Sphingolipids and ceramides of mouse aqueous humor: Comparative profiles from normotensive and hypertensive DBA/2J mice

Abstract

Purpose: To identify the sphingolipid and ceramide species and their quantitative differences between normotensive and hypertensive intraocular pressure states in DBA/2J mouse aqueous humor (AH).

Methods: Normotensive and hypertensive AH was sampled from mice by paracentesis. Lipid extraction was performed using modifications of the Bligh and Dyer method. Protein concentration was estimated using the Bradford colorimetric assay. Sphingolipids and ceramides were identified and subjected to ratiometric quantification using appropriate class specific lipid standards on a TSQ Quantum Access Max triple quadrupole mass spectrometer.

Results: The comparative profiles of normotensive and hypertensive DBA/2J mouse AH showed several species of sphingomyelin, sphingoid base, sphingoid base-1-phosphate (S1P) and ceramides common between them. A number of unique lipids in each of the above lipid classes were also identified in normotensive AH that were absent in hypertensive AH and vice versa.

Conclusion: A number of sphingolipid and ceramide species were found to be uniquely present in normotensive, but absent in hypertensive AH and vice versa. Further pursuit of these findings is likely to contribute towards expanding our understanding of the molecular changes associated with increased intraocular pressure (IOP) and glaucoma.

Keywords: Aqueous humor; Ceramides; Hypertensive; Mass spectrometry; Normotensive; Sphingolipids.

Fig. 1

Schematic depiction of sphingolipid metabolism pathway. Sphingolipids are synthesized de novo from serine plus palmitoyl CoA. Serine palmitoyltransferase catalyses the breakdown of serine plus palmitoyl CoA to sphinganine. Acylation of sphinganine by the enzyme dihydroceramide desaturase to produce dihydroceramide occurs and the final reaction to produce ceramide is catalyzed by dihydroceramide synthase. Ceramide undergoes one of three fates: (a) addition of headgroups to generate sphingomyelins, (b) glycosphingolipids and, (c) cleavage to sphingosine leading to sphingosine-1-phosphate. Boxed letters A, B and C indicate the location of the enzyme(s) in the overall scheme of metabolism responsible for conversion of indicated lipid species. Our identified lipid species and their potential conversion enzymes in these specific locations in the scheme have been indicated in Table 4 and Supplementary Table S3.

Fig. 2

Intraocular pressure (IOP) profile. IOP measurements for both genders of DBA/2J mice determined using a TonoLab instrument. Age of the animal at the time of tissue collection and IOP in mm of Hg is as indicated. Average IOP and standard deviation from a week of twice daily measurements have been indicated for each age and gender. Each group contained n=40 animals.

Fig. 3

(A) Comparison of average total amounts of sphingolipids and ceramides for normotensive and hypertensive states of both genders combined. Estimated average total amount of (B) sphingomyelin, (C) sphingoid base, (D) S1P and (E) ceramides in normotensive and hypertensive states are represented as filled and hollow bar graphs [(*p<0.05), differences are relative to the normotensive state].

Fig. 4

(A) Estimated average total amounts of sphingolipids and ceramides for normotensive and hypertensive states between both genders. Comparison of average total amounts of (B) sphingolipid classes between normotensive and hypertensive states are represented as filled and hollow bar graphs[(*p<0.05), differences are relative to the normotensive state].

Fig. 5

Heat map of fold change (fc) in common lipid species between normotensive and hypertensive states of both genders combined. The fold change is the ratio relative to normotensive state based on a log₂ scale. A negative sign precedes before modulus where the denominator is higher indicating higher amounts are present in normotensive than hypertensive state. Scale bar of fc used for given lipid species are as indicated[*|fc|≥ 5.0].