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Mechanisms of Ageing and Development 73 (1994) 137-143

Effect of ageing on erythrocyte aldose reductase and sorbitol dehydrogenase activity Masato Funasako*, Kazumi Fujimoto, Masahiro Taniji, Tadatoshi Suruda, Seiji Nakai, Hisashi Kobayashi, Yoshiyuki Sakagami, Masahiro Ohata Department of Internal Medicine, Wakayama Medical College Kihoku Hospital, 219 Myoji, Katsuragi-cho, Ito-gun, Wakayama Prefecture, 649-71 Japan

(Received 26 August 1993; revision received 16 November 1993; accepted 9 December 1993)

Abstract

We measured erythrocyte aldose reductase and sorbitol dehydrogenase activity in erythrocytes in healthy individuals aged from 16 to 91 years to determine the mechanism of age-dependent sorbitol accumulation. Erythrocyte aldose reductase activity increased significantly with age but ageing had no effect on sorbitol dehydrogenase activity. Age and the aldose reductase/sorbitol dehydrogenase ratio were positively correlated. These findings suggest that an increase in the ratio of aldose reductase to sorbitol dehydrogenase may contribute to the tissue accumulation of sorbitol in the elderly and may be a mechanism of a disease that is common in elderly individuals. Key words." Erythrocyte; Sorbitol; Aldose reductase; Sorbitol dehydrogenase; Polyol pathway; Ageing

1. Introduction Studies have suggested that tissue accumulation of sorbitol leads to the complications associated with diabetes mellitus (DM) [1-8]. Accumulation of sorbitol in the lens has been found to be associated with the development of cataracts [1-2] and sorbitol accumulation in nerve tissue is associated with neuropathy [3-4]. Cataracts, * Corresponding author. 0047-6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0047-6374(93)01423-6

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atherosclerosis and polyneuropathy are more common in elderly than younger individuals. We previously reported that sorbitol accumulation in red blood cells (RBCs) increased with ageing [9]. The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). AR catalyzes the reduction of glucose to sorbitol. Sorbitol is converted to fructose by SDH. In the present study, we attempted to elucidate the mechanisms of the age-dependent sorbitol accumulation by measuring the activity of AR and SDH, in RBCs in healthy individuals. 2. Materials and methods

2.1. Subjects We studied 82 subjects consisting of 28 males and 54 females between the ages of 16 and 91 years (mean 4- S.D.: 51.2 4- 22.0 years). The age distribution is presented in Table 1, showing that male and female individuals distribute uniformly and in a wide range. Subjects were either healthy residents of a nursing home or members of our staff.

2.2. Blood sampling and handling Venous blood was drawn in a heparinized syringe in the morning between meals. RBCs were isolated from blood samples by centrifugation, washed, and stored frozen until needed for assay.

2.3. Assay of AR AR activity was determined according to the method of Cabbie et al. [10]. Briefly, the hemolysate was diluted with 0.01 M phosphate buffer (pH 6.0) (buffer A), vigorously shaken and centrifuged at 1500 g. The supernatant was mixed with DE-52 solution (1 g/4 ml buffer A) and then centrifuged at 700 g. The precipitate was removed and extracted three times with buffer A. The AR-containing supernatant was incubated in a standard assay mixture consisting of 300 mM glyceraldehyde used as substrate, 0.3 M phosphate buffer, 0.3 M ammonium sulfate and 3 mM

Table I Age distribution Age (years)

s19 20-29 30-39 40-49 5~0-59 60,-69 70-79 80>

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Female

0 4 5 2 4 4 5 4

3 9 5 14 6 5 4 8

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~/-nicotinamide adenine dinucleotide phosphate-reduced form (NADPH). A decrease in fluorescence at ex. 350 nm, em. 440 nm was recorded at 37°C for 12 min using a fluorescence spectrophotometer (F-4000, Hitachi Co., Tokyo, Japan). A unit of enzyme activity was defined as the amount required to oxidize 1 /zmol NADPH/min at 37°C. The specific activity was expressed as unit of enzyme activity per gram of hemoglobin concentration (glib). 2.4. Assay o f S D H

SDH activity was determined according to the method of Barreto and Nonoyama [11]. In brief, the hemolyzate was mixed with DE-52 solution (50 g/150 ml) and centrifuged at 3000 rev./min for 10 min. The supernatant was incubated for 10 min with 60 mM NAD dissolved in 0.3 M Tris-HCl buffer (pH 9.0) containing 400 mg sorbitol/ml. A unit enzyme activity was defined as the amount required to deoxidize 1 t~mol of NADH/min at 37°C. The specific activity was expressed as unit of enzyme activity per g Hb. 2.5. Other measurements

Blood glucose (BG) was determined by the glucose oxidase method and sorbitol content of RBCs was determined according to the method of Malone et al. [12]. Stastical analysis was performed with Student's t-test. A level of P < 0.05 was accepted as statistically significant.

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M. Funasako et al./Mech. Ageing Dev. 73 (1994) 137-143

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3. Results Blood glucose ranged from 58 to 157 mg/dl (mean + S.D.: 92.3 4- 19.3). The broad range in values was attributed to a variation in blood sampling time. The mean sorbitol content in RBCs was 25.8 4- 7.2 nmol/g Hb, which was a little higher than that reported in a previous study [12], which can be explained by the fact that RBC Sorb values were known to be influenced by ageing [9] and BG levels [13]. AR activity in RBCs ranged from 11.1 to 94.3 mU/g Hb (59.2 4- 15.9 mU/g Hb). There was no significant difference in A R activity between males and females (57.4 ± 15.5 vs. 60.4 ± 15.5 mU/g Hb) (Fig. 1). Erythrocyte AR activity was significantly and positively correlated with age (r = 0.240, P < 0.05) (Fig. 2); The correlation was stronger in males (r = 0.396, P < 0.05) (Fig. 3) than in females. SDH activity in RBCs ranged from 165 to 400 mU/g Hb (276.1 4- 47.9 mU/g Hb). SDH activity was significantly higher in females than in males (283 4- 51.0 vs. 256 4- 31.3 mU/g Hb, P < 0.05) (Fig. 1). However, the enzyme activity showed no correlation with age in males and females (n = 46, r = -0.078, P > 0.5). The A R / S D H ratio was significantly and positively correlated with age (r = 0.415, e < 0.01) (Fig. 4). mU/gHb 100

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M. Funasako et al. / Mech. Ageing Dee. 73 (1994) 137-143

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4. Discussion Srivastava et al. [14] reported that 6-h incubation of RBCs from DM patients showing hyperglycemia exceeding 270 mg/dl activated AR activity in RBC. In a preliminary experiment, however, we found that postprandial hyperglycemia below 200 mg/dl 3 h after eating did not affect RBC AR or SDH activity in rats (data not shown). Consequently in the present study, we measured AR and SDH activity in RBCs obtained 2 - 3 h after breakfast. Sorbitol accumulation is believed to be partly responsible for diabetic complications such as cataracts, nephropathy, and polyneuropathy [1-8]. We previously found that sorbitoi content increased in RBCs with age [9]. The present results showed that activity of RBC AR was significantly and positively correlated with age, whereas activity of RBC SDH showed no change with age. More interestingly, the ratio of AR to SDH showed an age-dependent increase. These data suggest that an increase in the enzyme activity ratio is at least partly responsible for sorbitol accumulation in RBC in elderly subjects and that changes in the activity of these two

M. Funasako et al./ Mech. Ageing Dev. 73 (1994) 137-143

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enzymes may contribute to development of the disorders mentioned above, which are common in both diabetic patients and elderly individuals. Crabbe et al. [10] found no significant correlation between RBC AR and age. The discrepancy between their results and ours may be related to differences in characteristics of the study populations. Our subjects were healthy, non-diabetic individuals; Crabbe et al. studied a group that included diabetic patients. In addition, our subjects ranged in age from 18 to 91 years. The age range in their subjects was limited, making it difficult to reach a conclusion about age-related change in RBC AR. The mechanism of age-related increase in RBC AR is unclear. One possibility is that young RBCs may be increased in elderly people: young RBCs have a higher AR activity. Another possibility is related to impaired glucose tolerance in elderly individuals, who have higher blood glucose levels than younger individuals. Glucose has been found to activate R ~ s , susg~sting that hyperglycemia over 6 h, even if minimal, may activate RBC AR [14]. Reports [15,16] indicate that AR activity is increased in human tissues from diabetic patients compared with those from normal subjects. Further study is needed to explain the gender differences in the correlation between RBC AR and age and in RBC SDH activity. As we previously found that

M. Funasako et al. / Mech. Ageing Dev. 73 (1994) 137-143

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R B C s o r b i t o l w a s s i g n i f i c a n t l y c o r r e l a t e d w i t h age in males, b u t n o t in f e m a l e s [9], it is p o s s i b l e t h a t g o n a d a l h o r m o n e s i n f l u e n c e e n z y m e s o f R B C p o l y o l p a t h w a y .

5. Acknowledgment W e t h a n k Dr. T e r a j i m a , B i o - S i e n c e D e p a r t m e n t , R e s e a r c h Institute, O n o P h a r m a c e u t i c a l , O s a k a , J a p a n , for a d v i c e o n t e c h n i q u e s used to a n a l y z e p o l y o l enzymes.

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