Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina

• This manuscript describes a GC–MS method to quantify 26 LC-FAs and 14 VLC-PUFAs.
• This method can quantify VLC-PUFAs and n-3/n-6 ratios in individual human maculas.
• This method can quantify VLC-PUFAs and n-3/n-6 ratios in a pair of mouse retinas.
• LC-PUFA and VLC-PUFA contents of human and mouse retinas are compared.
• This method facilitates study of VLC-PUFA function in valuable research tissue.

Fatty acids (FAs), including long-chain and very long-chain polyunsaturated fatty acids (LC-PUFAs, C12–22; VLC-PUFAs, C24–38), play an important role in retinal function and health. Deficiencies in LC-PUFAs and VLC-PUFAs, as well as mutations in the enzyme responsible for elongation of very long-chain fatty acids (ELOVL4), have been associated with macular dystrophies and degenerations. Published analytical methods, including high-performance liquid chromatography coupled with mass spectrometry (HPLC–MS) and gas chromatography–MS (GC–MS), can quantify VLC-PUFAs but require at least an entire human retina which limits the ability to understand physiologically relevant variations in lipids that can occur at a regional level within the retina. Until now, quantification of VLC-PUFAs in just the human macula, the cone-rich region of the central retina responsible for high acuity vision, has not been feasible due to its small size (4–5 mm in diameter). In this study, we have developed a sensitive GC–MS method using newer generation enhanced GC–MS detector sensitivity which for the first time quantifies not only 14 VLC-PUFAs and 26 LC-FAs but also n-3/n-6 ratios of PUFAs in 4 mm punches of human retina or a single pair of mouse retinas. Our results showed that saturated LC-FAs are higher in the human peripheral retina than in the macula, while unsaturated LC-FAs are higher in the macula than in the peripheral retina. On the other hand, the VLC-PUFAs are higher in the peripheral retina compared to macula. There is no difference in n-3/n-6 ratios of PUFAs observed between human macula and peripheral retina, while mouse retina has almost ten times more VLC-PUFAs than human macula and peripheral retina (2.27% versus 0.25% and 0.32%, respectively) and much higher n-3/n-6 ratios compared to human retina (9:1 versus ∼0.9:1). This high sensitivity analytical technique provides a valuable new tool for studies on the role of FAs in the pathological processes of macular degenerations and dystrophies.