The Study Of Adequate Intake Of Vitamin A For Chinese Children Aged 4～9 Years Old

Vitamin A, also known as retinol, refers to a large class of substances with the structure of retinol and its biological activity, including the preformed vitamin A and provitamin A. It is an essential micronutrient for maintain the process of human life, extensively regulating the body's growth and development, and the proliferation and differentiation of organizations and cells. It is very important not only for visual, but also in maintaining the normal function of human tissues and organs, improve immune function and antioxidant.Vitamin A deficiency (VAD) can be divided into clinical vitamin A deficiency, subclinical vitamin A deficiency (SVAD) and suspicious subclinical vitamin A deficiency (suspicious SVAD). Of which SVAD and suspicious SVAD are slightly lack of vitamin A because insufficient intake of vitamin A, characterized by no obvious clinical symptoms and signs. But there are some researches showed that, even without the typical clinical manifestations of vitamin A deficiency, SVAD and suspicious SVAD can affect iron metabolism and immune function of children, then make children with iron decificency anemia or reduce their ability to resist infection, which will greatly increase the risk of illness and death.Vitamin A is one of the world's four major nutrient deficiency diseases recognized by the World Health Organization (WHO), the main susceptible population are children in developing countries. In China, according to the epidemiological investigations of the status of vitamin A deficiency in recent years, we found that China is the moderate SVAD country, and imbalanced in the geographical distribution. Now the severe vitamin A deficiency is rare in China, but the prevalence of SVAD is still very high, and VAD is still a major public health problem of chinese children.However, the basic research of vitamin A of children in our country is sorely lacking, specifically manifested in two aspects:one is that our current standards of recommended nutrient intake (RNI) of viatmin A for children are from Chinese Dietary Reference intakes(Chiese DRIs), these standards are developed by refering the standards of FAO/WHO (1988), we have no relevant research about vitamin A requirement of Chinese children. Because there are racial differences and different dietary pattern, it maybe not suitable to use the results of the developed countries as the standards for children in our country. From the results of current survey, the vitamin A dietary intakes of Chiese children were very low whether in urban or in rural areas, only 50%-60%percent of RNI. The reason for this phenomenon maybe that the vitamin A dietary intakes were really seriously low or the current RNI standard is really high, but it's unkown.The second is that the evaluation methods of vitamin A status were limited to the serum retinol levels and clinical symptoms, lack of studies which used more sensitive and reliable evaluation methods. There are many methods that can be used to evaluate the vitamin A status, the methods which used more internationally include:serum vitamin A levels, the relative dose response (RDR) test, modified relative dose response (MRDR) test, Deuterated-retinol-dilution (DRD) technique. Determination of serum vitamin A levels, although it is currently the most widely used method of evaluation, but it can not reflect the sub-clinical status of vitamin A deficiency because the homeostatically controlled of blood retinol concentrations in a well nourished population, and it can be vulnerable interfered by infection, protein status, lack of other nutrients and other factors. Because liver is the main organ where vitamin A stores, so the best indicator of vitamin A assessment should be the liver vitamin A storage. RDR, MRDR and DRD are all methods that indirectly reflect the state of the liver vitamin A storage. Of which RDR and MRDR is only a coarse indicator of liver vitamin A storage, can not quantitatively reflect the true level of liver vitamin A. DRD is the only direct method that can quantitatively measure body vitamkin A stores and liver vitamin A concentration. In recent years, there are studies using this method to assess the recommended dietary intake of vitamin A of the elderly and adults, but studies using this method in children have not been reported. The research that use DRD to evaluate the vitamin A status of children in our country is blank.All of these show that using more scientific methods to develop the vitamin A requirements and dietary intakes that fit for Chinese children is a key issue to improve our children's vitamin A status. Therefore, under the support from the funding of the National Science and Technology Support Project, we select 4-9 years old healthy children form Shiyan city as our subjects, and we firstly used the most advanced, most sensititive and reliable method--DRD technique to evaluate the children's vitamin A status, combining with the results of dietary surveys, the aim is to propose the adequate intake of vitamin A for our children aged 4-9 years old. The results obtained from this study will provide an important scientific basis for establishing the dietary reference intakes (DRIs) of vitamin A for our children, and will have important theoretical and practical significance for improving the vitamin A status for our children and even the children of whole Asian. At the same time, it will provide an important scientific basis for the further study of the physical requirements of Chinese children.The main content of this study include the following three parts:PartⅠThe survey and evaluation of children's nutrition and health statusObjective:To comprehensively understanding the nutrition and health status of children aged 4-9 years old in Shiyan city, making preparations for the further study of screening the vitamin A well nourished children.Methods:We selected the Experimental Kindergarten and Foreign Language School as the survey schools, for the kindergarten, we selected the middle class and senior class, for the primary school, we selected the second grade and third grade. From December 2009 to July 2010, the following researches carried out including a detailed questionnaire survey, physical examination, dietary survey and laboratory parameters detection. The contents of the questionnaire included basic family situation, the recent prevalence of disease, the eating habits, whether taking nutritional supplements; physical examination including height and weight measurement, and examination fo internal medicine and skin; dietary survey includes consecutive 3-day precisely weighed records and dieatry recalls, (dietary records by parents and other guardians). Laboratory tests including blood biochemical parameters, fat absorption test and fecal parasite examination.Results:There are 424 children attended the study, of which 231 were 4～6 years old, 193 were 7～9 years old, the percents were 54.5% and 45.5%.233 were boys and 191 were girls, the percents were 55.0% and 45.0%. Most of the parents with a higher level of education, half of them have a college or higher education, the most occupation was business, next was Professional and technical personnel, and office worker. The children's average household income was 52,307 yuan/year, the expenditure for food is about 17,161 yuan/year. Mean (±SD) of height was 119.92±11.10cm, mean (±SD) of weight was 24.16±6.09kg, the incidence of growth retardation was 2.66%, of which 4-6 age group was 4.52%,7～9 age group was 0.52%. The incidence of malnutrition was 0.24%, the rate of overweight was 15.01%, and the rate of obesity was 5.33%. Mean (±SD) of hemoglobin was 119.89±8.62g/L, the prevalence of anemia was 9.2%, Fat absorption test founded that 47 cases were positive, accounting for 12.0%, microscopic examination of intestinal parasites found one cases of positive eggs for roundworm. The results of other tests found no abnormalities. The dietary survey found that the intakes of energy and macronutrient were adequate, the proportion of energy from the three macronutrient was reasonable. But there is inadequate intake of nutrients, most serious of which were calcium, zinc and vitamin A, calcium intake accounted for 42.53%～71.68% of the RNI, zinc intake accounted for 67.73%～69.92% of the RNI, vitamin A intake accounted for 56.38%～58.59% of the RNI. Source of vitamin A, beta- carotene accounted for 36.44% of retinol equivalent, the result is in accordance with China's national conditions.Conclusion:From the results of physical examination and laboratory test, the overall developmental level of children aged 4～9 in Shiyan City was better, but the overweight and obesity rates were higher, so attention should be paid at these two points. The dietary survey found that a severe shortage intake of calcium, zinc and vitamin A, indicating that overnutrition and micronutrient deficiencies coexist in the investigation children. Part II The study of children's adequate intake of vitamin ASection 1 The first screening of subjects, evaluation and impact factor analysis of children's serum vitamin A levelsObjective:Using the serum retinol detection to screen the children with adequate serum vitamin A level, and using the univariate and multivariate analysis to filter the impact factor of children's serum vitamin A levels.Methods:An internal standard method of reversed-phase high performance liquid chromatography (RP-HPLC) were used. adding the volume of ethanol to blood sample for precipitation, extraction by N-hexane, the hexane layer was moved and dried by nitrogen evaporation, dissolved by absolute ethanol, then analyzed by HPLC. The analysis column was Hypersil ODS-C18 (4.6mmx250mm,5μm). The retinol was detected at 325nm with UV detector by using methanol/water (90:10 v/v) as mobile phase, the column temperature was 30℃, and the flow rate was lml/min. Protected from light of the whole process. Quantity was determined by internal standard method. The standards were multiply diluted as series application liquids, which analyzed by HPLC with the above chromatographic conditions, then use the ratio of retinol peak area and internal standard peak area as the abscissa, the concentration of retinol as the vertical axis, Make a calibration curve for quantitative.Results:The serum retinol levels of 403 children were between 0.39mmol/L to 4.66mmol/L, The mean (±SD) serum retinol concentration was 1.48±0.52 mol/L, the prevalence of vitamin A deficiency (VAD,<0.7 mol/L) was 3.0%, the prevalence of SVAD (0.7～1.05 mol/L) was 13.6%. Use the serum retinol value as the dependent variable to do univariate analysis, we found that gender, age, father's occupational groups, household income, household expenditure on food, leave days on sick in nearly six months, whether taking nutritional supplements, fat absorption test, height, weight and BMI index were statistically significant; divided the serum retinol into two categories (<1.05 mol/L group and≥1.05 mol/L group), then use this category variable as dependent variable, use the statistically significant factors as independent variable, use the Logistic regression model to do multivariate analysis, results showed that only age and fat absorption test were the factors affecting serum retinol, the older group with higher serum retinol values, and fat absorption test negative group with higher serum retinol values.Conclusion:After the first screening,83.4%(n=336) of the children were in good condition of the serum vitamin A level. The prevalenc of VAD and SVAD were better than the average level of the national survey in 2002. All of these showed that the overall situation of serum vitamin A level is good, but it is still important to focus on the prevention of children vitamin A subclinical deficiency.Section 2 Rescreening and assessment of vitamin A nutrition status of the selected children by RBP-RDR testObjective:Using retinol binding protein-relative dose response test (RBP-RDR test) to rescreen the selected children with adequate liver vitamin A level, to provide a basis for the further confirmation that the children with good vitamin A condition.Methods:According to the results of questionnaire, physical examination and laboratory test, those who with any of the following one shoud be removed:Ongoing acute or chronic disease; Having taken nutritional supplements within 3 months; Positive in the fat absorption test or parasite infection; CRP>8 mg/L; Serum level of retinol<1.05 mmol/L. From the largest to smallest to select the children with higher serum retinol concentration, there were 174 children selected to attend the RBP-RDR test. The method was double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and human retinol binding protein (RBP) ELISA kits were used for testing. Firstly, the anti-RBP antibody coated microtiter plate, made of solid phase antibody, then added standards and samples into the microtiter plate to form RBP* Anti-RBP complex, then washed away unbound proteins, and added anti-RBP-HRP to form anti-RBP-HRP*RBP*anti-RBP complex, then washed away unbound HRP, added the chromogenic substrate, finally detected the activity of the enzyme with a microplate reader. Results:There are 174 children attended the RDR test, of which 57 were 4～6 years old,117 were 7～9 years old, the percents were 32.8% and 67.2%.96 were boys and 78 were girls, the percents were 55.2% and 44.8%. Mean (±SD) of RDR value of 4～6 years old group was -33.22%±45.84%, mean (±SD) of RDR value of 7～9 years old group was-30.60%±78.99%.149 (85.6%) children were in good condition while 25 (14.4%) children were SVAD according to the result of RDR test. Of these SVAD children,16 were 4～6 years old,9 were 7～9 years old, the percents were 28.1%and 7.7%. The difference between two age groups was statistically significant (2 test, p<0.001). The difference between RDR≤20% group and RDR>20% group was statistically significant (t-test, p=0.711).Conclusion:After the second screening, it showed that 85.6%(n=149) children with good liver vitamin A storage. RBP-RDR test is simple, economical and sensitive. So it can be considered a good method for evaluating the liver vitamin A storage condition, but it still need further proof with more researches.Section 3 The study of adequate intake of vitamin A with the selected children by DRD testObjective:using deuterated-retinol-dilution (DRD) technique to further confirm the vitamin A status of these screened children, combined with the results of dietary surveys, then developed the the adequate intake of dietary vitamin A in 4～9 years old healthy children. And by the method of combining the 3 days DRD test and 18 days DRD test, whether the 3 days DRD test can be used to evaluate children's vitamin A status was explored.Methods:According to the results of the relative dose response test, picked the children with the RDR values close to 20% out, a total of 123 children were selected and divided into 3 days and 18 days groups to conduct the DRD trial.60 blood samples were choosed from the 123 blood samples for DRD determination. Two milliliters of CHCL3/CH3OH (2:1 in volume) was added to serum sample. The CHCL3 layer was collected. Two milliliters of hexane were added to the aqueous layere to re-extract fat soluble nutrients. The hexane layer was combined with the CHCL3 lyaer and evaporated under N2 on a N-EVAP evaporator. The residue was dissolved in 100μl of ethanol and 80μl was injected onto an HPLC system. Retinol fraction was collected by a fraction collector. The retinol HPLC fraction from the serum extract was extracted firstly by 2ml of CHCL3 and then by 2 ml of hexane. The CHCL3 and hexane extracts were combined and dried under N2. Derivatization reagent was added into the residue and heated at 70℃for 30 minutes, then analysed by a GC/MS system. The whole process was conducted in the red light. Because the endogenous retinol (Without deuterium) and deuterated retinol reached balance at 18 days, so the modified Bausch-Rietz DRD formula can be used to calculate the total body vitamin A; while the endogenous retinol (Without deuterium) and deuterated retinol didn't reach balance at 3 days, so the modified Bausch-Rietz DRD formula can not be used to calculate the total body vitamin A, the formula proposed by Dr. Tang et.al. was used. Firstly, the isotope abundance ratio of 21 days was calculated from the 3 days isotope abundance ratio, then the 21 days isotope abundance ratio was entered into the modified Bausch-Rietz DRD formula to calculate the total body vitamin A and liver vitamin A concentration.Results:Among the selected 60 children, there were 28 children in 3 days group and 32 in 18 days group, the percents were 46.6% and 53.4%. There were 38 boys and 22 girls, the percents were 63.8% and 36.2%. There were 22 children in 4～6 years old group and 38 children in 7～9 years old group, the percents were 36.2% and 63.8%. Two samples were unresponsive in the detection of enrichment of labeled vitamin A, so n=58. The total body store of vitamin A was 0.28±0.19 mmol retinol, and the liver vitamin A concentration was 0.33±0.25umol/g liver. Using correlation analysis to analyze the relationship between dietary intake of vitamin A and other major nutrients, and serum levels of vitamin A, total vitamin A and liver vitamin A concentrations. We found that positive correlations between vitamin A dietary intake and total body vitamin A (r=0.277, P=0.035) and liver vitamin A concentrations (r=0.270, P=0.040), while no correlation between dietary vitamin A intake and serum vitamin A levels (r=0.047, P=0.721). The analysis results ofβ-carotene just like the vitamin A, positive correlations were founded between vitamin A dietary intake and total body vitamin A (r=0.288 P=0.028) and liver vitamin A concentrations (r=0.419, P=0.001), while no correlation between dietary b-carotene intake and serum vitamin A levels (r=0.170, P=0.193). In addition to the negative correlation between carbohydrate and liver vitamin A concentrations (r=-0.304, P=0.020) was founded, there was no statistically significant results founded between other dietary intakes such as fat, protein and the three variables. In accordance with the range of reference values of liver vitamin A concentration (0.07μmol/g-1.05μmol/g), two children with lower (<0.07μmol/g) and one with higher (>1.05μmol/g) liver vitamin A reserve were excluded. According to the rest of the 55 children those in good condition, the calculated results are 476.9±196.7 mg RE/d (95%CI 384.4～568.95μg RE/d) for 4-6 years old group and 529.1±87.2 mg RE/d (95%CI 499.58-558.62μg RE/d) for 7-9 years old group.Conclusion:Based on the results of DRD test and the average dietary intake of vitamin A well-nourished children, we concluded that the adequate intakes of vitamin A are 450μg RE/d (95%CI,385～570μg RE/d) for 4-6 years old children and 550μg RE/d (95% CI,500～560μg RE/d) for 7-9 years old children.3 days DRD test also achieved good results, the total body vitamin A and liver vitamin A concentration between 3 days and 18 days had no statistically significant deferences. But by comparison of several 3 days DRD formulas, it was reflected that we needs further study to obtain a 3 days DRD formula just be recognized like the modified Bausch-Rietz DRD formula.PartⅢThe intervention and effect evaluation of children with vitamin A deficiencyObjective:In order to correct the serum retinol level of children with vitamin A deficiency, we provided vitamin A supplements to them and observed the intervention effects.Methods:we provided vitamin A supplements to children with serum retinol concentration<1.05μmol/L (12 in primary school and 55 in kindergarden), after 3 months, draw blood again from them and detected serum retinol concentrations again using the high performance liquid chromatography (the same method as section 1 of partⅡ). In addition to this, the diet of these children maintain to the previous state.Results:There were 67 children with serum retionl concentration lower than 1.05μmol/L, of which 55 children were 4-6 years old and 12 children were 7-9 years old, the percents were 82.1% and 17.9%.34 children were boys and 33 children were girls, the percents were 50.7% and 49.3%. After 3 months intervention with vitamin A supplements, we found that only one child still with serum retinol concentration lower than 1.05μmol/L, others were all higher than 1.05μmol/L, the recovery rate almost 99%. The mean (±SD) of serum retinol concentration before the intervention was 0.85±0.17μmol/L, and after the intervention it was 2.85±0.94μmol/L, it raised 2.00μmol/L after the intervention. The difference of the averages of serum retinol concentration between before and after intervention was statistically significant (t=14.86, P＜0.001) with the result of paired t test in accordance with the self-control design.Conclusion:From the recovery rate of the serum vitamin A level, taking vitamin A supplements in low dose but many times is an effective intervening measure to change the vitamin A nutritional status of children. But it should be pay attention to propaganda and education of nutritional knowledge to parents, guiding them the methods of rational nutrition and a balanced diet, the aim is to improve the children's dietary structure to consolidate the curative effect. Adequate vitamin A dietary intake is the most safe and effective way to correct VAD status, and it is the most economical and easy way too.