Optimization of Three-Dimensional Culture Conditions of HepG2 Cells with Response Surface Methodology Based on the VitroGel System.

Objective: This study optimizes three-dimensional (3D) culture conditions of HepG2 using response surface methodology (RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues. Method: HepG2 cell was 3D cultured on the VitroGel system. Cell viability was detected using Cell Counting Kit-8 (CCK-8) assay of HepG2 lived cell numbers. The proliferation of HepG2 cell and clustering performance was measured via fluorescence staining test. Albumin concentration in the culture medium supernatant as an index of HepG2 cell biological function was measured with ELISA kit. Independent factor tests were conducted with three key factors: inoculated cell concentration, cultured time, and dilution degree of the hydrogel. The preliminary results of independent factor tests were used to determine the levels of factors for RSM. Result: The selected optimal culture conditions are as follows: concentration of inoculated cells was 4.44 × 10 5/mL, culture time was 4.86 days, and hydrogel dilution degree was 1:2.23. The result shows that under optimal conditions, the predicted optical density (OD) value of cell viability was 3.10 and measured 2.978 with a relative error of 3.94%. Conclusion: This study serves as a reference for the 3D HepG2 culture and constructs liver tissues in vitro. Additionally, it provides the foundation for repeated dose high-throughput toxicity studies and other scientific research work.

Lipid Accumulation and IL-6 Production in L02 Hepatocytes Induced by Sodium Oleate: Dose and Time Dependence.

To explore interleukin-6 (IL-6) production and characterize lipid accumulation in L02 hepatocytes induced by sodium oleate. L02 hepatocytes were incubated with 0, 37.5, 75, 150, 300, 600, or 1,200 µmol/L sodium oleate for 24 h, and the supernatant was collected to detect the concentration of IL-6. L02 hepatocytes were incubated with 300, 150, 75, or 0 µmol/L sodium oleate for 0-24 h. The supernatant was collected for detection of IL-6 and free fatty acids. L02 hepatocytes treated with 300 µmol/L sodium oleate for 0-24 h were stained with Oil Red O. With extended sodium oleate incubation time, IL-6 levels increased, and free fatty acids decreased. After 24 h incubation, IL-6 levels increased as sodium oleate increased from 37.5 to 300 µmol/L ( P < 0.05 for 37.5 µmol/L, P < 0.01 for 75 µmol/L and P < 0.001 for concentrations 150 µmol/L or higher). Lipid accumulation increased as the sodium oleate concentration and incubation time increased. Oil Red O staining intensified with incubation time extending beyond 2 h. IL-6 production and lipid accumulation in L02 hepatocytes are influenced by sodium oleate in a dose- and time-dependent manner.

Distribution of High-sensitivity C-reactive Protein Status in an Urban Population in China.

OBJECTIVES: To evaluate the distribution by age and sex of serum high-sensitivity C-reactive protein (hsCRP) in an urban Chinese population and to provide a profile prediction for the risk of bacterial infection, inflammatory diseases, or tissue damages in the body. METHODS: Serum hsCRP was determined using the Roche Tina-quant immuno-turbidimetric assay on a Hitachi 7600-010 automatic biochemical analyzer (Roche Diagnostics) in 1,572 males and 1,800 females, including 78 pregnant women, who were derived from the National Health and Nutrition Survey in 2010-2012. RESULTS: The average hsCRP concentration in urban China was 0.68 mg/L for males and 0.65 mg/L for females. Significant differences in hsCRP were found among different age groups ( P < 0.05). Monitoring results showed no significant differences among the 6-11, 45-59, and ≥ 60-year-old groups in the comparison of hsCRP between males and females in large cities. However, hsCRP concentration was significantly higher in men aged 12-17 and 18-44 years than in women. CONCLUSION: The distribution of the hsCRP status of residents in large cities in China was influenced by age and gender, and the hsCRP levels of both sexes increased gradually with age. In addition, hsCRP concentration was higher in healthy pregnant women than in non-pregnant women. Basing on our results, we recommend that this parameter be included in future national and international screening for early detection of various illnesses.

Study on the Simultaneously Quantitative Detection for ß-Lactoglobulin and Lactoferrin of Cow Milk by Using Protein Chip Technique.

OBJECTIVE: To research a protein chip method which can simultaneously quantitative detect ß-Lactoglobulin (ß-L) and Lactoferrin (Lf) at one time. METHODS: Protein chip printer was used to print both anti-ß-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of ß-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. RESULTS: By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of ß-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of ß-L and Lf were 1:2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for ß-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant (r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024). CONCLUSION: A protein chip method of simultaneously quantitative detection for ß-L and Lf has been established and this method is worthy in further application.

Effect of NaFeEDTA-Fortified Soy Sauce on Anemia Prevalence in China: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

OBJECTIVE: To assess the effect of sodium iron ethylenediaminetetraacetate (NaFeEDTA)-fortified soy sauce on anemia prevalence in the Chinese population. METHODS: A systematic review was performed to identify potential studies by searching the electronic databases of PubMed, Cochrane Library, WHO Library, HighWire, CNKI, and other sources. The selection criteria included randomized controlled trials that compared the efficacy of NaFeEDTA-fortified soy sauce with that of non-fortified soy sauce. Anemia rates and hemoglobin levels were the outcomes of interest. Inclusion decisions, quality assessment, and data extraction were performed by two reviewers independently. A total of 16 studies met the inclusion criteria for anemia rate analysis, of which 12 studies met the inclusion criteria for hemoglobin analysis. All included studies assessed the effect of NaFeEDTA-fortified soy sauce on anemia rates and hemoglobin concentrations. RESULTS: After the intervention, the hemoglobin concentration increased and anemia rates decreased significantly as compared with the non-fortified soy sauce groups. For anemia rates, data from 16 studies could be pooled, and the pooled estimate odds ratio was 0.25 (95% CI 0.19-0.35). For hemoglobin concentrations, data from 12 studies could be pooled, and the pooled weighted mean difference was 8.81 g/L (95% CI 5.96-11.67). CONCLUSION: NaFeEDTA-fortified soy sauce has a positive effect on anemia control and prevention in the at-risk population.

Study on the method of quantitative analysis of serum ferritin and soluble transferrin receptor with protein microarray technology.

OBJECTIVE: To establish and evaluate a protein microarray method for combined measurement of serum ferritin (SF) and soluble transferrin receptor (sTfR). METHODS: Microarrayer was used to print both anti-SF antibodies I and anti-sTfR antibodies I on each protein microarray. Anti-SF antibodies II and anti-sTfR antibodies II were used as detection antibodies and goat antibodies coupled to Cy3 were used as antibodies III. The detection conditions of the quantitative analysis method for simultaneous measurement of SF and sTfR with protein microarray were optimized and evaluated. The protein microarray was compared with commercially available traditional tests with 26 serum samples. RESULTS: By comparison experiment, mouse monoclonal antibodies were chosen as the probes and contact printing was chosen as the printing method. The concentrations of SF and sTfR probes were 0.5 mg/mL and 0.5 mg/mL respectively, while those of SF and sTfR detection antibodies were 5 µg/mL and 0.36 µg/mL respectively. Intra- and inter-assay variability was between 3.26% and 18.38% for all tests. The regression coefficients comparing protein microarray with traditional test assays were better than 0.81 for SF and sTfR. CONCLUSION: The present study has established a protein microarray method for combined measurement of SF and sTfR.

[The nutrition status of children aged 6-23 months after three months of Wenchuan Earthquake in Beichuan and Lixian, Sichuan Province].

OBJECTIVE: This project aims to investigate the nutrition status of children aged from 6 to 23 months living in Beichuan and Lixian where were seriously affected by the earthquake in 2008. METHODS: The height, body weight and hemoglobin concentration were measured for 478 children from Leigu township, Qushan township in Beichuan county and Zagunao township, Xuecheng township and Putou township in Lixian county; the number of group 6 - 11, 12 - 17, 18 - 23 months were 190, 144, 144 respectively. The height for age (HAZ), the body weight for age (WAZ) and the weight for height (WHZ) were calculated respectively. The low weight, stunting, wasting prevalence, anemic rate were analyzed. RESULTS: The WAZ scores of 6 - 23 months males and females in Beichuan county and Lixian county ranged from -0.97 to -0.13 and from -0.67 to -0.23, HAZ scores ranged from -1.23 to -0.31 and from -1.25 to -0.38, respectively. The WAZ (-0.89 ± 1.16) and HAZ (-1.20 ± 1.60) of 18 - 23 months infants in Beichuan county were significantly different from the WAZ and HAZ of 6 - 11 months (WAZ: -0.32 ± 0.92; HAZ: -0.58 ± 0.98) and 12 - 17 months (WAZ: -0.47 ± 1.00; HAZ: -0.68 ± 1.34) infants (F values were 7.161 and 4.584, respectively; all P values < 0.05). The WAZ (-0.63 ± 1.03) and HAZ (-1.11 ± 1.15) of 18 - 23 months in Lixian county infants were significantly different from the WAZ and HAZ of 6 - 11 months (WAZ: -0.23 ± 0.93; HAZ: -0.51 ± 1.55) infants and 12 - 17 months (WAZ: -0.58 ± 0.52; HAZ: -0.80 ± 1.19) (F values were 3.156, 4.345, all P values < 0.05). The prevalence of low weight infants (18 - 23 months) these two counties were 15.6% (12/77) and 9.1% (6/66), respectively. The prevalence of stunting in Beichuan and Lixian have reached 26% (20/77) and 24.2% (16/66), respectively. The wasting rate of 18 - 23 months infants had reached 9.1% (7/77) in Beichuan. The total anemia prevalence among infants reached 49.6% (123/248) and 78.8% (178/226) in Beichuan and Lixian, respectively. The prevalence of moderate anemia have reached 7.7% (19/248) and 19.9% (45/226), respectively. CONCLUSION: After the earthquake in Beichuan, Lixian, the growth of infants aged 6 to 23 months old was below average. The prevalence of anemia was high. The malnutrition status of infant and young children aged from 18 to 23 months in Beichuan and Lixian was more serious, should be targeted of nutrition interventions.

Physical performance of migrant schoolchildren with marginal and severe iron deficiency in the suburbs of Beijing.

OBJECTIVES: To investigate relationship between iron deficiency of different degrees and physical performance and habitual activity of migrant schoolchildren at the age of 11-14 years. METHODS: Ninety one randomly selected schoolchildren were divided into three groups according to their iron status. Iron status including hemoglobin (Hb), serum ferritin (SF), serum iron (SI) and sTfR was determined. Physical performance tests included maximum oxygen consumption (VO2max) and maximum work time. Energy expenditure (EE) and daily physical activity were estimated by recording 24-h heart rate (HR). Dietary intake was assessed with frequency questionnaires, and physical activity level was estimated with frequency and physical activity questionnaires. RESULTS: Severe iron deficiency (IDA) impaired the aerobic capacity and habitual physical activity. When fat-free mass (FFM) was considered, VO2max (VO2max/FFM) was significantly lower in the iron-marginal group than in the iron-adequate groups among girls (P = 0.02), but such a deference was not found among boys (P = 0.28). Aerobic activity and EE at leisure were significantly lower in the severe iron deficient group than in the marginal iron deficient and iron adequate groups. Net HR at leisure time was correlated with Hb, log SF, body weight, and FFM (P < 0.05). CONCLUSION: The functional effect of iron deficiency on physical performance and habitual physical activity rely on the degree of current iron deficiency. Severe iron deficiency significantly impairs both aerobic capacity and habitual physical activity. Iron-marginal deficiency impairs VO2max/FFM in girls, rather than in boys.

Efficacy of different iron fortificants in wheat flour in controlling iron deficiency.

OBJECTIVE: To observe the different impacts of electrolytic iron, FeSO4, and NaFeEDTA on body iron store of anemic school students. METHODS: Four hundreds anemic students at the age of 11-18 years were divided into four groups. Of which, three consumed different iron fortificants from wheat flour as food vehicle for six months and one consumed non-fortified flour (control). The fortification level of electrolytic iron, FeSO4, and NaFeEDTA was 60 mg Fe/kg, 30 mg Fe/kg, and 20 mg Fe/kg, respectively. Blood samples were collected at 0, 2, 4, and 6 months and hemoglobin (Hb), serum ferritin (SF), and transferrin receptor (TfR) were measured. RESULTS: The hemoglobin levels in three intervention groups increased, the increments of Hb in the NaFeEDTA group were significantly higher than that in the other groups. SF and TfR levels increased in the tested groups and body iron store in the NaFeEDTA group was higher than that in the other groups. These parameters did not show any significant changes in the control group. CONCLUSION: NaFeEDTA and FeSO4 fortified wheat flour has positive impacts on iron status in anemic students and NaFeEDTA is more effective than FeSO4, while electrolytic iron is less effective in improving iron store in anemic students.

NaFeEDTA fortified soy sauce showed higher iron absorption rate in Chinese females.

OBJECTIVE: NaFeEDTA was considered as a promising iron fortificant for controlling iron deficiency anemia. Soy sauce is a suitable food carrier for iron fortification and is a popular condiment in China. Iron absorption rates of NaFeEDTA and FeSO4 were observed and compared in adult female subjects. METHODS: The stable isotope tracer method was used in Chinese females consuming a typical Chinese diet. Ten healthy young Chinese women were selected as subjects in the 15-day study. A plant-based diet was used based on the dietary pattern of adult women in the 1992 National Nutrition Survey. Six milligram of 54Fe in 54FeSO4 soy sauce and 3 mg 58Fe in Na58FeEDTA soy sauce were given to the same subjects in two days. Food samples and fecal samples were collected and analyzed. RESULTS: Iron absorption rates of NaFeEDTA and FeSO4 were 10.51% +/- 2.83 and 4.73% +/- 2.15 respectively. The 58Fe (NaFeEDTA) absorption was significantly higher than that of 54Fe (FeSO4) (P < 0.01). The iron absorption rate from NaFeEDTA was 1.2 times higher than that from FeSO4 in Chinese adult women consuming a typical Chinese diet. CONCLUSION: The higher absorption rate of NaFeEDTA suggested that NaFeEDTA would be a better iron fortificant used in soy sauce for the controlling of iron deficiency anemia in China.

[Capillary zone electrophoretic determination of NaFeEDTA in iron fortified soy sauce].

OBJECTIVE: An analytical method for separation and determination of sodium iron (III) ethylenediaminetetraacetate (NaFeEDTA) in iron fortified soy sauce by capillary zone electrophoresis (CZE) has been developed. METHODS: The electrolyte, which contains 30 mmol/L citric acid, 20 mmol/L Tris, 0.15 mmol/L CTAB and 0.05 mmol/L SDS, was used to improve the peak shape and reduce the retention time at the voltage of - 25.0 kV. The sample can be determined at UV 254 nm after diluted. RESULTS: In this method, which enjoyed a wide linear range and good precision, the RSD (n = 6) was 2.15%, the average recoveries for samples ranged from 94.3% - 101.2% and the detection limit of the NaFeEDTA in iron fortified soy sauce is 4 microg/ml (S/N = 3). CONCLUSION: The results of application of different kinds of soy sauce showed that the method was direct, accurate, fast and simple.