GLUCOSE CONCENTRATIONS AND GLYCOGEN LEVELS IN T H E AQUEOUS H U M O R O F F R E S H AND R E F R I G E R A T E D BOVINE EYES J. S T E V E N S A N D R E W S , P H . D .
Nashville, Tennessee
Interest in retaining the epithelial layer of donor corneal buttons is increasing. 1 Formerly, the mild but proven antigenicity of the epithelium 2 and its apparent superfluity in the presence of normal host epithelium led to the practice of discard ing the epithelium at the time of trans plantation. Interest in improving the transplantation result in difficult cases, such as alkali burns, 3 has led to a renewal of interest in retaining the epithelial layer on the donor tissue. Investigations with refrigerated rabbit eyes have shown that the metabolism of the epithelial layer is more sensitive to the time between donor death and enucleation for immediate refrigeration than is the endothelial layer. 1 Epithelial edema is a common sequela of human eye refrig eration 1 ' 4,5 and apparently coincides with the time of serious epithelial glycogen depletion in refrigerated human and rab bit eyes. 1 Concomitant with the develop ment of epithelial edema, mild trauma will denude the cornea 4 of at least its superficial layer. Since both viability and adherence of the epithelium to the cornea are important to successful grafting of a donor button with epithelium, we investigated the met abolic recovery of the epithelium of fresh and refrigerated bovine eyes.
slaughterhouse and divided randomly into two groups. One group of 12 was stored at 5°C and the other group was incubated in a moist chamber at 35°C after injecting 10 μπιοΐ of glucose uni formly labeled with radioactive carbon (14C) in 10 μΐ of saline into the anterior chamber. Two eyes were removed at 30minute intervals after incubation and the epithelia were removed by immersing the cornea in water at 90°C for 20 seconds. The tissue was placed in 1 ml of 30% potassium hydroxide for two hours, and the glycogen released from the digested tissue was precipitated overnight at 5CC with 2 ml of 95% ethanol. After washing the precipitate four times with ethanol and once with chloroform:methanol (2:1), the dried glycogen was hydrolyzed to glucose with 1 ml of 2N hydrochloric acid at 105°C for two hours. The glycogen and glucose content was assayed by an enzy matic method and liquid scintillation methods were used to determine glycogen and glucose radioactivity. Just before im mersing the cornea in the 90°C bath, a 150-μ1 sample of aqueous humor was withdrawn from the anterior chamber, frozen, and subsequently analyzed for glucose. Twenty-four hours after begin ning refrigeration, the eyes were removed from the refrigerator, held at ambient temperature for 30 minutes, and then sub jected to the incubation and analytical procedures. Zero time analyses were per formed on a separate batch of ten eyes, five of which were refrigerated for 24 hours and then allowed to warm for 30 minutes.
METHOD
Bovine eyes were purchased at a local From the Department of Ophthalmology, Vanderbilt School of Medicine, Nashville, Tennessee. This study was supported in part by research grant EY00048, National Eye Institute. Reprint requests to J. Stevens Andrews, Ph.D., Department of Ophthalmology, Vanderbilt Univer sity School of Medicine, Nashville, TN 37232.
RESULTS
The results of analyses of glucose in the 97
98
AMERICAN JOURNAL OF OPHTHALMOLOGY
REFRIGERATED
GLUCOSE
'FRESH
pmol/ml
4.0
GLUCOSE mg/IOOml
72
54
2.0
JULY, 1976
36
18
Fig. 1 (Andrews). Glucose con centration in the aqueous humor of fresh bovine eyes and eyes stored at 5°C for 24 hours. The incubation began immediately after injecting 10 μπιοΐ of glucose 14 C into the anterior chamber of each eye. Glu cose concentrations were deter mined at the times indicated. Zero time concentrations were measured before the addition of exogenous glucose. Each point represents the average of five different experi ments.
9
HOURS
aqueous humor were compiled (Fig. 1). While the glucose concentration in fresh eyes was low (approximately 1.6 μιηοΐ/ml or 30 mg/100 ml), the level in eyes that were refrigerated was lower (approxi mately 0.18 μιηοΐ/ml or 1.25 mg/100 ml). Despite this deficit, the disappearance of exogenous glucose from the aqueous humor was at approximately the same rate for both fresh and refrigerated eyes after the initial 30-minute incubation period. The initially slower disappearance rate of exogenous glucose from the aqueous humor of the refrigerated eye suggested that holding the refrigerated eyes at ambi ent temperature for 30 minutes before beginning the incubation may be insuffi cient for complete temperature reversal of those tissues lining the anterior chamber. This initial difference in glucose disap pearance provided a higher concentration of glucose in the aqueous humor of the refrigerated eye during the experimental period. Furthermore, during the last 30 minutes of the experiment, glucose disap pearance in the fresh eye slowed, whereas the rate of disappearance in the refrigerat ed eye continued at the same rate. This difference in glucose concentrations was
probably responsible for the differences in glycogen concentration and glucose incorporation into glycogen (Fig. 2). Another common feature between the two groups of eyes was the initial drop in glycogen concentration. 6 Statistical sig nificance between the epithelial glycogen levels in fresh and refrigerated eyes was not observed. There was, however, a trend toward net glycogen synthesis by the refrigerated epithelium between two and three hours of incubation, supported by continued high levels of glucose incor poration into glycogen. DISCUSSION
In the past, the absolute requirement of a viable endothelium for penetrating keratoplasty stimulated research into meth ods of endothelial preservation. The con cept that metabolic end products in the aqueous humor, such as lactate, had an irreversibly deleterious effect on endothe lial cells does not seem to apply to the conditions of short-term refrigeration. Extended periods of refrigeration, how ever, require more complex conditions of storage 7,8 to obtain good surgical results. On the other hand, the ability of the
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Nashville, Tennessee
Interest in retaining the epithelial layer of donor corneal buttons is increasing. 1 Formerly, the mild but proven antigenicity of the epithelium 2 and its apparent superfluity in the presence of normal host epithelium led to the practice of discard ing the epithelium at the time of trans plantation. Interest in improving the transplantation result in difficult cases, such as alkali burns, 3 has led to a renewal of interest in retaining the epithelial layer on the donor tissue. Investigations with refrigerated rabbit eyes have shown that the metabolism of the epithelial layer is more sensitive to the time between donor death and enucleation for immediate refrigeration than is the endothelial layer. 1 Epithelial edema is a common sequela of human eye refrig eration 1 ' 4,5 and apparently coincides with the time of serious epithelial glycogen depletion in refrigerated human and rab bit eyes. 1 Concomitant with the develop ment of epithelial edema, mild trauma will denude the cornea 4 of at least its superficial layer. Since both viability and adherence of the epithelium to the cornea are important to successful grafting of a donor button with epithelium, we investigated the met abolic recovery of the epithelium of fresh and refrigerated bovine eyes.
slaughterhouse and divided randomly into two groups. One group of 12 was stored at 5°C and the other group was incubated in a moist chamber at 35°C after injecting 10 μπιοΐ of glucose uni formly labeled with radioactive carbon (14C) in 10 μΐ of saline into the anterior chamber. Two eyes were removed at 30minute intervals after incubation and the epithelia were removed by immersing the cornea in water at 90°C for 20 seconds. The tissue was placed in 1 ml of 30% potassium hydroxide for two hours, and the glycogen released from the digested tissue was precipitated overnight at 5CC with 2 ml of 95% ethanol. After washing the precipitate four times with ethanol and once with chloroform:methanol (2:1), the dried glycogen was hydrolyzed to glucose with 1 ml of 2N hydrochloric acid at 105°C for two hours. The glycogen and glucose content was assayed by an enzy matic method and liquid scintillation methods were used to determine glycogen and glucose radioactivity. Just before im mersing the cornea in the 90°C bath, a 150-μ1 sample of aqueous humor was withdrawn from the anterior chamber, frozen, and subsequently analyzed for glucose. Twenty-four hours after begin ning refrigeration, the eyes were removed from the refrigerator, held at ambient temperature for 30 minutes, and then sub jected to the incubation and analytical procedures. Zero time analyses were per formed on a separate batch of ten eyes, five of which were refrigerated for 24 hours and then allowed to warm for 30 minutes.
METHOD
Bovine eyes were purchased at a local From the Department of Ophthalmology, Vanderbilt School of Medicine, Nashville, Tennessee. This study was supported in part by research grant EY00048, National Eye Institute. Reprint requests to J. Stevens Andrews, Ph.D., Department of Ophthalmology, Vanderbilt Univer sity School of Medicine, Nashville, TN 37232.
RESULTS
The results of analyses of glucose in the 97
98
AMERICAN JOURNAL OF OPHTHALMOLOGY
REFRIGERATED
GLUCOSE
'FRESH
pmol/ml
4.0
GLUCOSE mg/IOOml
72
54
2.0
JULY, 1976
36
18
Fig. 1 (Andrews). Glucose con centration in the aqueous humor of fresh bovine eyes and eyes stored at 5°C for 24 hours. The incubation began immediately after injecting 10 μπιοΐ of glucose 14 C into the anterior chamber of each eye. Glu cose concentrations were deter mined at the times indicated. Zero time concentrations were measured before the addition of exogenous glucose. Each point represents the average of five different experi ments.
9
HOURS
aqueous humor were compiled (Fig. 1). While the glucose concentration in fresh eyes was low (approximately 1.6 μιηοΐ/ml or 30 mg/100 ml), the level in eyes that were refrigerated was lower (approxi mately 0.18 μιηοΐ/ml or 1.25 mg/100 ml). Despite this deficit, the disappearance of exogenous glucose from the aqueous humor was at approximately the same rate for both fresh and refrigerated eyes after the initial 30-minute incubation period. The initially slower disappearance rate of exogenous glucose from the aqueous humor of the refrigerated eye suggested that holding the refrigerated eyes at ambi ent temperature for 30 minutes before beginning the incubation may be insuffi cient for complete temperature reversal of those tissues lining the anterior chamber. This initial difference in glucose disap pearance provided a higher concentration of glucose in the aqueous humor of the refrigerated eye during the experimental period. Furthermore, during the last 30 minutes of the experiment, glucose disap pearance in the fresh eye slowed, whereas the rate of disappearance in the refrigerat ed eye continued at the same rate. This difference in glucose concentrations was
probably responsible for the differences in glycogen concentration and glucose incorporation into glycogen (Fig. 2). Another common feature between the two groups of eyes was the initial drop in glycogen concentration. 6 Statistical sig nificance between the epithelial glycogen levels in fresh and refrigerated eyes was not observed. There was, however, a trend toward net glycogen synthesis by the refrigerated epithelium between two and three hours of incubation, supported by continued high levels of glucose incor poration into glycogen. DISCUSSION
In the past, the absolute requirement of a viable endothelium for penetrating keratoplasty stimulated research into meth ods of endothelial preservation. The con cept that metabolic end products in the aqueous humor, such as lactate, had an irreversibly deleterious effect on endothe lial cells does not seem to apply to the conditions of short-term refrigeration. Extended periods of refrigeration, how ever, require more complex conditions of storage 7,8 to obtain good surgical results. On the other hand, the ability of the
VOL. 82, NO. 1 GLUCOSE AND GLYCOGEN IN AQUEOUS HUMOR
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FRESH REFRIGERATED 6LYC0GEN LEVELS GLUCOSE INCORPORATION
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