We examined lipid composition of breast milk samples from healthy human volunteers representing Russian (Moscow) and Chinese (Shanghai) populations (n = 20), cows (n = 4), goats (n = 4), pigs (n = 4), domestic yaks (n = 2), rhesus monkeys (n = 2), and crab-eating monkeys (n = 2) (Additional file 5: Table S1). Furthermore, for each species, we measured a pooled sample containing equal volumes of milk from each individual.
Phylogenetically, examined species represent two orders of mammals: artiodactyla, or cloven-hoofed mammals, and primate. Within artiodactyla, our study contains representatives of two families: suidae (pig) and bovidae (cow, domestic yak, and goat). Primate species were represented by cercopithecidae (crab-eating and rhesus monkeys) and hominidae (human) (Fig. 1A).
Randomized milk samples were extracted, separated using liquid chromatography, and measured using untargeted mass spectrometry in positive ionization mode. The measurements yielded a total of 472 mass spectrometry features representing distinct hydrophobic compounds (lipids) with molecular weights below 1,200 Daltons (Da).
Visualization of the relationship among samples based on the abundance levels of these 472 detected lipids using multidimensional scaling (MDS) revealed good separation of species and phylogenetic groups (Fig. 1B). Furthermore, distances between species calculated using the normalized intensities of mass spectrometry signals generally agreed with the phylogenetic distances (Fig. 1C). Pig milk was the only obvious exception from this linear relationship, showing a greater difference to the bovidae species than expected from the phylogeny.
Of the 472 detected lipids 403 (85%) showed significant intensity differences among species (ANOVA, BH-corrected P < 0.05). Unsupervised clustering of these 403 species-dependent lipids based on their intensity profiles across samples yielded four clusters (Fig. 2A). Notably, lipid intensities within these clusters differed within the mammalian orders as much as between them. Particularly, the lipid composition of the pig milk stood out in three of the four clusters, while cluster 4 contained milk lipidome composition features shared between monkeys and bovids (Fig. 2B).
In agreement with previous knowledge, of all lipids detected in milk, the most of lipid was covered by a specific lipid class – triacylglycerides (TAGs). Interestingly, 76 lipid features computationally annotated as TAGs were present in all four clusters, covering the entire spectrum of lipid variation patterns (Additional file 6: Table S2). Furthermore, the average length and unsaturation extent of the fatty acid chains of these TAGs deferred among the clusters (Fig. 2C). For instance, cluster 2 TAGs contained long-chain polyunsaturated fatty acid residues, while cluster 4 TAGs preferentially contained medium-chain fatty acid residues (Fig. 2C; Additional file 1: Figure S1).
Notably, relative abundance analysis of detected TAGs revealed apparent intensity differences characteristic of each species. Specifically, cow milk contained more TAGs composed of long monounsaturated fatty acids. By contrast, goat milk contained more TAGs composed of medium-chain saturated and monounsaturated fatty acids. Pigs milk stood out from the rest of the species by having TAGs composed of long- and very long-chain polyunsaturated fatty acids. Monkey milk had more TAGs composed of medium-chain monounsaturated and polyunsaturated fatty acids. Finally, human milk tended to contain more TAGs with long-chain polyunsaturated fatty acids (Fig. 3A; Additional file 1: Figure S1).
We next specifically searched for lipids showing statistically significant intensity differences between humans and other three species’ groups represented in our study. Of the 472 detected lipids, 94 differed in intensity between humans and macaques, 23 of them annotated as TAGs (ANOVA, BH-corrected P < 0.05). Clustering of these lipids revealed a notable intensity increase in the human milk for a group of lipids, including nine TAGs with long- and very long-chain fatty acids (Fig. 4; Additional file 2: Figure S2).
Comparison between humans and bovids yielded significant intensity differences for 269 lipids, 61 of them annotated as TAGs (ANOVA, BH-corrected P < 0.05). Among them, 23 TAGs with long- and very long-chain polyunsaturated fatty acids showed increased intensities in human milk (Fig. 5B; Additional file 3: Figure S3).
Comparison between humans and pigs revealed significant intensity differences for 343 lipids, 64 of them annotated as TAGs (ANOVA, BH-corrected P < 0.05). Among them, 15 TAGs containing medium-chain saturated, monounsaturated, and polyunsaturated fatty acids were increased in the human milk, while the rest of TAGs containing long- and very long-chain polyunsaturated fatty acids were elevated in the pig milk (Fig. 4C; Additional file 4: Figure S4).