Comparative study of serine-plasmalogens in human retina and optic nerve: identification of atypical species with odd carbon chains.

The objective of this work was to detect and identify phosphatidylserine plasmalogen species in human ocular neurons represented by the retina and the optic nerve. Plasmalogens (vinyl-ether bearing phospholipids) are commonly found in the forms of phosphatidylcholine and phosphatidylethanolamine in numerous mammalian cell types, including the retina. Although their biological functions are unclear, the alteration of cellular plasmalogen content has been associated with several human disorders such as rhizomelic chondrodysplasia punctata Type 2 and primary open-angle glaucoma. By using liquid chromatography coupled to high-resolution and tandem mass spectrometry, we have identified for the first time several species of phosphatidylserine plasmalogens, including atypical forms having moieties with odd numbers of carbons and unsaturation in sn-2 position. Structural elucidation of the potential phosphatidylserine ether linked species was pursued by performing MS3 experiments, and three fragments are proposed as marker ions to deduce which fatty acid is linked as ether or ester on the glycerol backbone. Interpretation of the fragmentation patterns based on this scheme enabled the assignment of structures to the m/z values, thereby identifying the phosphatidylserine plasmalogens.

severe macular atrophy, macular hemorrhage, or grossly visible chorioretinal pathologic abnormality was included in the study. The vitreous body was carefully removed. The entire neural retina (n = 5) was carefully separated from the RPE/choroid with forceps. The optic nerves (n = 5) were excised by cutting tangential to the sclera after having removed the extraocular tissues. All samples were immediately stored at -80°C until further analyses.

Preparation of standard solutions and samples
PS standards were prepared at 10 µg/ml in methanol:chloroform (9:1). Lipids were extracted from retinas and optic nerves following the Folch's procedure ( 22 ). Phospholipids were purifi ed from total lipid extracts using silica cartridges (25 × 10 mm i.d.; Sep-pak, Waters S.A., Framingham, MA) as previously described ( 23 ). Before chromatographic analyses, the phosphorus content of the total phospholipid extract was determined according to the method developed by Bartlett and Lewis ( 24 ). The total phospholipid extracts were dried under nitrogen and diluted at a final concentration of 12.5 ng/µl in chloroform/methanol (1:1, v/v) under argon atmosphere. The extracted solutions were kept at Ϫ 20°C and were further diluted with methanol 1:1 directly before analysis. Aliquot from this solution (10 µl) containing 25% chloroform was injected on the column.

Liquid chromatography
An Accela 1250 liquid chromatograph (ThermoFisher Scientifi c, Bremen, Germany) equipped with an Agilent Poroshell 120 C18 column (fused-core, 2.7 m particle size, 2.1 × 150 mm) was used for separation of analytes. The applied gradient is shown in Table 2 . Solvent A was water enriched with 1% formic acid, solvent B was methanol enriched with 1% formic acid, and solvent C was isopropanol.

Mass spectrometric parameters
An LTQ-Orbitrap XL hybrid mass spectrometer (Thermo-Fisher Scientifi c, Bremen, Germany) was used for all high resolution and tandem analyses. Negative electrospray ionization with 3.5 kV capillary voltage was used to form ions at 400°C nebulizer temperature. Nebulizer and auxiliary gases were nitrogen at 40 and 20 units, respectively. Tube lens was adjusted to 80 V. Other parameters were the typical values optimized during calibration. Resolution of 100,000 (full width at half maximum) was applied in the m / z range of 200-1100, and profi le data acquisition was performed. A lock mass of m / z 255.23295 was used as a reference point for the internal mass correction. For this purpose, palmitic acid (100 mg/l in methanol) at 50 l/min was infused into the electrospray assembly via a T-element.
The tandem mass spectrometric experiments were carried out using the collision-induced dissociation fragmentation mode in the linear ion trap. Isolation width was m / z 2, normalized collision Recent technical advances, and particularly the development of tandem mass spectrometric methods, have allowed different groups to confi rm the presence of PS plasmalogens in bacteria ( 19 ), rat lung ( 19 ), and in human lens ( 20 ) or macrophages ( 21 ). PS plasmalogens in the lung and in the lens consist of classical structures with saturated and/or monounsaturated moieties in sn-1 and sn-2 positions of the glycerol, whereas PS plasmalogens from macrophages contain PUFA having at least 20 carbon atoms and three double bonds in the sn-2 position.
The objective of the present work was to identify and compare the apparent abundances of PS plasmalogen species present in human ocular neurons represented by the retina and the optic nerve. By using liquid chromatography coupled to high-resolution and tandem mass spectrometry, we have identifi ed for the fi rst time several species of PS plasmalogens, including atypical forms having moieties with odd numbers of carbons and unsaturation in sn-2 position. The potential origin and functions of these atypical ether-linked PS in ocular tissues are discussed. Eyes were collected from six human donors (bodies donated to science, four women and two men, mean age 83.2 ± 17.9 years, range 72-94 years; Table 1 ) within 19 h (median time, mean time 17.9 ± 7 h) after death and were handled in accordance with the guidelines of the Declaration of Helsinki. The bodies were refrigerated at 4°C within 24 h after death. Eyeballs were enucleated at the anatomy laboratory of the Saint Etienne School of Medicine (France). Enucleated eyeballs were immersed in a BSS (Alcon, Rueil Malmaison, France) at 4°C. Within 30 min after enucleation, a circular section at the pars plana was taken with a 18 mm diameter trephine, and the corneoscleral disc was removed for other studies. The posterior pole of the eyeball was placed on a back-lit table, and the retina was observed under an operating microscope to select the tissues further included in this study. No eye having large drusen, severe pigment epithelial alterations,   0  10  90  0  500  3  10  90  0  500  50  0  0  100  350  52.5  0  0  100  350  53  10  90  0  350  55  10  90  0  500  60 10 90 0 500

RESULTS
Ionization, chromatography, and fragmentation conditions were optimized using a PS standard mix (10 µg/ml). Negative ion mode proved to be approximately 30 times more sensitive for PS than positive ion mode; therefore, this mode was used in our entire work. In terms of chromatography, the above-described reversed-phase chromatographic conditions were chosen to reduce spectral complexity, in particular to resolve analyte pairs differing in one double bound. In such cases [e.g., PS(P-18:1/18:1) and PS(P-18:0/18:1)], the required mass resolution would exceed the currently available 100,000; therefore, the isotopomer envelope of the fi rst analyte could not be resolved from the second analyte. An example of such a case is given in Table 3, where the retention time of the potential PS(P-18:1/18:1) is 21 min and that of PS(P-18:0/18:1) is 22.5 min. By using the described chromatographic separation such pairs are suffi ciently resolved, enabling their distinct detection and quantifi cation. Furthermore, because phosphatidylserines commonly loose an 87 Da serine fragment independently from the fatty acid chains, the presence of serine moiety in potential PS lipids can be easily confi rmed based on the fragmentation pattern.
The preliminary investigations were performed in one representative retina and in one optic nerve sample.   Tables 3 and 4 , including the observed mass errors. In addition, the chromatographic peak areas obtained from the high-resolution chromatograms were extracted and were used to compare the apparent abundances of PS plasmalogen species ( Tables 3 and 4 ).

Structural elucidation of the potential PS ether-linked species was pursued by performing MS 3 experiments: After isolating and fragmenting the [M-H] Ϫ ions, the [M-H-87]
Ϫ fragments were again isolated and fragmented. The resulting MS 3 fragmentation patterns were then manually interpreted to determine which fatty acids are linked as ether or esters to the glycerol backbone. For this latter purpose, fi rst the fragmentation pattern of the hypothetical PS etherlinked lipids detected in the pooled retina and optic nerve   samples were studied in detail. Based on this, three fragments were proposed as marker ions to deduce which fatty acid is linked as ether or ester on the glycerol backbone ( Fig. 3 ). Interpretation of the fragmentation patterns according to this scheme enabled the assignment of structures to the m / z values to identify the PS plasmalogens. An example MS 3 fragmentation pattern is shown in Fig. 4 , which depicts the obtained fragments after isolating and fragmenting fi rst the [M-H] Ϫ ions then isolating and fragmenting the [M-H-87] Ϫ ions. This methodology does not allow distinction between isomers of fatty acids (e.g., straight vs. branched chains).
After the various PS plasmalogen species were detected and identifi ed, they were analyzed in the individual samples (5 retinas + 5 optic nerves). The averaged peak areas with the observed mass errors and retention times are given in Tables 3 and in
A striking difference was observed between the retina and the optic nerve concerning the PS species esterifi ed with arachidonic (20:4 n-6) acid. The profi les of these two ocular tissues were quite different because the optic nerve was characterized by a high level of PS(P-18:1/20:4),  Because the optic nerve is exclusively composed of myelinated axons, and subsequently comparable to the brain tissue, our data are consistent with previous studies revealing higher concentration of saturated phospholipids in the retina when compared with cerebral neurons ( 28 ). The functional implication of retinal PS plasmalogen species is unknown, but the abovementioned biochemical particularity of retinal tissue may be the result of a relatively low activity of alkyl-acyl phosphoglyceride desaturase, which catalyzes the conversion of alkyl-ether phospholipids into vinyl-ether phospholipids ( 29 ). Further, while the presence of PS lipids on the outer leafl et of the plasma membrane is a well established marker of apoptosis, to our knowledge, the affi nity of PS binding proteins, such as Annexin V, for ether-linked PS species has not been tested ( 30 ). Following the identifi cation of these species in multiple tissues, it would be interesting to pursue this further. Although it is well established that even-numbered fatty acids are predominant in vertebrates, odd-numbered fatty acids have been also identifi ed in various forms (e.g., sphingolipids, triglycerides, and glycerophospholipids) in numerous tissues, such as the testes, skin, and cataract tissue ( 31,32 ). These fatty acids can enter the food chain via synthesis by ruminant bacteria and plants ( 31,33 ). However, even in the absence of oddchain fatty acids in the animal diet, they have been identifi ed in signifi cant amounts in the sphingomyelin pool ( 34 ), and Nakano et al. (35) have shown that in rat the shorter-, odd-chain fatty acids might also get elongated by the liver. These fi ndings suggest that a mechanism may exist by which the detected odd-chain fatty acids may be synthesized in mammals. One such mechanism could be via ␣ oxidation of fatty acids. Apart from that, disorders of propionate catabolism (such as propionic academia) result in increased concentrations of odd-chain fatty acid in the plasma ( 36 ). This suggests that propionic acid can be used by mammalian systems to generate oddchain fatty acids. However, because the retinal tissue we analyzed was collected from elderly donors, these genetic disorders are highly unlikely as the cause of signifi cant presence of odd chain fatty acids. Instead, because a vitamin B12 defi ciency state has also been suggested to increase odd-chain fatty acids, we believe this to be a more likely explanation ( 36,37 ).
Because the overall abundance of the above-mentioned odd carbon number fatty acids in the sn-1 esterifi ed PS, PC, and PE pool was not determined, the biological signifi cance of this fi nding needs to be investigated further. Considering the existence of a plasmalogen-specifi c phospholipase A2 (iPLA2) ( 12,13 ) and the higher metabolic responsiveness and activity of retinal neurons when compared with those from the optic nerve, one can suggest the implication of PS (P-19:0/20:3) + PS (P-21:2/18:1) in cellular signaling after a release from cell membranes, followed by a possible metabolism into second messengers. Furthermore, as shown by several studies conducted in rats and human patients with metabolic diseases, other functions of odd carbon fatty acids in cells could be related to energy production (38)(39)(40).

CONCLUSIONS
In summary, this study confi rms the presence of various PS plasmalogen species in optic nerve and retina. The results revealed substantial differences in the abundance of these species in these samples, refl ecting the structural origin and functional difference between these two tissues. We also confi rmed the presence of odd-chain fatty acids in the PS plasmalogen species; however, the biological signifi cance of this latter fi nding needs to be further investigated.
The authors thank Pr Gilles Thuret and Pr Philippe Gain (Faculty of Medicine, Laboratory of Biology, Engineering and Imaging of Corneal Graft at Saint-Etienne, France) for collecting and providing retinas and optic nerves from human donors and Stéphane Grégoire, Lucy Martine, and Stéphanie Cabaret (Centre des Sciences du Goût et de l'Alimentation, Dijon, France) for preparing the samples prior to liquid chromatography.