Dynamic Changes in the Organophosphate Profile of the Experimental Galactose-Induced Cataract

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 triphosphate (ATP), adenosine diphosphate (ADP), inorganic orthophosphate, a-glycerophosphate, phosphorylated hexoses and trioses, nicotinamide adenine dinucleotide, uridine diphosphoglucose and uridine diphosphogalactose, glycerol3-phosphorylethanolamine and3-phosphorylcholine, and an unidentified phosphorus-containing molecule. The temporal sequences of metabolic 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 consumption of ATP, ivhich occurs as a sigmoidal function with time and which is typified by a characteristic half-life of 18 hr. Alpha-glycerophosphate accumulated at an increasing rate with time, whereas ADP, inorganic orthophosphate, and the other organophosphates ivere essentially 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

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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-

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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 organophosphate 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 (osmotic 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 diphosphosugars. 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

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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. AField-independent NMR units of ppm relative to the shift position of the 85% Pi reference at 25°. BCompound, as of this writing, is not identified with any known phosphorus-containing biomolecule. cMinor component not detectable in the intact tissue. "Three separate resonance signals are detected. EJ(P-H phosphorus-hydrogen) = 10.69 Hz. ••"Complex resonance band, the principal resonance signals of which come from the a-glycerophosphate triplet; J(POCH) =6.68 Hz. GThe hexose and pentose phosphates, principally the resonance triplet of inosine monophosphate; J(POCH) = 3.81 Hz. HJ(POCH) for the triplet = 5.63 Hz. 'Cannot be determined in the intact tissue as a separate resonance band but is combined with the Pi signal. JJ(POP,a/3) = 19.44 Hz; J(P0P,/3-y) = 19.44 Hz. KJ(POPa/3) = 22.44 Hz. •-Principal resonance signals of this band arise from the P,P'-diesterified pyrophosphate residues of NAD and NADH. 31 3l "Complex resonance band composed of the 2 sets of overlapping, p. p> ab NMR multiplets from uridine diphosphoglucose and uridine galactose.

points per spectrum 8192; acquisition time 0.82 nal standard used was the lens' natural glycerol sec; sweep width ±2500 Hz; filter-induced broad- 3-phosphorylcholine (GPC) resonance.' GPC is a ening 10 Hz. Signal-averaging, peak area integra- compound with a relatively constant chemical shift tions, and other mathematical manipulations of the for a phosphate (—0.13 ppm), which is not influ- transformed free-induction decay data were per- enced by variable physiologic pH, ionic strength, formed by the spectrometer's computer using ap- or countercation conditions.7 propriate subroutines of the NTCFT Fourier-trans- P-31 NMR spectroscopic analysis of the intact form NMR program. lens yields a spectrum of resonance peaks with PCA extracts were analyzed with and without discretely defined shift positions. These resonance proton decoupling and were spun at 18 Hz to en- shifts are determined by the physical and chemical hance signal resolution. PCA extract samples were characteristics of each phosphorus-containing func- analyzed in NMR microcell assemblies to enhance tional group. Thus the resonance shift position of signal intensities for quantitation of the minor lens each peak is a physiochemical marker for indi- metabolites. Extract samples were analyzed at 37° vidual organophosphate metabolites present in the C. Spectrometer conditions used throughout were crystalline lens.1 the same as those previously described.1 Mathematical analysis of dynamic lens changes The standard of 85% inorganic orthophosphoric in galactose-enriched medium. A least-squares re- acid was used for determining and reporting the gression analysis using a polynomial expression of 3 2 chemical shift data.6 The primary intact lens inter- the form y = Ax + Bx + Cx -I- D was employed

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Amount (% of total P in profile) In PCA extracts

Controls In intact Freshly 24 hr incubation 24 hr incubation in Experimental 24 hr incuba- lens excised in std. Earle's 30mM fructose tion in 30mM galactose

c 0.5 0.5 0.4 0.5 c 0.3 0.5 0.6 0.4 1.2 1.1 1.1 1.0 1.1 8.7 7.3 8.8 7.0 26.0 7.2 5.9 4.9 5.1 6.8 c 0.2 0.2 0.2 0.1 c 0.3 0.2 0.3 0.1 1 1.5 1.5 1.3 0.5 11.1 7.3 7.8 7.2 7.7 c 0.8 0.6 1.0 1.8 1.3 0.9 1.2 1.0 0.2 2.1 2.0 1.9 2.1 0.2 c 0.05 0.1 0.1 c 0.05 0.1 46.8 52.2 56.3 54.9 31.5 5.8 5.4 4.7 3.9 9.0 13.6 12.5 12.8 11.2 11.5 2.2 1.7 1.8 2.7 2.6

Table II. Coefficients for the expression, y = Ax3 + Bx2 + Cx + D, obtained in the linear regression analysis of lens time-course data* Component of time course B C 0 ATP 0-8 hr 0.0260 ± 0.00225 -0.799 dt 0.0145 4.49 ± 0.0844 47.0 dt 0.341 5-25 hr 0.000666 ± 0.000008 -0.00221 dt 0.00168 -1.413 ± 0.0334 57.1 dt 0.539 ADP, 0-25 hr -0.000900 ± 0.000067 0.0229 dt 0.00146 0.2167 ± 0.0288 4.36 dt 0.375 Pi, 0-25 hr -0.00266 ± 0.000032 0.101 dt 0.000701 -0.988 ± 0.0138 10.9 dt 0.185 Sugar phosphates, 0-25 hr 0.000326 ± 0.000058 0.00757 dt 0.00125 0.419 ± 0.0247 14.7 dt 0.322

*Ordinate is the % of total phosphorus detected, and abscissa is time in hours.

to fit time-course data for the incubation period so 30 mM fructose (osmotic control) in Earle's that the sum of the squared deviations about the buffers. The quantitative data obtained from curve was minimized. The third-degree expression these NMR spectra are given in Table I. Fig. was required for adequate approximation of the 2 shows the spectrum from a PCA extract of real time-course data obtained from the intact lens lenses after incubation in 30 mM galactose for tissue. By differentiation of the locus determined 25 hr. This spectrum is shown to illustrate from the regression analysis, the points in time corresponding to the periods of maximal and min- the resonance signals from the uridine diphosphosugars and from several unidentified imal change during the time course were calcu- 1 2> 8 lated for the affected metabolites. phosphates ' indicated by shift positions in the figure. Analysis of the integrated areas of Results the uridine diphosphosugar resonance band Fig. 1 illustrates the P-31 NMR spectra ob- verify that the uridine diphosphate (UDP)- tained from intact rabbit lenses before and glucose levels were reduced approximately during incubation with 30 mM galactose and one half of those of control whereas the

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GALACTOSEMIC LENS PCA EXTRACT

20 10 -10 -20 -30 PPM

Fig. 2. P-31 NMR spectrum from PCA extract of a rabbit lens after incubation in the galactose-rich medium at pH 7.4 at 37° C for 25 hr. The resonance position of the new unknown molecule is —5,37 ppm.

UDP-galactose levels were approximately phosphates.12' 13 Furthermore, it was not in- doubled in response to 30 mM galactose inorganic pyrophosphate, which also may give a cubation. A decrease in UDP-glucose and an single resonance line in this spectrum, since increase in UDP-galactose levels are consis- addition of pyrophosphate to the sample did tent with previous findings of other investiga- not result in uniform enhancement of this sig- tors.9"11 The PCA extract data provided more nal. (The pyrophosphate resonance occurs precise data with respect to minor phosphates at —7.25 ppm in this system.) Also, no other and illustrated that three minor metabo- inorganic polyphosphate could give rise to l4 lo lites, GPC? glycerol 3-phosphorylethanol- this resonance. ' Moreover, this signal did amine (GPE), and the dinucleotides, nicotin- not correspond to an alkyl phosphate ester, amide adenine dinucleotide (NAD) and nico- since the proton-coupled phosphorus spec- tinamide adenine dinucleotide phosphate trum gave no evidence for coupling to pro- (NADP), underwent concentration changes tons. The nature of the molecule that gave after incubation of the lenses in 30 mM galac- rise to this signal is not known at this time. tose. The changes in the phosphodiesters Molecules that come into resonance in this were rather pronounced. GPE was reduced region of the spectrum and that could account to 16% of the initial value, and GPC was re- for the —5.37 ppm signal are members of the duced to 10%. The —5.37 ppm peak corre- family of mixed anhydrides between carbox- sponds to an end-group phosphorus reso- ylic acids and phosphate or certain anhy- nance characterized by a single line under drides involving aromatic nitrogen heterocy- proton-coupled conditions, which precludes clic molecules. For example, the chemical its being in the family of nucleoside poly- shift of n-butyrylphosphate in butyric anhy-

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20-

1 i i I I 8 12 16 20 24 HOURS Fig. 3. Graphed time course of changes in the lens phosphate metabolites during lens incubation in 30 mM galactose-enriched Earle's buffer. DA7, Dinucleotides; aGP, a-glycerophosphate; NS, nucleosidediphosphosugars.

dride is —5.27 ppm, and the proton-coupled creased steadily with time after the introduc- resonance signal is a single sharp resonance tion of 30 mM galactose to the lenses, even- line. tually accumulating to levels approaching The extract spectrum also clearly illus- 40% of the total lens phosphorus. The other trated the resonances at 6, 10, and 18 ppm. phosphorus resonances in the spectrum ex- The structure of all these molecules is un- hibited little or no change during the galac- known. They do not correspond to any tose incubation. Cataract formation, observed phosphorus-containing molecules presently with the naked eye at 16 hr, was restricted to identified as constituents of biologic systems. the subcapsular and superficial cortical re- The signal from glucose-1-phosphate, which gions. Although the degree of cataract in- is particularly strong in galactose-treated lens creased with time, at 24 hr the cataract was PC A extracts is also indicated in Fig. 1. P-31 still restricted to the subcapsular and super- NMR spectra of control lens PC A extracts are ficial cortical regions. 1 illustrated elsewhere. The temporal data points for ATP, a-glyc- The 9 and 25 hr spectra of Fig. 1 illustrate erophosphate, ADP, and Pi depicted in Fig. the changes exhibited in the NMR data dur- 3 were least-squares fitted to a third-order ing lens incubation in 30 mM galactose. Such regression curve. The temporal changes in data are represented by the time-course plots the dinucleotides, nucleosidediphosphosug- shown in Fig. 3. ars, and the GPC curves derived from intact When healthy rabbit lenses are exposed to lens analyses did not yield maximal and min- buffer containing 30 mM galactose, the ATP imal values. The third-order expression was level initially increases and then declines satisfactory for the a-glycerophosphate, progressively with time. Only minor changes ADP, and Pi time-course curves; however, in lenticular ADP with time were evident, in the ATP curve could not be fitted to a single marked contrast to the metabolic changes in- polynomial expression. The ATP curve ap- duced by glucose deprivation.1 Similarly, Pi peared to have at least two major compo- levels were virtually unaffected except for a nents, both of which could be well fitted with well-defined minimum, early in the time the third-degree equation. Thus the ATP course. In contrast, a-glycerophosphate in- data were treated as two phases: an initial

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5- G-6-P

4- "AMP ^ .MP\

N ~ ~^^\ \ 3- Pi \\A ppm)

Shift \ \ emical o /-IMP 0-

-I- 1 1 1 1 1 1 i 10 PH Fig. 4. P-31 NMR-pH titration data employed in the identification of IMP and AMP in lens PCA extracts: solvent, aqueous saturated KC104 solution; temperature 37° C. These curves are reversible provided that the titrations are carried out with O.IN acid or base reagents of NMR spectral quality. NAD-2'-P, 2' phosphate of NADP; G-6-P, glucose-6-phosphate.

interval from 0 to 8 hr and a subsequent in- dependence on the pH environment of the terval overlapping the first, from 5 to 25 hr. phosphorus atom. This concept is illustrated The time increments during which the spe- in Fig. 4, where P-31-NMR-pH titra- cific lens metabolites underwent maximal tion data are given for several lens phos- and minimal rates of change were calculated, phates in water with potassium as the coun- and the changing values of the slope of each tercation at 37° C. Note that the phosphorus curve were defined. The coefficients for the chemical shifts for the phosphates illustrated regression equations are presented in Table resonate at higher fields as the pH was low- II along with their standard deviations. From ered, so that the nominal displacement per these equations the maximum rate of lens acid-base transition is 3 to 4 ppm. In regards ATP accumulation was calculated to occur at to the specific phosphates measured in Fig. 3.4 ± 0.01 hr, and the minimum in the Pi 4, the inosine-5'-monophosphate (IMP) and curve followed this at 4.2 ± 0.02 hr. The AMP resonance signals underwent nearly other components exhibited no maxima over identical behavior, and the titration curves the time course examined; however, the rate crossed at two points in the physiologic pH of change in the intensity of the a-glycero- range. With careful measurement these two phosphate resonance increased steadily over signals could be separated at pH values 10, the 25 hr time course of Fig. 3. The time- 7.6, and 3 and individually quantitated. Ob- course data of the control values exhibited no viously, rigid control of pH is essential in in- change except for a slight increase in ATP and terpreting these P-31 NMR data. Also, note decrease in Pi. that the shape of the Pi titration curve differs lntralenticular pH measurements. Phos- from that of the orthophosphate monoesters phate chemical shift values exhibit a marked illustrated in Fig. 4. This characteristic is a

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result of the contribution from the third weak quence of dynamic metabolic changes asso- acid proton of the molecule. ciated with elevated galactose as measured in Titration data such as given in Fig. 4 can be the intact rabbit lens. used in conjunction with intact tissue NMR Rabbit lenses incubated in 30 mM galac- spectra to determine intralenticular pH. For tose are characterized metabolically by an the mammalian lens, we have found three initial transitory rise of ATP and subsequent resonance signals useful for this purpose: Pi, progressive decline, an initial decrease in Pi, which has been used in muscle,16 microor- constant intralenticular pH, an accelerating ganisms,17 red blood cells,18 and adrenal increase in the level of a-glycerophosphate, gland tissue17; a-glycerophosphate, which in and an essentially constant level of ADP. The the rabbit lens resonates as a prominent sig- observation that ADP levels are unaffected nal that can be relied on to give a faithful by galactose is consistent with findings in indication of local pH environment, since galactose-fed rats reported by other inves- contributions to the spectrum from other tigators.10' u> 20> 21 Also consistent with the phosphates in this region are less than 10%; work of others is the long-term lowering of and the resonance at 6 ppm, which also ex- ATP levels in systems associated with high hibits a weak acid dissociation with the pK galactose loads.I0- 20' 21 Klethi10 also noted a value of 5.54. For both control and galactose 25% elevation in AMP levels and a 270% ele- treated lenses over the 25 hr incubation peri- vation in the levels of the uridine diphos- od, the pH value was invariant and exhibited phosugars in his elevated galactose feeding the value of 6.9 during the 25 hr incubations. study using the rat model. Our data give no This pH value was the same regardless of evidence for such overall changes, although which of the three phosphates was used to we did observe the relative enhancement of calculate intralenticular pH. The data are in- UDP-galactose over UDP-glucose. The AMP terpreted to indicate that in the lens the pH signal is a small component of the hexose was constant throughout 30 mM galactose band, and this band is enhanced by galactose and control incubations. Work on other tis- treatment. The resonance signals affected, sues has shown that phosphates residing in however, do not lie at the signal position of different pH pools will give rise to separate AMP. Our data appear to be similar to those resonance signals characteristic of that pool of Keiding and Mellemgaard,21 which show and can be used, for example, to deter- no change in AMP levels during a feeding mine mitochondrial phosphate and cytoplas- study employing the rat model. mic phosphate.19 The lens P-31 NMR data Ordinarily, in tissues under stress, relative indicate that all the Pi and the low-molecu- ATP levels and relative Pi and ADP levels are lar-weight phosphomonoesters sensed a inversely related, with the consumption of nearly equivalent pH environment. high-energy phosphate by various metabolic processes resulting in a proportional genera- Discussion tion of Pi and ADP. (See, for example, Fig. 1 Although previous research efforts have in Gadian et al.,22 where an example showing been directed toward elucidating the role of comparative spectra obtained from a per- dulcitol and reduced pyridine nucleotide fused beating rat heart are presented. This phosphate in galactose cataract formation, relationship was not evident after 4 hr of ga- little attention has been focused on the organ- lactose stress to the lens.) The early rise in ophosphates, particularly ATP and a-glycero- ATP during the first 4 hr of incubation with phosphate, and their roles in lens pathology. 30 mM galactose is accompanied by a de- Attempts to examine dynamic changes in lens crease in Pi and a slight decrease in ADP. In metabolites associated with sugar-induced the time course, the minimum level attained cataracts have been restricted to long-term by Pi lags behind the maximum attained by galactose feeding studies in the rat.9"11' 20> 21 ATP, indicating that one or more inter- The present study describes the pattern se- mediate steps must be occurring between the

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point at which ATP is utilized for phosphory- which precludes hydrolysis reactions as a lation and the point at which Pi is released mechanism for removing nucleotides. into the Pi pool. Our data are insufficient to ascertain the During the subsequent 20 hr period, the precise fate of the consumed nucleotide; rate of a-glycerophosphate accumulation ap- however, considering general mammalian proximates the rate of ATP loss, and this rate metabolic schemes, only three options for accelerates with time. (The precise point in catabolizing or storing nucleotides appear vi- time at which the rate of net ATP loss equals able: they can be stored as nucleotides, sal- the rate of a-glycerophosphate accumulation vaged as xanthine or hypoxanthine, or catab- is 16.6 hr.) Furthermore, during this time, olized to triose. Storage as nucleosides such there is no net change in the level of Pi or as inosine or adenosine would be unusual for ADP. Therefore net consumption of ATP a mammalian system and would present the must proceed so as to generate only a-glycero- lens with formidable toxicologic problems. phosphate, with the ADP that is generated in Storage as xanthine or hypoxanthine is ac- the course of the reaction sequence being re- ceptable; however, a key step in the conver- cycled to ATP and AMP through the adenyl- sion of nucleosides to xanthosine involves an ate kinase reaction. Neither AMP nor IMP NAD+-mediated oxidation, which would be (from the deamination of AMP) accumulates, significantly inhibited in a system saturated which is a finding consistent with reported with reducing equivalents and, thus, lacking effects in galactose-fed rats21; thus these re- an essential substrate for the reaction. sults suggest the conclusion that the lens nu- Catabolism of the purines to triose would cleotide pool is metabolized beyond the de- eliminate them as toxic factors; however, the amination of AMP to IMP. It should be noted purines are metabolically costly to regener- that one product of nucleotide catabolism is ate, and their loss from the lens system would ribose-5-phosphate, a precursor of a-glycero- preclude rapid functional recovery or, if se- phosphate. vere enough, would lead to irreversible tis- A key step in these catabolic reactions, sue damage. either from nucleotides or from galactose, is The intralenticular pH of rabbit lens rises the NADH-mediated reduction of dihydroxy- from a value of 6.9 to a value of 7.3 or greater acetone phosphate by glycerol-3-phosphate when the lenses are incubated in a glucose- dehydrogenase. The reaction requires 1 mole deficient pH 7.4 buffer.1 In similar work the of reducing equivalents per mole of triose, intralenticular pH falls to values below 6.4 and it provides a mechanism for restoring the when the lenses are incubated in a glucose- oxidizing potential of the pyridine dinucleo- sufficient pH 7.4 buffer that is also Ca++- tide system. The use of dihydroxyacetone deficient.23 In other parallel studies involving phosphate as a scavanger molecule for reduc- treatment by other sugars23 or steroid,24 the ing equivalents, however, has one particular intralenticular pH was found to change with disadvantage in the lens system. The product time depending on the system under study. molecule, a-glycerophosphate, is not me- Thus far, the only time-course studies that do tabolized further at a rate sufficient to pre- not involve intralenticular pH changes are vent its accumulation. The consequence is control studies, i.e., incubations in physio- that phosphate residues are removed from the logic Earle's buffer and incubation in Earle's lens phosphorylating system; that is, the intra- buffer containing 30 mM fructose. Incubation lenticular concentration of nucleotide must be with Earle's buffer containing 30 mM galac- reduced, since ADP, AMP, or IMP concen- tose is the first observed cataract model sys- trations do not change and these are the only tem that does not exhibit a pH change with other forms present in the lens in sufficient time. In view of the observation that the in- concentration to contribute significantly to tralenticular pH in the galactose-incubated the total nucleotide pool. Furthermore, the system remains constant for at least 25 hr, it concentration of Pi also does not change, can be said that the lens energy levels are

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ATP ., ADP Galactokinase2®'2^, Galactose • frGalactose-1-phosphate Glucose-1-P + UDP-galactose Galactose-1-uridyltransferase28 N •I 29 I UDPG epimerase I Glycolysis I UDP-glucose i a-glycerophosphate

Fig. 5

sufficient to maintain metabolic activity, i.e., cose, UDP-galactose, and glucose-1-phos- ion translocation, for this period of time. De- phate. On the basis of the metabolic pathways spite the constant intralenticular pH, how- for galactose known to be operant in the ever, cataract formation occurred. mammalian lens, the scheme shown in Fig. 5 The roles of GPE and GPC in cellular me- is consistent with the observed changes in tabolism have not been defined. Hypotheti- the distribution of organophosphates in the cally, these substances are purportedly simple rabbit lens in response to 30 mM galactose. end products of phospholipid metabolism; however, this concept does not necessarily fit REFERENCES with all the known data. For example, hydro- 1. Greiner JV, Kopp SJ, Sanders DR, and Glonek T: lysis of lecithin to GPC requires both a phos- Organophosphates of the crystalline lens: a nuclear pholipase A and phospholipase B activity, yet magnetic resonance spectroscopic study. INVEST there are tissues, such as the turtle heart and OPHTHALMOL VIS SCI 21:700, 1981. 2. Glonek T: Applications of 3IP NMR to biological sys- the toad gastrocnemius muscle, that are ex- tems with emphasis on intact tissue determinations. tremely rich in GPC but also contain no mea- In Phosphorus Chemistry Directed Towards Biol- sureable phospholipase B activity.25 In addi- ogy, Stec WJ, editor. New York, 1980, Pergamon tion, there are tissues with very active phos- Press, pp. 157-174. pholipase A and B enzymes that contain little 3. Dawson MJ, Gadian DG, and Wilkie DR: Contrac- tion and recovery of living muscles studied by 31P or no GPC. The data of Table I demonstrate nuclear magnetic resonance. J Physiol 267:703, 1977. that these two metabolites are, in fact, 4. Hollis DP, Nunnally RL, Jacobus WE, and Taylor GJ strongly affected by incubation in galactose- IV: Detection of regional ischemia in perfused beat- rich media. The most direct catabolic pathway ing hearts by phosphorus nuclear magnetic reso- for GPC involves hydrolysis by glycerol nance. Biochem Biophys Res Commun 75:1086, 1977. phosphorylcholine diesterase to a-glycerol- 3I 5. Glonek T and Marotta SF: P magnetic resonance of phosphate and choline, a-glycerolphosphate intact endocrine tissue: adrenal glands of dogs. Horm being the metabolite generated in the course Metab Res 10:420, 1978. of incubation in galactose-rich medium. Any 6. Glonek T: Aqueous tetrahydroxyphosphonium per- further speculation about the effects of galac- chlorate as a narrow-line 31-P NMR reference sub- tose on these metabolites must await eluci- stance. J Magnetic Reson 13:390, 1974. 7. Burt CT, Glonek T, and Barany M: Phosphorus-31 dation of their role in intact cellular systems. nuclear magnetic resonance detection of unexpected To conclude, the predominant changes in phosphodiesters in muscle. Biochemistry 15:4850, lens metabolism induced by 30 mM galactose 1976. incubation were the accumulation of a-glyc- 8. Kopp SJ, Glonek T, Erlanger M, Perry EF, Barany M, and Perry HM: Altered metabolism and function erophosphate, a parallel reduction in ATP of rat heart following chronic low level cadmium/lead content, and increased levels of UDP-glu- feeding. J Molec Cell Cardiol 12:1407, 1980.

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