Study design
As one part of the MING study which was designed to research the dietary and nutritional status of pregnant women, lactating mothers and young children aged 0–3 years living in urban areas of China, this study was focused on B-vitamins status in breast milk. A multi-stage sampling was applied in this cross-sectional study: firstly, three cities (Beijing is located in Northern China; Suzhou: Eastern China; Guangzhou: Southern China) were chosen according to the geographical location and status of economic development; secondly, one grade three and first-class hospital and one maternal & child hospital were selected from each city considering the scale of hospital and the feasibility of investigation; thirdly, 443 apparently healthy, well-nourished lactating women aged 18–42 years were recruited from registration records in aforementioned hospitals between October 2011 and February 2012, including 89 mothers of 5–11 d postpartum, 87 of 12–30 d postpartum, 89 of 31–60 d postpartum, 90 of 61–120 d postpartum, and 88 of 121–240 d postpartum. Study subjects were selected using the following criteria: 1) aged 18–45 years, 2) give birth to a single gestation, 3) having full-term and healthy child. Lactating women with self-reported diabetes, hypertension, cardiac diseases, and/or acute communicable diseases, or women who took hormone in recent 3 months, were ineligible for the study, as were those with postpartum depression or insufficient skills to understand study questionnaire. Figure 1 displays the recruitment flowchart from eligibility to sample analysis.
Data collection
Through face-to-face interviews, the structured questionnaire was used to collect socio-economic characteristics and lifestyle aspects of lactating women including maternal age, educational level, drinking, smoking, and family’s per capita income. The information of regular exercise defined as exercising more than once a week and lasting more than 30 min every time. Delivery mode and number of gestational weeks at delivery were also recorded. Additionally, anthropometric parameters such as height and weight were measured by the dedicated researchers. BMI (kg/m2) of mothers was calculated using height and current weight, and categorized as underweight (BMI < 18.5 kg/m2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), or obesity (≥30 kg/m2). In order to exclude those lactating mothers with hypertension or diabetes mellitus, the information of blood pressure and blood glucose were also collected from hospital records or maternal health records. Besides, gender information and date of birth of the baby was collected by telephone interview after the data collection since the data was not included in the initial questionnaires.
Milk collection
All human milk samples were collected in a dim light room in hospitals without direct sunlight exposure. After the women had breakfast, the samples were collected at the second feeding in the morning to avoid circadian influence on the outcomes (9–11 am). Before collection of milk, breasts of the subjects (which were emptied by mother herself during 6 am to 7 am) were warmed about 5–10 min by hot towel, then one full breast was emptied by trained investigators using an electric breast pump (Horigen HNR/X-2108ZB, Xinhe Electrical Apparatuses Co., Ltd., Guangzhou, China) and the milk was collected into a feeding bottle. After gently up-down shaking for ~10 times, a sample was taken (15 mL for 5–11 d postpartum, 40 mL for 12–30 d postpartum, 31–60 d postpartum, 61–120 d postpartum, and 121–240 d postpartum, respectively) and then shipped to the lab using portable incubator filled with ice bags within 1 h. The rest of the milk was returned to the mother for feeding to the infant. The samples were divided with 1 mL freezing tubes under the dim light on ice, labeled with subject number, and then frozen at -80 °C until analysis.
Analysis of B-vitamins in human milk
All of the milk samples were analyzed by Eurofins Technology Service (Suzhou) Co.Ltd in China with various analytical methods as mentioned below. Only vitamers listed in brackets were quantified in this study.
Methodology for quantifying Vitamin B
1
(thiamine), Vitamin B
2
(riboflavin), Vitamin B
3
(nicotinamide and nicotinic acid), and Vitamin B
6
(pyridoxal, pyridoxine and pyridoxamine): acidic hydrolysis followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based quantification.
One gram of sample was weighted in 15 mL centrifuge tube and hydrolyzed with 0.5 mL of hydrochloric acid (1 M) in autoclave for 30 min at 120 °C. Samples were cooled down and pH was then adjusted to 4.5 ± 0.5 by addition of HCl or NaOH. 0.01 g of taka-diastase was added to the samples, placed in water bath at 45 °C for 3 h. Sample volume was then adjusted to 10 mL with water and filtrated through 0.45 μm filter. 1 mL of sample extract was taken and isotopic labeled internal standards (thiamine-[13C4], riboflavin-[13C4, 15N2], nicotinic acid-d4, nicotinamide-d4, pyridoxine-d2, pyridoxal-[2H3] and pyridoxamine-[2H3] at a concentration of 200 μg/L) were added. Sample analysis was performed on a HPLC-MS/MS system. Liquid chromatography (LC) was performed using an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) LC system. Chromatographic separation was carried out with a Waters Acquity HSS T3 column (2.1 × 100 mm i.d., 1.8 μm). Column temperature was set to 30 °C and the autosampler remained stable at 15 °C. Ammonium formate aqueous solution (2.5 mM, solvent A) and acetonitrile (solvent B) served as mobile phase at a flow rate of 0.6 mL/min. The gradient process was: 0 min, 75% A; 1 min, 75% A; 1.1–10 min, 50% A; 10.1–15 min, 50% A; 15.1–16.5 min, 75% A; 16.6–0 min, 75% A. An API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) was used for detection of the chromatographic separation. Nestle Internal reference material (bovine milk-based infant formula) was used for method validation and quality control (QC) sample as well, reference values were established by Nestle Proficiency Test participated by Nestle laboratories and third party laboratories. Coefficient of variation of repeatability is ranged from 3.7 to 13% and Coefficient of variation of intermediate reproducibility is ranged from 5.4 to 12%. The internal reference samples were regularly included and analyzed in duplicate during analytical runs. The limit of detection (LOD) for thiamine, riboflavin, vitamin B3 and vitamin B6 were 0.65, 1, 1, and 0.65 (μg/100 g), respectively. Recovery rates of vitamin B1, vitamin B2, vitamin B3, and vitamin B6 were ranged from 92 to 107%. Cross-talking in scheduled multiple reaction monitoring (MRM) was not observed between internal standards and analytes. The result of Vitamin B3 is the sum of nicotinamide and nicotinic acid, and it is expressed as nicotinic acid (niacin). As the mol masses are nearly identical both masses are only summed up. The result for Vitamin B6 is reported as pyridoxine. Thus pyridoxal hydrochloride (correction factor 0.831) and pyridoxamine dihydrochloride (correction factor 0.702) are converted to pyridoxine.
Methodology for quantifying pantothenic acid, biotin, and folates: solvent extraction followed by microbiological assay based quantification.
One gram of sample was weighed and diluted with deionized water (40 mL). Sample extraction was then performed in a water bath at 95 °C for 30 min. After centrifugation (8000 g, 5 min), sample extracts could be further diluted with sterile water provided from the test kit if needed. Sample extracts were then pipetted on the commercially available microtiter plate VitaFast® (R-Biopharm Analysis System Trading Ltd, Beijing, China). After having applied all kit instructions including microorganism incubation at 37 °C in the dark for 20–24 h (pantothenic acid) or 44–48 h (biotin and folates), the turbidity of each vial was measured with a microtiter plate reader at 610–630 nm (alternatively at 540–550 nm), for the calculation of pantothenic acid, biotin, and folates concentration. The LOD values for pantothenic acid, biotin, and folates were 0.074, 0.080, and 0.160 (μg/100 g), respectively. The measurements of QC samples described above (internal reference samples) at predetermined intervals were regularly performed to obtain QC curve for the demonstration of method’s reliability. Recovery rates of pantothenic acid, biotin, and folates were ranged from 93 to 112%.
Dietary measurement
Experienced and trained researchers conducting dietary interviews obtained the information of dietary recall during the previous 24-h from participants by face-to-face interview when milk samples were collected. All of the foods intakes were coded and B-vitamins (including thiamine, riboflavin, and niacin intakes) were analyzed using a database according to Chinese Food Composition (CFC) tables 2004 & 2009 consist of 1773 food items [37, 38]. Considering the fact that vitamin B-6, biotin, and folic acid were detected in very limited food items in CFC, the Japan Food Composition (JFC) tables (2014) [39] was used to estimate intakes of them in lactating women. If the participants had consumed dietary supplements, they were asked to supply the basic supplement information, including the brand name, manufacturer, and actual consumption (daily dosage).
Statistical analysis
Socioeconomic characteristics of lactating women were described as count (percentage) for categorical variables and median value with interquartile range for continuous variables without normal distribution. Chi-squared tests (categorical variables) and Kruskal-Wallis tests (continuous variables) were used to compare participants’ characteristic according to stages of lactating period. Median (interquartile range) and mean ± standard deviation were calculated for each of B-vitamin according to research cities (Beijing, Suzhou, and Guangzhou cities) and stages of lactation (5–11 d postpartum, 12–30 d postpartum, 31–60 d postpartum, 61–120 d postpartum, and 121–240 d postpartum). Before the progress of analysis about B-vitamins, Shapiro-Wilk test was employed to determine whether B-vitamins in human milk had a normal distribution or not. Because of non-normal distribution in human milk vitamins, naturally logarithmic transformations were applied when doing analysis of variance (ANOVA) according to research cities and lactating stages. Multivariate linear regression analysis was used to describe the relationship between B-vitamins concentrations in breast milk (dependent variables) and socioeconomic characteristics (independent variable) of lactation women. A stepwise forward selection process was used in which the independent variables and confounders (maternal age, present BMI, education, family income, delivery mode, regular exercise, supplement intake, duration of breastfeeding, and city) were added to the models according to their significance (p value). Homogeneity and bias was showed in the analyses of the residuals of the final models. The associations between B-vitamins concentrations and diet characteristics such as dietary B-vitamins intakes were evaluated by partial-correlations adjusted for research cities and lactation stages. All statistical analyses were performed by using the SPSS software (Ver. 20.0) (SPSS Inc. Chicago, IL, USA), and the level of significance was set at p < 0.05 based on a two-sided calculation.