Free ammo acids in senile cataractous lenses: Possible osmotic etiology

Transcription

1 ree ammo acids in senile cataractous lenses: Possible osmotic etiology G. Winston Barber Comparison of free amino acids in freshly excised rabbit lenses and in lenses taken from the contralateral eyes after storage for 4 to 48 hours reveals essentially no postmortem changes. This suggests that human eyebank lenses may be considered as normal with respect to the free amino acid pool. Analysis of senile cataractous lenses, compared to eyebank lenses, reveals a wide range of amino acid concentrations, from much higher to much lower than eyebank lens levels. Amino acid concentrations in cataracts decrease with decreasing lens weight, visual acuity, and per cent protein. ree proteogenic amino acid concentrations decrease from levels well above those in eyebank lenses, while nonproteogenie amino acids decrease only from normal levels. Thus selective accumulation of free proteogenic amino acids seems to be an early event in the etiology of senile cataract. This conclusion suggests the following hypothetical mechanism of senile cataractogenesis. Inhibition of protein synthesis, ivith continued normal proteolysis, could account for selective increase in proteogenic amino acid levels. Increased normal hydrolysis or pathological autolysis are possible but not essential explanations. Increased intracellular osmotic pressure and resultant i?nbibition of water may cause the wellknown early intumescence of senile cataract. If damage to cell membranes results, lens protein may be lost by leakage rather than by autolysis. Consequent dilution of cytoplasm and disruption of cellular structure are sufficient explanation of opacification. JLhe intracellular fluid of the ocular lens contains a high concentration of soluble protein upon which, in part, transparency is dependent. 1 In addition, the individual amino acids from which proteins are synthesized (proteogenic amino acids) are present, as well as a number of other amino acids which are not protein constituents. Studies of several hundred normal lenses of various species have shown that the free amino acid pool is quite charac- rom Wills Eye Hospital and Research Institute, Philadelphia, Pa. Supported by Grant NB-446 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. Presented at the Annual eeting of the Association for Research in Ophthalmology, Clearwater, la., ay 1 to., teristic and constant for similar animals. Among a group of ten rabbits from the same source, for example, coefficients of variation for the individual pool constituents ranged from 15 to per cent. Somewhat greater variation is found with animals from different sources. In the rabbit, free amino acids are present in lens water at concentrations several times the plasma or aqueous humor levels (Table II), and lens concentrations are not appreciably affected by aging, fasting, or a protein-free diet. This constancy of normal lenses, as well as the importance of free amino acids as the precursors of lens protein, makes the lens free amino acid pool a most suitable subject for study. The constancy of amino acid levels reflects a very stable balance in the normal lens among uptake, excretion, synthesis, Downloaded rom: on 1/8/18

2 Volume 7 Number 5 ree amino acids in senile cataractous lenses 565 catabolism, incorporation into protein, and regeneration by hydrolysis of protein. This steady state is maintained for months in protein-deficient animals in spite of continuous negative systemic nitrogen balance and severe weight loss, and the lens continues to grow at a normal rate. In contrast, when study of the lenses of alloxandiabetic rabbits was undertaken, dramatic changes in free amino acid levels were immediately apparent. Decrease in concentrations of most constituents of the amino acid pool to almost the levels in aqueous humor takes place within a few days in severely hyperglycemic rabbits. This loss of free amino acids has also been found in alloxan-diabetic rats 4 and in pancreatectomized" as well as alloxan-diabetic rabbits." In our own studies, the extent of amino acid loss appeared to be proportional to the elevation of blood glucose and could be prevented by insulin therapy to maintain normal glucose levels. We have found completely comparable loss of free amino acids as an early event in galactose-fed rats and rabbits. Decrease in free amino acid levels was first reported for lenses of xylose-fed rats 7 and has also been reported for galactose-fed rats. s This common loss of amino acids, and quite possibly of other important metabolites, has found explanation in the studies of lens carbohydrate metabolism and of experimental cataract caused by elevated levels of glucose, galactose, or xylose which have been pursued in a number of laboratories. These have culminated in the proposal by Kinoshita 9 of a common osmotic, : i Asp Glu Gly Ala Val Leu ileu Phe Tyr Ser Thr Pro ml,, 1, n -.-- et Cys Lys His Arg Hyp But Cysta Tau Orn -e Tryp ig. 1. ree amino acid concentrations in lenses of alloxan diabetic rabbits, 1 to weeks after alloxan injection (first bar, groups of 5 animals); in lenses of normal rabbits (second bar, 6 groups of 1 animals); and cataractous lenses frbm diabetic rabbits, to 1 months after alloxan injection (third bar, 4 individual animals). The upper portion of each bar indicates the extreme range observed. Downloaded rom: on 1/8/18

3 566 Barber Investigative Ophthalmology October 1968 s -- 6hr lohr 1hr Uhl ig.. Tracing of a typical chromatogram of free amino acids in eyebank (normal) human lenses. Compounds identified with confidence are:, taurine; 7, urea; 1, methionine sulfone; 14, methionine sulfoxide; 5, aspartic acid; 16, asparagine; 17, threonine; 18, serine;, glutamic acid; 1, citrullin;, proline (at 44 nm.);, oxidized glutathione; 4, glycine; 5, alanine; 6, cysteine-glutathione disulfide; 7, butyrine; 8, valine; 9, cystine;, methionine;, cystathionine; 5, isoleucine; 7, leucine, 8; norleucine (added as standard); 4, tyrosine; 41, phenylalanine; 45, ethanolamine; 46, gamma-aminobutyric acid; 47, ammonia; 5, ornithine; 51, 1-methylhistidine; 5, histidine; 5, lysine; 54, -methylhistidine; 55, tryptophan; 56, carnosine (rare in human lenses); 57, homocarnosine; 58, arginine. Remaining peaks are essentially unidentified, but known compounds elute at the following positions: 1, cysteic acid, phosphoserine, S-sulfoglutathione; 4, ethanolamine phosphate; 8 and 1, S-(l,-dicarboxyethyl) glutathione; 1, S-( 1,-dicarboxyethyl) cysteine; 4, glutathione-cysteinylglycine disulfide; 9, cysteine-cysteinylglycine disulfide; 4, beta-aminoisobutyric acid; 44, homocystine; 45, ethanolamine, cysteamine-glutathione disulfide; 48, hydroxylysine; 49, hydroxytryptophan. Downloaded rom: on 1/8/18

4 Volume 7 Number 5 ree amino acids in senile cataractous lenses 567 8hr lohr 1hr hr o ig.. Cont'd 18hr hr mechanism for the three types of sugar cataract. This mechanism, involving intracellular accumulation of the polyol corresponding to the responsible sugar, induces rapid osmotic swelling, resulting in increased permeability of lenticular membranes. Whether or not loss of cellular protein accompanies the demonstrated loss of other soluble constituents does not appear to have been studied directly, but Sippel xa has noted in the aqueous humor of galactose-fed rats a sudden increase in protein concentration at the time that the lenses undergo sudden decrease in protein content. In contrast to the early loss of lens amino acids induced by diabetic hyperglycemia, the analysis of cataractous rabbit lenses after to 1 months of untreated diabetes revealed concentrations of free amino acids Downloaded rom: on 1/8/18

5 568 Barber In vestigative Ophthalmology October 1968 considerably higher than normal levels (ig. 1). But only the proteogenic amino acids were elevated in these cataractous lenses. Nonproteogenic amino acids were in general at even lower levels than in the precataractous lenses. This agrees with the concept of autolysis as a late manifestation of diabetic cataractogenesis. The study of human senile cataracts was undertaken to determine whether or not changes in free amino acids occur analogous to those found in sugar cataracts. ethods ree amino acids have been determined by column chromatographic analysis of deproteinized lens extracts. The method is essentially that of Spackman, Stein, and oore, 11 but utilizes the single-column, gradient elution technique of Piez and orris 1 '- in conjunction with a microcolumn similar to that described by Hamilton. 1 Column effluents are analyzed by AutoAnalyzer. 14 ' ir> With this system, an aqueous humor sample from a single eye can be analyzed, and a single human lens provides an optimal sample. Recoveries from standard amino acid mixtures are within ±5 per cent for quantities greater than.1 peq (equivalent to.6 to.1 /JEq per milliliter of lens water), and within ±1 per cent for quantities in the range.1 to.1 ixeq. Glutamine is converted quantitatively to pyrrolidonecarboxylic acid on this column (6 C.) and is therefore not measurable. Lenses from eyebank eyes were removed after 4 to 48 hours' storage of eyes in moist chambers at 4 C. Senile cataracts were obtained from the operating rooms of Wills Eye Hospital. Only intact, intracapsular lenses have been used. These have been placed in preweighed vials and delivered promptly to the laboratory. Lenses were freed from any adherent vitreous humor, weighed, and promptly homogenized in 5 ml. of water containing 1.5 mg. of dithiothreitol. Protein was precipitated by addition of trichloroacetic acid to 1 per cent, and the latter was removed byfiltration through Dowex--Chloride. 11 iltrates were evaporated at C. on a rotating evaporator and the residues were stored at - C. until analyzed. Water content of lenses has been taken as the difference between the wet lens weight and that of the trichloroacetic acidprecipitated protein dried to constant weight. No correction has been made for the weight of salts and other small solutes. or analysis of total lens protein, the trichloroacetic acid precipitates from eyebank lenses were extracted with hot 1 per cent trichloroacetic acid and the dried residues were heated with 6N hydrochloric acid in evacuated tubes for hours at 1 C. Aliquots were evaporated and chromatographed. Human aqueous humor was taken from cataractous eyes by paracentesis just before surgery. Samples were weighed and chromatographed directly. Heparinized plasmas taken from the same patients just after surgery were deproteinized and analyzed in the same manner as lenses. Results Human aqueous humor. Six samples from cataract patients have been analyzed. Comparison of amino acid concentrations with those in plasma samples from the same patients yielded ratios very similar to those obtained with normal rabbits (Table I), so it is probable that such samples are fairly representative of normal aqueous humor. Eyebank lens analyses. Completely normal human lenses are essentially unavailable, but 1 pairs of lenses from eyebank eyes, aged 5 to 8 years, have been analyzed. The composition of the free amino acid pool is reasonably constant, the coefficients of variation for most constituents being about per cent (Table II). This is just about the degree of variability we have found in random populations of experimental animals. The total of proteogenic amino acids varies within somewhat narrower limits. Values for glutathione are probably low, and the identity of the peak Downloaded rom: on 1/8/18

6 Volume 7 Number 5 ree amino acids in senile cataractous lenses 569 Table I. Amino acids in human aqueous humor Proteogenic Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Phenylalanine Tyrosine Serine Threonine Proline ethionine Half-cystine Lysine Histidine Arginine Tryptophan Total proteogenic Nonproteo genie Taurine Asparagine Citrullin Butyrine Ornithine 1-ethylhistidine -ethylhistidine Total nonproteogenic ean* neq/ml Concentration Rangef neq/ml ±.9 (S.D.) ±. (S.D.) Urea 'ean of 6 samples from cataract patients. f Extreme range. Jean of ratios for 6 cataract patients, plasma taken as 9 per cent water. {ean of ratios for 8 pools of 5 fed rabbits, plasma = 9 per cent water. I Dubious values, being ratios of two very low values. Ratios: aqueous /plasma Human t Rabbits^, eluted at the ophthalmic acid position is doubtful, for the dicarboxyethyl derivative of cysteine elutes at the same position and has been found in bovine lenses. 17 A tracing of a typical eyebank lens chromatogram is presented in ig.. Peak No. 6 was found in only two pairs of eyebank lenses, but is frequently present in cataracts. Peaks No. 49 and 56 are rarely present in either eyebank lenses or cataracts. Those components described as identified with confidence have been identified in rabbit lens extracts by isolation of column chromatographic fractions and comparison with authentic standards by paper chromatography. Postmortem changes in rabbit lenses. In contemplating the possibility of using lenses from eyebank eyes as controls for the study of free amino acids in cataractous lenses, it was necessary to determine what postmortem changes might be anticipated. reshly excised lenses from mature rabbits (6 months old) were therefore compared with lenses removed from the contralateral eyes after treatment as much Downloaded rom: on 1/8/18

7 57 Barber Investigative Ophthalmology October 1968 Table II. ree amino acids in eyebank lenses Proteogenic Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Phenylalanine Tyrosine Serine Threonine Proline Hydroxyprol ine ethionine Half-cystine Lysine Histidine Arginine Tryptophan Total proteogenic Nonproteo genie Taurine Butyrine Cystathionine Gamma-aminobutyric acid Ornithine 1-ethylhistidine -ethylhistidine Homocarnosine Total nonproteogenic Ophthalmic acid (?) Glutathione ean of 1 values ean of 8 highest Lens concentrations ean"* fieq/ml. of lens water S.D.f ean of 1 pairs of eyebank lenses f Standard deviation. i Ratio of mean eyebank lens concentration to mean human aqueous concentration. 5ean of ratios for 6 pools of 1 rabbits. Ratios of lens:aqueous Human\ Rabbits^ as possible like that experienced by eyebank eyes. The stored eyes were enucleated four hours post mortem and then kept for 4 or 48 hours in moist chambers at 4 C. The compared analyses of fresh and stored rabbit lenses indicated practically identical compositions of the free amino acid pools (ig. ). This absence of any significant postmortem change in rabbit lenses suggests that eyebank lenses may be regarded as normal human lenses with respect to free amino acid concentrations. This conclusion must be tempered, of course, by some reservation with respect to our ignorance of the effect on eyebank lenses of the cause of death or of any antecedent therapy. In contrast to amino acids, some loss of glutathione was noted after 48 hours. Lens-.aqueous humor concentration. The Downloaded rom: on 1/8/18

8 Volume 7 Number 5 ree amino acids in senile cataractous lenses 571 But Val Cysta et lieu Leu Tyr Phe Orn Lys His e JTau Urea GSH Glu HO Pro Asp Asp-NHj Thr Ser Pro Gly Ala Cys -t-1 ig.. Postmortem changes in free amino acids of rabbit lenses. Concentrations in pools of 1 freshly excised lenses (solid bars) and the pooled contralateral lenses after storage of eyes at 4 C. for 4 hours (first cross-hatched bar) or 48 hours (second cross-hatched bar). tentative values for human lens:aqueous humor ratios have been calculated for individual free amino acids from the mean concentrations found in twelve pairs of eyebank lenses and the mean concentrations in six samples of aqueous humor from cataract patients (Table II). The ratios are generally very similar to those found in normal rabbits, which might be taken as further support for the conclusion that eyebank lenses and aqueous humor from cataract patients are normal with respect to free amino acids. There are, however, some interesting differences from ratios found in rabbits. The ratio for glycine in human samples is much higher than in rabbits, which reflects the extremely low concentration in human aqueous humor. Ratios for lysine and arginine in human lens and aqueous humor are not significantly different from unity, which may have bearing on the unique behavior of these two constituents in cataractous lenses. Analysis of human senile cataracts. With the first group of six senile cataracts analyzed in a pilot study, a dichotomy resulted (ig. 4) similar to that found with lenses of diabetic rabbits (ig. 1). our lenses contained very low concentrations of all free amino acids, and two lenses had higher than normal concentrations of proteogenic amino acids. In the latter lenses, however, nonproteogenic amino acids were not depleted as in diabetic rabbit cataracts, but were present at essentially normal levels. Thus the resemblance to diabetic rabbit lenses is superficial, and it appeared that the order of events is actually reversed. A similar dichotomy of human cataracts with respect to amino acid levels has been observed by other investigators. 18 Since a complete analysis of the free amino acids in the lens provides too much information for convenient discussion, two parameters have been adopted which require clear definition: Total proteogenic Downloaded rom: on 1/8/18

9 57 Barber In vestigative Ophthalmology October 1968 H high range I low 1L1 n Asp Glu Gly Ala Val Leu ileu Phe Tyr Ser Thr Pro normal I B J J et Cys Lys His Arg Hyp But Cysta Tau Orn GABA Homo ig. 4. Amino acid concentrations in senile cataractous lenses, per cent of normal values for eyebank lenses. Extreme ranges found in 4 low amino acid lenses (cross-hatched bars) and in two high amino acid lenses (solid bars). Isoleucine Phenylalanine Leucine Tyrosine 1. V.5 1* ] % * Total PAA ig. 5. Correlations of concentrations in senile cataracts of individual proteogenic amino acids (ordinates) with the total concentration of proteogenic amino acids. Ranges (- one standard deviation) found in eyebank lenses are indicated by the dotted rectangles. The solid rectangles show ranges found in human aqueous humor. Downloaded rom: on 1/8/18

10 Volume 7 Number 5 ree amino acids in senile cataractous lenses 57 Table III. Nuclear-sclerotic senile cataracts Age Sex Visual* acuity Color} Weight (mo.) Estimated loss (mo.) Per cent water Total PAA (fieq/ml.) Total NPAA (fieq/ml.) GSH (fieq/ml.) C C C : C.. L.P C L.P. L.P. H.. C H.. C.. C.. H.. H L.P. H.. H.. H.. L.P L.P. L.P "Visual acuity is given as the decimal fraction of normal, or as counting fingers, hand motion, or light perception. fcolor is expressed on the scale: 1, very pale yellow;, yellow;, amber or very light brown; 4, brown: 5, darv brown Downloaded rom: on 1/8/18

11 574 Barber Investigative Ophthalmology October 1968 Table IV. Correlation of amino acid concentrations in cataracts Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Phenylalanine Tyrosine Serine Threonine Proline ethionine Cystine Lysine Histidine Arginine Taurine Cystathionine Ornithine Butyrine Total NPAA Glutathione Regression"*.7 PAA +.4. PAA S PAA PAA ^ PAA +..6 PAA +.. S PAA PAA ^ PAA PAA PAA -.8. PAA PAA +..9 PAA +.7. S PAA ^ PAA PAA -..1 PAA -.5. PAA PAA PAA PAA -.47 Eye bank fraction} Lens protein fraction\ "Regression of individual components against the total of proteogenic amino acid concentrations, least squares, 47 lenses. Ratio of mean concentration in eye bank lenses to mean total of proteogenic amino acid concentrations. t raction of the total amino acids in hydrolysate of lens protein (TCA precipitable), mean of 1 analyses, eyebank lenses. Table V. ree amino acids in tryptophan-deficient guinea pig lenses Weeks on diet No. Test 51 1 of animals Control} g. per lens Test Control ean concentration ratios Total PAA Total NPAA Test diet:.1 per cent tryptophan. 4 ]Control diet:. per cent tryptophan. J Ratios are of concentrations per milliliter of lens water, taken us 65 per cent of lens weight. of test:controlt Taurine GSH amino acids (PAA) means the sum of the concentrations in lens water of the 16 protein-derivable amino acids which are found in measurable quantity in the lens free amino acid pool. These are: aspartic acid, glutamic acid, glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, serine, threonine, proline, methionine, lysine, histidine, and arginine. Hydroxyproline, cystine, and tryptophan also occur, but not consistently, and only in traces. Total nonproteo genie amino acids (NPAA) means the sum of the concentrations of those free amino acids which are not protein constituents. or human lenses, this includes: taurine, alpha-aminobutyric acid, cystathionine, gamma-aminobutyric acid, ornithine, 1-methylhistidine, -methylhisti- Downloaded rom: on 1/8/18

12 Volume 7 Number 5 ree amino acids in senile cataractous lenses 575 Serine Proline 5 r*i f J * 1 15 v-r 1 IS Threonine Valine * ig. 6. See legend for ig. 5. ' * n- 1 IS 5 Total PAA Glutamic Acid Glycine Aspartic Acid JC 1. Alanine # " «A.'.} ig. 7. See legend for ig IS 5 Total PAA dine, and homocarnosine. Other compounds are variably present in traces but are not included in this total. Homocarnosine, incidentally, seems to be unique to human lenses, and gamma-aminobutyric acid is much more prominent than in animal lenses. Glutathione values for eyebank lenses did not seem reliable. Values for cataracts are reported (Table III), but their relationship to normal values is uncertain. An unidentified ninhydrin-positive substance (No. 6) which elutes between ispleucine (No. 5) and leucine (No. 7) is variably Downloaded rom: on 1/8/18

13 576 Barber Inoestigatioe Ophthalmology October 1968 ethionine Lysine. D- ;- _,.* V 5 1 IS 5 1 Histidine o. ig. 8. See legend for ig. 5. _ V Total PA A Arginine present in cataractous lenses. The occurrence of asparagine is also variable, but when it is present, No. 6 is absent, and vice versa. This observation is completely mysterious, and no correlation of either component with any other feature of cataractous lenses has been identified. No. 6 was also observed in two of the twelve pairs of eyebank lenses, but has never been seen in animal lenses. In the current study, 47 cataractous lenses have been analyzed, and in most of these patients, residual visual acuity was determined just before surgery. Wills Eye Hospital records were examined for any record of diabetes or other significant systemic disease or medication. All suspect lenses were rejected, but other medical histories were not available. Only cataracts classified as nuclear-sclerotic are included in this series, summarized in Table III. Pigmentation varied widely in these lenses, and in almost all the nucleus was more deeply colored than the cortex, which was clear in the great majority. No correlation of pigmentation with free amino acid pool composition could be discerned. The proteogenic amino acid concentrations in this population of lenses appear to span a range from about twice normal concentrations down to nearly the levels in aqueous humor. In contrast, nonproteogenic amino acids range downward only from normal levels. igs. 5 to 9 illustrate the ranges of concentrations found and demonstrate the correlation of the principal pool constituents with the total of proteogenic amino acid concentrations (total PAA). In these graphs, each point represents an individual cataractous lens. The dotted squares indicate the probable limits of eyebank lens concentrations, and the solid squares indicate the concentration ranges found in human aqueous humor. The parameters of the statistically determined regression lines for these plots are summarized in Table IV. The regression coefficients are remarkably similar to the mean mole fractions in eyebank lenses, and show some similarity to the fractional composition of eyebank lens protein. The significance of these similarities is not completely clear at the present stage of our knowledge of human lens proteins. It might be revealing, however, to compare these regression coefficients to the compositions of those proteins which are synthesized in mature lenses, and to the com- Downloaded rom: on 1/8/18

14 Volume 7 Number 5 ree amino acids in senile cataractous lenses 577 Cystathionine Taurine Total NPAA Ornithine js - Total PAA ig. 9. Correlations of concentrations in cataractous lenses of three individual nonproteogenic amino acids and of total NPAA (ordinates) with the total of proteogenic amino acids. re CD Total PAA Heq ml ig. 1. Correlation of glutathione concentrations in cataractous lenses with the total of proteogenic amino acids. The means for the 1 pairs of eyebank lenses, the 8 highest levels in eyebank lenses, and the highest levels are given by the numbered open circles. positions of the protein fractions broken down by normal hydrolysis (turnover) or by proteolytic enzymes. The data are therefore included in the hope that they may be useful in evaluating possible experimental approaches to reproduction of the observed course of free amino acid changes in cataracts. or the amino acids illustrated in ig. 7 maximum concentrations are not as high as would be expected, but these are the four proteogenic amino acids which rabbit lenses are capable of utilizing metabolically, 1 which may explain their failure to increase significantly in senile cataracts. Lysine and arginine (ig. 8) are also interesting exceptions from the general rule, for they neither increase as much as Downloaded rom: on 1/8/18

15 Oui ' sso (A Prleitly-Sinlth 188 «Colltm 1894 o Ir«Bank Age ig. 11. Weights of fresh postmortem human lenses; relationship to chronological age of donor '5 +-- ft Aquaow o ^-e o > o Total «o * PAA o _- o o o o Visual Ac ulty O.5 -. e c.r. H.. oi I.p. 15 ig. 1. Correlation of weight loss of senile cataracts, compared to normal weights from ig. 9, with the total of proteogenic amino acid concentrations. Visual acuity is indicated by the symbols: as the fraction of normal, as counting fingers (C..), or as either hand motion (H..) or light perception (L. P.). Total PAA ig. 1. Per cent water content of senile cataracts as a function of total proteogenic amino acid concentration. Downloaded rom: on 1/8/18

16 Volume 7 Number 5 ree amino acids in senile cataractous lenses 579 expected nor decrease significantly later. The explanation is not apparent, but in eyebank lenses the concentrations of these two components are not much higher than in aqueous humor. The behavior of lysine and arginine in diabetic lenses is similar (ig. 1). ig. 9 demonstrates that for the total of nonproteogenic amino acids, as well as for three of the components of this group, the high concentrations are about the same as in eyebank lenses. Thus, while most of the proteogenic amino acids decrease from greater than normal concentrations, nonproteogenic amino acids decrease only from normal levels. This is in agreement with the proposed chronology of amino acid changes in these cataractous lenses. Glutathione concentrations in cataracts tend downward with amino acids (ig. 1), but we are not sure of normal values for this compound, which did decrease somewhat in stored rabbit eyes (ig. ). Values in eyebank lenses varied widely. The highest concentration was about.5 /xeq per milliliter and several of the clearest cataracts contained about this same amount. Extrapolation of the regression line of ig. 1 to PAA = (the point in ig. 1 where A w = ) gives a glutathione concentration of about 5 /JEq per milliliter and this is about the maximum level found in two cataracts. Data for fresh, normal human lenses seem to be unavailable. Correlation of amino acid levels with lens weight, visual acuity, and water content. Loss of weight of cataractous lenses has been estimated by comparison with normal weights estimated from a graph of the meager data available in the literature for fresh postmortem human lenses. ~- The available data are such that exponential, logarithmic, linear, or power functions can be fitted equally well. Two leastsquares curves are illustrated in ig. 11, an exponential function analogous to that used by Kraus for the data of Jess on bovine lens weights: Lens weight (mg.) = 8 - x e" 19 x sc (yr) " and the linear regression function Lens weight (mg.) = x age (yr.) In the age range of cataract patients, the curves are practically identical for the present application. When the loss of weight of cataractous lenses compared to estimated normal lens weights is plotted against the total of proteogenic amino acid concentrations, a fair correlation is evident (ig. 1), although the actual relationship is probably not linear. or the data of ig. 1, the regression is A w = 1.68 x PAA - 57 Pearson's coefficient of correlation is.8 and the significance (N = 45) is p <.. If weight loss is similarly plotted as a function of the total of nonproteogenic amino acid concentrations, the regression is A w =. x NPAA - 55, the correlation coefficient is.47, and the significance is p <.. In ig. 1 residual visual acuity of the individual lenses is indicated by the symbols. Correlation of decreased acuity with decreased amino acid levels is evident. The relationship of water content of cataractous lenses to the total of proteogenic amino acid concentrations is shown in ig. 1. Discussion With decreasing amino acid concentrations, lens weight, visual acuity, and per cent protein all decrease. These facts appear to confirm the conclusion that in human senile cataract the elevation of proteogenic amino acids above normal concentrations precedes the stage of very low concentrations. Although this course of events is compatible with the concept of autolysis due to lowered intracellular ph 1 or to disruption of lysosomes, 5 what we would suggest here is that elevation of amino acid concentrations may be a contributing cause in the development of senile cataract, rather than a mere symptom of autolysis. If the regression line relating data on lens weight loss to the total of Downloaded rom: on 1/8/18

17 58 Barber Investigative Ophthalmology October 1968 proteogenic amino acid concentrations (ig. 1) is extrapolated to zero weight loss, a value of 4 /JEq total PAA per milliliter of lens water is obtained. The excess above the mean value for eyebank lenses should be equivalent in osmotic activity to 47 mg. per 1 ml. of sorbitol, which approaches concentrations found in lenses of diabetic animals. In such lenses, osmotic swelling is believed to be an important part of the cataractogenic process. 9 The osmotic effect of sorbitol, and of dulcitol in lenses of galactose-fed animals, is due to the difficulty with which these polyols escape when they are produced within lens cells. 9 That excessive levels of amino acids should also be osmotically effective in lenses is suggested by the observation that the concentrations in normal rabbit lenses do not decrease appreciably on incubation in amino acid-free media, and, with calf lenses incubated for hours in simple salt solution, less than per cent of the free amino acids escaped. Intumescence as an early event in the genesis of senile cataract is well known, but lenses are rarely available for study at this time. Our data suggest that that accumulation of amino acids should at least be considered as the possible cause of this intumescence. If, for example, water is absorbed as amino acids accumulate, so as to maintain normal osmotic pressure, lens weight should increase at the rate of.54 mg. for each microequivalent per milliliter of increase in total PAA. At the point where this relationship intersects the regression line of ig. 1, total PAA would have increased to 41 ^Eq per milliliter, lens weight would have increased by 16 mg., and lens water content would have increased 1 per cent. There is no implication here that these numbers precisely describe the early changes in senile cataract, but they do serve to illustrate the proposed mechanism and may give some idea of the possible magnitudes of changes. Resultant swelling could lead to opening of pores in cellular membranes, perhaps irreversibly, with consequent loss not only of amino acids, but possibly of other soluble constituents as well. Dilution of lens protein by simultaneous protein loss and water accumulation may in itself cause decreased transparency. 1 In his monograph of 194, Krause proposed swelling due to accumulation of autolysis products as the mechanism of rapidly developing cataracts. The proposal was based on the elevated levels of water 7 and of nonprotein amino nitrogen S in lenses of parathyroidectomized dogs and rabbits and naphthalene-poisoned rabbits, 9 and on the swelling observed in early diabetic cataract, which he erroneously assumed to be due to autolysis. Krause did not believe that amino acids formed by slow autolysis in senile cataract could accumulate to osmotically effective levels. The data presented here, however, demonstrate that such accumulation does occur, and there is one report of increased nonprotein amino nitrogen in "soft" cataracts, followed by lower levels in "hard" cataracts, as well as a report of higher amino nitrogen concentrations in cataractous than in normal lenses of horses. 1 In vivo turnover of at least one radioactive amino acid following incorporation into rabbit lens protein has been demonstrated, and protease activity at neutral ph has been described in homogenates of bovine lenses. Since lens protein was the substrate for this enzyme, a role in normal protein turnover was suggested. No attempt to demonstrate such activity in intact lenses appears to have been described. The observed formation of free amino acids as the only end products of proteolysis might be due to peptidase activity of the neutral proteinase, or to rapid activity of the widely studied lens aminopeptidase which does not act directly on lens protein. 1 In old age, the growth of the human lens is about 1 mg. per year. This is equivalent to net incorporation of about 1 /JEq of proteogenic amino acids per milliliter of lens water, and suggests a minimal rate at which amino acid concentration might in- Downloaded rom: on 1/8/18

18 Volume 7 Ntimber 5 ree amino acids in senile cataractous lenses 581 crease due to inhibition of synthesis. We have no idea how much greater the rates of actual protein synthesis and hydrolysis may be in human lenses, but we have observed incorporation of labeled amino acids into rabbit lens protein at a rate equivalent to synthesis of about 4 times as much protein as the net synthesis calculated from the growth rate of such lenses. 9 In bovine lens, incorporation of labeled amino acids indicated synthesis of about 6 times more protein than net synthesis. 5 Active transport is a second possible mechanism for amino acid accumulation as a result of inhibition of protein synthesis. or the nonutilizable alpha-aminoisobutyric acid, Kinsey and Reddy G found with cultured rabbit lenses a steady-state ratio of for concentrations in lens water and medium. This ratio decreased to 8.6 when the medium contained other amino acids in concentrations resembling aqueous humor, but ratios even this high are not found for proteogenic amino acids in normal lenses 7 (Table II). This may be due in part to utilization of pool amino acids for net synthesis of protein. If so, inhibition of such synthesis might permit active transport systems to achieve higher lens:aqueous concentration ratios. Thus we have two possible mechanisms for selective increase of proteogenic amino acid concentrations as a result of hypothetical inhibition of protein synthesis. This could occur by continued normal hydrolysis of protein, or by permitting the "pump-leak" mechanism 55 to reach higher amino acid concentrations. We have some very preliminary data which suggest that such a mechanism actually operates in the lenses of guinea pigs fed a tryptophandeficient diet, which presumably inhibits protein synthesis, 9 and has been shown to involve intumescence as an early lenticular effect. 4 In these lenses, which were pooled for analysis, selective increase in proteogenic amino acids has been observed (Table V). In any event, it is evident that pathologic autolysis due to lowered ph or to disruption of lysosomes is not an essential assumption as an explanation of elevated amino acid concentrations. It is not even necessary to postulate any increase in normal proteolysis, although this is an additional possible mechanism. Thus stimulation of the neutral proteinase by magnesium ion has been described, 41 and increased concentration of magnesium in senile cataracts has been reported. 4 Decreased permeability of lenticular membranes might also cause accumulation of free amino acids by inhibition of the leak-component of the "pump-leak" system, 8 but it is difficult to see how such a mechanism could affect only the proteogenic amino acids. Hydrolysis of protein and diffusion of amino acids were proposed by Burdon- Cooper 1 as an explanation for the decrease in weight and protein content of human senile cataracts. This mechanism is widely accepted, although it has never been proved. Impermeability of lenticular membranes to protein is an implicit tenet of the argument, but only capsules have been examined, and riedenwald 44 actually found these to be permeable to plasma proteins, more so in fact than the capillaries, and permeability of bovine capsules to bovine lens proteins has also been reported. 45 Although many negative reports exist, preoperative sensitivity to lens protein has been observed in cataract patients. Gifford, 4G for example, detected sensitivity in 11 of 86 cataract patients examined, and Burky and Woods 47 found sensitivity in 11 of 64 patients and in none of 75 controls. Sensitivity, of course, is a manifestation of reaction to an antigen, and a negative result does not prove absence of exposure. The positive findings, however, suggest that lens proteins leak out of cataractous lenses. It is possible therefore that proteolysis, normal or pathological, is not the sole mechanism of protein loss from cataractous lenses, and that protein is actually lost by leakage through damaged cell membranes. Apparently the possible pres- Downloaded rom: on 1/8/18

19 58 Barber Inuestigative Ophthalmology October 1968 ence of human lens protein in the aqueous humor of cataract patients has never been tested. Gel filtration chromatography of soluble protein of normal and cataractous human lenses has indicated selective loss of low molecular weight proteins during progression of cataract, whereas the fractions of highest molecular weight are much more resistant to loss. 4S ' 4 This observation is suggestive of leakage from the lens of intact protein. The apparent leakage of protein from lenses of galactose-fed rats has been cited, 1 and is supported by the recent report that the marked swelling of normal rat lenses incubated in distilled water is accompanied by leakage of protein into the medium. 5 Similar escape of intracellular protein accompanied swelling of calf lenses incubated in hypotonic media.' 19 In summation, it is proposed that osmotic swelling due to accumulation of proteogenic amino acids may cause direct leakage of soluble protein from the lens. Cellular disruption due to swelling and dilution of cytoplasm by both protein loss and water uptake may very well be sufficient explanation of the loss of transparency. 1 It is probably unnecessary to postulate any formation of insoluble material, but it has been pointed out that albuminoid becomes insoluble in homogenates of normal lenses only after dilution. 51 It is further proposed that the observed accumulation of proteogenic amino acids may be simply the result of a shift in the normal balance of lenticular protein synthesis and hydrolysis. If the hypothesis offered here should prove to have merit, it would appear logical to direct efforts to forestall or to delay cataractogenesis toward the provention of amino acid accumulation. This may require attempts to stimulate protein synthesis, or to inhibit hydrolysis. Opposite efforts would seem the logical approach to attempted production of the proposed mechanism in experimental animals. The first cataracts, the aqueous humor samples, and the original incentive were supplied by Dr. Irving H. Leopold. The larger series of cataracts and the visual acuity data were obtained by Dr. Peter Laibson. Eyebank lenses were supplied by iss Ada Padgett, and the technical assistance of r. Rene Sanchez was indispensable. REERENCES 1. Trokel, S.: The physical basis for transparency of the crystalline lens, INVEST. OPHTH. 1: 49, Barber, G. W.: ree amino acids in ocular fluids and the crystalline lens. In preparation.. Barber, G. W.: ree amino acids in the crystalline lens. Effects of diabetic hyperglycemia and of galactose. In preparation. 4. Patterson, J. W., Patterson,. E., Kinsey, V. E., and Reddy, D. V. N.: Lens assays on diabetic and galactosemic rats receiving diets that modify cataract development, INVEST. OPHTH. 4: 98, Reddy, D. V. N., Kinsey, V. E., and Nathorst- Windahl, G.: Comparison of amino acid transport in ocular structures of rabbits made diabetic by alloxan and pancreatectomy, IN- VEST. OPHTH. 5: 166, Reddy, D. V. N., and Kinsey, V. E.: Transport of amino acids into intraocular fluids and lens in diabetic rabbits, INVEST. OPHTH. : 7, van Heyningen, R.: etabolism of xylose by the lens.. Rat lens in vivo and in vitro, Biochem. J. 7: 197, Reddy, D. V. N.: Amino acid transport in the lens in relation to sugar cataracts, INVEST. OPHTH. 4: 7, Kinoshita, J. H.: Cataracts in galactosemia, INVEST. OPHTH. 4: 786, Sippel, T. O.: Changes in the water, protein, and glutathione contents of the lens in the course of galactose cataract development in rats, INVEST. OPHTH. 5: 568, Spademan, D. H., Stein, W. H., and oore, S.: Automatic recording apparatus for use in the chromatography of amino acids, Analyt. Chem. : 119, Piez, K. A., and orris, L.: A modified procedure for the automatic analysis of amino acids, Analyt. Biochem. 1: 187, Hamilton, P. B.: Ion-exchange chromatography of amino acids: a single column, high resolving, fully automatic procedure, Analyt. Chem. 5: 55, Skeggs, L. T., Jr.: An automatic method for colorimetric analysis, Am. J. Clin. Path. 8: 11, Barber, G. W.: ultiple Autoanalyzer mani- Downloaded rom: on 1/8/18

20 Volume 7 "Number 5 ree amino acids in senile cataractous lenses 58 folds for sulfur amino acid chromatography, in Automation in analytical chemistry, Technicon Symposia 1966, White Plains, N. Y., 1967, ediad Inc., Vol. 1, p Cleland, W. W.: Dithiothreitol, a new protective reagent for SH groups, Biochemistry : 48, Calam, D. H., and Waley, S. G.: Acidic peptides of the lens. 8. S-(«,/?-dicarboxyethyl) glutathione, Biochem. J. 86: 6, Dickinson, J. C, Durham, D. G., and Hamilton, P. B.: Ion exchange chromatography of free amino acids in aqueous fluids and lens of the human eye, INVEST. OPHTH. 7: 551, Barber, G. W., and Boyd, T..: etabolism of amino acids by the crystalline lens. In preparation.. Smith, P.: On the growth of the crystalline lens, Trans. Ophth. Soc. United Kingdom : 79, Collins, W. J.: Lectures on the anatomy and pathology of the eye, Lancet : 19, Scammon, R. E., and Hesdorffer,. B.: Growth in mass and volume of the human lens in postnatal life, Arch. Ophth. 17: 14, Krause, A. C.: The biochemistry of the eye, Baltimore, 194, The Johns Hopkins Press, p Krause, A. C.: Chemistry of the lens. III. Autolysis of the lenticular proteins, Arch. Ophth. 1: 61, Swanson, A. A.: The identification of lysosomal enzymes in bovine lens epithelium, Exper. Eye. Res. 5: 145, Kern, H. L.: Accumulation of amino acids by calf lens, INVEST. OPHTH. 1: 68, Evans, E., and Kern, R.: The relation of the parathyroid gland to cataract, Am. J. Ophth. 14: 19, ilano, A.: Comportamento del contenuto in aminoacidi del crystallino di coniglio dopo paratiroidectomia, Ann. di ottal. e clin. ocul. 66: 9, De Vecchi, I.: Ricerche sul contenuto in aminoacidi del crystallino nell'intossicazione naftalinica, Rassegna ital. d'ottal. 5: 55, De Vecchi, I.: Ricerche sul contenuto in aminoacidi del crystallino umano catarattoso, Boll, d'ocul. 16: 4, Labbe, H., and Lavagna,.: Sur la constitution chimique du cristallin normal et pathologique, Compt. rend. Acad. Sc. 18: 1186, Weber, D.: t)ber die mittlere Lebensdauer von Linseneiweissen, utersucht am lebendigen Kaninchen, Ber. deutsch. Ophth. Ges. 64: 96, Waley, S. G., and van Heyningen, R.: Neutral proteinases in the lens, Biochem. J. 8: 74, Spector, A.: Lens aminopeptidase. I. Purification and properties, J. Biol. Chem. 8: 15, Waley, S. G.: etabolism of amino acids in the lens, Biochem. J. 91: 576, Kinsey, V. E., and Reddy, D. V. N.: Studies on the crystalline lens. X. Transport of amino acids, INVEST. OPHTH. : 9, Reddy, D. V. N., and Kinsey, V. E.: Studies on the crystalline lens, IX. Quantitative analysis of free amino acids and related compounds, INVEST. OPHTH. 1: 65, Kinsey, V. E.: Studies on the crystalline lens. XIV. Kinetics of alpha-aminoisobutyric acid transport, Documenta Ophth. :, Nimni,. E., and Bavetta, L. A.: Dietary composition and tissue protein synthesis. I. Effect of tryptophan deficiency, Proc. Soc. Exper. Biol. & ed. 18: 8, von Salknann, L., Reid,. E., Grimes, P. A., and Collins, E..: Tryptophan-deficiency cataract in guinea pigs, Arch. Ophth. 6: 66, Anderson, E. I.: Dual cation activation of bovine lens autolysis, INVEST. OPHTH. 4: 181, Burge, W. E.: Analyses of the ash of the normal and the cataractous lens, Arch. Ophth. 8: 45, Burdon-Cooper, J.: Pathology of cataract: the hydrolysis theory, Ophth. Rev. : 19, riedenwald, J. S.: Permeability of the lens capsule with special reference to the etiology of senile cataract, Arch. Ophth. 4: 18, rancois, J., and Rabaey,.: Permeability of the capsule for the lens proteins, Acta ophth. 6: 87, Gifford, S..: Allergic and toxic properties of lens protein, J. A.. A. 85: 51, Burky, E. L., and Woods, A. C: Lens extract, its preparation and clinical use, Arch. Ophth. 6: 548, rancois, J., Rabaey,., and Stockmans, L.: Gel filtration of the soluble proteins from normal and cataractous human lenses, Exper. Eye Res. 4: 1, Charlton, J.., and van Heyningen, R.: An investigation into the loss of proteins of low molecular size from the lens in senile cataract, Exper. Eye Res. 7: 47, Pirie, A.: Difference in swelling and opacity formation between young and old lenses, Nature 16: 5, Waley, S. G.: The problem of albuminoid, Exper. Eye Res. 4: 9, Downloaded rom: on 1/8/18