Dynamic changes in the organophosphate profile of the experimental galactose-induced cataract Jack V. Greiner, Stephen J. Kopp, Donald R. Sanders, and Thomas Glonek Dynamic changes in lens organophosphate metabolites during 24 hr incubation in 30 mM galactose media were measured with phosphorus-31 nuclear magnetic resonance spectroscopy. The following phosphates were quantitated from the intact crystalline lens: adenosine triphos- phate (ATP), adenosine diphosphate (ADP), inorganic orthophosphate, a-glycerophosphate, phosphorylated hexoses and trioses, nicotinamide adenine dinucleotide, uridine diphosphoglu- cose and uridine diphosphogalactose, glycerol3-phosphorylethanolamine and3-phosphorylcho- line, and an unidentified phosphorus-containing molecule. The temporal sequences of meta- bolic events that define the dynamic rates of accumulation or depletion of lens organophosphates reveal that the first event in the decline of the tissue upon galactose incubation is a net con- sumption of ATP, ivhich occurs as a sigmoidal function with time and which is typified by a char- acteristic half-life of 18 hr. Alpha-glycerophosphate accumulated at an increasing rate with time, whereas ADP, inorganic orthophosphate, and the other organophosphates ivere essen- tially unchanged. Cataract formation in the subcapsular and superficial cortical regions was visible after 16 hr incubation in the experimental buffer. These findings support the hypothesis that alterations in the organophosphate levels of the lens are contributing factors to the initial formation of the experimental galactose cataract. (INVEST OPHTHALMOL VIS Sci 22:613-624, 1982.) Key words: experimental galactose cataract, organophosphates, crystalline lens, phosphorus-31 nuclear magnetic resonance spectroscopy he dynamics of energy metabolism in the events that precede cataract formation and intact crystalline lens during experimental therefore predispose the lens to it, have not cataractogenesis, particularly the metabolic been elucidated. Recently, Greiner et al.1 have, for the first time, demonstrated the feasibility of measuring the dynamic meta- From the Department of Pathology (Dr. Greiner) and the Nuclear Magnetic Resonance Laboratory (Drs. bolic activity of the intact crystalline rabbit Kopp and Glonek), Chicago College of Osteopathic lens by phosphorus-31 nuclear magnetic res- Medicine, and the Department of Ophthalmology, onance (P-31 NMR) techniques. The concen- University of Illinois Eye and Ear Infirmary (Drs. trations of the principal organophosphate me- Greiner and Sanders), Chicago, 111. tabolites detected in the intact lens, the intra- This study was supported in part by a Grant-In-Aid from the National Society to Prevent Blindness (Dr. lenticular pH, and the rates of metabolic Greiner) and core grant EY-01792 (Department of changes during glucose-free incubations were Ophthalmology, University of Illinois Eye and Ear reported.1 This method of NMR spectroscopic Infirmary) National Eye Institute, National Institutes analysis for intact tissues is a totally nonde- of Health. structive technique2 that permits simultane- Submitted for publication April 2, 1981. ous temperature-controlled tissue incuba- Reprint requests: Jack V. Greiner, Chicago Osteopathic Hospital, 5200 S. Ellis Ave., Chicago, 111. 60615. tions coupled with continuous monitoring 0146-0404/82/050613+12$01.20/0 © 1982 Assoc. for Res. in Vis. and Ophthal., Inc. 613 Downloaded from iovs.arvojournals.org on 09/23/2021 614 Greiner et al. Invest. Ophthalmol. Vis. Set. May 1982 RABBIT LENS WITH 30 mM GALACTOSE 30 mM FRUCTOSE i 20 0 -20 PPM 20 0 -20 PPM Fig. 1. For legend see facing page. of quantitative time-dependent metabolic provides the investigator with new informa- changes during defined increments of time.3'4 tion regarding rates of lens metabolic pro- The application of P-31 NMR to the study of cesses. The experimentally-induced galac- intact crystalline lens metabolism represents tose (sugar) cataract appeared to be a suitable a novel approach to the study of lens catarac- model to evaluate time-dependent metabolic togenic mechanisms by providing a dynamic events during actual cataract formation. Thus perspective of intact lens metabolism during this study describes the dynamic changes in cataract formation. As such, the NMR method lens organophosphate metabolite levels dur- Downloaded from iovs.arvojournals.org on 09/23/2021 Volume 22 Number 5 Organophosphate changes in galactose cataract 615 ing experimental incubation in a galactose- maintaining a constant volume. This procedure rich medium. prevented glucose depletion of the buffer bathing the lenses and generated constant P-31 NMR or- ganophosphate profiles. Detectable alterations in Methods the organophosphate profiles have been observed Surgery. Albino rabbits, weighing 2 to 3 kg, when the medium is changed at intervals exceed- were injected with a lethal dose of sodium pen- ing 2 hr.' Experimental and control group lenses tobarbital, and their eyes were enucleated. The were incubated for 25 hr in their respective eyes were opened at the posterior pole, the vitre- media. Ten lenses were analyzed in each group. ous humor was gently separated, the zonules were At consecutive 1 hr intervals during the incubation cut with curved blunt scissors, and the lenses were period, individual P-31 NMR profiles were ac- removed with a Parafilm-coated wire lens loupe. quired. Usable data were obtained in as little as 5 In vitro incubation. Lenses were freshly ex- min; however, continuous 1 hr periods were used cised, isolated, weighed, and placed together in a to signal average P-31 NMR data presented in this tared 12 mm NMR tube containing a volume of study. modified Earle's buffer (116.4 inM NaCl, 5.6 mM Lens perchloric acid extracts. Immediately dextrose, 5.4 mM KC1, 1.8 mM CaCl2, 1.4 mM after the incubation period, lenses were weighed, MgSO4, 0.9 mM NaH2PO4 • H2O, 26.4 mM frozen in liquid nitrogen, and prepared for per- 5 NaHCO3) at 37° C with a pH 7.4. This Earle's chloric acid (PCA) extraction. Lens PCA extrac- buffer has been shown to maintain lens clarity and tion and preparation procedures and P-31 NMR lens organophosphate metabolites for at least 24 hr calibrations and analyses were performed accord- of in vitro incubation.' The experimental medium ing to a previously described lens tissue extract was Earle's buffer containing 30 mM galactose in analysis.1 addition to 5.6 mM dextrose (325 mOsm). Lens P-31 NMR spectroscopy. A Nicolet NT-200 sys- incubations in Earle's buffer with added 30 mM tem equipped with deuterium stabilization, vari- fructose (325 mOsm) were performed as osmotic able temperature, and Fourier-transform capabili- controls. Lenses incubated in Earle's buffer (295 ties operating at 80.987663 mHz for P-31 was used mOsm) served as an additional control. All lenses in this study. A wide-bore Oxford superconducting were equilibrated for 2 hr in Earle's buffer at 37° C magnet (4.7 Tesla) was interfaced to the Nicolet prior to initiation of experimental and control in- system. Intact lenses were analyzed at 37° C under cubations. At 1 hr intervals during the incubation nonspinning, proton-coupled conditions. Typical period, used buffer (12 ml) was aspirated from the NMR scan conditions with 12 mm sample tubes base of the NMR tube, the lenses were washed were as follows: pulse width 9 /usec (45-degree three times with equivalent volumes of fresh buf- spin flip angle); acquisition time 200 /xsec; delay fer, and 12 ml of fresh buffer were added, thereby 200 fisec; number of scans 4K; number of data Fig. 1. P-31 NMR spectra of the intact rabbit lens at three time points during incubation in 30 mM galactose-enriched Earle's buffers and in 30 mM fructose-enriched Earle's buffer (osmo- tic control) at pH 7.4 at 37° C. Referring first to the 0 hr spectrum and all the control spectra, the resonances from left to right are as follows. The resonance band labeled SP has two principal components, the low-field (left) signal arises from the triose phosphates, the principal molecule of which is a-glycerophosphate; the upfield (right) resonance arises from pentose and hexose phosphates, the most important of which are IMP and AMP. The next prominent signal is that from Pi, followed by the resonance band of the phosphodiesters, labeled GPC in the figure (GPE and GPC are the principal phosphodiesters detected). In the ionized end-group phosphate region beginning at —5.6 ppm are located the y-phosphate resonance of ATP and the /3-phosphate resonance of ADP. Upfield of these are the esterified end-group phosphates, a-phosphate resonance for both ATP and ADP and the resonance band of the dinucleotides (DN) NAD and NADP. The small resonance labeled N arises from the nucleoside diphos- phosugars. In the lens the primary nucleoside diphosphosugars are uridine diphosphoglucose and uridine diphosphogalactose. The j8-phosphate resonance of ATP is the highest field signal in the lens spectrum. The chemical shift scale is given relative to the resonance position of 85% Pi, following the IUPAC convention.2 Downloaded from iovs.arvojournals.org on 09/23/2021 Invest. Ophthalmol. Vis. Sci. 616 Greiner et al. May 1982 Table I. Lens P-31 NMR profiles Chemical shift (ppmA) Phosphatic compound In intact lens In PCA extract Unknown8 c 18.05, 18.00, 17.81° Unknown8 c 10.80, 10.72, 10.57° Unknown8 5.90 6.001': Trioses 3.67 4.3(F Hexoses 3.19 3.78G NADP-2'-P c 3.50 Phosphoethanolamine c 3.41 Phosphocholine H 3.33" Pi 1.63 2.60 Glu-l-P c 2.311 GPE 0.40 0.82 GPC -0.13 -0.13 P-Cr c -3.10 Unknown8 c -5.36 ATP a,-10.65; 0,-19.24; y,-5.62 a,-10.92; 0,-21.45; y,-5.80J ADP a, -10.65; 0, -6.66 a,-10.61; /3,-6.11K Dinucleotides -11.34 -11.37L Nucleoside-diphosphosugars -12.89 12.63, -12.81M NADP-2'-P = 2' phosphate of NADP; Glu-l-P = glucose-1-phosphate; P-Cr = phosphocreatine.