Cerebrospinal fluid polyols in patients with diabetes mellitus

Clinica Chimica Acta, 44 (1973) 437-442 Q Elsevier Scientific Publishing Company,

CEREBROSPINAL

FLUID

Amsterdam

POLYOLS

IN

- Printed in The Netherlands

PATIENTS

WITH

437

DIABETES

MELLITUS

E. PITKdNEN

AND

CARITA

Fourth Department of Medicine, (Received

December

SERVO University Central Hospital. Helsinki (Finland)

14, 1972)

SUMMARY

A gas-liquid chromatographic method was used to study polyols in the cerebra.spinal fluid of zg diabetic patients and 12 non-diabetic subjects. The presence of several polyols was verified. The level of sorbitol was higher in diabetic patients than in the non-diabetic subjects. The degree of correlation between the level of sorbitol in cerebrospinal fluid and blood glucose was highly significant. On the other hand, the level of sorbitol was not elevated in the urine of the diabetic patients. The data suggest that the level of sorbitol in cerebrospinal fluid reflects a metabolic ment of the nervous tissue that occurs in patients with diabetes mellitus.

derange-

INTRODUCTION

Studies on diabetes mellitus, induced experimentally in alloxan-treated test animals, have provided data suggesting that an increased concentration of polyols is implied in the cataract formation of the lens tissue and that an accumulation of polyols may also be involved in the derangement of nerve function in these animals1-4. It is well grounded to postulate that a derangement in the polyol metabolism may also be involved in human diabetes mellitus. Changes in the urinary excretion of polyols in human diabetes mellitus have been described5. The present study was made on the assumption that an alteration in the metabolism of the nervous tissue, known to occur commonly in diabetic subjects, may be associated with a change in the content of cerebrospinal fluid polyols. MATERIALS

AND METHODS

Patients Twenty-nine diabetic patients were studied while they were being treated in medical wards. They were treated with insulin and with hypoglycemic agents. Seven patients without clinical diabetes but with a diabetic response to the glucose tolerance test were also included. It was checked that there was no underlying metabolic or hormonal state known to induce a positive result in the test. All the patients were

438

PITK.&NEN, SERVO

on a glucose- and saccharose-free diet. Obese patients were on caloric restriction. Twelve patients with a normal response to the oral glucose tolerance test were also studied. The patients showed no sign of an acute metabolic disease, and most of them were on a clinical routine check-up. No disease was detected. The patients were on a free diet and no attention was paid to the drugs administered. CLINICALMETHODS The glucose tolerance test was performed by oral administration of I g/kg body weight of partly hydrolyzed starch (Glucodyn, Medica Co., Helsinki). A fasting blood glucose level of more than 5.3 mmole/l, a peak value of more than 8.3 mmole/l or a z-h value of more than 6.6 mmole/l were considered abnormal. Cerebrospinal fluid (CSF) was taken by lumbar puncture. A local anesthetic was used. The CSF samples were immediately chilled to +4’, centrifuged to remove the cells, and stored at -25”. Finger-tip blood was used for blood glucose determinations. Two-hour urine samples after overnight fasting were collected. Laboratory methods

Glucose in the blood, CSF and urine were determined in an Autoanalyzer, using glucose oxidase reagent (Kabi AB, Stockholm, Sweden). In order to remove glucose, 0.3 mg of ATP and 0.05 mg of hexokinase (EC 2.7.1.1, Boehringer, Mannheim, Germany) were added to 0.25 ml of cerebrospinal fluid. The mixture was incubated at 37” until the Clinistix test (Ames Co., Bucks, England) turned negative. The samples were then deproteinized with perchloric acid, desalted with ion-exchange resins5 and dried under a stream of nitrogen. Details of the acetylation of the monosaccharides in the samples and of the gas-liquid chromatography (GLC) have been described elsewhere5. Desalted samples of CSF were also subjected to one-dimensional paper chromatography after the removal of glucoses. The areas of polyol were cut out and eluted in water. The sorbitol content of the eluates was measured with sorbitol dehydrogenase (EC 1.1.1.14, Boehringer)‘. The analysis of urinary polyols by GLC has been described in detail elsewhere5. Tests of s@iJicance

A t-test modified for small samples with different standard deviations was used to prove significant differences between two sample means8,9. Correlation between two samples was tested by calculating the linear regression coefficient Y. The signiflcance of Y was tested with a t-test. RESULTS The patients were divided into four groups according to type of illness with the following classification : Group I : Patients with overt clinical, insulin-dependent diabetes. Group II: Patients with ketosis-resistant (adult onset) diabetes treated with oral hypoglycemic drugs.

CSF

POLYOLS

IN DIABETES

439

MELLITUS

Group III: Patients with diabetic response in the glucose tolerance test (chemical diabetes) but without signs of clinical diabetes. Group IV : Non-diabetic patients with normal response in the glucose tolerance test. TABLE

I

MEAN VALUES

OF AGE,

BLOOD

GLUCOSE,

Group ~. Age

in years Blood glucose mmole/l CSF glucose mmole/l CSF sorbitol pmole/l

CSF

GLUCOSE,

Grou$

I

59 IO.5 f 3.2 6.5 + 2.0 45.3 f 34.0

AND

CSF

II

SORBITOL

Group

66 8.4 zt 3.9 4.8 * 2.1 44.2 & 16.1

IN

III

63 4.7 * 0.7 3.4 + 0.6 2I.2 + 5.1

THE

FOUR

GROUPS

Control group IV

66 4.3 * 0.7 3.3 * 0.5 17.2 k 4.6

The principal clinical parameters in each group are shown in Table I. The mean age in all groups was high and signs of atherosclerosis were observed in several patients. Severe diabetic complications were common in group I, especially in those patients who had suffered from manifest diabetes for more than IO years. Thirteen of the 15 patients in this group showed signs of clinical polyneuropathy, 5 of them had cataracts and 4 patients had a history of gangrene of peripheral extremities. In group II one patient had a history of gangrene and another patient had signs of polyneuropathy. In the 5 other patients no clinical signs of diabetic complications were detected. In group III two patients complained of neuralgia. Both of them were chronic alcoholics. With that exception, no sign of diabetic complications was detected in any of the patients. The results of the glucose and polyol measurement in CSF are summarized in Fig. I and Tables I and II. Peaks with retention times equal to erythritol, arabitol, mannitol, sorbitol and myo-inositol were detected in CSF. No trace of xylitol was

DIABETES

MELLITUS

A HEALTHY SUBJECT S

A R

MS

:i; TIME IN MINUTES

TIME IN MNUTES

Fig. 1. Gas-liquid chromatograms of acetylated derivatives of polyols in cerebrospinal fluid. A = arabitol, R = ribitol, M = mannitol, S = sorbitol, I = myo-inositol.

found. Similarly, spots suggesting the presence of tetritol, pentitol, hexitol and myoinositol were noted in the paper chromatography. Due to the small volume of the samples, only sorbitol was identified and quantitated with sorbitol dehydrogenase. There was a high degree of correlation between the sorbitol values measured enzymatitally and with GLC (r = 0.75, 9 = < 0.02). The mean level of sorbitol was significantly higher in groups I and II than in group IV ($ = < 0.01).

PITKiiNEN,

440 TABLE

SERVO

II

cONcENTRATION OF POLYOLS IN CEREBROSPINAI.FLUID Mean and standard deviation; ____.

,umole,/l. h’umber

Arabitol

Mannitol

Sorbitol

Myo-inositol

15 7 7 12

24.4 f 11.3 25.5 & 11.2 19.7f 7.3 19.0% 6.3

9.3 9.8 7.0 4.8

45.3 i 34.1 44.2 + 16.1 21.6* 5.1 17.2 & 4.6

102.0 & 171.6f 113.8 * 174.1 &

ofcases

Insulin-dependent diabetes (I) Adult onset diabetes (II) Chemical diabetes (III) Non-diabetic patients (IV)

i * * *

2.7 3.4 2.5 2.0

66.7 51.1 30.9 31.0

0

Fig. 2. The level of sorbitol in cerebrospinal fluid in relation to blood glucose (Y = 0.62, t = 4.90, p =
Only two patients in group I had low levels of CSF sorbitol. Both of them had severe cerebral circulatory disturbances and the blood glucose level was near normoglycemic at the time of the study. The level of sorbitol in CSF was in each case matched with the level of blood glucose and CSF glucose. The results are presented in Fig. 2. A high degree of correlation was seen between CSF sorbitol and blood glucose (Y = 0.62, t = 4.9, $J =: < 0.001). The degree of correlation was slightly less between CSF glucose and sorbitol (Y = 0.60, t = 4.7, $J = < 0.001). The mean values of arabitol, mannitol and myo-inositol did not seem to differ between the various groups.

Urine The polyol concentrations in urine are shown in Table III. The myo-inositol concentration was elevated in urine of diabetic patients. Patient-to-patient variation of mannitol concentration in urine was great, but the mean value was higher in diabetics than in non-diabetics. No difference in sorbitol concentrations was detected. TABLE

III

URINARY EXCRETIONOF PoLYOLS Mean and standard deviation;

Diabetic patients Non-diabetic patients

pmole/l.

Number Erythritol of cases

Rib&l

Arabitol

xy1ito1

Mannitol

So&o1

Myoinositol

10

5’2..1+35.6

111.7k50.1

26.3+24.1

138.71_81.5

33.2iIg.8

247.0+75.7

g

I8I.4&g2.5

237.2 & 95.9 61.5 + 25.0 174.2 & 54.8 59.0 & 44.6

65.1 i 22.9 29.4 i_ 12.9

78.3 f 40.0

CSF

POLYOLS IN DIABETES

MELLITUS

441

DISCUSSION

Both paper chromatography and GLC provided proof of the presence of several polyols in CSF. The data obtained suggested the presence of arabitol, mannitol, sorbitol and myo-inositol among other unidentified peaks and failed to detect xylitol. The outstanding feature emerging in the comparison of the diabetic and non-diabetic groups was the increased level of sorbitol in CSF in diabetic subjects, confirmed both in GLC and enzymatically. Evaluation of the sorbitol level with the clinical data showed that the level of CSF sorbitol was elevated in all except two cases of diabetic patients with neurological and microangiopathic involvement (group I) and slightly less elevated in patients with diabetes of short duration and with no obvious sign of diabetic complications. The level was increased in all patients who were hyperglycemic at the time the sample was taken. Only one measurement of CSF sorbitol was performed in each case, which does not permit a conclusion on how constant the elevation was in each case. The findings, especially in regard to group II, nevertheless suggest that the elevation of sorbitol precedes the appearance of the diabetic complications and suggest that the two phenomena may be related. The data do not limit the correlation only to neurological complications since very high levels of CSF sorbitol were also noted in patients with extensive microangiopathic but only minor neurologic involvement. On the other hand, the level of sorbitol did not seem to correlate with signs of atherosclerosis, which were common among patients in each group. In two cases with a high sorbitol level, the diabetic state was most probably secondary to relapsing pancreatitis and to a hyperplasia of the adrenal cortici. On the other hand, the sorbitol level was normal in patients with pathological glucose tolerance but with no sign of clinical diabetes mellitus. Some of these patients may have carried a genuine diabetic state in latent form. This provides no evidence for the possibility that the process leading to elevation role in diabetes mellitus per se.

of CSF sorbitol

plays an etiological

The degree of correlation was higher between CSF sorbitol and blood glucose than between CSF sorbitol and CSF glucose, providing evidence indicating that sorbito1 was formed in the nerve cells rather than in CSF cells. The probability that sorbitol in CSF was exogenous in origin (e.g. caused by sorbitol intake in food) was also unlikely because all the patients in groups I, II and III were on the same diet, but the sorbitol level wa.s not elevated in group III. In contrast to the findings in CSF, the urinary sorbitol excretion was not elevated. Although no numerical data are available on the renal clearance of sorbitol, it has been suggested that the renal threshold is very low or non-existenP. Accordingly, the low urinary concentration of sorbitol in the diabetic subjects provides further evidence indicating that sorbitol is formed in the nerve cells. According to earlier studies in urine polyols5 and to the present investigation, several differences between the polyol profile of urine and CSF can be noted. Xylitol has been demonstrated in the urine of both non-diabetic and diabetic patients; no trace of xylitol was detected in CSF. Mannitol and myo-inositol excretions were both elevated in the urine of diabetic patient9, but no elevation in the level of these polyols was seen in CSF. In contrast to the findings in CSF, the urinary excretion of polyols seems very poorly to reflect metabolic changes in the nervous tissue,

442

PITIdNEN,

SERVO

With regard to the close correlation of the level of sorbitol in CSF with the level of blood glucose, and to the experimental data1-4 which suggest a close correlation between the retention of sorbitol and the formation of pathological lesions in tissues, treatment to keep the blood sugar level normal in diabetic patients seems indicated. Although clinical data are conflicting it has been reported that careful treatment of the diabetic state postpones the formation of nervous complications in diabetes mellituslo. REFERENCES I R. VAN HEYNINGEN, Exp. Eye Res., I (1962) 396. 2 R. LEVARI, E. WERTHEIMER AND W. KORNBLUETH, Exp. Eye Res., 3 (1964) 99. 3 K. H. GABBAY, L. 0. MEROLA AND R. E. FIELD, Science, 131 (1966) 209. 4 M. A. STEWART, W. R. SHERMAN AND S. ANTHONY, Biochem. Biophys. Res. Comrnun., 22 (1966)

488. 5 E. PITKXEN, Clin. Chim. A&. 38 (1972) 221. 6 E. PITKANEN, A. PITKANEN AND J. PERHEENTUPA, Ann. Med. Exp. Fenniae, 42 (1963) 65. 7 H. ‘LI.BERGMEYER, W. GRUBER AND I. GUTMANN, Methoden der Enxymatischen Analyse, Band II, Verlag Chemie, Weinheim, 1970, D. 1292. 8 H. CRAMER, Mathematical Meth& oj Stat&s, Princeton University Press, Princeton, 1946. 9 M. R. SPIEGEL, Theory and Problems of Statistics, New York, 1961. IO E. GIBBELS, G. SCHLIEP, Fortschr. Neural. Psychiat., 39 (1971) 579.