LENS METABOLISM WITH T H E REVERSIBLE CATION SHIFT*

IV. T H E ABILITY OF VARIOUS MI

DANIEL J. HEINRICHS^ M.D., Portland,

Previous studies have indicated that the energy for active transport of cations across the lenticular barriers generally derives from the metabolism of glucose and to an unde­termined extent requires oxygen.1'2 This is not strictly true of younger lenses which, under the conditions imposed by the experi­mental technique, may maintain cation trans­port for a period in a medium devoid of glucose.3 (This is not observed if in addi­tion oxygen is deprived). However, the re­sults generally support the view that a con­tinuous supply of glucose delivered via the aqueous is essential to the lens. Whether certain metabolites are capable of replacing glucose is not certain.

The present studies were undertaken to determine whether various metabolites inter­mediate in major metabolic pathways when delivered to the lens in place of glucose could support cation transport across the limiting barriers in the lens.

MATERIAL AND METHODS

The methods employed have been de­scribed in greater detail elsewhere.3 Fresh rabbit lenses were placed in three ml. of a solution containing the substance under study, refrigerated at 0°C. for 41 to 44 hours, analyzed immediately, or incubated at 37°C. for an additional six hours. The basic solution was a modified Tyrode's solution containing added bicarbonate, glucose, and potassium.3 Where substitutions for glucose

* From the John E. Weeks Memorial Laboratory, Department of Ophthalmology, University of Ore­gon Medical School, Portland, Oregon. Supported by Grant B-187 from the National Institute of Neurological Diseases and Blindness, National In­stitutes of Health, Bethesda 14, Maryland.

t Part of a thesis submitted by Dr. Heinrichs to the Graduate Council of the University of Oregon Medical School in partial fulfillment of the require­ments for the degree of Master of Science.

STABOLITES TO REPLACE GLUCOSE

AND J O H N E. H A R R I S , M.D. Oregon

were made 0.01 M. concentrations were utilized. Generally, the two lenses from the same animal were compared, one of the pair being analyzed after refrigeration, the other after refrigeration and subsequent incuba­tion at 37°C. Results are generally ex­pressed as percent recovery of the normal cation distributions.3

Where the penetration into the lens of isotopically marked metabolites was to be determined the isolated lenses were placed in three ml. of media containing known amounts of C14 labeled substrate, in tracer quantities, refrigerated at 0 ° C . for 24 hours, and subsequently incubated at 37°C. for an additional six hours. At the end of this period the lenses were thoroughly rinsed with a medium of similar composition but devoid of isotope, weighed, frozen rapidly, and divided equatorially. The capsules were removed from the separate sections and trans­ferred onto one inch planchets of aluminum construction for counting with an end-window counter. The entire nucleus plus cortex was macerated, spread evenly over a one inch planchet, and counted after dry­ing for one hour at 105°C.

Standard curves were obtained by spread­ing decapsulated lenses on planchets in the aforementioned manner, adding known amounts of C14 labeled metabolite, thoroughly mixing, and counting as previously de­scribed. Standard curves for capsules were not obtained, but the assumption was made that the counting efficiency was in the same range as that of the macerated lens. Count­ing efficiency was determined by comparison with end-window counting of infinite thick­ness barium carbonate.*

* We wish to express our appreciation to Dr. John T. Van Bruggen and Miss Jean Scott for the valuable assistance given us during the course of this experiment.

LENS METABOLISM 359

Cation Concentration « K* ■ No* Meq/1000 gm. water

123.7 24.3

99.4 49.7

75 75

Fig. 1 (Heinrichs and Harris). Schematic repre­sentation of the changes in cation concentration of rabbit lenses during refrigeration at 0°C. and sub­sequent incubation at 37° C.

RESULTS

The effect on cation recovery of substituting for glucose various metabolites which may

be metabolized via the citric-acid cycle Glucose was replaced by various metabo­

lites which may be metabolized via the citric-acid cycle. Pyruvate, acetate, lactate, alpha-keto-glutarate, citrate, and oxaloacetate were added as a possible energy source.

With none of the above compounds was cation recovery observed to approach that obtained when glucose was added (figs. 2 and 3). Citrate appeared to exert a deleterious effect, cation recovery being virtually abol­ished, and a marked hydration of the lens observed (fig. 4) . Citrate is known to bind calcium effectively and the observed effect is probably similar to that seen when calcium is deleted from the incubating medium.4

The effect on cation recovery of substituting for glucose metabolites which may be me­

tabolized via the hexose-monophosphate shunt

Two compounds, d-ribose and gluconate, were substituted for glucose in the modified Tyrode's solution and cation recovery measured in the usual manner. Lenses of such a size as to require added glucose were utilized throughout. Neither ribose nor glu-

.01M Acetate

Fig. 2 (Heinrichs and Harris). Effect of replace­ment of glucose by lactate, pyruvate, or acetate on cation recovery of rabbit lenses during incubation at 37° C. following a cold-induced cation shift (each bar represents the average of at least 10 experi­ments involving 10 pairs of lenses).

conate supported cation recovery so effec­tively as did glucose (fig. 5) .

Incorporation of labeled substrates into the lens during incubation at 37°C.

The aforementioned results indicated quite clearly that the major metabolites of glucose

200mgm % .01M .01M Glucose «Keto Glutarote Oxoloacetate

Fig. 3 (Heinrichs and Harris). Effect of replace­ment of glucose by alpha-keto-glutarate or oxalo­acetate on cation recovery of rabbit lenses during incubation at 37°C. Following a cold-induced cation shift (each bar represents the average of at least 10 experiments involving 10 pairs of lenses).

360 D A N I E L J. H E I N R I C H S A N D J O H N E. H A R R I S

100

90

80

5-70 s 8 60

.N* 50

40

30

20

10

No

200mgm% Glucose

.01M Citrate

Fig. 4 (Heinrichs and Harr i s ) . Effect of replace­ment of glucose by citrate on cation recovery of rabbit lenses during incubation at 37 °C. Following a cold-induced cation shift (each bar represents the average of at least 10 experiments involving 10 pairs of lenses).

Fig. S (Heinrichs and Harr i s ) . Effect of replace­ment of glucose by ribose or gluconate on cation recovery of rabbit lenses during incubation at 37°C. following a cold-induced cation shift (each bar represents the average of at least 10 experiments involving 10 pairs of lenses).

when delivered to the lens were incapable of supporting lens functions as have been measured. Since it is known that glucose is preferentially moved across the lens surface it became imperative to determine whether the various metabolites moved into the lens with the same facility as did glucose. To test this hypothesis the movement of isotopically marked glucose into the lens was compared with that of the smallest metabolite tested, the two carbon residue, acetate, also isotopi­cally marked.

Approximately 4.5 to 6.0 percent of the activity of the added acetate was recovered in the lens structures (table 1). The majority of the activity was found in the lens sub­

stance, but significant activity was recovered in the capsule. The anterior capsule in each instance was more active by a factor of two to four than the posterior capsule. Per unit weight the activity of the capsule far ex­ceeded that of the lens substance.

On the other hand 8.5 to 9.5 percent of the activity of the added labelled glucose was incorporated into the lens at the end of the six hours of incubation at 37°C. following refrigeration at 0°C. for 24 hours (table 2). Again the activity of the anterior capsule exceeded that of the posterior capsule and, per unit weight, this activity of the capsule was far greater than that of the lens sub­stance.

TABLE 1 INCORPORATION OF ACTIVITY INTO THE LENS DURING INCUBATION AT 37°C. FOLLOWING REFRIGERATION

IN A MEDIUM CONTAINING C 1 4 LABELLED ACETATE

Experiment tiC Labelled

Acetate Added

Lens Substance

juC Recovered Anterior Capsule

Posterior Capsule

Total Percent

Incorporation

1 2 3 4

0.2 1.0 1.5 2.0

0.0076 0.0302 0.0564 0.0833

0.0040 0.0090 0.0121 0.0258

0.0008 0.0040 0.0024 0.0068

6.2 4.3 4.7 5.8

LENS METABOLISM 361

TABLE 2 INCORPORATION OF ACTIVITY INTO THE LENS DURING INCUBATION AT 37CC. FOLLOWING REFRIGERATION

IN A MEDIUM CONTAINING GLUCOSE UNIFORMLY LABELLED WITH C14

Experiment nC Labelled

Glucose Added

Lens Substance

ixC Recovered Anterior Posterior Capsule Capsule

Total Percent

Incorporation

0.4 1.0

0.0295 0.0696

0.0051 0.0115

0.0034 0.0050

9.5 8.6

DISCUSSION

It is obvious that the ability of any nu­trient to serve as a source of energy will require tha t :

1. The metabolic pathways necessary for its utilization be present.

2. The substance be delivered to the site of utilization in sufficient quantity.

The pathways in the lens by which glucose is metabolized and in which oxygen parti­cipates are still not known with certainty. The glucose consumption of the lens is known to be small.5 I ts oxygen consumption is so low that measurement by the usual Warburg techniques is extremely difficult and subjected to high error. Indeed, some6

have suggested that oxygen consumption is nonenzymatic but this does not appear to be true.2 Present knowledge of these pathways in the lens is best summarized as follows:

First , the studies of Green and his asso­ciates have demonstrated that the classical anaerobic pathway, the Embden-Meyerhof cycle, appears to be followed in lens homo­genates.7"9

Second, there is evidence that the cyto-chrome system of enzymes is present in the lens although in lesser concentration than in other tissues.10

Third, the participation of the citric-acid cycle in the lens is not certain. Bovine lens homogenates have been found to oxidize certain intermediates of the citric-acid cycle.11 However, the intact lens does not appear to oxidize isotopically labelled pyru-vate to carbon dioxide to any appreciable extent.12

Fourth, the hexose-monophosphate shunt

may be followed in the lens. Thus, certain supposed intermediates of this pathway, that is, glucose-6-phosphate and 6-phosphoglu-conate, are utilized by lens homogenates.12

Most of these data have of necessity been obtained from studies of lens homogenates. To what extent it can be quantitatively ap­plied to the intact structure is not certain. Indeed measurable changes in physiologic function as measured by cation transport attend even slight mechanical disturbance of the lens.13 Changes in metabolic pathways must be considered when structural integrity is disrupted. For example, when the lens cap­sule is nicked, the rate of oxygen consump­tion immediately rises.5 Fo r this reason any postulated mechanism must ultimately be proved to occur in the intact structure.

-The observations herein reported indicate that metabolites delivered to the lens do not support lens function as herein measured possibly because they do not penetrate to the site of utilization in sufficient quantity. The finding that glucose enters the lens more readily than acetate (on the basis of molecular size one would anticipate the op­posite) supports the hypothesis that glucose enters the lens by metabolic mediation.14 The failure of the various metabolites to support cation transport as well as glucose must, in the light of these experiments, be considered to be due largely to insufficient penetration of these metabolites into the lens. No definite conclusion concerning the specific path­ways of glucose degradation can be drawn from these studies.

S U M M A R Y

1. Compounds which may be metabolized

362 DANIEL J. HEINRICHS AND JOHN E. HARRIS

via the citric-acid cycle did not support cation recovery following the cold-induced cation shift in rabbit lenses as well as does glucose.

2. Compounds which may be metabolized via the hexose-monophosphate shunt did not support cation recovery following the cold-induced cation shift in rabbit lenses as well as does glucose.

DR. DANIEL J. HEINRICHS : The phosphorylated compounds intermediate in glucose metabolism have not been employed in our present studies. Previ­ously we have substituted for glucose, glucose-6-phosphate, and have observed that this compound does not support cation transport in the intact rabbit lens in vitro to the same extent as does glu­cose. However, additional studies utilizing various phosphorylated intermediates should indeed yield valuable information germane to the present studies.

We have restricted the present studies to the use of an artificial medium, a modified Tyrode's solu­tion. Certainly aqueous humor as an incubating

3. Glucose was incorporated into the intact rabbit lens in vitro to a greater extent during incubation at 37°C. following refrigeration than was acetate.

4. The significance of these findings is dis­cussed.

3181 S.W. Sam Jackson Park Road (1).

medium would provide an environment more closely resembling in vivo conditions. Perhaps in the future such studies utilizing aqueous humor as an incubat­ing medium may be possible.

We are anticipating additional studies involving the penetration of various metabolites into the lens. Studies are underway of the effect of insulin on the penetration of glucose across the lenticular barriers in vitro both in normal and alloxan diabetic rabbits.

I would like to express my appreciation to Dr. Wachtl for his discussion of the paper and his help­ful suggestions.

REFERENCES

1. Harris, J. E., Hauschildt, J. D., and Nordquist, L. T.: Lens metabolism as studied with the reversible cation shift: I. The role of glucose. Am. J. Ophth., 38 :141-147, 1954.

2. : Lens metabolism as studied with the reversible cation shift: II. The effect of oxygen and glutamic acid. Am. J. Ophth., 38:148-151, 1954.

3. Heinrichs, D. J., and Harris, J. E.: Lens metabolism as studied with the reversible cation shift: III. The effect of age. Arch. Ophth. In press.

4. Harris, J. E., Gehrsitz, L. B., and Nordquist, L. T.: The in-vitro reversal of the lenticular cation shift induced by cold or calcium deficiency. Am. J. Ophth., 36:39-49, 1953.

5. Ely, L. O.: Metabolism of the crystalline lens. II. Respiration of the intact lens and its separated parts. Am. J. Ophth., 32 :215-219, 1949.

6. Christiansen, G. S., and Leinfelder, P. J.: A critical study of lens metabolism: I. Nonenzymatic "respiration." Am. J. Ophth., 35:21-31, 1952.

7. Green, H., Bocher, C. A., and Leopold, I. H.: Anaerobic carbohydrate metabolism of the crystalline lens: I. Glucose and glucose-6-phosphate. Am. J. Ophth., 39:106-113, 1955.

8. : Anaerobic carbohydrate metabolism of the crystalline lens: II. Fructose diphosphate. Am. J. Ophth., 39:113-117, 1955.

9. : Anaerobic carbohydrate metabolism of the crystalline lens: III. Triosephosphate, phospho-glycerate, and phosphoenolpyruvate. Am. J. Ophth., 40:237-248, 1956.

10. Ely, L. O.: The cytochrome C content of bovine crystalline lens. A.M.A. Arch. Ophth., 47:717-719, 1952.

11. — •: The oxidation of lactic acid, pyruvic acid, and various members of the citric-acid cycle by bovine lens homogenates. Am. J. Ophth., 34:127-130, 1951.

12. Kinoshita, J. H.: Carbohydrate metabolism of the lens. Arch. Ophth., 54:360-368, 1955. 13. Harris, J. E., and Nordquist, L. T.: Factors effecting the cation and water balance of the lens. Tr.

XVII Internat. Cong. Ophth., 1954, pp. 1002-1012. 14. Harris, J. E., Hauschildt, J. D., and Nordquist, L. T.: Transport of glucose across the lens sur­

faces. Am. J. Ophth., 39 .161-169, 1955.

DISCUSSION