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Determination of Isatin, an Endogenous Monoamine Oxidase Inhibitor, in Urine and Tissues of Rats by HPLC
ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00274-7 All rights reserved
Gen. Pharmac. Vol. 30, No. 3, pp. 387–391, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Determination of Isatin, an Endogenous Monoamine Oxidase Inhibitor, in Urine and Tissues of Rats by HPLC Naoya Hamaue,1 Noriko Yamazaki,2 Masaru Minami,1* Toru Endo,1 Masahiko Hirahuji,1 Yoshio Monma1 and Hiroko Togashi3 1 Department of Pharmacology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido 061-02 [Tel: 81-1332-3-1211; Fax: 181-1332-3-1669; E-mail: [email protected]]; 2 Hokkaido University College of Medical Technology, Sapporo 060, and 3 Department of Pharmacology, Hokkaido University, School of Medicine, Sapporo 060, Japan
ABSTRACT. 1. We have previously identified isatin as one of the endogenous monoamine oxidase (MAO) inhibitors in the urine and the brain of stroke-prone spontaneously hypertensive rats (SHRSP), using gas chromatography-mass spectrometry (GC-MS). 2. In this study, we attempted to develop a convenient assay to determine isatin using high performance liquid chromatography with an ultraviolet detector (HPLC-UV). The standard curve for authentic isatin was linear at a range from 2 to 20 nmol per ml. The coefficient of variance was within 3% for both intra-assay and inter-assay. The sensitivity was 20 pmol per 10 ml of urine sample. 3. Isatin concentration correlated significantly and positively with endogenous MAO activity (tribulin-like activity) in both urine (r50.924, P,0.001) and kidney extracts (r50.862, P,0.01). There was a significant difference in urinary isatin between Wistar Kyoto rats (WKY) and SHRSP. Oral administration of isatin increased urinary isatin concentration and systolic blood pressure in WKY. 4. Determination of isatin using HPLC-UV may be useful for elucidating role of isatin in various conditions of stress and disease. gen pharmac 30;3:387–391, 1998. 1998 Elsevier Science Inc. KEY WORDS. Isatin, monoamine oxidase inhibitor, tribulin-like activity, urine and kidney of rats, HPLC-UV INTRODUCTION Endogenous monoamine oxidase (MAO) inhibitory components were first discovered in normal human urine by Glover et al. (1980), and one of these inhibitors was given the name tribulin (Sandler, 1982). Tribulin output in human urine is increased during various conditions of stress and anxiety (Clow et al., 1988). Several studies have previously shown that stress can induce a decrease in MAO activity and increases in monoamine levels. Cold immobilization stress has also been shown to be associated with an increase in the activity of the serotonergic system (Oxenkrug and McIntyre, 1985). Cold-restraint stress increases tribulin levels in the rat heart and kidney (Armomdo et al., 1988). With high blood pressure, plasma norepinephrine significantly increases and kidney MAO activity decreases in stroke-prone spontaneously hypertensive rats (SHRSPs) compared with those of Wistar Kyoto rats (WKYs) (Minami et al., 1988). Furthermore, we demonstrated that aromatic l-amino acid decarboxylase activity, which synthesizes dopamine, serotonin (5-HT) or both, was lower in the kidney of the SHRSP than in that of the WKY (Hamaue et al., 1992). Therefore, the metabolic enzyme activity of monoamines may be more important than the synthesizing enzyme activity in contributing to high blood pressure. Because tribulin-like activity is higher in the SHRSP than the WKY (Hamaue et al., 1992), lower MAO activity in the SHRSP may be due to the increase of tribulin-like activity. The chemical nature of tribulin was unknown until Glover et al. (1988) reported that isatin * To whom correspondence should be addressed. Received 8 April 1997; revised 25 April 1997; accepted 15 May 1997.
had properties similar to tribulin. They succeeded in identifying it in human urine by gas chromatography-mass spectrometry (GC-MS). We also identified isatin in purified extracts of SHRSP urine (Hamaue et al., 1992) and brain (Hamaue et al., 1994) by GC-MS. In this study, we attempted to develop a convenient alternative method for the determination of isatin by high performance liquid chromatography with an ultraviolet detector (HPLC-UV) to replace GC-MS determination (Hamaue et al., 1994). To elucidate the physiological role of isatin, we determined the isatin concentration in rat urine and kidney after exogenous isatin administration. MATERIALS AND METHODS
Animals and feeding Adult male WKYs (280–320 g) and SHRSPs (170–260 g) were fed separately in metabolic cages (KN-646, Natsume, Japan) with a rat laboratory chow (CE-2, Clea Japan, Tokyo, Japan). Rats were randomly assigned to groups receiving oral doses of 50 mg/kg or 200 mg/ kg of isatin (Sigma, St. Louis, MO, USA) or no isatin (nondrug control) for 4 weeks. The rats were kept on a light–dark cycle (light 08:00–20:00) in an air-conditioned room (22628C, 55–65% humidity). A 24-hr urine sample was collected 2 and 4 weeks after treatment.
Tribulin extraction and sample preparation Rat kidneys were homogenized in 2 M HCl and homogenates were centrifuged at 2500 rpm for 10 min. Supernatants were extracted with ethylacetate and the extracts were evaporated to dryness with
388
FIGURE 1. Chromatogram of authentic isatin (a) and urine extract (b) separated by HPLC-UV: (a) amount of authentic isatin injected in a 40-ml volume was 80 pmol; (b) 10 ml of urine extract dissolved in 0.1 M HCl after extraction and evaporation was injected.
nitrogen gas. To assay MAO inhibitory activity, the residues were dissolved in 0.1 M phosphate buffer (pH 7.4). Rat urine samples were adjusted at pH 1.0 with 6 M HCl. Urinary creatinine concentration was determined by the method of Jaffe et al. (1886) and was diluted with water to give a constant creatinine concentration of 0.3 mg/ml. One milliliter of the diluted urine was added with 1 ml of 4 M HCl and 2.5 ml of ethylacetate. The mixtures were shaken and centrifuged at 3000 rpm for 5 min. The urine mixture was extracted twice with ethylacetate. The ethylacetate layers were separated and evaporated to dryness with nitrogen gas. The residues were redissolved in a volume of 0.1 M phosphate buffer (pH 7.4) equal to that of the original urine volume. Blanks, in which water replaced urine, did not display MAO inhibitory activity. Inhibition of rat liver mitochondrial MAO by the urine and kidney extracts was determined as described in detail by Elsworth et al. (1986).
N. Hamaue et al.
FIGURE 2. Standard curve for authentic isatin determined by using HPLC-UV.
Statistical analysis Values were expressed as the mean6SD. The Students t-test was used to analyze differences between two groups. When more than two groups were compared, the significance of the differences between groups was evaluated by analysis of variance and, when applicable, was followed by Tukey’s test. The Bonferroni adjustment was used for testing two points (Wallenstein et al., 1980). P,0.05 was considered to be statistically significant. RESULTS
Reproducibility and coefficient of variation for HPLC-UV determination of authentic isatin Figure 1(a) shows a chromatogram of the authentic isatin separated by HPLC-UV. As shown in Figure 2, the standard curve for authentic isatin was linear at a range from 2 to 20 nmol/ml. With the use of the aforedescribed isatin assay, recovery was approximately 96% at this concentration range. The coefficient of variance (CV) for the determination was approximately 3% for both intraassay and interassay. The isatin concentration at 20 pmol/10 ml of urine sample could be measured (the limitation of determination).
Isatin determination by HPLC-UV The HPLC apparatus consisted of a LC pump (LC-6A, Simadzu, Japan), an injector (Model 7125, Reodyne, USA) with a 100-ml loop, an ultraviolet spectrophotometer (L-4000, Hitachi, Japan) and a chromatographic data processor (CR-3A, Simadzu, Japan). The spectrophotometer was set at 242 nm. The molecular extinction coefficient of isatin was 2.33103 mol/l/mm at 242 nm. All separations were performed at ambient temperature on a 25034.6 mm (i.d.) column packed with a particle diameter of 5 mm (Tsk gel ODS80TM, TOSOH, Japan). The HPLC mobile phase was adjusted to pH 2.5 with 55 mM phosphate buffer, 15% acetonitrile and 0.1 mM sodium octylsulfate. The flow rate was 1.0 ml/min. Authentic isatin (Sigma, USA) was dissolved in 0.1 M HCl. Evaporated samples were dissolved in 0.1 M HCl, filtered through a Millipore filter (pore size 0.45 mM) and then injected into the HPLC.
Correlation between isatin concentration and tribulin-like activity in the urine and the kidney of WKY Figure 1(b) shows a chromatogram of urine extract separated by HPLC-UV. Isatin concentrations in the rat urine and kidney were determined by using HPLC-UV, and their tribulin-like activities were compared. As shown in Figure 3, isatin concentration correlated significantly and positively with tribulin-like activity in both urine (r50.924, n581, P,0.001) and kidney extracts (r50.862, n514, P,0.01) of WKY.
Urinary isatin excretion in WKY and SHRSP As shown in Figure 4, the urinary isatin concentration of SHRSPs was significantly higher than that of WKYs.
Isatin, MAO Inhibitor
389
FIGURE 3. Correlation between isatin concentration and tribulin-like activity in the urine and the kidney of WKYs.
Effect of exogenously administered isatin on the isatin concentration in urine and kidney extracts of rats
Endogenous MAO inhibitory components were first discovered in normal human urine by Glover et al. (1980), and one of these inhibitors was given the name tribulin (Sandler et al., 1982). Two types of endogenous MAO inhibitor have been described: one is a peptide; the other is a nonpeptide (Elsworth et al., 1986). Tribulin is an endogenous, low-molecular-weight, nonpeptide MAO inhibitor
(Elsworth et al., 1986). Through the use of purified endogenous inhibitor from normal human urine, the chemical nature of tribulin was investigated, and isatin was first identified in tribulin by GCMS (Glover et al., 1988). Indeed, isatin had properties similar to those of tribulin (Glover et al., 1988). We also determined isatin in endogenous MAO inhibitors in SHRSP urine, brain and kidney by using the GC-MS method and clarified the relation between isatin and the high blood pressure of SHRSPs (Hamaue et al., 1992, 1994). The GC-MS method is useful for the identification of isatin; however, a great deal of time is required for the extraction, induction and determination of isatin by this method. In this study, we developed a convenient method for the determination of isatin in the urine and tissue of rats by using HPLC-UV. The recovery of this assay was approximately 96% (in authentic study) at a range from 2 to 20 nmol/ml and was reproducible. CV for the determination was approximately 3%. A chromatogram ob-
FIGURE 4. Urinary isatin concentration in SHRSPs and WKY rats.
FIGURE 5. Urinary isatin concentration after oral administration of isatin for 4 weeks to WKY rats.
As shown in Figure 5, the urinary isatin concentration significantly increased with exogenous isatin administration in a dose-dependent manner. Also, isatin concentration in the WKY kidney was increased after oral administration of isatin compared with nondrug WKY controls (Fig. 6). DISCUSSION
390
N. Hamaue et al. Urinary isatin excretion of SHRSPs was significantly greater than that of WKYs (Fig. 4). We previously reported that the inhibition of liver MAO activities was significantly greater in SHRSP urine extract than in that of WKYs (Hamaue et al., 1992). These findings suggest that the development of high blood pressure in SHRSPs may be involved in the increased monoamine concentrations induced by isatin. In conclusion, the present study demonstrated that HPLC-UV determination of isatin is a simple and accurate method. SUMMARY
FIGURE 6. Isatin concentration in the kidney after oral administration of isatin for 4 weeks to WKY rats. tained from urine extract separated by HPLC-UV showed a broad, but almost the same, shape compared with that of authentic isatin (Fig. 1). As shown in Figure 1, the retention time to the peak in the chromatogram of urine extract coincided well with that of authentic isatin. No peak was observed in chromatograms obtained from extracts of urine and kidneys. Exogenously administered isatin increased urinary and kidney levels of isatin in a dose-dependent manner. Moreover, the isatin concentration measured by this assay correlated significantly and positively with tribulin-like activity (MAO inhibitory activity), suggesting the specificity of that method. Functionally, isatin expresses approximately 77.4613.1% of its tribulin-like activity as MAO inhibitory activity. Isatin competitively inhibits the MAO activity of rat liver homogenate in a dosedependent manner and is a potent MAO inhibitor that is more active against MAO-B the MAO-A (Hamaue et al., 1992). Previous reports have shown that stress can induce a decrease in MAO activity. It is well known that exposure of adults rats to chronic environmental stress induces a reduction in MAO activity (Maura et al., 1974) and the development of chronic hypertension (Maura et al., 1979). We recently reported that isatin increased tribulin-like activity. Isatin also produced a significant and dosedependent increase in urinary norepinephrine excretion in WKYs. Further, the systolic blood pressure of WKYs that received an isatin dose of 200 mg/kg (124.365.45 mmHg, n58, P,0.01 vs. nondrug control WKY) was significantly higher than that of the nondrug control WKY (114.365.28, n58). Systolic blood pressure correlated significantly with urinary tribulin-like activity 4 weeks after treatment of WKYs with isatin (Hamaue et al., 1996). Urinary tribulin excretion increases under various conditions of stress and anxiety (Clow et al., 1988). Tribulin, which acts on central benzodiazepine receptors, has been proposed as an anxiety-promoting substance (Sandler et al., 1982). Peripheral benzodiazepine receptors are located in the mitochondrial center membrane with which MAO also is associated (Anholt, 1986). Cold-restraint stress increases tribulin in the rat heart and kidney (Armando et al., 1988). Although the physiological function of peripheral benzodiazepine receptors is still unknown, they are associated with ion-transport systems in both the heart and the kidney (Basile and Skolnick, 1988; Mestre et al., 1986). Isatin may, therefore, have a role in the ion-transport system in these tissues through action on mitochondrial MAO activity.
Endogenous MAO inhibitory components were first discovered in normal human urine. Two types of endogenous MAO inhibitor have been described: one is a peptide; the other is a nonpeptide. One of these inhibitors has been given the name tribulin. Tribulin is an endogenous, low-molecular-weight, nonpeptide MAO inhibitor. We previously identified isatin as one of the chemical structures of tribulin in both the urine and the brain of SHRSPs by using GCMS. In this study, we developed a convenient assay to determine isatin by using HPLC-UV. The recovery, the coefficient of variance and the sensitivity of the assay are promising. Isatin concentration correlated positively with tribulin-like activity in both urine (r50.924, P,0.001) and kidney extracts (r50.862, P,0.01) by using HPLC-UV. This assay to determine isatin may be useful in elucidating roles of isatin in various conditions of stress and disease. References Anholt R. R. (1986) Mitochondrial benzodiazepine receptors as potential modulators of inter-mediatory metabolism. Trends. Pharm. Sci. 6, 506– 511. Armando I., Levin G. and Barontini M. (1988) Stress increases benzodiazepine receptor ligand-monoamine oxidase inhibitory activity (tribuline) in tissues. J. Neural Transm. 71, 29–37. Basile A. S. and Skolnick P. (1988) Tissue specific regulation of “peripheraltype” benzodiazepine receptor density after chemical sympathectomy. Life Sci. 42, 273–283. Clow A., Glover V., Weg M. W., Walker P. L., Sheehan D. V., Carr D. V. and Sandler M. (1988) Urinary catecholamine metabolite and tribulin output during lactate infusion. Br. J. Psychiatry 152, 122–126. Elsworth J. D., Dewar D., Glover V., Goodwin B. L., Clow A. and Sandler M. (1986) Purification and characterization of tribulin, an endogenous inhibitor of monoamine oxidase and benzodiazepine receptor binding. J. Neural Transm. 67, 45–56. Glover V., Reveley M. A. and Sandler M. (1980) A monoamine oxidase inhibitor in human urine. Biochem. Pharmac. 29, 467–470. Glover V., Halket J. M., Watkins P. J., Clow A., Goodwin B. L. and Sandler, M. (1988) Isatin: identity with the purified endogenous monoamine oxidase inhibitor tribulin. J. Neurochem. 51, 656–659. Hamaue N., Minami M., Kanamaru Y., Togashi M., Monma Y., Ishikura M., Mahara R., Yamazaki N., Togashi H., Saito H. and Parvez S. H. (1992) Endogenous monoamine oxidase (MAO) inhibitor (tribulin-like activity) in the brain and urine of stroke-prone SHR. Biog. Amines 8, 401– 412. Hamaue N., Minami M., Kanamaru Y., Ishikura M., Saito H. and Parvez S. H. (1994) Identification of isatin, an endogenous MAO inhibitor, in the brain of stroke-prone SHR. Biog. Amines 10, 99–110. Hamaue N., Minami M., Hirafuji M., Monma Y., Yamazaki N., Togashi H., Saito H. and Parvez S. H. (1996) Significance of isatin, an endogenous MAO inhibitor, on blood pressure control and monoamine levels. Biog. Amines 12, 395–405. Jaffe M. (1886) Uever den Niederschlag, welchen Pikrinsa¨re in normalem Harn erzeugt und u¨ber eine neue Reaction des Kreatinins. Physiol. Chem. 10, 391. Maura G., Vaccari A., Gemignani A. and Cugurra F. (1974) Development of monoamine oxidase activity after chronic environmental stress in the rat. Environ. Physiol. Biochem. 4, 64–79. Maura G., Versace P. and Paudice P. (1979) Stress-induced hypertension and COMT activity in rats. Pharmac. Res. Commun. 11, 537–543. Mestre M., Garriot T., Neliat G., Uzau A., Renault C., Dubroeueq M. C.,
Isatin, MAO Inhibitor Gueremy C., Doble A. and LeFur G. (1986) PK11195, an antagonist of peripheral type benzodiazepine receptors, modulates BAYK8644 sestive but not b or H2-receptor sensitive voltage operated calcium channels in the guinea pig heart. Life Sci. 39, 329–339. Minami M., Senjo M., Togashi H., Yoshioka M., Saito H., Kawaguchi H., Takebayashi K. and Parvez H. (1988) Kidney glutathione S-transferase activity and kidney monoamine oxidase activity in stroke cases of SHRSP. Biog. Amines 5, 517–526.
391 Oxenkrug G. and McIntyre I. (1985) Stress-induced synthesis of melatonin: possible involvement of the endogenous monoamine oxidase inhibitor (tribulin). Life Sci. 37, 1743–1746. Sandler M. (1982) The emergence of tribulin. Trends Pharmac. Sci. 3, 471– 472. Wallenstein S., Zucker C. L. and Fleiss J. L. (1980) Some statistical methods useful in circulation research. Circ. Res. 47, 1–9.
Gen. Pharmac. Vol. 30, No. 3, pp. 387–391, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Determination of Isatin, an Endogenous Monoamine Oxidase Inhibitor, in Urine and Tissues of Rats by HPLC Naoya Hamaue,1 Noriko Yamazaki,2 Masaru Minami,1* Toru Endo,1 Masahiko Hirahuji,1 Yoshio Monma1 and Hiroko Togashi3 1 Department of Pharmacology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido 061-02 [Tel: 81-1332-3-1211; Fax: 181-1332-3-1669; E-mail: [email protected]]; 2 Hokkaido University College of Medical Technology, Sapporo 060, and 3 Department of Pharmacology, Hokkaido University, School of Medicine, Sapporo 060, Japan
ABSTRACT. 1. We have previously identified isatin as one of the endogenous monoamine oxidase (MAO) inhibitors in the urine and the brain of stroke-prone spontaneously hypertensive rats (SHRSP), using gas chromatography-mass spectrometry (GC-MS). 2. In this study, we attempted to develop a convenient assay to determine isatin using high performance liquid chromatography with an ultraviolet detector (HPLC-UV). The standard curve for authentic isatin was linear at a range from 2 to 20 nmol per ml. The coefficient of variance was within 3% for both intra-assay and inter-assay. The sensitivity was 20 pmol per 10 ml of urine sample. 3. Isatin concentration correlated significantly and positively with endogenous MAO activity (tribulin-like activity) in both urine (r50.924, P,0.001) and kidney extracts (r50.862, P,0.01). There was a significant difference in urinary isatin between Wistar Kyoto rats (WKY) and SHRSP. Oral administration of isatin increased urinary isatin concentration and systolic blood pressure in WKY. 4. Determination of isatin using HPLC-UV may be useful for elucidating role of isatin in various conditions of stress and disease. gen pharmac 30;3:387–391, 1998. 1998 Elsevier Science Inc. KEY WORDS. Isatin, monoamine oxidase inhibitor, tribulin-like activity, urine and kidney of rats, HPLC-UV INTRODUCTION Endogenous monoamine oxidase (MAO) inhibitory components were first discovered in normal human urine by Glover et al. (1980), and one of these inhibitors was given the name tribulin (Sandler, 1982). Tribulin output in human urine is increased during various conditions of stress and anxiety (Clow et al., 1988). Several studies have previously shown that stress can induce a decrease in MAO activity and increases in monoamine levels. Cold immobilization stress has also been shown to be associated with an increase in the activity of the serotonergic system (Oxenkrug and McIntyre, 1985). Cold-restraint stress increases tribulin levels in the rat heart and kidney (Armomdo et al., 1988). With high blood pressure, plasma norepinephrine significantly increases and kidney MAO activity decreases in stroke-prone spontaneously hypertensive rats (SHRSPs) compared with those of Wistar Kyoto rats (WKYs) (Minami et al., 1988). Furthermore, we demonstrated that aromatic l-amino acid decarboxylase activity, which synthesizes dopamine, serotonin (5-HT) or both, was lower in the kidney of the SHRSP than in that of the WKY (Hamaue et al., 1992). Therefore, the metabolic enzyme activity of monoamines may be more important than the synthesizing enzyme activity in contributing to high blood pressure. Because tribulin-like activity is higher in the SHRSP than the WKY (Hamaue et al., 1992), lower MAO activity in the SHRSP may be due to the increase of tribulin-like activity. The chemical nature of tribulin was unknown until Glover et al. (1988) reported that isatin * To whom correspondence should be addressed. Received 8 April 1997; revised 25 April 1997; accepted 15 May 1997.
had properties similar to tribulin. They succeeded in identifying it in human urine by gas chromatography-mass spectrometry (GC-MS). We also identified isatin in purified extracts of SHRSP urine (Hamaue et al., 1992) and brain (Hamaue et al., 1994) by GC-MS. In this study, we attempted to develop a convenient alternative method for the determination of isatin by high performance liquid chromatography with an ultraviolet detector (HPLC-UV) to replace GC-MS determination (Hamaue et al., 1994). To elucidate the physiological role of isatin, we determined the isatin concentration in rat urine and kidney after exogenous isatin administration. MATERIALS AND METHODS
Animals and feeding Adult male WKYs (280–320 g) and SHRSPs (170–260 g) were fed separately in metabolic cages (KN-646, Natsume, Japan) with a rat laboratory chow (CE-2, Clea Japan, Tokyo, Japan). Rats were randomly assigned to groups receiving oral doses of 50 mg/kg or 200 mg/ kg of isatin (Sigma, St. Louis, MO, USA) or no isatin (nondrug control) for 4 weeks. The rats were kept on a light–dark cycle (light 08:00–20:00) in an air-conditioned room (22628C, 55–65% humidity). A 24-hr urine sample was collected 2 and 4 weeks after treatment.
Tribulin extraction and sample preparation Rat kidneys were homogenized in 2 M HCl and homogenates were centrifuged at 2500 rpm for 10 min. Supernatants were extracted with ethylacetate and the extracts were evaporated to dryness with
388
FIGURE 1. Chromatogram of authentic isatin (a) and urine extract (b) separated by HPLC-UV: (a) amount of authentic isatin injected in a 40-ml volume was 80 pmol; (b) 10 ml of urine extract dissolved in 0.1 M HCl after extraction and evaporation was injected.
nitrogen gas. To assay MAO inhibitory activity, the residues were dissolved in 0.1 M phosphate buffer (pH 7.4). Rat urine samples were adjusted at pH 1.0 with 6 M HCl. Urinary creatinine concentration was determined by the method of Jaffe et al. (1886) and was diluted with water to give a constant creatinine concentration of 0.3 mg/ml. One milliliter of the diluted urine was added with 1 ml of 4 M HCl and 2.5 ml of ethylacetate. The mixtures were shaken and centrifuged at 3000 rpm for 5 min. The urine mixture was extracted twice with ethylacetate. The ethylacetate layers were separated and evaporated to dryness with nitrogen gas. The residues were redissolved in a volume of 0.1 M phosphate buffer (pH 7.4) equal to that of the original urine volume. Blanks, in which water replaced urine, did not display MAO inhibitory activity. Inhibition of rat liver mitochondrial MAO by the urine and kidney extracts was determined as described in detail by Elsworth et al. (1986).
N. Hamaue et al.
FIGURE 2. Standard curve for authentic isatin determined by using HPLC-UV.
Statistical analysis Values were expressed as the mean6SD. The Students t-test was used to analyze differences between two groups. When more than two groups were compared, the significance of the differences between groups was evaluated by analysis of variance and, when applicable, was followed by Tukey’s test. The Bonferroni adjustment was used for testing two points (Wallenstein et al., 1980). P,0.05 was considered to be statistically significant. RESULTS
Reproducibility and coefficient of variation for HPLC-UV determination of authentic isatin Figure 1(a) shows a chromatogram of the authentic isatin separated by HPLC-UV. As shown in Figure 2, the standard curve for authentic isatin was linear at a range from 2 to 20 nmol/ml. With the use of the aforedescribed isatin assay, recovery was approximately 96% at this concentration range. The coefficient of variance (CV) for the determination was approximately 3% for both intraassay and interassay. The isatin concentration at 20 pmol/10 ml of urine sample could be measured (the limitation of determination).
Isatin determination by HPLC-UV The HPLC apparatus consisted of a LC pump (LC-6A, Simadzu, Japan), an injector (Model 7125, Reodyne, USA) with a 100-ml loop, an ultraviolet spectrophotometer (L-4000, Hitachi, Japan) and a chromatographic data processor (CR-3A, Simadzu, Japan). The spectrophotometer was set at 242 nm. The molecular extinction coefficient of isatin was 2.33103 mol/l/mm at 242 nm. All separations were performed at ambient temperature on a 25034.6 mm (i.d.) column packed with a particle diameter of 5 mm (Tsk gel ODS80TM, TOSOH, Japan). The HPLC mobile phase was adjusted to pH 2.5 with 55 mM phosphate buffer, 15% acetonitrile and 0.1 mM sodium octylsulfate. The flow rate was 1.0 ml/min. Authentic isatin (Sigma, USA) was dissolved in 0.1 M HCl. Evaporated samples were dissolved in 0.1 M HCl, filtered through a Millipore filter (pore size 0.45 mM) and then injected into the HPLC.
Correlation between isatin concentration and tribulin-like activity in the urine and the kidney of WKY Figure 1(b) shows a chromatogram of urine extract separated by HPLC-UV. Isatin concentrations in the rat urine and kidney were determined by using HPLC-UV, and their tribulin-like activities were compared. As shown in Figure 3, isatin concentration correlated significantly and positively with tribulin-like activity in both urine (r50.924, n581, P,0.001) and kidney extracts (r50.862, n514, P,0.01) of WKY.
Urinary isatin excretion in WKY and SHRSP As shown in Figure 4, the urinary isatin concentration of SHRSPs was significantly higher than that of WKYs.
Isatin, MAO Inhibitor
389
FIGURE 3. Correlation between isatin concentration and tribulin-like activity in the urine and the kidney of WKYs.
Effect of exogenously administered isatin on the isatin concentration in urine and kidney extracts of rats
Endogenous MAO inhibitory components were first discovered in normal human urine by Glover et al. (1980), and one of these inhibitors was given the name tribulin (Sandler et al., 1982). Two types of endogenous MAO inhibitor have been described: one is a peptide; the other is a nonpeptide (Elsworth et al., 1986). Tribulin is an endogenous, low-molecular-weight, nonpeptide MAO inhibitor
(Elsworth et al., 1986). Through the use of purified endogenous inhibitor from normal human urine, the chemical nature of tribulin was investigated, and isatin was first identified in tribulin by GCMS (Glover et al., 1988). Indeed, isatin had properties similar to those of tribulin (Glover et al., 1988). We also determined isatin in endogenous MAO inhibitors in SHRSP urine, brain and kidney by using the GC-MS method and clarified the relation between isatin and the high blood pressure of SHRSPs (Hamaue et al., 1992, 1994). The GC-MS method is useful for the identification of isatin; however, a great deal of time is required for the extraction, induction and determination of isatin by this method. In this study, we developed a convenient method for the determination of isatin in the urine and tissue of rats by using HPLC-UV. The recovery of this assay was approximately 96% (in authentic study) at a range from 2 to 20 nmol/ml and was reproducible. CV for the determination was approximately 3%. A chromatogram ob-
FIGURE 4. Urinary isatin concentration in SHRSPs and WKY rats.
FIGURE 5. Urinary isatin concentration after oral administration of isatin for 4 weeks to WKY rats.
As shown in Figure 5, the urinary isatin concentration significantly increased with exogenous isatin administration in a dose-dependent manner. Also, isatin concentration in the WKY kidney was increased after oral administration of isatin compared with nondrug WKY controls (Fig. 6). DISCUSSION
390
N. Hamaue et al. Urinary isatin excretion of SHRSPs was significantly greater than that of WKYs (Fig. 4). We previously reported that the inhibition of liver MAO activities was significantly greater in SHRSP urine extract than in that of WKYs (Hamaue et al., 1992). These findings suggest that the development of high blood pressure in SHRSPs may be involved in the increased monoamine concentrations induced by isatin. In conclusion, the present study demonstrated that HPLC-UV determination of isatin is a simple and accurate method. SUMMARY
FIGURE 6. Isatin concentration in the kidney after oral administration of isatin for 4 weeks to WKY rats. tained from urine extract separated by HPLC-UV showed a broad, but almost the same, shape compared with that of authentic isatin (Fig. 1). As shown in Figure 1, the retention time to the peak in the chromatogram of urine extract coincided well with that of authentic isatin. No peak was observed in chromatograms obtained from extracts of urine and kidneys. Exogenously administered isatin increased urinary and kidney levels of isatin in a dose-dependent manner. Moreover, the isatin concentration measured by this assay correlated significantly and positively with tribulin-like activity (MAO inhibitory activity), suggesting the specificity of that method. Functionally, isatin expresses approximately 77.4613.1% of its tribulin-like activity as MAO inhibitory activity. Isatin competitively inhibits the MAO activity of rat liver homogenate in a dosedependent manner and is a potent MAO inhibitor that is more active against MAO-B the MAO-A (Hamaue et al., 1992). Previous reports have shown that stress can induce a decrease in MAO activity. It is well known that exposure of adults rats to chronic environmental stress induces a reduction in MAO activity (Maura et al., 1974) and the development of chronic hypertension (Maura et al., 1979). We recently reported that isatin increased tribulin-like activity. Isatin also produced a significant and dosedependent increase in urinary norepinephrine excretion in WKYs. Further, the systolic blood pressure of WKYs that received an isatin dose of 200 mg/kg (124.365.45 mmHg, n58, P,0.01 vs. nondrug control WKY) was significantly higher than that of the nondrug control WKY (114.365.28, n58). Systolic blood pressure correlated significantly with urinary tribulin-like activity 4 weeks after treatment of WKYs with isatin (Hamaue et al., 1996). Urinary tribulin excretion increases under various conditions of stress and anxiety (Clow et al., 1988). Tribulin, which acts on central benzodiazepine receptors, has been proposed as an anxiety-promoting substance (Sandler et al., 1982). Peripheral benzodiazepine receptors are located in the mitochondrial center membrane with which MAO also is associated (Anholt, 1986). Cold-restraint stress increases tribulin in the rat heart and kidney (Armando et al., 1988). Although the physiological function of peripheral benzodiazepine receptors is still unknown, they are associated with ion-transport systems in both the heart and the kidney (Basile and Skolnick, 1988; Mestre et al., 1986). Isatin may, therefore, have a role in the ion-transport system in these tissues through action on mitochondrial MAO activity.
Endogenous MAO inhibitory components were first discovered in normal human urine. Two types of endogenous MAO inhibitor have been described: one is a peptide; the other is a nonpeptide. One of these inhibitors has been given the name tribulin. Tribulin is an endogenous, low-molecular-weight, nonpeptide MAO inhibitor. We previously identified isatin as one of the chemical structures of tribulin in both the urine and the brain of SHRSPs by using GCMS. In this study, we developed a convenient assay to determine isatin by using HPLC-UV. The recovery, the coefficient of variance and the sensitivity of the assay are promising. Isatin concentration correlated positively with tribulin-like activity in both urine (r50.924, P,0.001) and kidney extracts (r50.862, P,0.01) by using HPLC-UV. This assay to determine isatin may be useful in elucidating roles of isatin in various conditions of stress and disease. References Anholt R. R. (1986) Mitochondrial benzodiazepine receptors as potential modulators of inter-mediatory metabolism. Trends. Pharm. Sci. 6, 506– 511. Armando I., Levin G. and Barontini M. (1988) Stress increases benzodiazepine receptor ligand-monoamine oxidase inhibitory activity (tribuline) in tissues. J. Neural Transm. 71, 29–37. Basile A. S. and Skolnick P. (1988) Tissue specific regulation of “peripheraltype” benzodiazepine receptor density after chemical sympathectomy. Life Sci. 42, 273–283. Clow A., Glover V., Weg M. W., Walker P. L., Sheehan D. V., Carr D. V. and Sandler M. (1988) Urinary catecholamine metabolite and tribulin output during lactate infusion. Br. J. Psychiatry 152, 122–126. Elsworth J. D., Dewar D., Glover V., Goodwin B. L., Clow A. and Sandler M. (1986) Purification and characterization of tribulin, an endogenous inhibitor of monoamine oxidase and benzodiazepine receptor binding. J. Neural Transm. 67, 45–56. Glover V., Reveley M. A. and Sandler M. (1980) A monoamine oxidase inhibitor in human urine. Biochem. Pharmac. 29, 467–470. Glover V., Halket J. M., Watkins P. J., Clow A., Goodwin B. L. and Sandler, M. (1988) Isatin: identity with the purified endogenous monoamine oxidase inhibitor tribulin. J. Neurochem. 51, 656–659. Hamaue N., Minami M., Kanamaru Y., Togashi M., Monma Y., Ishikura M., Mahara R., Yamazaki N., Togashi H., Saito H. and Parvez S. H. (1992) Endogenous monoamine oxidase (MAO) inhibitor (tribulin-like activity) in the brain and urine of stroke-prone SHR. Biog. Amines 8, 401– 412. Hamaue N., Minami M., Kanamaru Y., Ishikura M., Saito H. and Parvez S. H. (1994) Identification of isatin, an endogenous MAO inhibitor, in the brain of stroke-prone SHR. Biog. Amines 10, 99–110. Hamaue N., Minami M., Hirafuji M., Monma Y., Yamazaki N., Togashi H., Saito H. and Parvez S. H. (1996) Significance of isatin, an endogenous MAO inhibitor, on blood pressure control and monoamine levels. Biog. Amines 12, 395–405. Jaffe M. (1886) Uever den Niederschlag, welchen Pikrinsa¨re in normalem Harn erzeugt und u¨ber eine neue Reaction des Kreatinins. Physiol. Chem. 10, 391. Maura G., Vaccari A., Gemignani A. and Cugurra F. (1974) Development of monoamine oxidase activity after chronic environmental stress in the rat. Environ. Physiol. Biochem. 4, 64–79. Maura G., Versace P. and Paudice P. (1979) Stress-induced hypertension and COMT activity in rats. Pharmac. Res. Commun. 11, 537–543. Mestre M., Garriot T., Neliat G., Uzau A., Renault C., Dubroeueq M. C.,
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