Sphingolipidomics of Bovine Pink Eye: A Pilot Study

Abstract

Sphingolipids are essential structural components of tear film that protect the surface of the eye from dehydration. A detailed analysis of the effects of pink eye infections on the sphingolipidome in cattle has not previously been undertaken. We recently published a new assay utilizing high-resolution mass spectrometric monitoring of the chloride adducts of sphingolipids that provides enhanced sensitivity and specificity. Utilizing this assay, we monitored decreases in the levels of tear film ceramides with short-chain fatty acids, hydroxy-ceramides, phytoceramides, and hydroxy-phytoceramides. Dihydroceramide levels were unaltered and increased levels of ceramides with long-chain fatty acids (24:0 and 24:1) were monitored in cattle with pink eye. The data from this pilot study (n = 8 controls and 8 pink eye) demonstrate a major disruption of the lipid tear film layer in pink eye disease, that can result in severe eye irritation and damage.

Keywords: Infectious Bovine Keratoconjunctivosous (IBK); bovine pink eye; sphingolipids.

Figure 1

Tear film levels of ceramides (Cer d18:1/x) in controls and cows with pink eye (P. Eye). The peak intensities of individual ceramides are expressed as a ratio of the peak intensity of ceramide d18:1/16:0. *, significant t-test statistics: 16:1 (0.013); 18:1 (0.021); 22:0 (0.027); 24:1 (0.039).

Figure 2

Tear film levels of hydroxy-ceramides (Hydroxy-Cer d18:1/x) in controls and cows with pink eye (P. Eye). The peak intensities of individual hydroxy-ceramides are expressed as a ratio of the peak intensity of ceramide d18:1/16:0. *, significant t-test statistics: 16:0 (0.023); 18:0 (0.014); 18:1 (0.020); 20:1 (0.0083).

Figure 3

Tear film levels of phytoceramides (Phyto-Cer) in controls and cows with pink eye (P. Eye). The peak intensities of individual phytoceramides are expressed as a ratio of the peak intensity of ceramide d18:1/16:0. *, significant t-test statistics: 34:0-O3 (0.024), 36:0-O3 (0.016), 36:1-O3 (0.01), 38:0-O3 (0.011), 40:0-O3 (0.040), 42:0-O3 (0.043), 44:0-O3 (0.036), 46:0-O3 (0.012), 46:1-O3 (0.034), 48:0-O3 (0.0086), and 48:1-O3 (0.026).

Figure 4

Tear film levels of hydroxy-phytoceramides (CerX-O4) in controls and cows with pink eye (P. Eye). The peak intensities of individual hydroxy-phytoceramides are expressed as a ratio of the peak intensity of ceramide d18:1/16:0. *, significant t-test statistics: 38:0-O4 (0.0064), 40:0-O4 (0.0056), 40:1-O4 (0.0070), 42:0-O4 (0.0075), 42:1-O4 (0.0048), 44:0-O4 (0.013), 44:1-O4 (0.015), 46:0-O4 (0.0030), 48:0-O4 (0.10), and 48:1-O4 (0.018).

Figure 5

Tear film levels of di-hydroxy-phytoceramides (CerX-O5) in controls and cows with pink eye (P. Eye). The peak intensities of individual di-hydroxy-phytoceramides are expressed as a ratio of the peak intensity of ceramide d18:1/16:0. *, significant t-test statistics: 42:0-O5 (0.021) and 44:0-O5 (0.0090).

Figure 6

Schematic of sphingolipid metabolism. Hydroxylation of free sphingolipid bases is catalyzed by monooxygenases (SUR2) and of sphingolipid bases in dihydroceramides via hydroxylases (DES2). The introduction of hydroxylation at position 2 of the fatty acid substituents of ceramides occurs via de novo synthesis with a hydroxy fatty acid (CERS, ceramide synthase) and via lipid modification by ceramide fatty acyl 2-hydroxylase (FA2H; phytoceramide → 2-hydroxy-phytoceramide).