Diagnostic Profiling Analysis of Polyols in Urine Samples of Patients with Various Diseases Performed by Capillary Gas Chromatography

ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6 657

Hidehiko HAGA*, Sachiko KAMEI**, Akiyuki OHKUBo** and Terumi NAKAJIMA*

*Department of Analytical Chemistry , Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo 113, Japan **Department of Laboratory Medicine , Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan

A capillary gas chromatographic method, based on trifluoroacetylated polyols, was applied to a study of urinary polyols in normal subjects and in patients with various diseases. Polyol excretion patterns during fasting periods and circadian variations were studied in normal subjects. Twenty-four hour urine sample sets of normal subjects showed almost constant polyol profiles, suggesting the existence of a polyol regulation system in the body. Excretion patterns of 10 polyols were studied in 100 specimens of 24-h urine samples from patients hospitalized with various diseases. Polyol profiles showed patterns characteristic of pathological states of the diseases: such as diabetes mellitus, chronic renal failure, and chronic liver diseases. The possibility of diagnosis of several diseases by urinary polyol profiles is presented.

Keywords Urinary polyols, capillary gas chromatography, diabetes mellitus, renal failure, liver diseases

The clinical significance of polyols in human physio- intake, medication and exogenous polyols especially, logical fluids and tissues has been drawing attention adding more difficulties to the study of urinary polyols. recently. Several recent studies have revealed that However, it has not been known to what extent food urinary levels of some polyols may vary in diseases and medication influence urinary polyol excretion. associated with carbohydrate metabolism derangements We recently established a specific, suitable and new such as diabetes mellitus and uremia.'-3 The abnormal rapid capillary gas chromatographic method for the occurrence of various polyols in diseases with a specific profiling analysis of urinary polyols as their trifluoro- enzyme deficiency such as pentosuria4 and galactosemias acetyl derivatives.6 By this method, eleven polyols can has also been reported. However, comprehensive be determined simultaneously. detailed studies of urinary polyols in patients with In this study, by applying our method, we first various diseases have not been reported yet, partly precisely investigated patterns of urinary polyol excre- because a convenient analytical method for that tion profiles in terms of changes in fasting state, purpose has not been available. The possible sources of circadian variation and 24 h urine samples with normal urinary polyols can be divided into the following four subjects to determine normal ranges. categories: 1) part of polyols contained in food or drugs Secondly, we examined the diagnostic possibility of may be absorbed and excreted into urine without profiling analysis of urinary polyols by our method by metabolic modification; 2) polyols produced by gastro- randomly measuring 100 specimens of 24 h urine samples intestinal microorganisms may be absorbed and excreted from hospitalized patients, submitted to our clinical in urine without further modification; 3) substances laboratory center for routine urinalysis. from digested and absorbed food or drugs may be metabolized into polyols in the body; 4) endogenous constituents in the body may be metabolized into Experimental polyols. Many of the polyols have been known to occur in Urine samples nature and they may also occur in food constituents. Normal urine samples were collected over a 24-h Moreover, sorbitol and xylitol are used as substitutes period from nine subjects with no known abnormalities for saccharose. Mannitol and sorbitol are used as on a free diet (only restricting any drugs and alcoholic ingredients in several drugs. It has been known that beverages). One hundred patient urine samples were urinary excretion of polyols may be influenced by food taken randomly from 24-h urine samples of hospitalized 658 ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6 patients which were submitted for routine urinalysis to were measured without modifying the original method, our clinical laboratory center. During the collection however, in case of diabetic urine whose glucose period, no preservative was used, but a sample was kept concentration was extremely high (more than 5 g/ dl), at 4° C and stored in a deep-freezer as soon as urinary the amount of Dowex 1X8 resin was increased volume was measured. Patient urine samples with only accordingly to remove excessive glucose from the urine. reference numbers were applied to analysis on a blind basis concerning patient information. Patient information such as age, sex and diagnosis was retrieved by the Results and Discussion computer system in the hospital, after the determination of urinary polyols was completed. Urinarypolyol excretion during fasting The medication history of patients with abnormal Urinary excretion of eleven polyols was measured in polyol values was obtained from doctors in charge by one male and one female over 20 h during fasting. sending them questionnaires. Excretion patterns of erythritol, arabitol, xylitol, mannitol, sorbitol and myoinositol in a male, for example, are Materials shown in Fig. 1. Polyol excretion decreased gradually All the materials and apparatus used for the after the last food intake, but it increased again after determination of polyols in this study were the same 14 h. The other polyols (including iditol) also showed ones as described in our previous paper.6 Urinary similar excretion patterns as in Fig. 1. These patterns glucose and creatinine were measured by a Hitachi 726 suggest that polyols are closely related with glyco- Autoanalyzer. genolysis during fasting which has been reported to begin 14 h after the last food intake, to maintain blood Sample preparation glucose level.' The origins of urinary polyols are not One milliliter of urine was treated by the method.6 well known yet; it has been considered that diet, host When polyol concentrations were very low due to metabolism and metabolism of gastrointestinal micro- increased urinary volume, 2 ml of urine was used and organisms may be their sources. These results suggested treated by the same method. Most of the urine samples that some amount of polyols are derived from intrinsic carbohydrate metabolism.

Fig. 1 Urinary polyol excretion during fasting. Last meal was taken at 19:00 (7 p. m., one hour before the first voiding). Fig. 2 Circadian variation of polyols in urine. ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6 659

Circadian variation of urinary polyol excretion voidings over 24 h. Circadian variation of ten polyols To our knowledge, no precise investigation on the in urine from a normal subject is shown in Fig. 2. circadian variation of urinary polyol excretion has been Polyol excretion varied very much in terms of time, reported yet. We determined urine samples from four person and kind of polyol. No significant circadian healthy men on a free diet (only restricting any drugs rhythm was found; however, it seemed that polyol and alcoholic beverages) and by unscheduled (voluntary) excretion increased soon after the meal. When we

Fig. 3 Urinary polyol profiles from normal subjects by unscheduled voiding over 24 h. Al, A2, A3 etc. indicate the orders of voidings in each subject. Abbreviations: E, erythritol; T, threitol; F, fucitol; R, ribitol; A, arabitol; X, xylitol; M, mannitol; S, sorbitol; G, galactitol; I, myoinositol. 660 ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6 looked at the polyol profile in the same person (normal subject C), it varied very much from one time to other time, as well as in other three normal subjects (Fig. 3). However, there was a tendency that erythritol, arabitol and mannitol were excreted in larger amounts, and fucitol, xylitol and galactitol in smaller amounts.

Normal 24-h urinary polyol excretion The polyol profiles of 24-h urine samples from normal subjects on a free diet are shown in Fig. 4. In this case, all the profiles showed a very similar pattern, which was different from the patterns of urine by unscheduled voidings. The origins of urinary polyols are still uncertain, as mentioned above. Nevertheless, we found that the polyol excretion in normal 24-h urine was within a certain range with small standard deviations. Namely, it can be said that some homeostasis exists in urinary polyol excretions. In other words, polyols are under the regulation of carbohydrate metabolism in the body. Accordingly, we judged that 24-h urine specimens would be more suitable than spot urine samples to compare the polyol profiles of urine from different kinds of diseases. All polyol levels except for myoinositol were similar to the previously reported values.l,g-13 Myoinositol levels (5.03±3.74 mg/d) were about one sixth of the Fig. 4 Polyol profiles of 24-h urine from 4 normal subjects. values (29.8±4.9 mg/ d) reported by Clements et al.10 Abbreviations are the same as in Fig. 3. The cause of this difference is uncertain but it may be

Table 1 Polyols excreted in 24-h urine (mg/g of creatinine)

a. mean. b. ±S.D. *, p<0.05; **, p<0.01; N.S., not significant compared with the normal group. Ery, erythritol; Thr, threitol; Fuc, fucitol; Rib, ribitol; Ara, arabitol; Xyl, xylitol; Man, mannitol; Sor, sorbitol; Gal, galactitol; Ino, myoinositol. ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6 661

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Patient 24-h urinary polyol excretion Correlation coefficientsbetween urinary volume and polyols As a first attempt to determine the diagnostic Correlation coefficients between urinary volume and possibility of our profiling analysis of urinary polyols, polyols are shown in Table 4. Relatively high correla- to avoid bias, we analyzed one hundred urine samples tion was found between urinary volume and two from patients on a blind basis, and after the completion polyols (fucitol and mannitol). In case of mannitol, it is of analysis we retrieved patient information by using reasonable since mannitol has diuretic activity.14 the computer system in our hospital. Among 100 samples, 41 samples lacked some information about the Correlation coefficientsbetween glucose and polyols patient, usually about urinary volumes and diagnoses. Correlation coefficients between glucose and polyols Thus, 59 samples were utilized for statistical considera- are shown in Table 5. High correlation was found tions. between glucose and myoinositol in the groups of Twenty four-hour urinary excretion of ten polyols hepatic diseases and hepatoma, which suggested the (mg/ g of creatinine) from patients with various diseases significance of the liver in glucose metabolism. is shown in Table 1. Most of the patient polyol excretion expressed in terms of creatinine showed Correlation coefficientsamong polyols statistically significant differences from the normal Correlation coefficients among polyols are shown in value. This seemed mainly due to the decreased Table 6. A relatively high correlation was found among excretion values of creatinine in patients. erythritol, fucitol, ribitol and arabitol. Twenty four-hour urinary polyol excretion expressed in mg/ d is shown in Table 2. In this table, abnormal Polyol profiles of 24 h urine in various diseases polyol values are not so many as those in Table 1. Urinary polyol levels of each disease group were These results forced us to give more careful considera- compared with those of healthy subjects by Wilcoxon's tion to the correlation between polyols and creatinine. rank-sum test, as shown in Table 2. Polyol patterns are Besides the ten polyols in Table 2, a trace amount of also shown in graphs in Fig. 5. Although we used urine iditol was detected in some patients (0-6.1 mg/d). from patients without any restriction of food and medication, polyol profiles of each group showed a Correlation coefficientsbetween creatinine and polyols characteristic pattern. Correlation coefficients between creatinine and polyols 1. Diabetes mellitus are shown in Table 3. Relatively high correlation was Myoinositol was significantly increased (p<0.01) as found between creatinine and both erythritol and previously reported 1,2,9,10, 12, due to impaired intracellular fucitol. However, correlation between creatinine and transport of myoinositol in diabetes mellitus.10 Ribitol other polyols was low. These correlation coefficients and arabitol were decreased (p<0.05) though its

Table 3 Correlation coefficients between creatinine and polyols

a. p<0.05. b. p<0.01. c. p<0.001. ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6 663

Table 4 Correlation coefficients between urinary volume and polyols

a. p<0.05. b. p<0.01. C. p<0.001.

Table 5 Correlation coefficients between glucose and polyols

a. p<0.05. b. p<0.01. c. p<0.001.

physiological meaning is uncertain. Mannitol levels 3. Renal failures were markedly increased in average, although it was not Myoinositol was significantly increased (p<0.01) as statistically significant because of the large standard previously reported"3", due to the impaired renal deviation, as previously described by Pitkanen et al.' oxidation of myoinositol to D-glucuronate in chronic 2. Liver diseases renal failure.15'16 All the other polyols except for Myoinositol was significantly increased (p<0.01). threitol were significantly (p<0.01 or p<0.05) decreased. Mannitol and sorbitol were markedly incresed in This result was similar to that of Niwa et a1.3 in which average, although the amounts were not statistically mannitol and sorbitol were slightly elevated from the significant because of the large standard deviation. normal level. Since the urinary excretion of creatinine 664 ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6

in renal failure. 4. Cancer Arabitol was significantly (p<0.01) decreased and myoinositol increased (p<0.05). Mannitol was markedly increased in average. As this group included 8 cases of hepatoma, this group was separated into two subgroups (hepatoma and other cancers). 4-a Liver cancer Arabitol was significantly (p<0.01) decreased and myoinositol increased (p<0.05). Mannitol and sorbitol levels were extremely high in liver cancer, although they were not statistically significant because of the large standard deviation. The polyol profile of this subgroup was very similar to that of hepatic diseases as shown in Fig. 5 and suggested that they are related with common impaired hepatic function. 4-b Other cancers Fucitol, ribitol (p <0.05) and arabitol (p <0.01) were significantly decreased, and myoinositol increased (p< 0.05). Mannitol was slightly increased. The polyol profile of this subgroup was clearly different from that of liver cancer. 5. Other diseases Myoinositol was significantly increased (p<0.01). Except for the slight increase of xylitol and mannitol, there was no change in the other polyols.

Otherfindings in abnormal polyol excretion Beside the statistical evaluation of the groups above, we also looked more precisely into some cases with abnormal urinary excretion of polyols in view of their relationship with diseases. 1. Arabitol In three cases (osteosarcoma, combustio bullosa and pneumonia, respectively), urinary arabitol excretion was increased (144.69, 106.88, 178.02 mg/ d, respectively). Although it was not confirmed, this may be due to candidal infection in immuno-compromised hosts since Bernard et a1.13 reported that excess arabitol was in this group was 0.46 g/ d, which was significantly produced by medically important species of Candida (p <0.01) lower than that of normal group (1.58 g/ d), and consequently was useful for the diagnosis of the decrease of urinary excretion of these polyols is candidiasis. considered to be due to impaired glomerular filtration ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6 665

2. Xylitol in the body. One patient with gastric ulcer who excreted an Urinary polyol profiles in patients with various extremely large amount of xylitol (1.1 g/d) was found diseases were investigated precisely by using 24 h urine to have received an intravenous infusion of xylitol. samples. When we expressed polyol values in mg/ d This seemed to be due to relatively slow metabolism of rather than mg/g of creatinine, polyol profiles showed xylitol in the body.'' This case, of course, was excluded characteristic patterns according to their pathological from the statistical evaluation. states. 3. Mannitol Although we used urine from patients without any Fifteen patients excreted highly increased amounts restriction of food and medication, polyol profiles of of mannitol (more than 100 mg/ d). Among them, 12 diabetes mellitus and renal failures were similar to those patients were associated with hepatic insufficiency such of formally reported ones.'-3,8-" Polyol profiles of liver as liver cancer, hepatitis and diabetes mellitus. Some of diseases and cancer were investigated for the first time. them were found to have received intravenous infusions Polyol profiles of liver diseases showed very similar of glucose. There were also 21 cases who excreted patterns to those of liver cancer, and not to cancers extremely decreased amounts of mannitol, less than other than liver cancer. Their patterns were very 2 mg/ d. Among them, no mannitol was detected at all abnormal in the points that mannitol and sorbitol in 16 cases (the detection limit of mannitol was about fluctuated very much, that is, with large standard 0.3 mg/d). Most of these patients (11 cases) were also deviations. Further study is needed in patients with associated with liver insufficiencies such as liver cancer, liver diseases. Influences of glucose infusion on urinary hepatitis and diabetes mellitus. Five patients were with polyol excretion should be studied more precisely. cancer other than liver cancer such as rectum cancer, We showed the diagnostic possibility of various malignant lymphoma, multiple myeloma, osteosarcoma, diseases by polyol profiles of 24 h urine. Further study lymphatic leukemia. This extremely decreased mannitol of urinary polyol profiles in other different diseases was noteworthy, since urinary mannitol excretion never may also reveal specific patterns characteristic to decreased to the non-detectable level in healthy subjects respective diseases. as described above. Some of the patients with very low mannitol level were also found to be treated with intravenous infusions of glucose. Correlation of manni- References tol excretion and glucose infusion should be studied further. 1. E. Pitkanen, Clin. Chim. Acta, 38, 221 (1972). 4. Sorbitol 2. J. F. Aloia, J. Lab. Clin. Med., 82, 809 (1973). Two patients were found to be treated with intravenous 3. T. Niwa, N. Yamamoto, K. Maeda, K. Yamada, T. Ohki infusions of sorbitol. However, none of them showed and M. Mori, J. Chromatogr., 277, 25 (1983). abnormal increases of sorbitol excretion in urine. This 4. 0. Touster and S. 0. Harwell, J. Biol. Chem., 230, 1031 seemed to be due to very rapid metabolism of sorbitol (1958). in the body; it was oxidized to fructose and then 5. W. W. Wells, T. A. Pittman and T. J. Egan, J. Biol. Chem., 239, 3192 (1964). utilized in the glycolytic pathway.18,19 6. H. Haga and T. Nakajima, Biomed. Chromatogr., 3, 68 (1989). Our study in healthy subjects revealed the following 7. R. A. Harris and D. W. Crabb, "Textbook of Biochemistry phenomena on urinary polyols. with Clinical Correlations", ed. T. M. Devlin, p. 531, John 1) During fasting, all urinary polyols decreased rapidly Wiley & Sons, New York, 1986. after the last food intake, however, they increased again 8. C. D. Pfaffenberger, J. Szafranek, M. G. Horning and E. about 14 h later. This suggested that polyols were also C. Horning, Anal. Biochem., 63, 501 (1975). produced along with glucose in the course of glyco- 9. W. H. Daughaday and J. Lamer, J. Clin. Invest., 33, 326 genolysis. (1954). 2) Under the condition of free diet and free urinary 10. R. S. Clements and R. Reynertson, Diabetes, 26, 215 voidings, there was no distinct circadian rhythm in (1977). 11. E. Pitkanen, Clin. Chim. Acta, 71, 461 (1976). urinary polyols, and urinary polyol profiles varied very 12. G. Gregersen, B. Bertelsen, H. Harbo, E. Larsen, J. R. much from one time to another and from one subject to Anderson, A. Helles, M. Schmiegelow and J. E. J. another. Christensen, Acta Neurol. Scand., 67, 164 (1983). 3) Twenty four-hour urine samples, on the other 13. E. M. Bernard, B. Wong and D. Armstrong, J. Infect. hand, showed a relatively constant polyol profile in Dis.,151, 711 (1985). each subject, and there was a certain range ("normal 14. F. D. Moore, "Metabolic care of the surgical patients", p. range") in each polyol. 690, W. B. Aunders Co., Philadelphia, 1959. In other words, urinary polyol profiles of normal 15. C. F. Howard and L. Anderson, Arch. Biochem. Biophys., subjects showed a relatively constant pattern despite 118, 332 (1967). 16. Y. M. Wang and J. van Eys, N. Engl. J. Med., 282, 892 various kinds of food intake in usual life, which suggested that there might be a regulatory system in (1970). 17. K. H. Bassler, G. Stein and W. Belzer, Biochem. Z., 346, polyol metabolism to maintain homeostasis in polyols 666 ANALYTICAL SCIENCES OCTOBER 1990, VOL. 6

171 (1966). Publishing Company, Inc., Westport,1977. 18. 0. Touster and D. R. D. Shaw, Physiol. Rev., 42, 181 (1962). (Received March 22, 1990) 19. B. K. Dwivedi, "Carbohydrates and health", ed. L. F. (Accepted June 27, 1990) Hood, E. K. Wardrip and G. N. Bollenback, p. 27, AVI