Exp. Eye Res. (1991) 52, 253-260 Phosphatic Intermediate Metabolites of the Porcine Ocular Tunica Fibrosa SHENIN SACHEDINA”, JACK V. GREINERb~~~ THOMAS GLONEK”” “MR Laboratory, Chicago College of Osteopathic Medicine, Chicago, IL, bHowe Laboratory of Ophthalmology, Harvard Medical School, The Cornea Service, Massachusetts Eye and Ear Infirmary, Division of Ophthalmology, Beth Israel Hospital, and the Eye Research Institute of Retina Foundation, Boston, MA, U.S.A. (Received 25 September 1989 and accepted in revised form 16 July 1990) This study compares porcine scleral and cornea1 phosphorylated intermediatemetabolites determined using31P NMR. These tissues comprise the tunica fibrosa (outer coat) of the eye.Since the sclera does not possess epithelia asdoes the cornea, comparative analysis of these tissues includedexamination of the cornea with and without its epithelia. The phosphorylated intermediates detected include: dihydroxy- acetone phosphate, hexose 6-phosphates, a-glycerolphosphate, /&glycerolphosphate, ethanolamine and choline phosphates. nucleoside mono-, di-, and t&phosphates,inorganic orthophosphate, glycerol 3- phosphoryl-ethanolamine and -choline, phosphoglycans, phosphocreatine, nucleoside diphosphosugars, dinucleotides, and four uncharacterized (unknown) signals. Metabolicindices, comprised of individual or grouped metabolites. were calculatedto further compare and contrast metabolites and to provide more pathway specific metabolic interrelations for phosphorylated metabolites. Significant differences exist between the cornea1 stroma and the sclerain 16 of the 22 phosphorylated metabolites determined, whereasdifferences exist between the whole cornea and the sclerain six of the 22 phosphorylated metabolites. Considering all metabolite levels and metabolicindices in aggregate,the sclera is most similar to the whole cornea and least similar to the cornea1 stroma. Key words : sclera : cornea : phosphaticmetabolites ; 31P nuclear magnetic resonance spectroscopy : ocular metabolism : intermediate metabolism ; phosphorus. 1. Introduction The ocular tunica Ebrosa is the outermost coat of the eye. The anterior one-sixth of the tunica Ebrosa is composed of the transparent avascular cornea and the posterior five-sixths is composed of the white, mini- mally-vascularized sclera. The large contribution of collagen comprising the cornea and sclera provides a relatively tough ocular coat; thus, these tissues serve to maintain the shape of the eye. In addition, the cornea serves to refract and focus light entering the eye ; the sclera serves to limit the entrance of light. Phosphatic metabolite profiles determined by 31P nuclear magnetic resonance (NMR) of the normal cornea have been reported (Greiner et al., 1983; Greiner, Braude and Glonek, 1985 ; Greiner, Lass and Glonek, 1989a, b). Age differences (Glonek et al., 1987) as well as differences in disease (Greiner, Lass and Glonek, 1985) have also been reported in the cornea ; however, no detailed metabolic examination of phosphatic intermediate metabolites has been performed comparing the cornea to the sclera. This is especially important when considering recent reports on altered metabolism in keratoconus (Greiner et al., 1989), which could be regarded as an anterior staphyloma of the outer coat of the eye, and when * For correspondence at: MR Laboratory, Chicago College of Osteopathic Medicine, 5200 S. Ellis Ave., Chicago, IL 60615, U.S.A. 00144835/91/030253+08 $03.00/O 18 considering a similar posterior staphyloma of the posterior pole (sclera) of the outer coat of the eye in high myopia. The purpose of this study is: (1) to determine similarities and differences in the phosphatic inter- mediate metabolite profiles of the tissues comprising the tunica fibrosa (cornea and sclera) of the eye using 31P NMR; and (2) to develop and utilize metabolic indices in order to compare and contrast pathway- specific metabolic interrelationships that can be used in studying disease processes. 2. Materials and Methods Cornea1 and Scleral Samples Freshly enucleated porcine eyes (n = 40) were obtained from a local abattoir within 1 hr post mortem and maintained in physiological saline solution. Analysis of cornea and sclera was conducted on ten samples consisting of four tissue specimens each. After irrigating the surface of the globe with fresh physio- logical saline, extraocular tissues, eg. conjunctiva, Tenon’s capsule and extraocular muscles, were excised from its surface using a No. 15 Bard-Parker scalpel blade. Corneas were excised using a No. 15 Bard- Parker scalpelblade, by entering the anterior chamber and cutting the cornea circumferentially with fine corneal-scleral scissors. The excised corneas were then 0 1991 Academic Press Limited HER 52