Dynamic changes in the organophosphate profile of the experimental galactose-induced cataract

Transcription

1 Dynami hanges in the organophosphate profile of the experimental galatose-indued atarat Jak V. Greiner, Stephen J. Kopp, Donald R. Sanders, and Thomas Glonek Dynami hanges in lens organophosphate metabolites during 24 hr inubation in 30 mm galatose media were measured with phosphorus-31 nulear magneti resonane spetrosopy. The following phosphates were quantitated from the intat rystalline lens: adenosine triphosphate (ATP), adenosine diphosphate (ADP), inorgani orthophosphate, a-glyerophosphate, phosphorylated hexoses and trioses, niotinamide adenine dinuleotide, uridine diphosphogluose and uridine diphosphogalatose, glyerol3-phosphorylethanolamine and3-phosphorylholine, and an unidentified phosphorus-ontaining moleule. The temporal sequenes of metaboli events that define the dynami rates of aumulation or depletion of lens organophosphates reveal that the first event in the deline of the tissue upon galatose inubation is a net onsumption of ATP, ivhih ours as a sigmoidal funtion with time and whih is typified by a harateristi half-life of 18 hr. Alpha-glyerophosphate aumulated at an inreasing rate with time, whereas ADP, inorgani orthophosphate, and the other organophosphates ivere essentially unhanged. Catarat formation in the subapsular and superfiial ortial regions was visible after 16 hr inubation in the experimental buffer. Thesefindingssupport the hypothesis that alterations in the organophosphate levels of the lens are ontributing fators to the initial formation of the experimental galatose atarat. (INVEST OPHTHALMOL VIS Si 22: , 1982.) Key words: experimental galatose atarat, organophosphates, rystalline lens, phosphorus-31 nulear magneti resonane spetrosopy he dynamis of energy metabolism in the intat rystalline lens during experimental ataratogenesis, partiularly the metaboli From the Department of Pathology (Dr. Greiner) and the Nulear Magneti Resonane Laboratory (Drs. Kopp and Glonek), Chiago College of Osteopathi Mediine, and the Department of Ophthalmology, University of Illinois Eye and Ear Infirmary (Drs. Greiner and Sanders), Chiago, 111. This study was supported in part by a Grant-In-Aid from the National Soiety to Prevent Blindness (Dr. Greiner) and ore grant EY (Department of Ophthalmology, University of Illinois Eye and Ear Infirmary) National Eye Institute, National Institutes of Health. Submitted for publiation April 2, Reprint requests: Jak V. Greiner, Chiago Osteopathi Hospital, 5200 S. Ellis Ave., Chiago, events that preede atarat formation and therefore predispose the lens to it, have not been eluidated. Reently, Greiner et al. 1 have, for the first time, demonstrated the feasibility of measuring the dynami metaboli ativity of the intat rystalline rabbit lens by phosphorus-31 nulear magneti resonane (P-31 NMR) tehniques. The onentrations of the prinipal organophosphate metabolites deteted in the intat lens, the intralentiular ph, and the rates of metaboli hanges during gluose-free inubations were reported. 1 This method of NMR spetrosopi analysis for intat tissues is a totally nondestrutive tehnique 2 that permits simultaneous temperature-ontrolled tissue inubations oupled with ontinuous monitoring /82/ $01.20/ Asso. for Res. in Vis. and Ophthal., In. 613 Downloaded From: on 01/20/2019

2 614 Greiner et al. Invest. Ophthalmol. Vis. Set. May 1982 RABBIT LENS WITH 30 mm GALACTOSE 30 mm FRUCTOSE i PPM PPM Fig. 1. For legend see faing page. of quantitative time-dependent metaboli hanges during defined inrements of time. 3 ' 4 The appliation of P-31 NMR to the study of intat rystalline lens metabolism represents a novel approah to the study of lens ataratogeni mehanisms by providing a dynami perspetive of intat lens metabolism during atarat formation. As suh, the NMR method provides the investigator with new information regarding rates of lens metaboli proesses. The experimentally-indued galatose (sugar) atarat appeared to be a suitable model to evaluate time-dependent metaboli events during atual atarat formation. Thus this study desribes the dynami hanges in lens organophosphate metabolite levels dur- Downloaded From: on 01/20/2019

3 Volume 22 Number 5 Organophosphate hanges in galatose atarat 615 ing experimental inubation in a galatoserih medium. Methods Surgery. Albino rabbits, weighing 2 to 3 kg, were injeted with a lethal dose of sodium pentobarbital, and their eyes were enuleated. The eyes were opened at the posterior pole, the vitreous humor was gently separated, the zonules were ut with urved blunt sissors, and the lenses were removed with a Parafilm-oated wire lens loupe. In vitro inubation. Lenses were freshly exised, isolated, weighed, and plaed together in a tared 12 mm NMR tube ontaining a volume of modified Earle's buffer (116.4 inm NaCl, 5.6 mm dextrose, 5.4 mm KC1, 1.8 mm CaCl 2, 1.4 mm MgSO 4, 0.9 mm NaH 2 PO 4 H 2 O, 26.4 mm NaHCO 3 ) at 37 C with a ph 7.4. This Earle's buffer has been shown to maintain lens larity and lens organophosphate metabolites for at least 24 hr of in vitro inubation.' The experimental medium was Earle's buffer ontaining 30 mm galatose in addition to 5.6 mm dextrose (325 mosm). Lens inubations in Earle's buffer with added 30 mm frutose (325 mosm) were performed as osmoti ontrols. Lenses inubated in Earle's buffer (295 mosm) served as an additional ontrol. All lenses were equilibrated for 2 hr in Earle's buffer at 37 C prior to initiation of experimental and ontrol inubations. At 1 hr intervals during the inubation period, used buffer (12 ml) was aspirated from the base of the NMR tube, the lenses were washed three times with equivalent volumes of fresh buffer, and 12 ml of fresh buffer were added, thereby maintaining a onstant volume. This proedure prevented gluose depletion of the buffer bathing the lenses and generated onstant P-31 NMR organophosphate profiles. Detetable alterations in the organophosphate profiles have been observed when the medium is hanged at intervals exeeding 2 hr.' Experimental and ontrol group lenses were inubated for 25 hr in their respetive media. Ten lenses were analyzed in eah group. At onseutive 1 hr intervals during the inubation period, individual P-31 NMR profiles were aquired. Usable data were obtained in as little as 5 min; however, ontinuous 1 hr periods were used to signal average P-31 NMR data presented in this study. Lens perhlori aid extrats. Immediately after the inubation period, lenses were weighed, frozen in liquid nitrogen, and prepared for perhlori aid (PCA) extration. 5 Lens PCA extration and preparation proedures and P-31 NMR alibrations and analyses were performed aording to a previously desribed lens tissue extrat analysis. 1 P-31 NMR spetrosopy. A Niolet NT-200 system equipped with deuterium stabilization, variable temperature, and Fourier-transform apabilities operating at mhz for P-31 was used in this study. A wide-bore Oxford superonduting magnet (4.7 Tesla) was interfaed to the Niolet system. Intat lenses were analyzed at 37 C under nonspinning, proton-oupled onditions. Typial NMR san onditions with 12 mm sample tubes were as follows: pulse width 9 /use (45-degree spin flip angle); aquisition time 200 /xse; delay 200 fise; number of sans 4K; number of data Fig. 1. P-31 NMR spetra of the intat rabbit lens at three time points during inubation in 30 mm galatose-enrihed Earle's buffers and in 30 mm frutose-enrihed Earle's buffer (osmoti ontrol) at ph 7.4 at 37 C. Referring first to the 0 hr spetrum and all the ontrol spetra, the resonanes from left to right are as follows. The resonane band labeled SP has two prinipal omponents, the low-field (left) signal arises from the triose phosphates, the prinipal moleule of whih is a-glyerophosphate; the upfield (right) resonane arises from pentose and hexose phosphates, the most important of whih are IMP and AMP. The next prominent signal is that from Pi, followed by the resonane band of the phosphodiesters, labeled GPC in the figure (GPE and GPC are the prinipal phosphodiesters deteted). In the ionized end-group phosphate region beginning at 5.6 ppm are loated the y-phosphate resonane of ATP and the /3-phosphate resonane of ADP. Upfield of these are the esterified end-group phosphates, a-phosphate resonane for both ATP and ADP and the resonane band of the dinuleotides (DN) NAD and NADP. The small resonane labeled N arises from the nuleoside diphosphosugars. In the lens the primary nuleoside diphosphosugars are uridine diphosphogluose and uridine diphosphogalatose. The j8-phosphate resonane of ATP is the highest field signal in the lens spetrum. The hemial shift sale is given relative to the resonane position of 85% Pi, following the IUPAC onvention. 2 Downloaded From: on 01/20/2019

4 616 Greiner et al. Invest. Ophthalmol. Vis. Si. May 1982 Table I. Lens P-31 NMR profiles Chemial shift (ppm A ) Phosphati ompound Unknown 8 Unknown 8 Unknown 8 Trioses Hexoses NADP-2'-P Phosphoethanolamine Phosphoholine Pi Glu-l-P GPE GPC P-Cr Unknown 8 ATP ADP Dinuleotides Nuleoside-diphosphosugars In intat lens H a,-10.65; 0,-19.24; y,-5.62 a, ; 0, In PCA extrat 18.05, 18.00, , 10.72, ' : 4.3(F 3.78 G " a,-10.92; 0,-21.45; y,-5.80 J a,-10.61; /3,-6.11 K L 12.63, M NADP-2'-P = 2' phosphate of NADP; Glu-l-P = gluose-1-phosphate; P-Cr = phosphoreatine. A Field-independent NMR units of ppm relative to the shift position of the 85% Pi referene at 25. B Compound, as of this writing, is not identified with any known phosphorus-ontaining biomoleule. Minor omponent not detetable in the intat tissue. "Three separate resonane signals are deteted. E J(P-H phosphorus-hydrogen) = Hz. "Complex resonane band, the prinipal resonane signals of whih ome from the a-glyerophosphate triplet; J(POCH) =6.68 Hz. G The hexose and pentose phosphates, prinipally the resonane triplet of inosine monophosphate; J(POCH) = 3.81 Hz. H J(POCH) for the triplet = 5.63 Hz. 'Cannot be determined in the intat tissue as a separate resonane band but is ombined with the Pi signal. J J(POP,a/3) = Hz; J(P0P,/3-y) = Hz. K J(POPa/3) = Hz. -Prinipal resonane signals of this band arise from the P,P'-diesterified pyrophosphate residues of NAD and NADH. "Complex resonane band omposed of the 2 sets of overlapping, 31 p. 3l p > a b NMR multiplets from uridine diphosphogluose and uridine galatose. points per spetrum 8192; aquisition time 0.82 se; sweep width ±2500 Hz; filter-indued broadening 10 Hz. Signal-averaging, peak area integrations, and other mathematial manipulations of the transformed free-indution deay data were performed by the spetrometer's omputer using appropriate subroutines of the NTCFT Fourier-transform NMR program. PCA extrats were analyzed with and without proton deoupling and were spun at 18 Hz to enhane signal resolution. PCA extrat samples were analyzed in NMR miroell assemblies to enhane signal intensities for quantitation of the minor lens metabolites. Extrat samples were analyzed at 37 C. Spetrometer onditions used throughout were the same as those previously desribed. 1 The standard of 85% inorgani orthophosphori aid was used for determining and reporting the hemial shift data. 6 The primary intat lens internal standard used was the lens' natural glyerol 3-phosphorylholine (GPC) resonane.' GPC is a ompound with a relatively onstant hemial shift for a phosphate ( 0.13 ppm), whih is not influened by variable physiologi ph, ioni strength, or ounteration onditions. 7 P-31 NMR spetrosopi analysis of the intat lens yields a spetrum of resonane peaks with disretely defined shift positions. These resonane shifts are determined by the physial and hemial harateristis of eah phosphorus-ontaining funtional group. Thus the resonane shift position of eah peak is a physiohemial marker for individual organophosphate metabolites present in the rystalline lens. 1 Mathematial analysis of dynami lens hanges in galatose-enrihed medium. A least-squares regression analysis using a polynomial expression of the form y = Ax 3 + Bx 2 + Cx -I- D was employed Downloaded From: on 01/20/2019

5 Volume 22 Number 5 Organophosphate hanges in galatose atarat 617 Amount (% of total P in profile) In PCA extrats Controls In intat lens Freshly exised 24 hr inubation in std. Earle's 24 hr inubation in 30mM frutose Experimental 24 hr inubation in 30mM galatose Table II. Coeffiients for the expression, y = Ax 3 + Bx 2 + Cx + D, obtained in the linear regression analysis of lens time-ourse data* Component of time ourse ATP 0-8 hr 5-25 hr ADP, 0-25 hr Pi, 0-25 hr Sugar phosphates, 0-25 hr ± ± ± ± ± B dt dt dt dt dt *Ordinate is the % of total phosphorus deteted, and absissa is time in hours. C 4.49 ± ± ± ± ± dt dt dt dt dt to fit time-ourse data for the inubation period so that the sum of the squared deviations about the urve was minimized. The third-degree expression was required for adequate approximation of the real time-ourse data obtained from the intat lens tissue. By differentiation of the lous determined from the regression analysis, the points in time orresponding to the periods of maximal and minimal hange during the time ourse were alulated for the affeted metabolites. Results Fig. 1 illustrates the P-31 NMR spetra obtained from intat rabbit lenses before and during inubation with 30 mm galatose and 30 mm frutose (osmoti ontrol) in Earle's buffers. The quantitative data obtained from these NMR spetra are given in Table I. Fig. 2 shows the spetrum from a PCA extrat of lenses after inubation in 30 mm galatose for 25 hr. This spetrum is shown to illustrate the resonane signals from the uridine diphosphosugars and from several unidentified phosphates 1 ' 2> 8 indiated by shift positions in the figure. Analysis of the integrated areas of the uridine diphosphosugar resonane band verify that the uridine diphosphate (UDP)- gluose levels were redued approximately one half of those of ontrol whereas the Downloaded From: on 01/20/2019

6 Invest. Ophthahnol. Vis. Set. May Greiner et al. GALACTOSEMIC LENS PCA EXTRACT PPM Fig. 2. P-31 NMR spetrum from PCA extrat of a rabbit lens after inubation in the galatose-rih medium at ph 7.4 at 37 C for 25 hr. The resonane position of the new unknown moleule is 5,37 ppm. UDP-galatose levels were approximately doubled in response to 30 mm galatose inubation. A derease in UDP-gluose and an inrease in UDP-galatose levels are onsistent with previous findings of other investigators. 9 " 11 The PCA extrat data provided more preise data with respet to minor phosphates and illustrated that three minor metabolites, GPC? glyerol 3-phosphorylethanolamine (GPE), and the dinuleotides, niotinamide adenine dinuleotide (NAD) and niotinamide adenine dinuleotide phosphate (NADP), underwent onentration hanges after inubation of the lenses in 30 mm galatose. The hanges in the phosphodiesters were rather pronouned. GPE was redued to 16% of the initial value, and GPC was redued to 10%. The 5.37 ppm peak orresponds to an end-group phosphorus resonane haraterized by a single line under proton-oupled onditions, whih preludes its being in the family of nuleoside poly- phosphates. 12 ' 13 Furthermore, it was not inorgani pyrophosphate, whih also may give a single resonane line in this spetrum, sine addition of pyrophosphate to the sample did not result in uniform enhanement of this signal. (The pyrophosphate resonane ours at 7.25 ppm in this system.) Also, no other inorgani polyphosphate ould give rise to this resonane.l4' lo Moreover, this signal did not orrespond to an alkyl phosphate ester, sine the proton-oupled phosphorus spetrum gave no evidene for oupling to protons. The nature of the moleule that gave rise to this signal is not known at this time. Moleules that ome into resonane in this region of the spetrum and that ould aount for the 5.37 ppm signal are members of the family of mixed anhydrides between arboxyli aids and phosphate or ertain anhydrides involving aromati nitrogen heteroyli moleules. For example, the hemial shift of n-butyrylphosphate in butyri anhy- Downloaded From: on 01/20/2019

7 Volume 22 Number 5 Organophosphate hanges in galatose atarat i 12 HOURS Fig. 3. Graphed time ourse of hanges in the lens phosphate metabolites during lens inubation in 30 mm galatose-enrihed Earle's buffer. DA 7, Dinuleotides; agp, a-glyerophosphate; NS, nuleosidediphosphosugars. i 16 I 20 I 24 dride is 5.27 ppm, and the proton-oupled resonane signal is a single sharp resonane line. The extrat spetrum also learly illustrated the resonanes at 6, 10, and 18 ppm. The struture of all these moleules is unknown. They do not orrespond to any phosphorus-ontaining moleules presently identified as onstituents of biologi systems. The signal from gluose-1-phosphate, whih is partiularly strong in galatose-treated lens PC A extrats is also indiated in Fig. 1. P-31 NMR spetra of ontrol lens PC A extrats are illustrated elsewhere. 1 The 9 and 25 hr spetra of Fig. 1 illustrate the hanges exhibited in the NMR data during lens inubation in 30 mm galatose. Suh data are represented by the time-ourse plots shown in Fig. 3. When healthy rabbit lenses are exposed to buffer ontaining 30 mm galatose, the ATP level initially inreases and then delines progressively with time. Only minor hanges in lentiular ADP with time were evident, in marked ontrast to the metaboli hanges indued by gluose deprivation. 1 Similarly, Pi levels were virtually unaffeted exept for a well-defined minimum, early in the time ourse. In ontrast, a-glyerophosphate inreased steadily with time after the introdution of 30 mm galatose to the lenses, eventually aumulating to levels approahing 40% of the total lens phosphorus. The other phosphorus resonanes in the spetrum exhibited little or no hange during the galatose inubation. Catarat formation, observed with the naked eye at 16 hr, was restrited to the subapsular and superfiial ortial regions. Although the degree of atarat inreased with time, at 24 hr the atarat was still restrited to the subapsular and superfiial ortial regions. The temporal data points for ATP, a-glyerophosphate, ADP, and Pi depited in Fig. 3 were least-squares fitted to a third-order regression urve. The temporal hanges in the dinuleotides, nuleosidediphosphosugars, and the GPC urves derived from intat lens analyses did not yield maximal and minimal values. The third-order expression was satisfatory for the a-glyerophosphate, ADP, and Pi time-ourse urves; however, the ATP urve ould not be fitted to a single polynomial expression. The ATP urve appeared to have at least two major omponents, both of whih ould be well fitted with the third-degree equation. Thus the ATP data were treated as two phases: an initial Downloaded From: on 01/20/2019

8 620 Greiner et al. Invest. Ophthalmol. Vis. Set. May G-6-P ppm) 4-3- "AMP ^. MP \ N ~ ~ ^ ^ \ Pi \\ \ A Shift emial o 0- \ \ /-IMP -I PH Fig. 4. P-31 NMR-pH titration data employed in the identifiation of IMP and AMP in lens PCA extrats: solvent, aqueous saturated KC10 4 solution; temperature 37 C. These urves are reversible provided that the titrations are arried out with O.IN aid or base reagents of NMR spetral quality. NAD-2'-P, 2' phosphate of NADP; G-6-P, gluose-6-phosphate. i interval from 0 to 8 hr and a subsequent interval overlapping the first, from 5 to 25 hr. The time inrements during whih the speifi lens metabolites underwent maximal and minimal rates of hange were alulated, and the hanging values of the slope of eah urve were defined. The oeffiients for the regression equations are presented in Table II along with their standard deviations. From these equations the maximum rate of lens ATP aumulation was alulated to our at 3.4 ± 0.01 hr, and the minimum in the Pi urve followed this at 4.2 ± 0.02 hr. The other omponents exhibited no maxima over the time ourse examined; however, the rate of hange in the intensity of the a-glyerophosphate resonane inreased steadily over the 25 hr time ourse of Fig. 3. The timeourse data of the ontrol values exhibited no hange exept for a slight inrease in ATP and derease in Pi. lntralentiular ph measurements. Phosphate hemial shift values exhibit a marked dependene on the ph environment of the phosphorus atom. This onept is illustrated in Fig. 4, where P-31-NMR-pH titration data are given for several lens phosphates in water with potassium as the ounteration at 37 C. Note that the phosphorus hemial shifts for the phosphates illustrated resonate at higher fields as the ph was lowered, so that the nominal displaement per aid-base transition is 3 to 4 ppm. In regards to the speifi phosphates measured in Fig. 4, the inosine-5'-monophosphate (IMP) and AMP resonane signals underwent nearly idential behavior, and the titration urves rossed at two points in the physiologi ph range. With areful measurement these two signals ould be separated at ph values 10, 7.6, and 3 and individually quantitated. Obviously, rigid ontrol of ph is essential in interpreting these P-31 NMR data. Also, note that the shape of the Pi titration urve differs from that of the orthophosphate monoesters illustrated in Fig. 4. This harateristi is a Downloaded From: on 01/20/2019

9 Volume 22 Number 5 Organophosphate hanges in galatose atarat 621 result of the ontribution from the third weak aid proton of the moleule. Titration data suh as given in Fig. 4 an be used in onjuntion with intat tissue NMR spetra to determine intralentiular ph. For the mammalian lens, we have found three resonane signals useful for this purpose: Pi, whih has been used in musle, 16 miroorganisms, 17 red blood ells, 18 and adrenal gland tissue 17 ; a-glyerophosphate, whih in the rabbit lens resonates as a prominent signal that an be relied on to give a faithful indiation of loal ph environment, sine ontributions to the spetrum from other phosphates in this region are less than 10%; and the resonane at 6 ppm, whih also exhibits a weak aid dissoiation with the pk value of For both ontrol and galatose treated lenses over the 25 hr inubation period, the ph value was invariant and exhibited the value of 6.9 during the 25 hr inubations. This ph value was the same regardless of whih of the three phosphates was used to alulate intralentiular ph. The data are interpreted to indiate that in the lens the ph was onstant throughout 30 mm galatose and ontrol inubations. Work on other tissues has shown that phosphates residing in different ph pools will give rise to separate resonane signals harateristi of that pool and an be used, for example, to determine mitohondrial phosphate and ytoplasmi phosphate. 19 The lens P-31 NMR data indiate that all the Pi and the low-moleular-weight phosphomonoesters sensed a nearly equivalent ph environment. Disussion Although previous researh efforts have been direted toward eluidating the role of dulitol and redued pyridine nuleotide phosphate in galatose atarat formation, little attention has been foused on the organophosphates, partiularly ATP and a-glyerophosphate, and their roles in lens pathology. Attempts to examine dynami hanges in lens metabolites assoiated with sugar-indued atarats have been restrited to long-term galatose feeding studies in the rat. 9 " 11 20> 21 ' The present study desribes the pattern sequene of dynami metaboli hanges assoiated with elevated galatose as measured in the intat rabbit lens. Rabbit lenses inubated in 30 mm galatose are haraterized metabolially by an initial transitory rise of ATP and subsequent progressive deline, an initial derease in Pi, onstant intralentiular ph, an aelerating inrease in the level of a-glyerophosphate, and an essentially onstant level of ADP. The observation that ADP levels are unaffeted by galatose is onsistent with findings in galatose-fed rats reported by other investigators. 10 u> 20> ' 21 Also onsistent with the work of others is the long-term lowering of ATP levels in systems assoiated with high galatose loads. I0-20 ' 21 Klethi 10 also noted a 25% elevation in AMP levels and a 270% elevation in the levels of the uridine diphosphosugars in his elevated galatose feeding study using the rat model. Our data give no evidene for suh overall hanges, although we did observe the relative enhanement of UDP-galatose over UDP-gluose. The AMP signal is a small omponent of the hexose band, and this band is enhaned by galatose treatment. The resonane signals affeted, however, do not lie at the signal position of AMP. Our data appear to be similar to those of Keiding and Mellemgaard, 21 whih show no hange in AMP levels during a feeding study employing the rat model. Ordinarily, in tissues under stress, relative ATP levels and relative Pi and ADP levels are inversely related, with the onsumption of high-energy phosphate by various metaboli proesses resulting in a proportional generation of Pi and ADP. (See, for example, Fig. 1 in Gadian et al., 22 where an example showing omparative spetra obtained from a perfused beating rat heart are presented. This relationship was not evident after 4 hr of galatose stress to the lens.) The early rise in ATP during the first 4 hr of inubation with 30 mm galatose is aompanied by a derease in Pi and a slight derease in ADP. In the time ourse, the minimum level attained by Pi lags behind the maximum attained by ATP, indiating that one or more intermediate steps must be ourring between the Downloaded From: on 01/20/2019

10 622 Greiner et al. Invest. Ophthalmol. Vis. Si. May 1982 point at whih ATP is utilized for phosphorylation and the point at whih Pi is released into the Pi pool. During the subsequent 20 hr period, the rate of a-glyerophosphate aumulation approximates the rate of ATP loss, and this rate aelerates with time. (The preise point in time at whih the rate of net ATP loss equals the rate of a-glyerophosphate aumulation is 16.6 hr.) Furthermore, during this time, there is no net hange in the level of Pi or ADP. Therefore net onsumption of ATP must proeed so as to generate only a-glyerophosphate, with the ADP that is generated in the ourse of the reation sequene being reyled to ATP and AMP through the adenylate kinase reation. Neither AMP nor IMP (from the deamination of AMP) aumulates, whih is a finding onsistent with reported effets in galatose-fed rats 21 ; thus these results suggest the onlusion that the lens nuleotide pool is metabolized beyond the deamination of AMP to IMP. It should be noted that one produt of nuleotide atabolism is ribose-5-phosphate, a preursor of a-glyerophosphate. A key step in these ataboli reations, either from nuleotides or from galatose, is the NADH-mediated redution of dihydroxyaetone phosphate by glyerol-3-phosphate dehydrogenase. The reation requires 1 mole of reduing equivalents per mole of triose, and it provides a mehanism for restoring the oxidizing potential of the pyridine dinuleotide system. The use of dihydroxyaetone phosphate as a savanger moleule for reduing equivalents, however, has one partiular disadvantage in the lens system. The produt moleule, a-glyerophosphate, is not metabolized further at a rate suffiient to prevent its aumulation. The onsequene is that phosphate residues are removed from the lens phosphorylating system; that is, the intralentiular onentration of nuleotide must be redued, sine ADP, AMP, or IMP onentrations do not hange and these are the only other forms present in the lens in suffiient onentration to ontribute signifiantly to the total nuleotide pool. Furthermore, the onentration of Pi also does not hange, whih preludes hydrolysis reations as a mehanism for removing nuleotides. Our data are insuffiient to asertain the preise fate of the onsumed nuleotide; however, onsidering general mammalian metaboli shemes, only three options for atabolizing or storing nuleotides appear viable: they an be stored as nuleotides, salvaged as xanthine or hypoxanthine, or atabolized to triose. Storage as nuleosides suh as inosine or adenosine would be unusual for a mammalian system and would present the lens with formidable toxiologi problems. Storage as xanthine or hypoxanthine is aeptable; however, a key step in the onversion of nuleosides to xanthosine involves an NAD + -mediated oxidation, whih would be signifiantly inhibited in a system saturated with reduing equivalents and, thus, laking an essential substrate for the reation. Catabolism of the purines to triose would eliminate them as toxi fators; however, the purines are metabolially ostly to regenerate, and their loss from the lens system would prelude rapid funtional reovery or, if severe enough, would lead to irreversible tissue damage. The intralentiular ph of rabbit lens rises from a value of 6.9 to a value of 7.3 or greater when the lenses are inubated in a gluosedefiient ph 7.4 buffer. 1 In similar work the intralentiular ph falls to values below 6.4 when the lenses are inubated in a gluosesuffiient ph 7.4 buffer that is also Ca ++ - defiient. 23 In other parallel studies involving treatment by other sugars 23 or steroid, 24 the intralentiular ph was found to hange with time depending on the system under study. Thus far, the only time-ourse studies that do not involve intralentiular ph hanges are ontrol studies, i.e., inubations in physiologi Earle's buffer and inubation in Earle's buffer ontaining 30 mm frutose. Inubation with Earle's buffer ontaining 30 mm galatose is the first observed atarat model system that does not exhibit a ph hange with time. In view of the observation that the intralentiular ph in the galatose-inubated system remains onstant for at least 25 hr, it an be said that the lens energy levels are Downloaded From: on 01/20/2019

11 Volume 22 Number 5 Organophosphate hanges in galatose atarat 623 ATP Galatokinase 2 ' 2^,., ADP Galatose frgalatose-1-phosphate Gluose-1-P + UDP-galatose I I I Glyolysis I i a-glyerophosphate Galatose-1-uridyltransferase 28 N UDPG epimerase 29 UDP-gluose Fig. 5 suffiient to maintain metaboli ativity, i.e., ion transloation, for this period of time. Despite the onstant intralentiular ph, however, atarat formation ourred. The roles of GPE and GPC in ellular metabolism have not been defined. Hypothetially, these substanes are purportedly simple end produts of phospholipid metabolism; however, this onept does not neessarily fit with all the known data. For example, hydrolysis of leithin to GPC requires both a phospholipase A and phospholipase B ativity, yet there are tissues, suh as the turtle heart and the toad gastronemius musle, that are extremely rih in GPC but also ontain no measureable phospholipase B ativity. 25 In addition, there are tissues with very ative phospholipase A and B enzymes that ontain little or no GPC. The data of Table I demonstrate that these two metabolites are, in fat, strongly affeted by inubation in galatoserih media. The most diret ataboli pathway for GPC involves hydrolysis by glyerol phosphorylholine diesterase to a-glyerolphosphate and holine, a-glyerolphosphate being the metabolite generated in the ourse of inubation in galatose-rih medium. Any further speulation about the effets of galatose on these metabolites must await eluidation of their role in intat ellular systems. To onlude, the predominant hanges in lens metabolism indued by 30 mm galatose inubation were the aumulation of a-glyerophosphate, a parallel redution in ATP ontent, and inreased levels of UDP-gluose, UDP-galatose, and gluose-1-phosphate. On the basis of the metaboli pathways for galatose known to be operant in the mammalian lens, the sheme shown in Fig. 5 is onsistent with the observed hanges in the distribution of organophosphates in the rabbit lens in response to 30 mm galatose. REFERENCES 1. Greiner JV, Kopp SJ, Sanders DR, and Glonek T: Organophosphates of the rystalline lens: a nulear magneti resonane spetrosopi study. INVEST OPHTHALMOL VIS SCI 21:700, Glonek T: Appliations of 3I P NMR to biologial systems with emphasis on intat tissue determinations. In Phosphorus Chemistry Direted Towards Biology, Ste WJ, editor. New York, 1980, Pergamon Press, pp Dawson MJ, Gadian DG, and Wilkie DR: Contration and reovery of living musles studied by 31 P nulear magneti resonane. J Physiol 267:703, Hollis DP, Nunnally RL, Jaobus WE, and Taylor GJ IV: Detetion of regional ishemia in perfused beating hearts by phosphorus nulear magneti resonane. Biohem Biophys Res Commun 75:1086, Glonek T and Marotta SF: 3I P magneti resonane of intat endorine tissue: adrenal glands of dogs. Horm Metab Res 10:420, Glonek T: Aqueous tetrahydroxyphosphonium perhlorate as a narrow-line 31-P NMR referene substane. J Magneti Reson 13:390, Burt CT, Glonek T, and Barany M: Phosphorus-31 nulear magneti resonane detetion of unexpeted phosphodiesters in musle. Biohemistry 15:4850, Kopp SJ, Glonek T, Erlanger M, Perry EF, Barany M, and Perry HM: Altered metabolism and funtion of rat heart following hroni low level admium/lead feeding. J Mole Cell Cardiol 12:1407, Downloaded From: on 01/20/2019

12 624 Greiner et al. Invest. Ophtlialmol. Vis. Si. May Klethi J and Mandel P: Uridine diphosphate hexoses of lenses of rats on a high galatose diet. Biohim Biophys Ata 57:379, Klethi J: Les nuleosides polyphosphates du rystallin: leur evolution au ours de la differeniation, du vieillissement et de la surharge en galatose. Do Ophthalmol 29:1, Nordmann J, Mandel P, and Klethi J: Les anomalies initiates in metabolisme gluidique dans la atarate experimentale par surharge en galatose. Do Ophthalmol 16:199, Glonek T, Kleps RA, Van Wazer JR, and Myers TC: Carbodiimide-intermediated esterifiations of the inorgani phosphates and the effet of tertiary amine base. Bioinorg Chem 5:283, Glonek T, Van Wazer JR, and Myers TC: Carbodiimide-mediated esterifiations and ondensations of phosphori aids dissolved in various alohols. Phosphorus Sulfur 3:137, Glonek T, Costillo AJR, Myers TC, and Van Wazer JR: Phosphorus-31 nulear magneti resonane studies on ondensed phosphates. III. Polyphosphate spetra. J Phys Chem 79:1214, Glonek T, Van Wazer JR, Mudgett M, and Myers TC: Cyli metaphosphates from hydrolysis of the produts from phosphori aid ondensation with arbodiimides. Inorg Chem 11:567, Burt CT, Glonek T, and Barany M: Analysis of phosphate metabolites, the intraellular ph, and the state of adenosine triphosphate in intat musle by phosphorus nulear magneti resonane. J Biol Chem 251:2584, Ogawa S, Shuhnan RG, Glynn P, Yamane T, and Navon G: On the measurement of ph in Esherihia oli by 31 P nulear magneti resonane. Biohim Biophys Ata 502:45, Moon RB and Rihards JH: Determination of intraellular ph by 31 P magneti resonane. J Biol Chem 248:7276, Ogawa S, Rottenberg H, Brown TR, Shulman RG, Castillo CL, and Glynn P: High-resolution 31 P nulear magneti resonane study of rat liver mitohondria. Pro Natl Aad Si USA 75:1796, Kinoshita JH: Catarats in galatosemia. INVEST OPHTHALMOL 4:786, Keiding S and Mellemgaard L: Dose dependene of galatose atarat in the rat. Ata Ophthalmol 50:174, Gadian DG, Hoult DI, Radda GK, Seeley PJ, Chane B, and Barlow C: Phosphorus nulear magneti resonane studies on normoxi and ishemi ardia tissue. Pro Natl Aad Si USA 73:4446, Glonek T, Greiner JV, Kopp SJ, and Sanders DR: The intralentiular ph during ataratogenesis in the intat lens as determined by phosphorus-31 nulear magneti resonane spetrosopy. INVEST OPH- THALMOL VIS SCI 20(ARVO Suppl.):89, Greiner JV, Kopp SJ, and Glonek T: Dynami hanges in the organophosphate profile upon treatment of the rystalline lens with dexamethasone. INVEST OPHTHALMOL VIS SCI (in press). 25. Chalovih JM, Burt CT, Danon MJ, Glonek T, and Barany M: Phosphodiesters in musular dystrophies. Ann NY Aad Si 317:649, Hayman S, Lou MF, Merola LO, and Kinoshita JH: Aldose redutase ativity in the lens and other tissues. Biohim Biophys Ata 138:474, Gupta JD, Irvine S, and Harley JD: Speies variation in galatokinase ativity of erythroytes, lens and liver. Aust J Exp Biol Med Si 50:511, Talman EL: Mehanism for galatose-1-phosphate arual in galatose atarat. II. Effet of galatose- 1-phosphate uridylyl transferase and UDP gluose pyrophosphorylase ativities. Physiol Chem Phys 1: 255, MLaren DS and Halasa A: Nutritional and metaboli atarat. In Catarat and Abnormalities of the Lens, Bellows JG, editor. New York, 1975, Grime & Stratton, In., p Downloaded From: on 01/20/2019