Advances in metabolomics and lipidomics using LC-MS/MS have enabled the accurate quantification of complex lipid molecules in milk, as well as in very small amount of infant serum or plasma [26-29]. Such lipidomic measurements can provide markers for tissue composition , and have been proven to be related to many important clinical endpoint events in children and adults [31-33]. Therefore, using these sophisticated and detailed analytical methods in conjunction with appropriate bioinformatics strategies should provide a better understanding of the physiological role of complex lipids in early life.
According to our LC-MS/MS results, in terms of the total contents of major lipid subsets, the concentrations of 10 out of the 43 lipid subclasses were found to be persistently higher in preterm milk compared to term milk over the course of lactation, with the FAHFA and DAG classes contributing the greatest differences. The difference between preterm milk of different GA stages and term milk is relatively consistent; in other words, the composition of preterm milk did not visibly resemble that of term milk as GA increased. In terms of individual lipid species, differential lipids (VIP > 1, p < 0.05) between preterm and term milk began to show discrepancies with increasing GA of the neonate and the progression of lactation. As mentioned in the background section, we paid special attention to EPT and VPT milk. Interestingly, compared with their FT counterparts, several PLs from the PE class (18:1/22:1, 18:1/20:1, 14:1/26:4 and 16:0/16:0) and the DAG species (12:0/22:5, 16:0/22:6 and 14:1/18:1) in VPT colostrum were significantly higher. In mature milk, FA and FAHFA lipids comprised the 10 most upregulated substances in both the EPT and VPT groups (P<0.0001 for all).
DAG is one of the primary lipid subpopulations in living systems, and acts as a second messenger in multicellular activities. It has been reported that DAG can accelerate the β-oxidation of fatty acids and affect the expression of genes related to lipid metabolism, thereby reducing TAG levels in serum and liver [34, 35]. In our current study, we found the total concentrations of DAG to be markedly increased in both preterm colostrum and mature milk, and several individual DAG species, such as DAG 12:0/22:5, 16:0/22:6 and 14:1/18:1 were significantly elevated in the VPT colostrum. The high levels of DAG species in preterm human milk may be linked to enhanced long-term health outcomes; for instance, reduced obesity, diabetes, and cardiovascular disease. Interestingly, Xu, L et al.  reported that DAG levels were significantly reduced in the colostrum of mothers who gave birth prematurely. Discrepancies between the present study and that of Xu, L et al. might stem from the significantly smaller sample size (n= 6, in both the preterm group and term groups) and greater GA of premature delivery (32–36 weeks) in the latter. More research is needed to validate our findings.
FAHFAs have come to be recognized as one of the bioactive lipids present in the HM with anti-inflammatory, anti-diabetic effects; they enhance glucose tolerance and the secretion of insulin and glucagon-like peptide 1 (GLP-1) while reducing inflammation [36-39]. Lee, J. et al.  also reported the role of FAHFAs in the gut, regulating both innate and adaptive immune responses in a model of colitis. So far, FAHFAs have only been detected in the serum and adipose tissue of mice and humans , and adipose tissue represents a major site for FAHFAs synthesis [36, 37]. Brezinova M et al.  performed a lipidomic analysis of milk samples obtained from lean and obese mothers, and compared the PAHSA (an FAHFA species containing palmitic acid) levels of these groups. They found, for the first time, that lipids from the FAHFA family exist in human colostrum/transient milk, and that the concentration of FAHFA in HM was negatively correlated with maternal weight. As can be seen from the above, FAHFAs hold great biological significance, but little research has been done on FAHFAs in HM, except the above. For the first time, we found that the levels of FAHFAs in preterm HM at different GAs were significantly higher than in term HM, with the levels of several FAHFA species (16:1/22:4, 22:3/22:4, 2:0/17:2, 20:2/20:1, 18:2/14:1, 16:1/22:4, 20:3/22:5, 12:0/18:2 and 18:1/18:2) remarkably higher in either EPT or VPT mature milk compared with their FT counterparts. Our findings may indicate that FAHFAs play an important role in improving the metabolism and promoting the intestinal maturation of premature infants. More maternal–child cohort studies and animal experiments will be needed to further explore the role and the mechanism of action of FAHFAs in HM.
PLs are a class of lipids with important biological functions, despite only accounting for 1% of total milk lipids . They are involved in the digestion, absorption and transport of TAG, making up the milk fat globule membrane and providing important bioactive components, such as choline, and the long-chain polyunsaturated fatty acids fatty acids (LC-PUFAs) docosahexaenoic acid (DHA) and arachidonic acid (AA), which are crucial for the neural and visual development and growth of the neonate [43-46]. The glycerophospholipids (GPL) PC and PE, and the sphingolipid (SL) SM, are the three major PL classes found in HM. So far, omics has rarely been used to thoroughly study the PLs in both preterm and term milk from different gestation ages and lactation stages.
Here, we show that the total concentration of PE was increased in both preterm colostrum and mature milk, and several individual PE species, such as PE 18:1/22:1, 18:1/20:1, 14:1/26:4 and 16:0/16:0, were significantly elevated in the VPT colostrum. PE, also known as cephalin, is an important part of the nerve cell membrane. It regulates all the metabolic activities of nerve cells and affects a series of important functions of nervous tissue, such as cell permeability, myelin formation and mitochondrial operation . Our results highlight the important role that the PE in HM plays in the early nervous system development of premature infants. Furthermore, LC-PUFAs are often found as FA moieties of PE, and contribute to the neurological as well as visual development of the neonate [47,48]. The transport of LC-PUFA from mother to infant mainly occurs through the placenta in late pregnancy. The dramatically higher content of PE observed in both preterm colostrum and mature milk in our findings suggests that the PE in HM may be an important channel through which premature infants with low GAs can obtain sufficient LC-PUFA through breastfeeding. Furthermore, in the present study, the observed differences in PE between preterm and term colostrum were almost equally prominent in mature milk, which diverges from the observations of Ingvordsen Lindahl, I.E. , wherein the effects of gestation subsided in mature milk. Discrepancy might be influenced by the sample preparation process, especially the addition of different internal standards prior to lipid extraction. More homogenization and standardized HM lipidomic studies starting from sample collection and storage are needed in the future.
In addition, we found that preterm colostrum and mature milk contain higher concentrations of PC than their term counterpart. PC is an important source of choline, along with SM, accounting for approximately 17% of the choline in HM . Research has shown that choline is essential to fetal development [50,51]. The three- to fourfold higher choline concentration in fetal vs. term infant plasma corresponds to the fetus’s higher growth rate and choline requirements [52,53]. Plasma choline, however, rapidly and prematurely decreases after preterm birth . Low plasma choline levels may contribute to the impaired lean body mass growth of preterm infants. Recently, in a randomized controlled trial conducted by Bernhard W et al. , supplementation with 30 mg/kg/day additional choline increased plasma choline to near-fetal concentrations and improved DHA homeostasis in preterm infants. High concentrations of choline in human breast milk ensure the adequate supply of choline to the breastfed preterm infant, which help to preserve fetal concentrations of plasma choline in the perinatal period , thus playing a key role in tissue muscle accumulation, as required for the catch-up growth of premature infants after birth.