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Tryptophan glycoconjugate as a novel marker of renal function
Tryptophan Glycoconjugate as a Novel Marker of Renal Function Reiko Takahira, MD, Katsuhiko Yonemura, MD, PhD, Osamu Yonekawa, MD, PhD, Kunihiro Iwahara, MS, Takashi Kanno, MD, PhD, Yutaka Fujise, PhD, Akira Hishida, MD, PhD PURPOSE: Neither serum creatinine concentration nor creatinine clearance assess renal function accurately. Serum creatinine concentration is affected by muscle mass, and the creatinine clearance overestimates the glomerular filtration rate because of tubular secretion of creatinine. The present study was designed to determine whether serum concentrations of 2-(␣mannopyranosyl)-L-tryptophan (MPT), a tryptophan glycoconjugate, can be used as a marker of renal function. METHODS: Clearances of MPT and of inulin were compared in normal rats and in rats with cisplatin-induced acute renal failure. We also compared the clearances of MPT and of creatinine with inulin clearance in 25 patients with chronic renal disease. Serum concentrations of MPT and creatinine as a function of MPT clearance were determined in 108 patients with chronic renal disease. RESULTS: There was strong linear correlation between clear-
ances of MPT and inulin in rats (r ⫽ 0.97) and humans (r ⫽ 0.87), indicating that renal handling of MPT is similar to that of inulin. In humans, linear regression analyses indicated that MPT was a better indicator of inulin clearance than was creatinine clearance. At the same level of renal function, serum creatinine concentrations tended to be lower in patients with less muscle mass (as indicated by a urinary creatinine excretion ⬍1,000 mg in 24 hours) than in those who excreted ⬎1,000 mg in 24 hours, whereas serum MPT concentrations were not affected by creatinine excretion. CONCLUSION: MPT clearance can replace inulin clearance in the clinical setting. The serum MPT concentration is an accurate measure of renal function even in patients with diminished muscle mass, and thus is a better indicator of renal function than is the serum creatinine concentration. Am J Med. 2001; 110:192–197. 䉷2001 by Excerpta Medica, Inc.
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sure to radiolabeled materials and the need for exogenous administration. For these reasons, a more definitive and convenient measure of assessing renal function is desirable. We recently isolated a hydrophilic tetrahydro--carboline from human urine (16) that is endogenously synthesized in most vertebrates and has a tryptophanlike fluorescence spectrum. Gutsche et al (17) have reported that this substance is 2-(␣-mannopyranosyl)-L-tryptophan (MPT), a tryptophan c-glycoside with a molecular weight of 366 Da. MPT is found in serum and cerebrospinal fluid, but its metabolism and physiologic role are not yet known. The structure— characterized by a sugar moiety bound to the carbon—indicates that MPT is a stable substance in the body. We observed that serum MPT concentrations increased progressively as renal function declined in patients with chronic renal failure. Therefore, this study was designed to determine whether the measurement of the serum MPT concentration might be useful for assessing renal function in patients.
n accurate measure of renal function is of great importance in evaluating the progression of chronic renal failure and the effects of therapeutic strategies (1–7). Sequential measurements of serum creatinine concentration and creatinine clearance are inadequate as markers for assessing the glomerular filtration rate in patients with chronic renal failure, however, as the serum creatinine concentration is greatly influenced by muscle mass and the ingestion of high-protein meals (8 –11). Furthermore, tubular secretion of creatinine (12) varies inversely with the glomerular filtration rate (13,14). The slope of the reciprocal of the serum creatinine concentration is often misleading as a measure of the progression in chronic renal failure because of overestimation or underestimation of the rate of progression (15). Although clearance of radiolabeled materials closely approximates the glomerular filtration rate (7), these measurements have inherent problems, including expo-
From the First Department of Medicine (RT, AH), Hemodialysis Unit (KY), Department of Laboratory Medicine (KI, TK), and Department of Chemistry (YF), Hamamatsu University School of Medicine, and the Seirei Hamamatsu General Hospital (OY), Hamamatsu, Japan. Supported in part by the Clinical Pathological Research Foundation of Japan and in part by a Grant-in-Aid for Scientific Research for the Japanese Ministry of Education, Science and Culture (07672486). Requests for reprints should be addressed to Katsuhiko Yonemura, MD, PhD, Hemodialysis Unit, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431-3192, Japan. Manuscript submitted May 16, 2000, and accepted in revised form September 27, 2000. 192
䉷2001 by Excerpta Medica, Inc. All rights reserved.
SUBJECTS AND METHODS Animal Study Ten male Sprague-Dawley rats weighing 280 to 300 g had free access to standard rat chow and water. Cisplatin (5 mg/kg of body weight) or saline was injected intravenously. Five days after injection, clearance studies were performed. A tracheostomy tube and jugular venous 0002-9343/01/$–see front matter PII S0002-9343(00)00693-8
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Figure 1. MPT [2-(␣-mannopyranosyl)-L-tryptophan] clearance plotted as a function of inulin clearance in rats with acute renal failure induced by cisplatin and in untreated rats. There was strong positive correlation between clearances of MPT and inulin (y ⫽ 0.97 x ⫹0.037, r ⫽ 0.97, P ⬍0.0001).
Next, we enrolled 108 patients (58 men and 50 women, mean age 50 ⫾ 14 years) with chronic renal disease. We performed 24-hour urine collections to determine whether measurement of serum MPT concentration could assess renal function, independent of muscle mass. Creatinine clearances ranged between 6 and 215 mL/min per 1.73 m2. The correlations of urinary excretion of creatinine and MPT with body weight were determined. Concentrations of creatinine and MPT in serum and urine collected during 24 hours were measured. Serum concentrations of MPT and creatinine as a function of MPT clearance were determined. The patients were divided into two groups—those with a urinary creatinine excretion of ⱖ1,000 mg per 24 hours (n ⫽ 54) and those with a urinary creatinine excretion of ⬍1,000 mg per 24 hours (n ⫽ 54) as an estimate of muscle mass (10). Blood and urine samples were stored at ⫺70⬚C until assayed to determine creatinine, inulin, and MPT concentrations.
catheter were inserted under anesthesia with pentobarbital sodium (40 mg/kg of body weight intraperitoneally). A carotid artery catheter was used to collect blood samples. The left ureter was cannulated for urine collection through a flank incision. The [14C]inulin clearance was determined (18). In brief, [14C]inulin dissolved in saline (0.5 Ci/mL; Amersham, Arlington Heights, Illinois) was infused at a constant rate (2.5 mL/h) through the jugular venous catheter. After a 40-minute equilibration period, two 20-minute clearance studies were performed. Blood samples were collected at the midpoint of each clearance period to measure MPT and radioactivity of [14C]inulin.
Human Studies The protocols were approved by the Ethics Committee of Hamamatsu University School of Medicine. Informed consent was obtained from each patient. We enrolled 25 patients with chronic renal disease (13 men and 12 women, mean [⫾ SD] age 49 ⫾ 16 years). None of the patients had a history of voiding difficulties; complete bladder emptying after voluntary voiding was confirmed with ultrasonography. The cause of chronic renal disease was chronic glomerulonephritis in 15 patients, nephrosclerosis in 6, diabetic nephropathy in 1, Alport’s syndrome in 1, chronic tubulointerstitial nephritis in 1, and polycystic kidney disease in 1 patient. After an overnight fast, each patient ingested tap water (20 mL/kg of body weight). Patients remained supine throughout the observation period. Inulin clearance was determined (19). A priming dose of inulin (Wako, Osaka, Japan), dissolved in 5% dextrose, was injected and followed by continuous infusion that was calculated to maintain a steady serum concentration of 10 mg/dL. After an equilibration period of 60 minutes, three 20minute clearance studies were performed. Blood samples were collected at the midpoint of each clearance period.
Figure 2. Clearances of (A) creatine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] plotted as a function of inulin clearance in 25 patients with chronic renal disease. The clearances of both creatinine and MPT were significantly correlated with inulin clearance. However, the regression line of MPT clearance (y ⫽ 1.09 x ⫹9.09, r ⫽ 0.87, P ⬍0.0001) as a function of inulin clearance was much closer to the line of identity than that of creatinine clearance (y ⫽ 1.84 x ⫹16.6, r ⫽ 0.81, P ⬍0.0001). The dotted lines represent the line of identity.
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Table 1. Demographic and Clinical Characteristics of 54 Patients with Urinary Creatinine Excretion ⱖ1,000 mg in 24 Hours and 54 Patients with ⬍1,000 mg in 24 Hours Urinary Creatinine Excretion ⱖ1,000 mg/24 Hours
Urinary Creatinine ⬍1,000 mg/24 Hours
P Value
39/15 49.4 ⫾ 12.7 22.4 ⫾ 2.32 52.1 ⫾ 27.1 480 ⫾ 417 1.72 ⫾ 1.53 1285 ⫾ 243
19/35 50.6 ⫾ 14.6 20.6 ⫾ 2.48 33.7 ⫾ 25.9 776 ⫾ 680 2.22 ⫾ 1.89 770 ⫾ 152
0.0001 0.648 0.0002 0.0008 0.008 0.139 ⬍0.0001
Men/women Age (years) Body mass index (kg/m2) MPT clearance (mL/min/1.73 m2) Serum MPT (nmol/L) Serum creatinine (mg/dL) Urinary creatinine (mg/24 hours) MPT ⫽ 2-(␣-mannopyranosyl)-L-tryptophan.
Measurements 14
Radioactivity of [ C]inulin in serum and urine in the animal study was measured using 50 L of each sample added to 10 mL of aqueous counting scintillant ACS II (Amersham, Arlington Heights, Illinois). Radioactivity was determined in a Beckman LC-500TA scintillation counter (Beckman, Fullerton, California). Creatinine concentrations in serum and urine were determined by the enzymatic method using a Hitachi 7350 autoanalyzer (Hitachi, Tokyo, Japan). Inulin was determined using the anthrone method (21). The intraassay coefficient of variation was 1.3%, with an interassay coefficient of variation of 6.3%. MPT concentrations were determined by high-performance liquid chromatography (HPLC) (16). In brief, 500 L serum was mixed with 400 L cold hydrogen chloride solution (0.1 M) and ascorbic acid (10 g/L), and 100 L of 60% perchloric acid. This mixture was placed on ice for 30 minutes and then centrifuged at 2,000g for 15 minutes at 4⬚C. Supernatant was then filtered through a 0.45-m membrane filter. A 100 L aliquot of the filtrate was used for HPLC analysis. The HPLC system consisted of a model 600E system controller (Waters Associates, Milford, Massachusetts), a model 7125 injector (Rheodyne, Berkeley, California) equipped with a 100 L sample loop, a model FP-210 spectrofluorometer (JASCO, Tokyo, Japan), and a model RC-250 data processor (JASCO). A 4-mm ⫻ 25-cm column prepared with Finepak SIL C18-T5 (5-m particle size, JASCO) was used. Renal clearances of creatinine, inulin, and MPT were adjusted to a body surface area of 1.73 m2.
Statistical Analyses
Continuous values are presented as mean ⫾ SD. The correlation between variables was assessed by linear regression analysis; the slopes and y intercepts between groups were compared using analysis of covariance. Continuous variables were compared using Student’s t tests. Dichotomous variables were compared using chi-square tests. Analyses were performed with a standard software package (Statview 4.1; Abacus Concepts, Berkeley, Califor194
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nia). The nonlinear regressions in Figures 4 and 5 were determined with a standard software package (Microsoft Excel; Microsoft Corporation)
RESULTS Animal Study There was a strong positive correlation between clearances of MPT and [14C]inulin (r ⫽ 0.97, P ⬍0.0001) in normal rats and in rats with cisplatin-induced renal failure (Figure 1). The slope of the regression line was 0.97.
Human Study
Clearances of both MPT (r ⫽ 0.87) and creatinine (r ⫽ 0.86) were significantly correlated with inulin clearance (Figure 2). Although the slopes of the regression lines were not significantly different (P ⬎0.1), there was a significant difference in the y intercept between the two lines (P ⬍0.001).
Effects of Urinary Creatinine Excretion MPT clearance was about 35% lower and serum MPT concentration was about 60% greater in patients whose urinary creatinine excretion was ⬍1,000 mg per 24 hours (Table 1). Serum creatinine concentration was not significantly different between the two groups. Urinary excretions of both creatinine and MPT were significantly correlated with body weight (Figure 3), but the correlation between excretion of MPT and body weight (r ⫽ 0.32) was lower than that between excretion of creatinine and body weight (r ⫽ 0.66). Serum creatinine concentrations were lower in patients with urinary creatinine excretion of ⬍1,000 mg per 24 hours than those with urinary creatinine excretion of ⱖ1,000 mg in 24 hours even at the same MPT clearance (Figure 4). However, serum MPT concentrations were similar in the two groups at the same MPT clearance. Although the reciprocal of the serum creatinine concentration was correlated with MPT clearance (Figure 5A), the slopes and the y intercepts of the regression lines were statistically different between the two groups (P ⬍0.001).
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Figure 3. Correlation between urinary excretion of (A) creatinine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] and body weight in 108 patients. Urinary excretions of both creatinine and MPT were significantly correlated with body weight. However, the correlation between excretion of MPT and body weight (y ⫽ 214 x ⫹8,234, r ⫽ 0.32, P ⫽ 0.001) was weaker than that between urinary excretion of creatinine and body weight (y ⫽ 26.8 x ⫺459, r ⫽ 0.66, P ⬍0.0001).
By contrast, when the reciprocal of the serum MPT concentration was correlated with the MPT clearance, there were no significant differences in either the slope (P ⬎0.1) or the y intercept (P ⬎0.1) between these two groups.
DISCUSSION An ideal method for the assessment of renal function involves determining the clearance of an endogenous substance that is freely filtered through the glomerulus but neither secreted nor reabsorbed by the tubule. Although creatinine clearance is generally used for assessing the glomerular filtration rate, it overestimates the true rate because tubular secretion of creatinine varies inversely with the glomerular filtration rate (12,13). In addition, renal tubular secretion of creatinine is inhibited by med-
Figure 4. Serum concentrations of (A) creatinine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] plotted as a function of MPT clearance in patients with urinary creatinine excretion ⱖ1,000 or ⬍1,000 mg per 24 hours. Serum creatinine concentrations were significantly lower in the 54 patients with urinary creatinine excretion ⬍1,000 mg/24 hours (open circles and dashed line; y ⫽ 27.6 x ⫺0.90, r ⫽ 0.93) than in 54 patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 42.2 x ⫺0.92, r ⫽ 0.95). Serum MPT concentrations were similar in patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 9,930 x ⫺0.87, r ⫽ 0.95) and those with urinary creatinine excretion ⬍1,000 mg per 24 hours (open circles and dashed line; y ⫽ 8,719 x ⫺0.86, r ⫽ 0.94).
ications such as calcitriol or cimetidine (22–26), which are often used by patients with chronic renal failure. The gold standard for the measurement of the glomerular filtration rate is renal clearance of inulin. However, the utility of inulin is limited because of the technical difficulties inherent in its measurement and the need for exogenous administration. Therefore, a more definitive and convenient measure for assessing renal function would be worthwhile. In this study, we have shown that MPT clearance was almost identical to inulin clearance in experimental animals, indicating a similar renal handling of the two molecules. Thus, MPT may be an ideal endogenous substance to assess the glomerular filtration rate. Endogenous MPT
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Figure 5. Reciprocals of serum concentrations of (A) creatinine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] plotted as a function of MPT clearance, by urinary excretion of creatinine. The reciprocal of the serum concentration of creatinine was significantly correlated with MPT clearance in patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 0.016 x ⫹0.09, r ⫽ 0.91, P ⬍0.0001) and those with urinary creatinine excretion ⬍1,000 mg per 24 hours (open circles and dashed line; y ⫽ 0.024 x ⫹0.07, r ⫽ 0.91, P ⬍0.0001). The reciprocal of the serum concentration of MPT was significantly correlated with MPT clearance in patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 4.97 ⫻ 10⫺5 x ⫹0.001, r ⫽ 0.91, P ⬍0.0001) and those with urinary creatinine excretion ⬍1,000 mg per 24 hours (open circles and dashed line; y ⫽ 5.01 ⫻ 10⫺5 x ⫹0.001, r ⫽ 0.85, P ⬍0.0001).
clearance in humans correlated strongly with inulin clearance; the regression line of MPT clearance plotted as a function of inulin clearance had a slope close to 1.0 and an intercept close to 0. In accordance with previous studies (12,13,27,28), we observed that creatinine clearance overestimated inulin clearance. These findings suggest that MPT clearance may be preferable to creatinine clearance as a measure of the glomerular filtration rate. Indeed, endogenous MPT clearance may be able to replace inulin clearance in the clinical setting, especially if in vitro studies establish that MPT is transported across the renal tubules. Creatinine is generated from the nonenzymatic conversion of creatine and phosphocreatine in muscle (29) in proportion to muscle mass. Thus, the serum creatinine 196
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concentration is affected by muscle mass. We observed that the serum creatinine concentration was lower in subjects with less muscle mass (as measured by urinary excretion of creatinine) than in those with greater muscle mass, even at the same glomerular filtration rate. By contrast, serum MPT concentrations were not affected by muscle mass. Although the metabolism and the physiologic role of MPT remain to be clarified, these findings suggest that MPT is not primarily synthesized in muscle. Thus, serum MPT concentration may be a better indicator of renal function than the serum creatinine concentration, especially in patients with reduced muscle mass, such as women, the elderly, and those with muscle atrophy. There are some limitations, however, to the determination of the serum MPT concentration. It can be measured only by the HPLC method, which is time consuming and expensive. A new and more convenient measuring technique is being developed, however. Additional studies focusing on whether serum MPT concentration is affected by a variety of conditions, including sepsis, malnutrition, and critical illness, are needed. Because MPT is derived from tryptophan, it is reasonable to hypothesize that serum concentration and urinary excretion of MPT may be affected by dietary protein intake. However, the profiles and conformations of tryptophan conjugates that originate from food are quite different from those of MPT (17). In conclusion, both the serum concentration and the endogenous clearance of MPT appear to be accurate measures of renal function. Endogenous MPT clearance may be able to replace inulin clearance as the gold standard for measuring the glomerular filtration rate.
REFERENCES 1. Maschio G, Alberti D, Janin G, et al. Effect of the angiotensinconverting-enzyme inhibitor benazepril on the progression of chronic renal insufficiency. N Engl J Med. 1996;334:939 –945. 2. Lewis EJ, Hunsicker LG, Bain RP, Rohde RD, for the Collaborative Study Group. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. N Engl J Med. 1993;329:1456 –1462, . 3. Hannedouche T, Landais P, Goldfarb B, et al. Randomized controlled trial of enalapril and  blockers in non-diabetic chronic renal failure. BMJ. 1994;309:833– 837. 4. Ihle BU, Becker GJ, Whitworth JA,et al. The effect of protein restriction on the progression of renal insufficiency. N Engl J Med. 1989;321:1773–1777. 5. Zucchelli P, Zuccala` A, Borghi M, et al. Long-term comparison between captopril and nifedipine in the progression of renal insufficiency. Kidney Int. 1992;42:452– 458. 6. Mitch WE, Walser M, Steinman TI, et al. The effect of a keto acidamino acid supplement to a restricted diet on the progression of chronic renal failure. N Engl J Med. 1984;311:623– 629. 7. Walser M, Hill SB, Ward L, Magder L. A crossover comparison of progression of chronic renal failure: ketoacids versus amino acids. Kidney Int. 1993;43:933–939. 8. Jacobsen FK, Christensen CK, Mogensen CE, et al. Pronounced
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increase in serum creatinine concentration after eating cooked meal. BMJ. 1979;1:1049 –1050. Jacobsen FK, Christensen CK, Mogensen CE, Heilskov NSC. Evaluation of kidney function after meals. Lancet. 1980;1:319. Laville M, Hadj-Aissa A, Pozet N, et al. Restrictions on use of creatinine clearance for measurement of renal functional reserve. Nephron. 1989;51:233–236. Herrera J, Rodrı´guez-Iturbe B. Stimulation of tubular secretion of creatinine in health and in conditions associated with reduced nephron mass: evidence for a tubular functional reserve. Nephrol Dial Transplant. 1998;13:623– 629. Arendshorst WJ, Selkurt EE. Renal tubular mechanisms for creatinine secretion in the guinea pig. Am J Physiol. 1970;218:1661–1670. Walser M, Drew HH, LaFrance ND. Creatinine measurements often yield false estimates of progression in chronic renal failure. Kidney Int. 1988;34:412– 418. Shemesh O, Golbetz H, Kriss JP, Myers BD. Limitations of creatinine as a filtration marker in glomerulopathic patients. Kidney Int. 1985;28:830 – 838. Walser M, Drew HH, LaFrance ND. Reciprocal creatinine slopes often give erroneous estimates of progression of chronic renal failure. Kidney Int. 1989;36(suppl 27):S81–S85. Horiuchi K, Yonekawa O, Iwahara K, et al. A hydrophilic tetrahydro--carboline in human urine. J Biochem. 1994;115:362–366. Gutsche B, Grun C, Scheutzow D, Herderich M. Tryptophan glycoconjugates in food and human urine. Biochem J. 1999;343:11–19. Hishisa A, Yonemura K, Ohishi K, et al. The effect of saline loading on uranium-induced acute renal failure in rats. Kidney Int. 1988;33: 942–946. Florijn KW, Barendregt JNM, Lentjes EGWM, et al. Glomerular
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filtration rate measurement by “single-shot” injection of inulin. Kidney Int. 1994;46:252–259. Heymsfield SB, Arteaga C, McManus C, et al. Measurement of muscle mass in humans: validity of the 24-hour urinary creatinine method. Am J Clin Nutr. 1983;37:478 – 494. Scott TA, Melvin EH. Determination of dextran with anthrone. Analyt Chem. 1953;25:1653–1661. Bertoli M, Luisetto G, Ruffatti A, et al. Renal function during calcitriol therapy in chronic renal failure. Clin Nephrol. 1990;33:98 – 102. Perez A, Raab R, Chen TC, et al. Safety and efficacy of oral calcitriol (1,25-dihydroxyvitamin D3) for the treatment of psoriasis. Br J Dermatol. 1996;134:1070 –1078. Zaltzman JS, Whiteside C, Cattran DC, et al. Accurate measurement of impaired glomerular filtration rate using single-dose oral cimetidine. Am J Kidney Dis. 1996;27:504 –511. Ixkes MC, Koopman MG, van Acker BA, et al. Cimetidine improves GFR-estimation by the Cockcroft and Gault formula. Clin Nephrol. 1997;47:229 –236. Walser M. Assessing renal function from creatinine measurements in adults with chronic renal failure. Am J Kidney Dis. 1998;32:23– 31. Berlyne GM. Endogenous creatinine clearance and the glomerular filtration rate. Am Heart J. 1965;70:143–145. Carrie BJ, Golbetz HV, Michaels AB, Myers BD. Creatinine: an inadequate filtration marker in glomerular diseases. Am J Med. 1980;69:177–182. Borsook H, Dubnoff JW. The hydrolysis of phosphocreatine and the origin of urinary creatinine. J Biol Chem. 1947;168:493–510.
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ances of MPT and inulin in rats (r ⫽ 0.97) and humans (r ⫽ 0.87), indicating that renal handling of MPT is similar to that of inulin. In humans, linear regression analyses indicated that MPT was a better indicator of inulin clearance than was creatinine clearance. At the same level of renal function, serum creatinine concentrations tended to be lower in patients with less muscle mass (as indicated by a urinary creatinine excretion ⬍1,000 mg in 24 hours) than in those who excreted ⬎1,000 mg in 24 hours, whereas serum MPT concentrations were not affected by creatinine excretion. CONCLUSION: MPT clearance can replace inulin clearance in the clinical setting. The serum MPT concentration is an accurate measure of renal function even in patients with diminished muscle mass, and thus is a better indicator of renal function than is the serum creatinine concentration. Am J Med. 2001; 110:192–197. 䉷2001 by Excerpta Medica, Inc.
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sure to radiolabeled materials and the need for exogenous administration. For these reasons, a more definitive and convenient measure of assessing renal function is desirable. We recently isolated a hydrophilic tetrahydro--carboline from human urine (16) that is endogenously synthesized in most vertebrates and has a tryptophanlike fluorescence spectrum. Gutsche et al (17) have reported that this substance is 2-(␣-mannopyranosyl)-L-tryptophan (MPT), a tryptophan c-glycoside with a molecular weight of 366 Da. MPT is found in serum and cerebrospinal fluid, but its metabolism and physiologic role are not yet known. The structure— characterized by a sugar moiety bound to the carbon—indicates that MPT is a stable substance in the body. We observed that serum MPT concentrations increased progressively as renal function declined in patients with chronic renal failure. Therefore, this study was designed to determine whether the measurement of the serum MPT concentration might be useful for assessing renal function in patients.
n accurate measure of renal function is of great importance in evaluating the progression of chronic renal failure and the effects of therapeutic strategies (1–7). Sequential measurements of serum creatinine concentration and creatinine clearance are inadequate as markers for assessing the glomerular filtration rate in patients with chronic renal failure, however, as the serum creatinine concentration is greatly influenced by muscle mass and the ingestion of high-protein meals (8 –11). Furthermore, tubular secretion of creatinine (12) varies inversely with the glomerular filtration rate (13,14). The slope of the reciprocal of the serum creatinine concentration is often misleading as a measure of the progression in chronic renal failure because of overestimation or underestimation of the rate of progression (15). Although clearance of radiolabeled materials closely approximates the glomerular filtration rate (7), these measurements have inherent problems, including expo-
From the First Department of Medicine (RT, AH), Hemodialysis Unit (KY), Department of Laboratory Medicine (KI, TK), and Department of Chemistry (YF), Hamamatsu University School of Medicine, and the Seirei Hamamatsu General Hospital (OY), Hamamatsu, Japan. Supported in part by the Clinical Pathological Research Foundation of Japan and in part by a Grant-in-Aid for Scientific Research for the Japanese Ministry of Education, Science and Culture (07672486). Requests for reprints should be addressed to Katsuhiko Yonemura, MD, PhD, Hemodialysis Unit, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431-3192, Japan. Manuscript submitted May 16, 2000, and accepted in revised form September 27, 2000. 192
䉷2001 by Excerpta Medica, Inc. All rights reserved.
SUBJECTS AND METHODS Animal Study Ten male Sprague-Dawley rats weighing 280 to 300 g had free access to standard rat chow and water. Cisplatin (5 mg/kg of body weight) or saline was injected intravenously. Five days after injection, clearance studies were performed. A tracheostomy tube and jugular venous 0002-9343/01/$–see front matter PII S0002-9343(00)00693-8
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Figure 1. MPT [2-(␣-mannopyranosyl)-L-tryptophan] clearance plotted as a function of inulin clearance in rats with acute renal failure induced by cisplatin and in untreated rats. There was strong positive correlation between clearances of MPT and inulin (y ⫽ 0.97 x ⫹0.037, r ⫽ 0.97, P ⬍0.0001).
Next, we enrolled 108 patients (58 men and 50 women, mean age 50 ⫾ 14 years) with chronic renal disease. We performed 24-hour urine collections to determine whether measurement of serum MPT concentration could assess renal function, independent of muscle mass. Creatinine clearances ranged between 6 and 215 mL/min per 1.73 m2. The correlations of urinary excretion of creatinine and MPT with body weight were determined. Concentrations of creatinine and MPT in serum and urine collected during 24 hours were measured. Serum concentrations of MPT and creatinine as a function of MPT clearance were determined. The patients were divided into two groups—those with a urinary creatinine excretion of ⱖ1,000 mg per 24 hours (n ⫽ 54) and those with a urinary creatinine excretion of ⬍1,000 mg per 24 hours (n ⫽ 54) as an estimate of muscle mass (10). Blood and urine samples were stored at ⫺70⬚C until assayed to determine creatinine, inulin, and MPT concentrations.
catheter were inserted under anesthesia with pentobarbital sodium (40 mg/kg of body weight intraperitoneally). A carotid artery catheter was used to collect blood samples. The left ureter was cannulated for urine collection through a flank incision. The [14C]inulin clearance was determined (18). In brief, [14C]inulin dissolved in saline (0.5 Ci/mL; Amersham, Arlington Heights, Illinois) was infused at a constant rate (2.5 mL/h) through the jugular venous catheter. After a 40-minute equilibration period, two 20-minute clearance studies were performed. Blood samples were collected at the midpoint of each clearance period to measure MPT and radioactivity of [14C]inulin.
Human Studies The protocols were approved by the Ethics Committee of Hamamatsu University School of Medicine. Informed consent was obtained from each patient. We enrolled 25 patients with chronic renal disease (13 men and 12 women, mean [⫾ SD] age 49 ⫾ 16 years). None of the patients had a history of voiding difficulties; complete bladder emptying after voluntary voiding was confirmed with ultrasonography. The cause of chronic renal disease was chronic glomerulonephritis in 15 patients, nephrosclerosis in 6, diabetic nephropathy in 1, Alport’s syndrome in 1, chronic tubulointerstitial nephritis in 1, and polycystic kidney disease in 1 patient. After an overnight fast, each patient ingested tap water (20 mL/kg of body weight). Patients remained supine throughout the observation period. Inulin clearance was determined (19). A priming dose of inulin (Wako, Osaka, Japan), dissolved in 5% dextrose, was injected and followed by continuous infusion that was calculated to maintain a steady serum concentration of 10 mg/dL. After an equilibration period of 60 minutes, three 20minute clearance studies were performed. Blood samples were collected at the midpoint of each clearance period.
Figure 2. Clearances of (A) creatine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] plotted as a function of inulin clearance in 25 patients with chronic renal disease. The clearances of both creatinine and MPT were significantly correlated with inulin clearance. However, the regression line of MPT clearance (y ⫽ 1.09 x ⫹9.09, r ⫽ 0.87, P ⬍0.0001) as a function of inulin clearance was much closer to the line of identity than that of creatinine clearance (y ⫽ 1.84 x ⫹16.6, r ⫽ 0.81, P ⬍0.0001). The dotted lines represent the line of identity.
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Table 1. Demographic and Clinical Characteristics of 54 Patients with Urinary Creatinine Excretion ⱖ1,000 mg in 24 Hours and 54 Patients with ⬍1,000 mg in 24 Hours Urinary Creatinine Excretion ⱖ1,000 mg/24 Hours
Urinary Creatinine ⬍1,000 mg/24 Hours
P Value
39/15 49.4 ⫾ 12.7 22.4 ⫾ 2.32 52.1 ⫾ 27.1 480 ⫾ 417 1.72 ⫾ 1.53 1285 ⫾ 243
19/35 50.6 ⫾ 14.6 20.6 ⫾ 2.48 33.7 ⫾ 25.9 776 ⫾ 680 2.22 ⫾ 1.89 770 ⫾ 152
0.0001 0.648 0.0002 0.0008 0.008 0.139 ⬍0.0001
Men/women Age (years) Body mass index (kg/m2) MPT clearance (mL/min/1.73 m2) Serum MPT (nmol/L) Serum creatinine (mg/dL) Urinary creatinine (mg/24 hours) MPT ⫽ 2-(␣-mannopyranosyl)-L-tryptophan.
Measurements 14
Radioactivity of [ C]inulin in serum and urine in the animal study was measured using 50 L of each sample added to 10 mL of aqueous counting scintillant ACS II (Amersham, Arlington Heights, Illinois). Radioactivity was determined in a Beckman LC-500TA scintillation counter (Beckman, Fullerton, California). Creatinine concentrations in serum and urine were determined by the enzymatic method using a Hitachi 7350 autoanalyzer (Hitachi, Tokyo, Japan). Inulin was determined using the anthrone method (21). The intraassay coefficient of variation was 1.3%, with an interassay coefficient of variation of 6.3%. MPT concentrations were determined by high-performance liquid chromatography (HPLC) (16). In brief, 500 L serum was mixed with 400 L cold hydrogen chloride solution (0.1 M) and ascorbic acid (10 g/L), and 100 L of 60% perchloric acid. This mixture was placed on ice for 30 minutes and then centrifuged at 2,000g for 15 minutes at 4⬚C. Supernatant was then filtered through a 0.45-m membrane filter. A 100 L aliquot of the filtrate was used for HPLC analysis. The HPLC system consisted of a model 600E system controller (Waters Associates, Milford, Massachusetts), a model 7125 injector (Rheodyne, Berkeley, California) equipped with a 100 L sample loop, a model FP-210 spectrofluorometer (JASCO, Tokyo, Japan), and a model RC-250 data processor (JASCO). A 4-mm ⫻ 25-cm column prepared with Finepak SIL C18-T5 (5-m particle size, JASCO) was used. Renal clearances of creatinine, inulin, and MPT were adjusted to a body surface area of 1.73 m2.
Statistical Analyses
Continuous values are presented as mean ⫾ SD. The correlation between variables was assessed by linear regression analysis; the slopes and y intercepts between groups were compared using analysis of covariance. Continuous variables were compared using Student’s t tests. Dichotomous variables were compared using chi-square tests. Analyses were performed with a standard software package (Statview 4.1; Abacus Concepts, Berkeley, Califor194
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nia). The nonlinear regressions in Figures 4 and 5 were determined with a standard software package (Microsoft Excel; Microsoft Corporation)
RESULTS Animal Study There was a strong positive correlation between clearances of MPT and [14C]inulin (r ⫽ 0.97, P ⬍0.0001) in normal rats and in rats with cisplatin-induced renal failure (Figure 1). The slope of the regression line was 0.97.
Human Study
Clearances of both MPT (r ⫽ 0.87) and creatinine (r ⫽ 0.86) were significantly correlated with inulin clearance (Figure 2). Although the slopes of the regression lines were not significantly different (P ⬎0.1), there was a significant difference in the y intercept between the two lines (P ⬍0.001).
Effects of Urinary Creatinine Excretion MPT clearance was about 35% lower and serum MPT concentration was about 60% greater in patients whose urinary creatinine excretion was ⬍1,000 mg per 24 hours (Table 1). Serum creatinine concentration was not significantly different between the two groups. Urinary excretions of both creatinine and MPT were significantly correlated with body weight (Figure 3), but the correlation between excretion of MPT and body weight (r ⫽ 0.32) was lower than that between excretion of creatinine and body weight (r ⫽ 0.66). Serum creatinine concentrations were lower in patients with urinary creatinine excretion of ⬍1,000 mg per 24 hours than those with urinary creatinine excretion of ⱖ1,000 mg in 24 hours even at the same MPT clearance (Figure 4). However, serum MPT concentrations were similar in the two groups at the same MPT clearance. Although the reciprocal of the serum creatinine concentration was correlated with MPT clearance (Figure 5A), the slopes and the y intercepts of the regression lines were statistically different between the two groups (P ⬍0.001).
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Figure 3. Correlation between urinary excretion of (A) creatinine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] and body weight in 108 patients. Urinary excretions of both creatinine and MPT were significantly correlated with body weight. However, the correlation between excretion of MPT and body weight (y ⫽ 214 x ⫹8,234, r ⫽ 0.32, P ⫽ 0.001) was weaker than that between urinary excretion of creatinine and body weight (y ⫽ 26.8 x ⫺459, r ⫽ 0.66, P ⬍0.0001).
By contrast, when the reciprocal of the serum MPT concentration was correlated with the MPT clearance, there were no significant differences in either the slope (P ⬎0.1) or the y intercept (P ⬎0.1) between these two groups.
DISCUSSION An ideal method for the assessment of renal function involves determining the clearance of an endogenous substance that is freely filtered through the glomerulus but neither secreted nor reabsorbed by the tubule. Although creatinine clearance is generally used for assessing the glomerular filtration rate, it overestimates the true rate because tubular secretion of creatinine varies inversely with the glomerular filtration rate (12,13). In addition, renal tubular secretion of creatinine is inhibited by med-
Figure 4. Serum concentrations of (A) creatinine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] plotted as a function of MPT clearance in patients with urinary creatinine excretion ⱖ1,000 or ⬍1,000 mg per 24 hours. Serum creatinine concentrations were significantly lower in the 54 patients with urinary creatinine excretion ⬍1,000 mg/24 hours (open circles and dashed line; y ⫽ 27.6 x ⫺0.90, r ⫽ 0.93) than in 54 patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 42.2 x ⫺0.92, r ⫽ 0.95). Serum MPT concentrations were similar in patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 9,930 x ⫺0.87, r ⫽ 0.95) and those with urinary creatinine excretion ⬍1,000 mg per 24 hours (open circles and dashed line; y ⫽ 8,719 x ⫺0.86, r ⫽ 0.94).
ications such as calcitriol or cimetidine (22–26), which are often used by patients with chronic renal failure. The gold standard for the measurement of the glomerular filtration rate is renal clearance of inulin. However, the utility of inulin is limited because of the technical difficulties inherent in its measurement and the need for exogenous administration. Therefore, a more definitive and convenient measure for assessing renal function would be worthwhile. In this study, we have shown that MPT clearance was almost identical to inulin clearance in experimental animals, indicating a similar renal handling of the two molecules. Thus, MPT may be an ideal endogenous substance to assess the glomerular filtration rate. Endogenous MPT
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Figure 5. Reciprocals of serum concentrations of (A) creatinine and (B) MPT [2-(␣-mannopyranosyl)-L-tryptophan] plotted as a function of MPT clearance, by urinary excretion of creatinine. The reciprocal of the serum concentration of creatinine was significantly correlated with MPT clearance in patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 0.016 x ⫹0.09, r ⫽ 0.91, P ⬍0.0001) and those with urinary creatinine excretion ⬍1,000 mg per 24 hours (open circles and dashed line; y ⫽ 0.024 x ⫹0.07, r ⫽ 0.91, P ⬍0.0001). The reciprocal of the serum concentration of MPT was significantly correlated with MPT clearance in patients with urinary creatinine excretion ⱖ1,000 mg per 24 hours (closed circles and solid line; y ⫽ 4.97 ⫻ 10⫺5 x ⫹0.001, r ⫽ 0.91, P ⬍0.0001) and those with urinary creatinine excretion ⬍1,000 mg per 24 hours (open circles and dashed line; y ⫽ 5.01 ⫻ 10⫺5 x ⫹0.001, r ⫽ 0.85, P ⬍0.0001).
clearance in humans correlated strongly with inulin clearance; the regression line of MPT clearance plotted as a function of inulin clearance had a slope close to 1.0 and an intercept close to 0. In accordance with previous studies (12,13,27,28), we observed that creatinine clearance overestimated inulin clearance. These findings suggest that MPT clearance may be preferable to creatinine clearance as a measure of the glomerular filtration rate. Indeed, endogenous MPT clearance may be able to replace inulin clearance in the clinical setting, especially if in vitro studies establish that MPT is transported across the renal tubules. Creatinine is generated from the nonenzymatic conversion of creatine and phosphocreatine in muscle (29) in proportion to muscle mass. Thus, the serum creatinine 196
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concentration is affected by muscle mass. We observed that the serum creatinine concentration was lower in subjects with less muscle mass (as measured by urinary excretion of creatinine) than in those with greater muscle mass, even at the same glomerular filtration rate. By contrast, serum MPT concentrations were not affected by muscle mass. Although the metabolism and the physiologic role of MPT remain to be clarified, these findings suggest that MPT is not primarily synthesized in muscle. Thus, serum MPT concentration may be a better indicator of renal function than the serum creatinine concentration, especially in patients with reduced muscle mass, such as women, the elderly, and those with muscle atrophy. There are some limitations, however, to the determination of the serum MPT concentration. It can be measured only by the HPLC method, which is time consuming and expensive. A new and more convenient measuring technique is being developed, however. Additional studies focusing on whether serum MPT concentration is affected by a variety of conditions, including sepsis, malnutrition, and critical illness, are needed. Because MPT is derived from tryptophan, it is reasonable to hypothesize that serum concentration and urinary excretion of MPT may be affected by dietary protein intake. However, the profiles and conformations of tryptophan conjugates that originate from food are quite different from those of MPT (17). In conclusion, both the serum concentration and the endogenous clearance of MPT appear to be accurate measures of renal function. Endogenous MPT clearance may be able to replace inulin clearance as the gold standard for measuring the glomerular filtration rate.
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