Developmental Patterns of Fecal Bile Acids in Healthy Neonates and Children.

BACKGROUND Normal profiles of FBAs in healthy neonates and children in Kunming city and surrounding areas in China have not been previously determined. The objective of this study was to determine a developmental pattern of fecal bile acids (FBAs) in healthy neonates and children. MATERIAL AND METHODS A cross-sectional study was performed on 238 healthy neonates and children recruited in the First Affiliated Hospital of Kunming Medical University, China from October 2015 to September 2016. Secreted primary and secondary FBAs in fresh feces were quantitated by liquid chromatography mass spectrometry (LC-MS). Amounts of FBAs in feces were compared among various age groups. RESULTS Trace amounts of cholic acid and chenodiol acid of primary FBAs were detectable at day 3 after birth, with a significant increase from day 3 to day 7. The primary FBAs gradually decreased from day 25 to the age of 6 years old. In contrast, a significant amount of glycochenodeoxycholic acid was detected on day 3 but decreased to a trace amount by day 7 and onwards. Primary FBAs appeared to maintain a high level, accounting for 98% of total FBAs, with no significant changes from day 7 to day 25 after birth. They gradually decreased from 90% to 10% from age 6 months to 6 years old. While the secondary FBAs were barely detected in neonates, only accounting for 2% of total FBAs, they were gradually elevated to 90% of total FBAs from age 6 months to 6 years old. CONCLUSIONS The liver can effectively synthesize primary bile acids 7 days after birth, and fecal primary bile acids tend to be stable after the neonate stage. Secondary bile acids continuously increase along with the maturation of intestinal flora, which reaches a relatively stable level at around 3 years old.

[Levels of short-chain fatty acids in enterobacteria-related metabolites in the feces of infants with cholestatic hepatopathy].

OBJECTIVE: To compare the levels of short-chain fatty acids in enterobacteria-related metabolites in feces between infants with cholestatic hepatopathy and healthy infants. METHODS: Thirty infants with cholestatic hepatopathy were enrolled in this study as the disease group, while 30 healthy infants were enrolled as the control group. Fecal specimens were collected from the disease group before and after treatment and from the control group. Gas chromatography was used to quantitatively determine the content of short-chain fatty acids in the feces of both groups including acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid. RESULTS: There were no significant differences in the concentrations of acetic acid and propionic acid between the control and disease groups before and after treatment, as well as no significant changes in the two markers in the disease group after treatment (P>0.05). The disease group had a significantly increased concentration of butyric acid after treatment (P<0.05). The concentrations of isobutyric acid and isovaleric acid in the control group were significantly higher than those in the disease group before and after treatment (P<0.05). CONCLUSIONS: Intestinal protein metabolites in infants with cholestatic hepatopathy are significantly different from those in healthy infants, whereas there is no significant difference with respect to carbohydrate metabolites.

[Significance of detection of biomarker fecal bile acids in the diagnosis and treatment of childhood Henoch-Schönlein purpura].

OBJECTIVE: To investigate the changes and clinical significance of biomarker fecal bile acids (BA) in children with Henoch-Schönlein purpura (HSP). METHODS: Nineteen children with HSP and twenty-seven healthy children were enrolled in this study. The stool samples were obtained at the acute and remission phases. Fecal BA levels were measured by high performance liquid chromatography mass spectrometry (HPLC-MS). RESULTS: The fecal cholic acid level in the HSP remission group was significantly higher than in the HSP acute group and the healthy control group (P<0.016). The fecal chenodeoxycholic acid level in the HSP remission group was significantly higher than in the healthy control group (P<0.016). The levels of fecal secondary colonic bile acids, deoxycholic acid and lithocholic acid, in the HSP acute and remission groups were significantly lower than in the healthy control group(P<0.05, P<0.016 respectively). No significant differences were found in the levels of fecal urosodeoxycholic acid among the three groups (P>0.05). CONCLUSIONS: Fecal secondary colonic bile acids, deoxycholic acid and lithocholic acid, are in decrease in children with HSP at the acute stage, which may be involved in the pathogenesis and treatment outcomes of HSP.

Evaluating Intestinal Permeability by Measuring Plasma Endotoxin and Diamine Oxidase in Children with Acute Lymphoblastic Leukemia Treated with High-dose Methotrexate.

BACKGROUND AND AIM: The incidence of acute lymphoblastic leukemia (ALL) is highest in childhood malignant tumor in China. The high-dose methotrexate (HDMTX) treatment is very effective in ALL, and it can improve event-free survival rate. However, while executing the anti-tumor effect, it produces highly toxic effects on rapidly dividing cells which are normal. It seems probable that the HDMTX treatment injures intestinal mucosal barrier. The changes of intestinal mucosal barrier can be evaluated through measuring the level of plasma endotoxin and diamine oxidase (DAO). METHOD: Blood samples were collected from 30 normal children and 30 children with ALL at 1h, 24h, 44h and 68h after HDMTX. The levels of plasma endotoxin and DAO were measured at 1h, 24h, 44h and 68h after HDMTX with spectrophotometry. The levels of endotoxin and DAO were also measured in 4 different courses in 7 children with ALL. RESULTS: The levels of plasma endotoxin and DAO at 1h, 24h, 44h and 68h after HDMTX were higher than in normal children (P<0.01). The levels of plasma endotoxin and DAO at 24h and 44h after HDMTX were both higher than at 1h and 68h (P<0.01). There was no significant difference found in the measured results of plasma endotoxin and DAO at 1h and 68h after HDMTX (P>0.05). There was no significant difference found in the increased levels of endotoxin and DAO at 1h, 24h, 44h and 68h after HDMTX in 4 different courses of 7 children with ALL(P>0.05). CONCLUSION: By measuring the level of plasma endotoxin and DAO in children with ALL and during HDMTX chemotherapy, the results suggest that there is increased intestinal permeability.

[Effects of hyperglycaemia in pregnancy and relevant factors on neonatal body composition].

OBJECTIVE: To compare body composition at birth in the appropriate-for-gestational-age infants of women with gestational diabetes mellitus (GDM) and normal glucose tolerance and determine the influencing factors of body composition in infants of women with GDM and normal glucose tolerance. METHODS: A study was conducted on 160 appropriate-for-gestational-age infants (90 males and 70 females) of women with gestational diabetes mellitus (GDM group) and 284 appropriate-for-gestational-age infants (139 males and 145 females) of women with normal glucose tolerance (control group). Anthropometric measurements were obtained within 24 to 48 hours of birth. Multiple stepwise regression was used to determine the correlating factors of fat mass, percent of body fat and fat free mass mass. RESULTS: There were no significant difference in gestational age, birth weight, length, body mass index, circumferences of head, chest and upper arm, biceps, abdominal superficial skin fold between two groups (all P > 0.05), but GDM group was characterized by higher skin folds of triceps and subscapular and flank versus control group(all P = 0.000). GDM group had greater fat mass but decreased fat free mass versus control group ((585 ± 59) vs (480 ± 74) g, 17.8% ± 0.8% vs 14.7% ± 1.9%, (2685 ± 127) vs (2784 ± 109) g, all P = 0.000). Stepwise regression showed that maternal fasting glucose level of oral glucose tolerance test and pre-gravid body mass index correlated with fat mass and percent of body fat. Fasting glucose level had the strongest correlation with fat mass and percent of body fat (P = 0.004, 0.006). Gestational age and maternal height correlated with fat free mass in GDM group (P = 0.040, 0.013). On the other hand, maternal weight gain correlated with fat mass (P = 0.015), fasting glucose level and maternal prepartal weight were correlated with percent of body fat (P = 0.002, 0.043) and pre-gravid body mass index had correlation with fat free mass in control group (P = 0.004). CONCLUSIONS: The appropriate-for-gestational-age infants of women with GDM have increased fat mass and percent of body fat, but decreased fat free mass. Maternal fasting glucose level of oral glucose tolerance test, pre-gravid body mass index, weight gain and maternal prepartal weight are influencing factors of body composition in neonates.

[Effect of breastfeeding on childhood overweight in the offspring of mothers with gestational diabetes mellitus].

OBJECTIVE: To investigate whether breastfeeding can reduce the risk of childhood overweight in the offspring of mothers with gestational diabetes mellitus (GDM). METHODS: Follow-up was performed on 1189 offspring of mothers with GDM between January 2003 and December 2009. The influence of the manner and duration of breastfeeding between 0 to 3 months after birth on the risk of childhood overweight in the offspring of mothers with GDM was analyzed by logistic regression. RESULTS: After correcting confounding factors such as pre-pregnancy BMI, gestational weight gain, gestational blood sugar, sex, birth weight, age and farther's body weight, it was found that the risk of childhood overweight in the offspring who received exclusive breastfeeding during the first 3 months after birth was lower than in the artificial feeding group (OR: 0.479, 95%CI: 0.256-0.897). Offspring who were breastfed for 0 to 3 months, 4 to 6 months and over 6 months had a lower risk of childhood overweight than the artificial feeding group (OR: 0.456, 95%CI: 0.233-0.827; OR: 0.29, 95%CI: 0.103-0.817; OR: 0.534, 95%CI: 0.280-0.970), offspring who were breastfed for 4 to 6 months had a lower risk of childhood overweight than those who were breastfed for 0 to 3 months (OR: 0.372, 95%CI: 0.129-0.874), and offspring who were breastfed for more than 6 months did not show significantly lower risk of overweight than those who were breastfed for less than 6 months (OR: 0.769, 95%CI: 0.470-1.258). CONCLUSIONS: Within 3 months of birth, breastfeeding, especially exclusively, may reduce the risk of childhood overweight in the offspring of mothers with GDM. Within 6 months of birth, the risk of childhood overweight decreases as the duration of breastfeeding increases, but prolonging the duration of breastfeeding cannot necessarily reduce the risk of childhood overweight after postnatal six months.

[Effects of high-dose methotrexate therapy on intestinal bacterial flora in children with acute lymphoblastic leukemia].

OBJECTIVE: To investigate the effects of high-dose methotrexate (HDMTX) therapy on intestinal bacterial flora in children with acute lymphoblastic leukemia (ALL). METHODS: Thirty-six children with ALL of pre-and post-HDMTX therapy and 36 control children were enrolled. The bacterial DNA in stool was extracted. The primers for Bacillus bifidus and Escherichia coli with the 16SrRNA/DNA sequence of bacteria were designed. The bacteria were identified through general PCR. The standard curve of both bacterial DNA was produced using a series of dilution of accurately quantified bacterial DNA. The unknown samples were measured by 16SrRNA/DNA-targeted PCR. The amount of stool Bacillus bifidus and Escherichia coli among 36 control children and 36 children with ALL of pre- and post-HDMTX therapy were obtained by using the standard curves. RESULTS: Bacillus bifidus logarithmic absolute value of the first day before treatment, of third day after treatment, of seventh day after treatment in patients with ALL and the control was 7.24 +/- 0.33, 6.00 +/- 0.27, 6.59 +/- 0.33, and 9.49 +/- 0.41, respectively (P < 0.01). Escherichia coli logarithmic absolute value of the first day before treatment, of third day after treatment, of seventh day after treatment in patients with ALL and the control was 6.62 +/- 0.42, 5.96 +/- 0.42, 7.02 +/- 0.41, and 7.52 +/- 0.43, respectively (P < 0.01). The logarithm of the amount of stool Bacillus bifidus and Escherichia coli in control group was higher in ALL group (F = 739.61, 88.67, P < 0.01). There were significant difference (P < 0.01) in the logarithm of the amount of stool Bacillus bifidus and Escherichia coli between pre-therapy and post-therapy group. The logarithm of the bacterium was very low on third day after treatment, but gradually increased on the seventh day after treatment. CONCLUSIONS: (1) HDMTX therapy has great effects on intestinal flora of Bacillus bifidus and Escherichia coli and significantly reduced the bacteria in children with ALL. (2) Probiotics had significantly decreased in ALL group than in control group.

[Immunogenetic analysis of human leukocyte antigen DRB1, DQB1 locus among Han ethnic children with Helicobacter pylori infection in Kunming].

OBJECTIVE: To explore the immunogenetic features of human leukocyte antigen DRB1, DQB1 locus and children with Helicobacter pylori (H. pylori) infection in Han ethnic population in Kunming and its association with digestive diseases and H. pylori to better understand the immunogenetic features of the H. pylori infection. METHODS: Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used to study the HLA-DRB1, DQB1 allelic frequency distribution on 35 children with H. pylori infection and 37 healthy controls in Han ethnic population in Kunming. RESULTS: Allelic frequencies of HLA-DRB1 * 0901, DQB1 * 03032 in the H. pylori infection group were lower than those of the healthy control group (7.14% vs. 31.08%, chi(2) = 13.16, Pc < 0.012; 5.71% vs. 25.68%, chi(2) = 10.68, Pc = 0.007) but the rest alleles' frequencies did not show significant differences. CONCLUSION: These result suggested that HLA-DRB1 * 0901, DQB1 * 03032 might protect the H. pylori infection in Han ethnic population in Kunming.

[Possible association between HLA-HRB1 and DQB1 genes frequency and susceptibility or resistance to Helicobacter pylori infection in Kunming Yi ethnic group children].

OBJECTIVE: To study if there is any association between frequency of HLA-DRB1 and DQB1 genes and susceptibility or resistance to Helicobacter pylori (Hp) infection among children of Yi ethnic group in Kunming for understanding the immunogenetic features of the digestive diseases associated with Hp infection. METHODS: Peripherial blood samples were collected from 156 children of Yi ethnic group in a primary school in Kunming city by cluster sampling and the blood Hp-IgG tests (ELISA) were performed. The samples were divided into two groups (Hp-IgG-positive group and Hp-IgG-negative group) according to the blood Hp-IgG test results. There were 61 children in Hp-IgG-positive group and 95 children in Hp-IgG-negative group. Forty children who were chosen from each group by simple random sampling underwent (13)carbon-urea breath test ((13)C-UBT). Thirty-three children who were Hp-IgG-positive and (13)C-UBT-positive were defined as currently Hp- infected group; 39 children who were Hp-IgG-negative and (13)C-UBT-negative were defined as Hp-non-infected group. DNA specimens were extracted from the lymphocytes of their peripheral blood samples. HLA-DRB1 and DQB1 DNA typing was performed by using polymerase chain reaction with sequence specific primers (PCR-SSP). HLA-DRB1, DQB1 allelic frequency distribution among currently Hp infected and non-infected children was compared. RESULTS: HLA-DRB1 * 12 gene frequency among children in Hp non-infected group was higher than that in the currently Hp-infected group (42.31% vs. 14.52%, P < 0.001, Pc < 0.012); however, HLA-DRB1 * 11 gene frequency in the Hp-non-infected group was lower than that in the currently Hp-infected group (3.85% vs. 12.9%, P < 0.05, Pc > 0.05). HLA-DQB1 * 0301 gene frequency in the Hp non-infected group was higher than that in the currently Hp-infected group (55.13% vs. 32.26%, P < 0.007, Pc < 0.05); however, HLA-DQB1 * 04 gene frequency in the Hp non-infected group was lower than that in currently Hp infected group (2.56% vs. 11.29%, P < 0.05, Pc > 0.05). CONCLUSIONS: HLA-DRB1 * 12 and HLA-DQB1 * 0301 gene may be associated with protection against Hp infection in Kunming Yi ethnic group children. Further studies with larger sample size are needed to clarify if HLA-DRB1 * 11 and HLA-DQB1 * 04 are associated with susceptible gene to Hp infection.

[Analysis on molecular characteristic of VP7 and NSP4].

OBJECTIVE: To explore the molecular characteristics and molecular variation of human rotavirus (HRV) strains and to understand the relationship between clinical characteristics and epidemiology of different HRV-VP7 and NSP4. METHODS: Double-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 were amplified with RT-PCR. Then cDNAs were sequenced and compared with 4 human rotavirus NSP4 (Wa, KUN, AU-1, Hochi)) and 3 animal rotavirus NSP4 (EW, OSU, SA11) available in the GenBank while the epidemic strains of human rotavirus isolated in different areas of China were compared, using the Clustal-mp, DNAssist, MEGA2 software. The G serotype of VP7 was analysed by PCR. RESULTS: Serotype G1 was prevalent in 2002 while serotype G3 was the prevalent in Kumming in 2003. The NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 belonged to Wa with highly conservative amino acid. Samples isolated in the same years but not in the same area shared higher homology. Symptoms associated with heavy diarrhea did not seem to be associated with NSP4 molecular variation (P > 0.05). CONCLUSION: Obvious variations of VP7 typing were seen in the same season, as well as in different areas and years. Due to the stable nature of NSP4, it seem to be a better candidate for vaccine production, than VP7.