Title : Human milk as a model of functional foods identification of the 500 major Triacylglycerol Regioisomers lifts the Veil
Human milk is a natural and optimal model for infant formulas. Lipids form a major class of components, energy source and versatile protectants, not only in human milk, but also in milk of other mammals (Mammalia) and of egg-laying mammals (Prototheria). Further, lipids in eggs, spawn and roe of various animal species of the phylum Chordata play a key-role among all the nutrients. Quantitatively the major class of lipids is triacylglycerols (TAG) with three fatty acids (FA) of various chain lengths and an assembly of double bonds (DB) attached to glycerol. The lipid fraction of human milk contains a pool of over 1000 different TAGs but 40 major ones cover 2/3 of all the TAGS. From a nutritional point of view the three stereospecific (sn) positions of FAs in the glycerol backbone are biologically not equal. Absorption of short and medium chain (8:0-12:0) FAs is enhanced when located in the sn-3 position. Palmitic acid (16:0) located in the sn-2- position gains digestion and is absorbed as monoacylglycerol (MAG). This also promotes mineral absorption, bone strength and wellbeing of gut microbiota. Lauric (12:0) and linoleic [18:2(n-6)] acids have beneficial microbicidal effects. Gastric lipase produces 1,2-diacyl-sn-glycerols which act as second messengers in stomach. Human milk contains also small but proper amounts of α-linolenic [18:3(n-3)], arachidonic [20:4(n6)], eicosapentenoic [20:5(n-3) and docosahexaenoic [22:6(n-3)] acids. Rumenic acid (c9,t11-18:1) is beneficial for cardiovascular health. The lipid pool of human milk has not a constant composition. It depends on many factors such as gestation at birth, time after postpartum, diet, diurnal rhythm, metabolic status and genetic background. The fat content of milk also increases during breastfeeding. Further, phospholipids and cholesterol have significant roles in wellbeing of an infant. Compared with milks of other mammalian species, human milk is rich in oleic [18:1(n-9)], linoleic and α-linolenic acids and in a series of PUFAs with 20 and 22 carbons. The two key-factors in human milk lipids are the composition of FAs and their regioisomeric sn-positions in the glycerol backbone. Analysis of TAGs of natural fats has traditionally involved isolation and purification procedures followed by enzymatic and multiphase chromatographic and mass spectrometric (MS) steps. Development of chromatographic resolution and a number of MS methods for TAG analysis has reached many hyphenated solutions. On the top, we developed a highly sensitive method to analyze ratios of TAG regioisomers in human milk. UPLC resolution of lithiated TAGs followed by daughter scan MS/ MS of positive ions revealed several position-indicating ions. Reference TAGs containing C14–C20 FAs showed good linear response. Analysis of Finnish and Chinese pooled human milk samples revealed hundreds of TAGs. Over 60 mol% of the TAGs were quantified with standard deviation <17%. 16:0 was typically in the sn-2 position when present together with C18 FAs. When together with FAs 10:0, 12:0, 14:0, 20:1 and 20:2, the sn-2 preference of 16:0 was less clear. Oleic acid occupied typically the primary positions but when together with FAs 20:1, 20:2, 18:2, 14:1, 12:0 or 10:0 the positioning of 18:1(n-9) did not follow the rule.