Triacylglycerols (TG) and glycerophospholipids (GPL) are essential lipid molecules found in food and play vital roles in human physiology. The diversity of fatty acids (FAs) and the presence of regioisomers and stereoisomers cause significant challenges to their structural analysis. In natural samples, the distribution of FAs on the glycerol backbone is not random, and the properties of isomers can vary considerably despite structural similarities. These structural variations can affect the physical properties or nutritional significance of fats and oils, such as the melting point and bioavailability of TGs and GPLs.

This thesis focuses on the structural analysis of enantiomeric TGs and the investigation of the bioavailability of FAs from regio- and enantiopure TGs. Using reference compounds, the research begins with a comprehensive study of the chiral chromatographic separation of TG enantiomers. A total of 33 TG enantiomer pairs, each comprising three different fatty acyls, were analyzed using recycling chiral high-performance liquid chromatography (HPLC), of which 26 pairs were successfully separated. The retention time at separation exhibited a negative correlation with the sn-3 carbon number of the first eluted enantiomer and a positive correlation with the carbon number difference between the outer positions. TGs with one saturated fatty acid (SFA) and one unsaturated fatty acid (UFA) at the outer positions were easier to separate, whereas TGs with two SFAs or two UFAs at sn-1/3 positions were more difficult to separate. Additionally, the results revealed that all three fatty acyls significantly influenced the chromatographic behavior in terms of carbon chain length and the number of double bonds (DBs). These results highlight the importance of steric hindrance and intermolecular interactions in determining the efficiency of the separation mechanism. This systematic study enhanced the understanding of the chiral chromatographic retention behavior and elution order of TG enantiomers.

The second part of this thesis investigated the nutritional and biological implications of fatty acyl positional isomers of TGs through an animal trial by exploring how the positional distribution of docosahexaenoic acid (DHA) in TGs influences the bioavailability and metabolic fate of DHA. Regio- and enantiopure TGs with DHA at the sn-1, sn-2, or sn-3 positions were synthesized and fed to rats for 4 weeks of intervention. During this time, body weight and food intake were monitored to assess physiological responses.

After the intervention, biological samples: plasma, visceral fat, and organs including the heart, liver, brain, lung, testis, spleen, eye, and kidney were collected for analysis. The FA composition of total lipids was analyzed. Additionally, polar and neutral lipids from plasma, liver, and kidney were fractionated, and FA composition was analyzed separately. Using a novel mass spectrometric methodology recently developed by our group and advanced data processing software, the molecular species and regioisomer composition of phosphatidylcholines (PC) in these tissues were identified, revealing the effects of positional distribution of FAs on lipid composition and metabolism. The results demonstrated significant differences in DHA content in the plasma and visceral fat between groups fed TGs with DHA at the sn-1 and sn-3 positions. Historically, the effect of positional distribution has been evaluated mainly through FA content and composition in tissues. Therefore, this study provides fresh insights into the impact of the sn-position of DHA in dietary TGs on GPL molecular species and regioisomer distribution for the first time. Furthermore, this research is the first to demonstrate that n-3 PUFA deficient diet altered GPL molecular species composition in organs, and DHA supplementation counteract these changes.

In conclusion, this thesis offers valuable contributions to understanding the composition and metabolism of chiral TGs, from analytical methodologies to biological evaluation. The study provides critical insights into the chromatographic behavior of TG enantiomers and highlights the impact of FA positional distribution on lipid chemistry and biology. These findings serve as foundational references for future stereospecific studies of natural lipids.