Int J Biometeorol (1995) 38:57-59 © Springer-Verlag 1995

Yoshinori Ohtsuka. Noriyuki Yabunaka Ichiro Watanabe - Hiroshi Noro Hiroyuki Fujisawa • Yuko Agishi

Thermal stress and diabetic complications

Received: 25 February 1994; Revised: 19 September 1994; Accepted: 20 September 1994

Abstract Activities of erythrocyte aldose reductase were compared in 34 normal subjects, 45 diabetic pa- tients, and nine young men following immersion in water at 25, 39, and 42 ° C. Mean basal enzyme activity was 1.11 (SEM 0.12) U/g Hb and 2.07 (SEM 0.14) U/g Hb in normal controls and diabetic patients, respectively (P<0.0001). Activities of the enzyme showed a good correlation with hemaglobin A 1 (HbAI) concentrations (P<0.01) but not with fasting plasma glucose concentra- tions. After immersion at 42 ° C for 10 min, enzyme ac- tivity was increased by 37.6% (P<0.01); however, the ac- tivity decreased by 52.2% (P<0.005) after immersion for 10 min at 39 ° C and by 47.0% (P<0.05) at 25 ° C. These changes suggest that heat stress might aggravate diabetic complications, and body exposure to hot environmental conditions is not recommended for diabetic patients.

Key words Polyol pathway • Diabetes mellitus Water immersion • Heat stress

Introduction

The polyol pathway has been reported to be strongly re- lated to the complications of diabetes, such as neuropa- thy, retinopathy, cataract, and nephropathy (Malone et al. 1984; Hotta et al. 1985). It consists of two steps in which glucose is first reduced to sorbitol by the enzyme aldose reductase in an NADPH-dependent reaction; the sorbitol then is converted to fructose by sorbitol dehydrogenase in an NAD+-dependent reaction (see Fig. 1). The contribu- tion of the polyol pathway to glucose metabolism is very small and the activity is independent of insulin. As the ambient glucose concentration rises, the activity of the

Y. Ohtsuka • Y. Agishi Department of Rehabilitation and Physical Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan

Y. Ohtsuka ( ~ ) • N. Yabunaka. I. Watanabe • H. Noro H. Fujisawa Noboribetsu Branch Hospital, Hokkaido University School of Medicine, Noboribetsu 059-04, Japan

polyol pathway increases (Malone et al. 1980). One of the mechanisms responsible for the complications of dia- betes has been thought to be the enhanced activity of al- dose reductase (Hotta et al. 1991), leading to the cellular accumulation of sorbitol and fructose which do not readi- ly diffuse across the cell membrane, and resulting in os- motic imbalance, depletion of myoinositol, and cellular death (Finegold et al. 1983; Kador and Kinoshita 1985; Cheng and Gonzalez 1986). Such enzyme activity has been examined in various tissues and the activity in eryth- rocytes found to be quite well correlated with that from other tissues and with the extent of diabetic complications (Crabbe et al. 1980; Malone et al. 1980; Hotta et al. 1985).

Patients with peripheral circulatory disorder from dia- betic neuropathy and angiopathy usually feel relieved from symptoms such as pain and numbness after bath- ing. However, it is unclear whether or not the water tem- perature is important, moreover, no experiment has been done to investigate the influence of thermal stress on the polyol pathway. In the present study, to evaluate the ef- fect of thermal stress on diabetic complications, we im- mersed men in water at three different temperatures, 25, 39, and 42 ° C, and investigated their erythrocyte aldose reductase activity.

Materials and methods

Reagents

DL-glyceraldehyde was purchased t~om Wako Pure Chemical In- dustries. NADPH was from Sigma (St. Louis Mo., USA). All oth- er reagents used were of analytical grade.

Subjects and experimental protocol

Basal aldose reductase activity was determined in 45 (22 male and 23 female) type II diabetic patients (mean age 57.0 years, SEM

D-gluco se+NADPH+H + aldose ~ SorbitoI+NADP + reductase

+ sorbitol Sorbitol+NAD > D- fructose+NADH+H +

dehydrogenase

Fig. 1 The polyol pathway

58

1.9 years) and 34 (19 male and 15 female) normal subjects (mean age 34.4 years, SEM 1.8 years). The effect of water temperature on the enzyme activity was investigated at 25, 39, and 42°C in nine normal male students (mean age 20.1 years, SEM 0.2 years). After 5-10 min rest in a chair at room temperature (27 ° C), venous blood was drawn, and the subjects were then immersed in a sitting position with their legs outstretched in a water bath for 10 rain. A second blood sample was taken immediately after the end of the immersion. Water immersion at the different temperatures was carried out with at least a 1-week interval between immersions. The Ethics Committee of the Hokkaido University School of Med- icine approved this experiment and informed consent was obtained from all subjects.

Preparation of blood samples

Venous blood collected in 1 mg/ml ethylenediaminetetraacetic ac- id was passed through a column of a-cellulose and microcrystal- line cellulose (1:1, w/w) to remove platelets and leukocytes (Beut- ler et al. 1976). Erythrocytes were washed three times with cold isotonic saline, then frozen at -70 ° C and thawed to induce hemol- ysis.

Estimation of enzyme activity

The hemolysate prepared as described above was used for assays of aldose reductase activity, which was determined with glyceral- dehyde as a substrate. The assay system, was as described previ- ously (Das and Srivastava 1985) with slight modifications, and contained 50 mmol/1 potassium phosphate (pH 6.0), 5 mmol/1 2- mercaptoethanol, 1 retool/1 lithium sulfate, 0.1 mmol/1 NADPH, and diluted hemolysate with or without (as a blank) 10 mmol/1 DL- glyceraldehyde in a total volume of 0.5 ml. The reaction was start- ed by addition of the substrate and the decrease in absorbance at 340 nm recorded at 37°C for 20 min. Enzyme activity was de- fined as the number of micromoles of NADPH oxidized per minute at 37 ° C. The hemoglobin A[ (HbA1) concentration of each hemolysate sample was measured by the cyanmethemoglobin method.

Statistical analysis

The Welch t-test was used for the analysis of basal enzyme activi- ty and paired t-test for the study of thermal stress, together with linear regression analysis of enzyme activity and HbA 1 concentra- tion. A probability level of 0.05 or less was used to indicate statis- tical significance.

Results

Basal aldose reductase activity

Basal activities of aldose reductase are shown in Fig. 2. The mean enzyme activity in control subjects was 1.11 (SEM 0.12) U/g Hb, compared to 2.07 (SEM 0.14) U/g Hb in diabetic patients (P<0.0001). Figure 3 shows a sig- ni f icant correlat ion (r=0.556, P<0.01) be tween enzyme activities and the levels of HbA] in the diabetic patients. There was no statistically signif icant relat ionship be- tween the enzyme activities and the levels of fasting plasma glucose (data not shown).

"2" - r

=o

"o =0 =0 0

"10

v P<O.OOOl

o

0 0 0

; 1 ° 0

| Control Diabetic

Fig. 2 Basal activity of aldose reductase in 34 normal subjects (Control) and 45 type II diabetic patients (Diabetic). The squares and errors bars indicate mean and SEM (Fib Hemoglobin)

...~ 2

O0 r=0.556 o P=e 1 ~ 0 0 0 0 0 p<O.01

"o

O ~ " i 0 I I

0 5 10 15 20 HbAI(~)

Fig. 3 Relationship between aldose reductase activities and hemo- globin A 1 (HbA1) concentrations in the diabetic patients. The re- gression line was drawn by computer

2.0

'3" 1.5

1.0 " o

P

0.5

0.0

Q

b

Before After Fig. 4 Effect of immersion temperature on the activity of erythrocyte aldose reductase. Enzyme activities were measured before and after water immersion for 10 min at 42 ° C (©), 39 ° C (0) and 25 ° C (V). Data are expressed as mean (SEM). ap<0.01, bp<0.005, cp<0.05

Effect of thermal stress

Mean enzyme activity was increased from 1.09 (SEM 0.19) to 1.50 (SEM 0.25) U/g Hb (P<0.01) by immers ion

at 42 ° C. The mean activity decreased from 0.92 (SEM 0.08) to 0.44 (SEM 0.08) U/g Hb (P<0.005) and from 1.08 (SEM 0.14) to 0.54 (SEM 0.09) U/g Hb (P<0.05) at 39 ° C and 25 ° C respectively (Fig. 4).

59

Discussion

The polyol pathway plays an important role in the devel- opment of complications in diabetes. Elevated aldose re- ductase activity in erythrocytes from diabetic patients has been reported (Crabbe et al. 1980; Hamada et al. 1991), in accordance with our present results (Fig. 2). In the study of Crabbe et al. (1980) and in this work, no correlation between enzyme activity and age or sex was observed. Therefore, the observed difference in enzyme activities was not thought to be brought about by the dif- ference of ages between these two groups, tn the present study, the enzyme activities showed a good correla- tion with the concentrations of HbA~ but not with the concentrations of fasting plasma glucose, indicating that the activity of aldose reductase may be regulated by the relatively long-term concentrations of plasma glu- cose.

Activation of erythrocyte aldose reductase in man af- ter oral intake of glucose has been reported by Lyons et al. (1991). Glucose consumption led to a transient activa- tion of this enzyme, which paralleled the rise and subse- quent fall in blood glucose concentrations. In the present study (data not shown) and in a previous study (Agishi 1985), no significant changes in the levels of blood glu- cose were observed after immersion at 25, 39, and 42 ° C for 10 min; therefore, it is unlikely that blood glucose concentration altered the aldose reductase activity during thermal stress.

According to Hotta et al. (1991) adrenaline has been found to increase the sorbitol content of the rabbit retina, an effect that was markedly reduced by an inhibitor of aldose reductase (ONO-2235-{(E)-3-carboxymethyl-5- [(E)-2-methyl-3-phenylpropenylidene]rhodanine }). Ad- renaline was thought to augment the aldose reductase ac- tivity. We have observed elevation of the plasma adrena- line concentration only after immersion at 42 ° C (Agishi 1985). Thus, one of the reasons for the increased enzyme activity after immersion at 42 ° C is suggested to be the raised adrenaline concentration.

An elevated concentration of extracellular sodium chloride has been reported to increase both aldose reduc- tase activity and the sorbitol concentration in renal med- ullary cells in order to maintain osmotic pressure (Bag- nasco et al. 1987). Plasma renin and antidiuretic hor- mone (ADH) activities have been found to be increased after immersion at 42 ° C but suppressed at 25 ° C (Agishi 1985). Thus not only static water pressure but also water temperature is likely to be crucial for extracellular water distribution. A change in the extracellular sodium con- centration, which is responsible for osmotic pressure, is thought to be another important factor in determining the enzyme activity.

In a preliminary study (Ohtsuka et al. 1994) carried out under the same experimental conditions, we have found that mean rectal temperature increased from 36.94 (SEM 0.07) °C to 37.73 (SEM 0.07) °C (P<0.05) only after the immersion at 42 ° C. Therefore, enhancement of the enzyme activity by heat stress is thought to occur not

only on the outer skin surface but in the body generally. Since no significant hormonal changes have been ob- served at 39°C (Agishi 1985), further experiments are required to elucidate the mechanism of this thermal ef- fect.

Water immersion is a useful technique for applying thermal stress in humans, and the changes observed in the present study may also occur under other conditions such as in saunas or in stressful occupational environ- ments. Although we did not measure the intracellular sorbitol concentrations, it is hypothesized that an incre- ment of aldose reductase activity may lead to the devel- opment of complications in diabetes. In conclusion, it is better for diabetic patients to avoid hot environments.

Acknowledgements We thank Yoshiko Iijima for excellent tech- nical assistance.

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