Introduction

The consumption of total trans fatty acids increases the risk of heart diseases and may affect the growth and development of infants [1,2,3]. Human studies have revealed that trans fatty acid consumption raises plasma cholesterol and triacylglycerol level. Arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3) are considered essential fatty acids for pre-term infants. These long-chain polyunsaturated fatty acids (LC-PUFA) can be endogenously synthesized if diet contains linoleic acid (LA, 18:2n-6) and alpha-linolenic acid (ALA, 18:3n-3) through a pathway of desaturation and elongation. Inverse correlations are found between trans fatty acid consumption and LC-PUFA in the venous cord blood lipids in full-term infants [4]. Although adverse effect of trans fatty acids on infant growth and development has not been established, it must exercise caution in this aspect.

Hong Kong is a city where the East meets the West. Diet westernization is taking place due to extensive cultural exchanges, industrialization, and urbanization [5, 6]. With improvement in educational attainment and an increase in job opportunities, there is an increase in the percentage of Hong Kong women joining the labor force in the past 20 years [7]. A smaller family size, recently due to a decrease in fertility rates for working couples, and an increase in opportunity cost of women’s time, may lead to eat outside home more frequently and increase the demand of convenient processed foods, such as fast foods, and ready-made meals [7, 8].

Breast milk is the primary nutrient source for infants and serve as an indicator for dietary practices of the lactating mothers. It is known that humans do not produce trans fatty acids and maternal dietary trans fatty acids are able to transfer to the breast milk [9]. In this regard, the presence of trans fatty acids in breast milk is mainly come from maternal diet [10]. Our previous research in 1995 showed that the total trans fatty acids in breast milk of Hong Kong lactating women accounted for 0.88% total milk lipids [11]. Accompanied with diet westernization in Hong Kong and an increase in consumption of convenient processed foods, trans fat intake may increase accordingly, thus possibly leading to an increase in breast milk trans fatty acid content. Although the fatty acid composition of human milk has been well reported in various countries [12,13,14,15,16,17], there is very limited updated information on human milk fatty acid composition of Hong Kong Chinese lactating women in the recent years. The aims of the present study were (i) to quantify trans fatty acids and LC-PUFA in diets of Hong Kong lactating mothers using a method of 3-day food records; (ii) to quantify trans fatty acids and LC-PUFA in breast milk of Hong Kong lactating women using a method of gas chromatography; and (iii) to compare the current breast milk trans fatty acid composition with that obtained in 1995 [11].

Materials and methods

Study design

A 3-day food record method including 2 weekdays and 1 weekend day within a week was used to collect dietary intake data. Breast milk samples were collected to investigate its fatty acid composition. Data collection started in September 2017 and completed in December 2018. Ethical approval was obtained from the New Territories East Cluster Clinical Research Ethics Committee of the Chinese University of Hong Kong prior to initiation of the study. A signed consent was obtained from all lactating women who agreed to participate in this study.

Subjects and recruitment

Pregnant women were recruited mainly from obstetrics department in hospitals in Hong Kong. During their antenatal checkups, invitation letter and study information were distributed. After the delivery, those who were interested in participating the study were contacted to arrange home visits and provide training on documenting food record and materials for collecting breast milk. A signed consent was collected at the first home visit. Inclusion criteria of subjects included: (i) they were able to complete a 3-day food record at week 4 and 6 after the delivery; (ii) they were willing to donate breast milk sample after completion of week 4 and 6 food records; (iii) they had no chronic diseases that require food restriction or follow specific diets; and (iv) they had no breast disease such as mastitis.

Dietary data collection and analyses

The lactating women were instructed how to document their food intake by a registered dietitian. Each subject was required to record and describe the names, consumption amount, time, and location, where the foods and drinks were consumed. No food record was conducted 3 days before and after festivals that might affect one’s regular eating habits (e.g., Mid-autumn Festival or Chinese New Year). Each lactating mother recorded her food intake for 3 days, 2 weekdays, and 1 weekend day, at week 4 and 6 after delivery. Thus, a total of 6 days of food records from each subject was collected.

Estimation of trans fat intake and nutrient intake using dietary data

Dietary data analyses were performed using a food composition database, Nutrition Data System for Research (NDSR) (Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN, USA). When a consumed food that could not be found in the NDSR database, a similar food would be used as an alternative, or created a new food item by using ingredients that are available in the NDSR database. The data were then exported to the Statistical Package for the Social Sciences version 23.0 (SPSS Inc, Chicago, IL, USA) for analysis. The mean 3-day food record of week 4 and 6 was accordingly computed. Results were compared with the recommendations for lactating women of the Chinese Nutrition Society (CNS) and the United States (US) Institute of Medicine (IOM). The recommendations by the World Health Organization (WHO) and Food and Agricultural Organization of the United Nations were also used when appropriate.

Milk collection and lipid extraction

Each participant expressed 15 ml of her milk using a manual milk pump on the assigned dates at week 4 and 6 after the delivery. The participants were instructed to collect the milk samples at the end of feeding in the morning. Babies were breastfed on demand and mothers look for signs of hungry and feed their babies whenever babies were hungry. Therefore, there was no a fixed time schedule for collecting the milk. Each time after collection, milk sample was placed in a residue-free tube and stored immediately into a freezer at −20 °C. Milk samples were thawed at room temperature (25 °C) before the analyses. Total fat from 6 g sample was extracted using 15 milliters of CHCl3/MeOH (2:1, vol/vol) containing 6 mg/ml triheptadecanoin as an internal standard to quantify total milk fat.

Gas chromatographic analyses of milk fatty acids

The milk lipids were converted to fatty acid methyl esters (FAME) using a mixture of 14% BF3/MeOH reagent (Sigma-Aldrich Co., St Louis, MO, USA) and toluene (1:1, vol/vol) at 90 °C for 45 min under nitrogen. FAME were analyzed on a SP-2560 flexible fused-silica capillary column (100 m × 0.25 mm i.d., 0.2 μm film thickness; Supelco, Inc., Bellefonte, PA, USA) in a gas chromatograph (Hewlett-Packard 6890 Series with auto injector; Agilent, Palo Alto, CA, USA) equipped with a flame-ionization detector. Peaks were identified by comparison with authentic fatty acid standards (GLC reference standard GLC-674; Nu-Chek-Prep, Inc., Elysian, MN, USA). Column temperature was programmed from 150 °C to 180 °C at a rate of 0.5 °C/min, and then to 210 °C at a rate of 3 °C/min. Injector and detector temperatures were 220 °C and 220 °C, respectively. Hydrogen was used as the carrier gas at a head pressure of 20 psi. A typical GC chromatogram was shown in Supplementary Fig. 1.

Calculation of trans fat intake on its percentage in breast milk lipids

The trans fat intake by each lactating woman was also calculated on the basis of trans fatty acid content in breast milk, using a method of Craig-Schmidt et al. [18], who found that the trans fatty acid in the mother diet of previous day was well correlated with that in the breast milk. The trans fatty acid intake by mothers could be estimated using two equations shown below (Supplementary Fig. 2):

$${{Y}} = - 0.011{\mathrm{X}}^2 + 0.707{\mathrm{X}};$$
(1)
$${{T}}_{{\mathrm{intake}}} = \left( {{{E}}_{{\mathrm{total}}} \times {{E}}_{{\mathrm{fat}}} \times {{X}}} \right) \div {\mathrm{9}}$$
(2)

where Y is the percentage of trans fatty acids in breast milk fat; X is the percentage of trans fatty acids in the dietary fat of previous day consumed by mother; Tintake is the trans fatty acid intake of the mother; Etotal is mother’s total energy intake; Efat is mother’s energy percentage of fat intake; and 9 is kcal value per gram fat.

Statistical analysis

Data were expressed as mean ± standard deviation. Analysis of variance followed by Student’s t-test (two tailed) where applicable was used for statistical evaluation of significant differences between groups. A p value < 0.05 was considered statistically significant.

Results

Participants

Total 72 lactating women were recruited in the present study. Among them, 60 met all of the inclusion criteria. The mean age of the participants was 33 years old, with a range of 23–44 years old. Characteristics of the participants were shown in Table 1.

Table 1 Characteristics of participants.
Full size table

Intakes of energy, carbohydrate, protein, and dietary fiber by Hong Kong lactating women in 2018

Mean energy intake was found to be 2069 kcal/day, lower than the estimated energy requirement (EER) of 2300–2900 kcal by CNS (Table 2). Mean carbohydrate intake was 215 g, accounting for 43% of total energy. As CNS only has estimated the average requirement (EAR) available for total grams of carbohydrate, IOM RDA was used for assessment. The mean carbohydrate intake among these Hong Kong lactating women was very close to the RDA of 210 g/day. Dietary data showed that 88% of the carbohydrate was from refined grains, whereas the mean intake of added sugar was ~27 g, which was about 5% of the total energy within the AMDRs (Table 2). Mean protein intake was 123 g, accounting for 24% of total energy intake. Compared with that set by IOM AMDR (10–35% energy), an average of 24% of the energy from protein among these lactating mothers was within the recommendation. The intake of animal proteins was prevalent, compared with that of vegetable protein in these women. Dietary fiber intake was 15 g, with 3 g of soluble fiber and 12 g of insoluble fiber (Table 2). Mean intake of dietary fiber was lower than the AI of 29 g/day set by IOM. Hong Kong Department of Health (DH) recommends daily fiber intake of at least 25 g. This indicates that the intake of dietary fiber by these Hong Kong lactating women was insufficient.

Table 2 Calculated intakes of carbohydrate (CHO), protein (Pro), total fat, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), trans fatty acids, using database Nutrition Data System for Research (NDSR) compared with the relevant recommendations.
Full size table

Calculation of intakes of cholesterol, total fat and trans fat by Hong Kong lactating women in 2018 using NDSR

Results from NDSR showed that Hong Kong lactating women had a mean intake of 477 mg cholesterol. CNS does not have a recommendation for cholesterol. IOM recommends daily cholesterol intake to be as low as possible. From the result of the 3-day food records, mean intakes of total fat, saturated fatty acids (SFA), trans-fatty acids, monounsaturated fatty acids (MUFA), and PUFA were 79, 24, 1.2, 29, and 18 g, respectively. Energy from total fat was 33%, exceeding the CNS recommendation of 20–30%, but within 20–35% recommended by IOM (Table 2). Energy from SFA was ~10% energy, which was at the borderline of the CNS recommendation of <10% in a day. Result from NDSR analysis showed that the Hong Kong lactating women consumed 1.15 g trans fat daily (0.50% energy), which was below WHO’s recommendation of <1.0% total energy. Table 2 also showed the intakes of other fatty acids collected by 3-day food records.

Calculation of trans fat intake by Hong Kong women in 2018 using gas chromatographic analysis

Results from gas chromatographic analysis showed total fat and trans fat in breast milk were 22.89 and 0.24 g/kg breast milk (Table 3). The gas chromatographic analysis found that trans fatty acids accounted 1.08% total milk fat. Using the first equation Y = −0.011X2 + 0.707X, an average of 1.52% trans fatty acids as total milk fat was calculated. Using the second equation Tintake = (Etotal × Efat × X) ÷ 9 described above [18], the Hong Kong lactating women consumed 1.20 g trans fat (Table 3), which was close to 1.15 g calculated using database NDSR (Table 2). Figure 1 shows a good correlation of Hong Kong lactating women’s trans fat intake calculated using database NDSR and its intake calculated using the percentage of trans fatty acids in the breast milk determined by gas chromatographic analysis [18].

Table 3 Trans fatty acid intake by Hong Kong Chinese lactating women in 2018a.
Full size table
Fig. 1: Estimation of trans fat intake by two methods.
figure 1

Correlation of trans fat intake (g) of Hong Kong lactating women calculated by using data base Nutrition Data System for Research (NDSR) and using the equations described in “Materials and methods” adapted from [18] (p < 0.01).

Full size image

Comparison of milk fatty acid composition between week 4 and 6

Milk fatty acid composition in week 4 and 6 was shown in Table 4. In general, no differences were seen except for several minor fatty acids. The breast milk samples from week 4 had slightly but significantly higher amounts of 20:2n-6, 20:3n-6, and total n-6 long chain fatty acids than those from week 6. In contrast, the breast milk samples from week 4 had slightly but significantly lower amounts of total trans fatty acids mainly including18:1(Δ7–Δ11)t and 16:1Δ7t (Table 4).

Table 4 Milk fatty acid composition (wt% total fatty acids) of Hong Kong Chinese women at different stages of lactation in 2018a.
Full size table

Comparison of milk fatty acid composition between 2018 and 1995

The average fatty acid composition of milk pooled from week 4 and 6 of lactation in Hong Kong Chinese lactating women in 2018 was shown in Table 4. For comparison, the data of Hong Kong Chinese lactating women in 1995 were also shown in Table 5 [11]. A number of differences in the milk fatty acid profiles were observed between 1995’s and 2018. The breast milk in 2018 contained higher amounts of total trans-fatty acids and total n-3 fatty acids than those from 1995 (Table 5). Regarding the major saturated and mono-unsaturated fatty acids, the breast milk samples in 2018 had lower amounts of palmitic acid (PA, 16:0), stearic acid (SA, 18:0), and oleic acid (OA, 18:1n-9). Regarding the major n-6 fatty acids, no difference in LA (18:2n-6) and ARA (20:4n-6) were seen between 2018 and 1995. Regarding the major n-3 fatty acids, the breast milk samples from 2018 had higher amounts of ALA (18:3n-3) and EPA (20:5n-5) than those from 1995 (P < 0.01). DHA (22:6n-3) in the breast milk samples derived from 2018 was marginally higher than that from 1995 (P = 0.06).

Table 5 Milk fatty acid composition (wt% total fatty acids) of Hong Kong Chinese women at different stages of lactation in 2018 compared with that in 1995a.
Full size table

Discussion

Extensive consumption of trans fat from partially hydrogenated vegetable oils is associated with the risk of heart diseases in adults [1, 2]. Excessive consumption of trans fat by mothers may also affect the growth and development of infants [3]. To find out trans fat intake by Hong Kong people, we estimated the trans fat intake using NDSR, finding that Hong Kong lactating women consumed an average of 1.15 g trans fat daily in 2018 (Table 2). The present study was the first of its kinds to estimate the trans fat intake by lactating women based on the percentage of trans fatty acids in the breast milk using Eqs. 1 and 2 described in “Materials and Methods”, finding that Hong Kong lactating women consumed an average of 1.20 g trans fat each day (Table 3). Results demonstrated the two methods were well correlated and reliable (p < 0.01; Fig. 1). A total daily trans fat consumption of 1.15–1.20 g trans fat accounted for 0.50–0.52% total calories, which was a half of WHO’s recommendation of <1.0% total energy. If the data on trans fat intake by lactating women could be extrapolated to the rest members of their family or general population at this time, it would be concluded that the relative trans fat intake by Hong Kong people was low.

Trans fat intake across the lactation in Hong Kong lactating women was not constant. It was interesting to notice that daily trans fat intake increased from 0.92 g in week 4 to 1.39 g in week 6 using NDSR (Table 2), while it increased from 1.05 g in week 4 to 1.35 g in week 6 using Eqs. 1 and 2 (Table 3). This was probably due to changes in dietary pattern. Usually, Chinese lactating women stay and eat at home in the first month after delivery. Thereafter, they start to eat outside home, leading to an increase in consumption of trans fat-enriched foods. Compared with a value of 0.88% trans fatty acids in total lipids of breast milk in 1995, the trans fat content in breast milk in 2018 slightly but significantly increased to 1.08%, an 27% increase (Table 5). In this regard, the trans fat content in breast milk of Hong Kong lactating women was much lower than that recently reported from Malaysia (2.95% milk lipids) and Brazil (1.5% milk lipids), but higher than that from New Zealand (0.86% total milk lipids) [12, 16, 17].

The milk fatty acid composition in 2018 samples of Hong Kong lactating women was slightly different from that in 1995 samples. Regarding the saturated and MUFA, Hong Kong lactating women in 2018 had slightly lower contents of PA (16:0), SA (18:0) and OA (18:1n-9) but had a slight higher content of palmitioleic acid (16:1n-7) (Table 5). Among n-6 PUFA, no differences in LA (18:2n-6) and ARA (20:4n-6) were seen between 2018 and 1995. However, these minor n-6 PUFA including 18:3n-6, 20:2n-6, 22:4n-6, and 22:5n-6 in breast milk samples of 2018 were slightly higher than those of 1995. When total n-6 PUFA in the breast milk of 2018 were compared with those of 1995, no significant difference was seen (Table 5). Regarding n-3 PUFA, Hong Kong lactating women in 2018 had slightly higher contents of ALA (18:3n-3) and EPA (20:5n-3) in the breast milk compared with those in 1995. As a whole, the breast milk samples collected in 2018 had an intake of total n-3 PUFA 18% higher than that collected in 1995. The changes in these minor fatty acids of 2018 breast milk samples suggested that dietary fats consumed by Hong Kong lactating women in 2018 were slightly different from those in 1995.

Human breast milk fat contains various fatty acids derived from three sources: maternal fat stores, maternal dietary lipids, and de novo synthesis in the mammary gland [19,20,21,22,23]. Contents of PUFA in breast milk vary with countries [12,13,14,15,16,17]. Hong Kong Chinese lactating women have a higher ALA (18:3n-3) content of 1.52% in total milk lipids compared with those in Brazil, Malaysia, Switzerland, New Zealand, and Spain [12, 15,16,17, 20]. DHA (22:6n-3) in the breast milk originates from either maternal diet or de novo synthesis. Hong Kong lactating women have a DHA content of 0.66% in total breast milk lipid comparable to that among the lactating women in Malaysia and South Korea [13, 16], but much higher than that in Brazil, Switzerland, New Zealand, Canada, and Spain, probably due to high consumption of sea foods in Hong Kong [12, 15, 17, 19, 20] (Supplementary Table 1). Hong Kong people prefer vegetable oils like corn oil, sunflower oil, and peanut oil as cooking oils. This was reflected from a higher content of LA (18:2n-6) in the breast milk lipids of Hong Kong lactating women compared with that in the breast milk of Malaysia, Switzerland, New Zealand, Canada, and Spain [12, 15, 16, 19, 20]. ARA (20:4n-6) in the breast milk originates from either maternal diet or de novo synthesis. As LA is the precursor for the synthesis of ARA, it was expected that Hong Kong Chinese lactating women had a higher ARA content in the breast milk compared with those in Brazil, New Zealand, Switzerland, South Korea, Canada, and Spain [12, 13, 15, 17]. Breast milk contains five major saturated and MUFA including PA (16:0), OA (18:1n-9), and SA (18:0), myristic acid (14:0), and lauric acid (12:0). The contents of these saturated fatty acids in the breast milk lipids of Hong Kong lactating women were similar to those in the breast milk lipids among the other countries [12,13,14,15,16,17].

A strength of the current study was the first time to estimate the trans fat intake by Hong Kong lactating women using the two methods, namely NDSR database and calculation based on the percentage of trans fatty acids in the breast milk. Our results demonstrated that these two methods were well correlated and both methods were able to provide a reliable and accurate estimation of the trans fat intake by the lactating women. The second strength of the current study was to collect the breast milk twice at both week 4 and 6, ensuring the consistency and no bias of milk sampling. The third strength of the present study was to compare the fatty acid composition of breast milk collected in 2018 with that in 1995, demonstrating the fats consumed by Hong Kong Lactating women were slightly different from those in 1995 because the former had a relative high content of n-3 PUFA. However, the present study had three limitations. First, there was no a fixed time schedule for collecting the breastmilk due to avoid the disruption of on-demand feeding. Inconsistent timing of collecting milk sample might affect the results of milk fatty acid composition as the breast milk composition could change during each feeding. Second, all the lactating women were instructed to collect their milk sample at the end of feeding in the morning and were trained to record their dietary intake using 3-day food record, however, there was no actual measure on their compliance. Third, the blood samples of mothers were no taken so that the association between the trans fat concentrations in maternal plasma with that in breast milk could not be explored.

In conclusion, Hong Kong lactating women consumed a diet in which total trans fats accounted for 0.50–0.52% total calories. This trans fat intake remains in line with WHO’s recommendation that total trans fat intake should not be over than 1.0% total energy. Compared with Western countries, the breast milk of Hong Kong Chinese lactating women had relatively higher contents of LA, ARA, ALA, and DHA, most likely due to the popularity in consuming vegetable oils and sea foods by Hong Kong lactating women.