who possess predominantly pattern B LDL particles (small LDL) are more prone to develop the insulin resistance syndrome. Thus, the presence of these 3 biomarkers in Mexican children supports the high prevalence of diabetes type II in the older population.
Diet and physical activity effects on chronic disease. We have shown in this study that plasma LDL-C concentrations are normal in children from this region at the ages of 812 y. Thus, it appears that the high plasma LDL-C concentrations observed in a high percentage of the adult population are a result of the diet that is typical in this region of Mexico. The intake of total fat is much higher than reported values for children who are at high risk for cardiovascular disease (CVD) (30). Epidemiological data clearly indicate that a strong positive relationship exists between the percentage of energy obtained from saturated fatty acids and CHD incidence (31). A fluctuation in LDL-C of 0.120 mmol/L (4.6 mg/dL) can be expected for every 1% change in saturated fat intake with relation to the percentage of total energy consumed (32). Therefore, the high prevalence of CHD in this population can in part be explained by the elevated levels of plasma LDL-C resulting from the high intake of saturated fat, which starts at a very early age. In addition, children from this group had a moderate level of physical activity as measured by pedometer. When compared to other reported values in children using a similar instrument, the number of steps taken per day was much lower (33) in this group of Mexican children. Human studies have shown that increased physical activity achieved via walking results in improved plasma lipoprotein profiles (34); thus, the low levels of activity present in these children may contribute to the detrimental lipid profiles observed in the adult population (2,3,5).
In summary, this study shows for the first time that the high prevalence of risk factors for chronic disease present in the adult population in this area of Mexico is also seen in this selected group of children. In addition, our results from this study suggest that there is a multifactorial component for the presence of CHD and type II diabetes that involves both genetics and lifestyle.
| FOOTNOTES |
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3 Abbreviations used: apo, apolipoprotein; CHD, coronary heart disease; HDL-C, HDL cholesterol; HOMA, homeostasis model assessment; IR, insulin resistance; LDL-C, LDL cholesterol; NCEP, National Cholesterol Education Program; PMSF, phenyl methyl sulfonyl fluoride; TG, triglyceride. ![]()
Manuscript received 6 August 2004. Initial review completed 27 September 2004. Revision accepted 4 October 2004.
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