In recent years, the attention to the concept of early-life metabolic programming and the future health of growing infants was remarkably rising. Early environmental factors, such as chemical exposure, diet, and nutritional supplements might have after-effects on human biology and long-term health. Human milk is recommended as the best source of nutrients for infants from 0 to 6 months [1
]. It provides not only metabolic nutrients, but also functional elements such as vitamins, minerals, oligosaccharides, and various protective factors that could give long-term effects on physical, mental [3
] and immune system development [1
Among these functional compounds, vitamins, particularly, have fundamental roles for the proper growth and development of infants. Vitamin A is one of the most important micronutrients affecting vision, the immune system, lung development, and maturation [6
]. Vitamin D is involved in the calcium absorption, mineralization of the skeleton, and the prevention of rickets in children [9
]. Vitamin E is a strong antioxidant that inhibits lipid peroxidation and protects cell membranes and lipoproteins from free radicals [8
]. The water-soluble vitamin B complex is a co-enzyme of numerous biochemical reactions and has various functions in the human body [13
]. In addition, lutein is a major carotenoid in the human eye [17
]. It is also a dominant carotenoid in the infant’s brain as well, and plays an important role in cognitive development [18
It has been well noticed that the deficiency of vitamin or lutein could lead infants to improper development, diseases, and even cause death [5
]. Vitamin deficiency of an infant occurs due to their limited amount of vitamin reserves at birth and the rapid consumption during their early age of growth. Since vitamin supplement depends only on the exogenous source, the amount of vitamins in human milk was essential for infant health and development. Moreover, the concentration of retinol in human milk is also used as a biomarker of vitamin A deficiency in lactating women and children up to 71 months [21
The amount and types of vitamins and lutein in human milk are closely related to the mother’s diet, nutritional status, and lactation duration [22
]. Up to now, vitamin contents in breast milk were studied in several developed countries, however, the information about vitamin status in Asian mother’s milk is still rare. Moreover, the different analytical methods contribute to the strong variation of vitamin content between countries. Here, we attempt to collect mother’s milk from four Asian countries in different climate zones, including the temperate zone (Northeastern China), sub-tropical zone (South-Korea), and tropical zone (Vietnam and Pakistan). These mothers also consumed different styles of food, such as Muslim food in the Pakistan population and Asian cuisine for the others. Vitamin concentrations in human milk were studied and compared between countries.
2. Materials and Methods
2.1. Sample Collection
Human milk samples were collected from Korea, China, Pakistan, and Vietnam from 2017 to 2018. This study was approved by the Institutional Review Board of Chungnam National University (Korea), Maeil Dairies. Co., University of Medicine and Pharmacy at Ho Chi Minh City (Vietnam) and University of Agriculture (Faisalabad, Pakistan). Informed consent was obtained from all participants and methods were performed in accordance with the relevant guidelines and regulations. In this study, the number of human milk samples from Korea, China, Vietnam, and Pakistan were 254, 137, 92, and 97 samples, respectively.
Human milk was directly collected in a sterilized 50 mL conical tube (Corning, NY, USA), by hand press or breastmilk pump. The total volume of maternal milk donated from each mother was 50–150 mL. Then, the sample was delivered to the laboratory with a gel ice pack in well-insulated containers, to maintain a low temperature. The human milk sample was stored at −80 °C before analysis. The sample was rejected from analysis if the storage time was longer than 2 months.
Vitamin standards that include thiamin hydrochloride, riboflavin, nicotinic acid and nicotinamide, calcium-D-pantothenate, pyridoxin-hydrochloride, biotin, folic acid, cyanocobalamin and phylloquinone, were purchased from AccuStandard, New Haven, CT, USA. The following standards and reagents were obtained from Sigma-Aldrich, (Seoul, Korea): pyridoxal-hydrochloride (B6), all-trans retinol, all-trans tocopherol (E), ergocalciferol (D2), cholecalciferol (D3), all-trans lutein, butylated hydroxytoluene (BHT), HPLC grade solvent (methanol, ethanol, acetonitrile, and hexane). The HPLC grade MTBT (tert-Butyl methyl ether) was purchased from Thermo Fisher, Korea. Moreover, 7-dehydrocholesterol (DHC) was purchased from Cayman Chemical (MI, USA). Potassium hydroxide, ascorbic acid and sodium chloride were purchased from Deajung. Co. Ltd. (Busan, Korea).
2.3. Water-Soluble Vitamin Analysis
2.3.1. Sample Preparation
The milk sample (0.5 mL) was mixed with 1 mL of ethanol and sonicated for 20 min. Then, 2 mL of hexane (containing 0.025% BHT) was added to the sample and shaken for 20 min. The mixture was centrifuged (3600× g, 15 min). The aqueous layer was collected and mixed with 0.5 mL ice ethanol (−80 °C, 1 h). The supernatant obtained after centrifugation (21,000× g, 20 min) was quickly evaporated to remove ethanol. The sample was cleaned up by solid phase extraction using C18 cartridge (Sep-Pak C18, 200 mg sorbent, Waters, UK). The cartridge was activated and conditioned by methanol and distilled water. After sample loading, water-soluble vitamins were eluted by 6 mL of methanol: water (1:1). The elution was completely evaporated at 37 °C. The sample was reconstituted and diluted in acetonitrile: water (3:7) solution (containing 0.1% formic acid) before the injection into LC-MS/MS. The number of human milk samples in China, Korea, Pakistan and Vietnam were 111, 155, 97 and 92, respectively.
2.3.2. LC-MS/MS Conditions
Vitamin B-complexes were analyzed by using ultra performance liquid chromatography tandem mass spectrometry (6460 Triple Quadrupole System, Agilent, CA, USA). The analytical column was Glycan BEH Amide (2.1 × 100 mm, 0.35 mm, Waters, Hertfordshire, UK) and was kept at 30 °C during analysis. The solvents were 50% acetonitrile (solvent A) and 90% acetonitrile (solvent B), each of which contained 0.1% formic acid and 10 mM ammonium formate. The flow rate was 0.3 mL/min and the gradients between the time points were as follows: 0–2 min, B = 99.9%; 2–6 min, B = 99.9–70% B; 6–12 min B = 70–30%; 12–14 min, B = 30–10%; 14–20 min, B = 10%; 21–30 min, B = 99.9%. The MS was performed in positive mode with the MS conditions, as follows: gas temperature, 300 °C; gas flow, 8 L/min; nebulizer pressure, 45 psi; sheath gas temperature, 300 °C; sheath gas flow, 12 L/min; capillary voltage, 4000 V (positive); nozzle voltage, 1000 V (positive). The injection volume was 1 μL. The MS/MS parameter and MRM transitions of analytes are summarized in Table S1
The standard curves were set up with the solution of thiamin, riboflavin, nicotinic acid, pyridoxine, pyridoxal ranged from 0.1 to 200 µg/L. The standard solution of pantothenic acid, nicotinamide ranged from 50 to 5000 µg/mL and 0.1 to 500 µg/mL, respectively. The r2 of the standard curve for all standard materials ranged from 0.994 to 0.999. The limit of quantitations (LOQ) for all vitamins were 0.1–0.6 ppb, except vitamin B9 (4.5 ppb) and B12 (1.8 ppb).
2.4. Fat-Soluble Vitamins and Lutein Analysis
2.4.1. Samples Preparation
The milk sample (2 mL) was put into a 15 mL centrifuge tube. Then, 4 mL of ethanol (0.1% (w/v) BHT), 1 mL of NaCl solution (2%, w/v), 1 mL of ascorbic acid solution (4%) and 1 mL of KOH solution (60%, w/v) were subsequently added. The mixture was saponification at 70 °C for 60 min in shaking water-bath. After that, sample was quickly cooled in ice water. The sample was double extracted in 5 mL hexane. Both hexane fractions were combined and washed with NaCl (0.1%) to remove KOH. Then, hexane was evaporated under vacuum condition (40 °C, 30 min). The residual was reconstituted in 100 µL of isopropanol: hexane (75:25, 0.025% BHT). The sample was filtrated through a 0.2 µm and injected into HPLC.
2.4.2. HPLC Conditions
Fat-soluble vitamin was quantified by HPLC coupled with UV detector (1260 Agilent, California, USA). Analytes were separated using a C30 YMC Carotenoids column (5 µm, 4.6 × 250 mm, YMC Korea Co. ltd., Gyeonggi-do, Korea) at 30 °C. The mobile phase A consisted of methanol: acetonitrile: water (4:5:1) and mobile phase B was methyl tert-butyl ether. The flow rate of the mobile phase was 1 mL/min. The gradient profile was as follows (t in min): t0, B = 5%; t20, B = 25%; t25, B = 40%; t29, B = 70%; t33, B = 90%; t39, B = 90%; t40, B = 5%; t50, B = 5%. Fat-soluble vitamins were detected at various wavelengths: retinol: 325 nm, vitamin D: 265 nm, vitamin E: 220 nm, vitamin K1: 246 nm, and lutein: 445 nm. The limit of quantitation (LOQ) of the method was 0.6–2.7 ppb for all fat-soluble vitamins, except vitamin E (10.5 ppb).
2.5. Statistical Analysis
The data were expressed as mean ± standard deviation. The one-way ANOVA and Scheffe’s multiple range test (p < 0.05) were used to define the significant difference between groups. The correlation between variables was determined by Pearson’s product-moment correlation coefficient (r-value). Stata/SE (version 12.1) has been used for the statistical analysis and the generation of resulting figures.
Vitamins belong to micronutrients and are essential for infant growth and development. Breast milk contains almost all the vitamins that a healthy full-term baby needs, although the supplementation of vitamin D and K is still recommended for infants. However, not all babies can be breastfed, for various reasons, which makes infant formula inevitable. To supply sufficient and balanced vitamins for infant’s needs, the reference information of vitamin concentration in human milk is necessitated for infant formula setting up.
Vitamin concentrations in breast milk were not consistent widely in many publications. It is partly due to the complex structures and isomers of vitamins and the subsequent diversity of the numbers and types of vitamin standards. Vitamin B occurs in human milk under various forms and derivatives. It was reported that vitamin B1
is present in human milk under the forms of free thiamin, thiamin-monophosphate, and thiamin-pyrophosphate. Similarly, vitamin B2
was founded as free riboflavin and flavin adenine-dinucleotide [19
], or vitamin B3
naturally presented as nicotinic acid and nicotinamide in human milk. Vitamin B6
mainly occurs as pyridoxal and a small amount of pyridoxine + pyridoxamine [24
]. Vitamin B9
in human milk consisted of unmetabolized folic acid (23%), 5-methyl-tetrahydrofolate (55%), and other reduced folates (tetrahydrofolate (THF), 5-formyl-THF and 5, 10-methenyl-THF) [25
], and so on. In the same way, the fat-soluble vitamin also had various forms and isomers, such as vitamin E (alpha, beta, and gamma-tocopherol), vitamin K (phylloquinone, menaquinones) [26
], vitamin D metabolites [21
-lutein isomer [27
In this study, thiamin (free form), riboflavin (free form), niacin (nicotinamide and nicotinic acid), D-pantothenic acid, vitamin B6
(pyridoxal and pyridoxine), biotin, folic acid (free form) and cyanocobalamin were analyzed. Although the concentrations of all vitamin metabolites were not able to be covered in one study, we attempt to make a comparison of our human milk vitamin data obtain from four Asian countries and previously published data of the same metabolites (Table 4
). Apparently, each vitamin concentration found from various studies varied in the wide range. On the other hand, the level of vitamin obtained in this study was reasonable and within a similar range of previous studies. In this study, human milk was randomly collected during the lactation stage, from both supplementation users and non-users. Vitamin K value in this study was quite high, however, it should be noted that the value was only calculated from the sample observed the vitamin K (LOQ = 2.7 µg/L). There was 30 to 54% of mother’s milk in which vitamin K was not found. Taking these samples into consideration, the concentration of vitamin K in Asian human milk became 12.6 ± 17.0 µg/L (median: 6.0 µg/L).
Although we could not cover all of the vitamins, a general comparison of vitamin concentration in human milk between Asian countries would be valuable. The vitamin B level in the Vietnamese population was usually significantly lower than those in breast milk from other countries, particularly, riboflavin (B2
), pantothenic acid (B5
), and pyridoxine (B6
). Pakistani maternal milk was noticed with a low concentration of folic acid. It was reported that thiamin, riboflavin, and pyridoxine in the mother were also strongly influenced by the mother’s diet or supplementation [15
]. Hence, a recommendation of supplementation or improving vitamin B in the diet seems to be required for Vietnamese and Pakistani mothers.
Breastmilk is a great source of lutein for infants. The lutein concentration in breastmilk may vary from 10 to 100 μg/L, which is several times higher than that in cow milk (approximate 5–15 μg/L) [29
]. Particularly, lutein originating from breast milk plays an important role in visual processing in early life [30
]. It is the predominant carotenoid in adults and infant brains [31
], especially in the neocortex area, and also a key functional component in the neural retina [32
]. Recently, research on rhesus macaques indicated that lutein supplementation of infant formula significantly increased serum and tissue lutein concentrations compared to the unsupplemented formula, however, both of them were still lower than those in breastfed infants.
Interestingly, lutein in Chinese maternal milk was higher than in other countries. The average of lutein concentrations in human milk from Korean, Pakistani, and Vietnamese mothers was 40–50 µg/L while 66.1 µg/L in Chinese maternal milk. It was also observed that lutein in China maternal milk had a high content, up to the median of 93.1 µg/L [33
]. It might be due to the difference in the mother’s diet, since the breast milk lutein concentration depended on maternal intake [34
Based on the data we observed, it was still difficult to conclude whether the human milk of Asian mothers could provide enough vitamin for their baby or not. However, the data in this study also clearly indicates that vitamins B12
, K, and D were not sufficient in human milk. Vitamin K intake for the first 0–6 months is recommended at 5 mg/day [51
]. Based on the obtained data, approximately 43–53% of infants in Korea, China, and Pakistan and 75% of Vietnam did not get enough vitamin K. Vitamin B12
and D levels of other countries were also reported as low as the range of pmol/L [35
Under the circumstances, providing additional vitamin B12
and D through the mother’s diet or supplements seems to be necessary. It was reported that B12
supplementation of the mother during pregnancy and early lactation could improve vitamin B12
status in breast milk and infant [37
]. Moreover, the mother supplemented with a high dose of vitamin D was able to increase a slight amount of vitamin D in her breast milk [43
]. In this regard, the WHO strongly recommended giving vitamin supplementation for breastfed infants [51
]. It is recommended that they supplement with 400 IU per day of vitamin D, beginning in the first few days of life [53
]. Additionally, it has been recommended that mothers should pay more attention to their diets or provide vitamin K supplementation to their baby [51
Vitamin A deficiency is one of the most common health problems in the world. In addition, retinol concentration of mother’s milk of less than 1.05 μmol/L is used as a biomarker of vitamin A deficiency (VAD) in lactating women and children up to 71 months [51
]. The percentage of the indicator (milk retinol <1.05 μmol/L) ≥25%, from 10 to 25%, or <10%, is considered as severe, moderate, and mild VAD, respectively [51
]. Interestingly, retinol in Vietnamese maternal milk was richer than in other countries. The Korean and Chinese populations had a high percentage of milk retinol <1.05 µmol/L, with 44%, followed by Pakistan (23%) and Vietnam (16%). These data indicated that Korea and China mother and infant are at high risk of VAD.
A comprehensive comparison of fat and water-soluble vitamins in breast milk between countries has been carried out in this work. The data exhibited a general view of vitamin status in Asian maternal milk. It could provide useful information for mother and baby care that may be used as a reference to establish the best infant formula for Asian babies.