Clinical usefulness of measuring urinary polyol excretion by gas-chromatography/mass-spectrometry in type 2 diabetes to assess polyol pathway activity

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Abstract

Introduction: Decreased myo-inositol levels and increased activity of the polyol pathway have been proposed to play a role in causing diabetic microvascular complications. There are few clinical methods for examining the activity of the polyol pathway in diabetic patients. We assessed the effect of changes in glycemic control on polyol pathway activity by measuring urinary polyol excretion. Materials and methods: Gas-chromatography/mass-spectrometry (GC/MS) was used to assess the urinary excretion of glucose and polyols (myo-inositol, sorbitol, and fructose) in 50 patients who had type 2 diabetes without nephropathy and 20 healthy subjects. Results: In the diabetic patients with poor glycemic control, urinary sorbitol levels were significantly increased and urinary myo-inositol excretion was approximately 6.5-fold higher than in healthy controls (33.0 ± 6.5 vs 221.7 ± 45.9 mg/day, mean ± SE, P < 0.01). During strict glycemic control, some patients (Group A) showed simultaneous disappearance of glucosuria and normalization of the urinary excretion of myo-inositol (<50 mg/day) and, while others (Group B) showed delayed normalization of urinary myo-inositol excretion. Group B showed significantly higher urinary myo-inositol, sorbitol, and fructose excretion than Group A at the time of disappearance of glucosuria. These findings suggest that patients in Group B may have increased polyol pathway activity. Conclusion: Even though short-term strict glycemic regulations were established in long-standing hyperglycemic diabetic patients, to normalize the once-exaggerated polyol pathway activities, it was essential to maintain glucosuria-free conditions for some period. Quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol pathway activity.

Introduction

Diabetic complications are characterized by glucose toxicity, vascular dysfunction, and metabolic abnormalities. Enhancement of polyol pathway activity caused by hyperglycemia is thought to be a major cause of these complications. Aldose reductase activity has been found in many organs, in which glucose is utilized independently of insulin. Several biochemical mechanisms by which polyol pathway activation could produce diabetic complications have been proposed. These mechanisms include an increase of the intracellular sorbitol concentration, altered NADPH/NADP+ and NADH/NAD+ rations, and an increase of de novo diacylglycerol production, leading to protein kinase C activation [1], [2]. Since sorbitol only crosses the cell membrane with difficulty, its accumulation causes osmotic stress, thereby causing morphological changes and cellular damage [3]. The intracellular accumulation of sorbitol also causes depletion of other intracellular organic osmolytes, such as myo-inositol and taurine [4]. There is evidence that a decrease of the intracellular myo-inositol content during hyperglycemia is involved in the development of diabetic complications [5], [6], [7], [8], [9], [10]. Greene et al. [11] reported that streptozotocin-induced diabetic rats with hyperglycemia for two weeks showed a reduction of both sciatic nerve conduction velocity and myo-inositol contents, and that strict glycemic control with insulin reversed these changes. A decreased in the myo-inositol content of retinal endothelial cells produces a marked increase of adhesions between these cells and the extracellular matrix, thereby altering retinal permeability [10]. These findings have revealed the involvement of polyol pathway activation in the development of complications in diabetic animals. However, there is less evidence for a link between the development of diabetic microangiopathy and polyol pathway activation in diabetic patients.

For clinical evaluation of polyol pathway activity in diabetes, the metabolites of this pathway have been measured in red blood cells or surgically excised tissues, such as nerves and muscles. [6], [12], [13]. These studies have shown that the sorbitol content of these tissues is correlated with that of nerve fibers and kidneys, while no such relationship is observed for myo-inositol and fructose. Therefore it is important to quantitate myo-inositol and fructose levels in the end organs of diabetic complications. Renal tubular cell abundantly express aldose reductase because of the high osmotic environment required for urinary concentrating processes. Two putative mechanisms for increasing urinary myo-inositol excretion in diabetes are as follows. (1) Hyperglycemia increases the urinary glucose level, leading to a reduction of urinary myo-inositol reabsorption by competitive inhibition of transport in the renal tubules [6], [8]. (2) Inhibition of myo-inositol uptake is caused by an increase in the intracellular concentration of glucose metabolites such as sorbitol and fructose, which are critically important mediators for reduction of intracellular myo-inositol [21]. In the present study, we simultaneously measured several organic osmolytes such as sorbitol, fructose and myo-inositol in urine by gas-chromatography/mass-spectrometry (GC/MS), and examined links between the levels of these metabolites and the levels of glucosuria in diabetic patients, including diabetic complications. We also investigated the effect of glycemic control on urinary organic osmolytes, especially myo-inositol.

Section snippets

Subjects

Urine samples were collected from 50 patients with type 2 diabetes mellitus (type 2 DM) and 20 normal subjects at our hospital. The myo-inositol, sorbitol and fructose contents were determined in 24 h urine specimens. The 20 normal subjects were 11 men and 9 women, while the 50 patients with type 2 DM comprised 26 men and 24 women. The clinical characteristics of the type 2 DM patients are shown in Table 1. All of the patients fulfilled the criteria of the National Diabetes data group for type

Results

As shown in Fig. 1, 24 h urinary myo-inositol and sorbitol excretion was significantly increased in the diabetic subjects compared with control subjects (P < 0.05). The urinary myo-inositol excretion was 221.7 ± 45.9 mg/day and 33.0 ± 6.5 mg/day in the diabetic subjects and the control subjects, respectively. The diabetic patients also had a significantly larger urinary sorbitol excretion (77.2 ± 24.6 mg/day) than the control subjects (19.1 ± 7.4 mg/day), but there was no significant difference in

Discussion

It has been shown that the myo-inositol content is reduced in muscle, brain, peripheral nerve, and renal medullary cells in the hyperglycemic state [6], [7], [8], [9]. The decreased organ myo-inositol content in diabetes is clinically important [6], because diabetic microvascular complications are related to the decrease of myo-inositol [5], [16], [17], [18]. In this study, we found that urinary myo-inositol excretion was higher in diabetic patients compared with normal subjects (Fig. 1). The

Acknowledgements

This work was supported by grants from the Suzuken Memorial Foundation and from Kyowa Hakko Kogyo, Japan. We thank Dr. Isamu Matumoto and Dr. Chun-hua Zhang (Division of Human Genetics, Medical Research Institute, Kanazawa Medical University) for their comments and suggestions regarding the design and interpretation of the GC/MS studies. We are also indebted to Mrs. Naoko Honda for her excellent technical assistance. Parts of this study will be presented at the 59th Annual Meeting and

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