Coupled Enzymatic Assay for the Determination of Sucrose

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

244, 103–109 (1997)

AB969865

Coupled Enzymatic Assay for the Determination of Sucrose Earle W. Holmes Departments of Pathology and Molecular and Cellular Biochemistry, Loyola University Stritch School of Medicine, Maywood, Illinois 60153

Received September 12, 1996

An enzymatic spectrophotometric assay for the determination of sucrose in unextracted samples of serum and urine was developed. The method entailed the coupling of invertase-catalyzed sucrose hydrolysis with a fructose dehydrogenase-catalyzed oxidation of the liberated fructose. The latter reaction generated reducing equivalents that were transferred to a tetrazolium salt with a concomitant increase in absorbance at 570 nm. The assay, which was carried out in microtiter plates, had a minimum detectable sucrose concentration of 0.03 mmol/liter and run-to-run and withinrun coefficients of variation of 7.5 and 6.7%, respectively, and showed a good correlation with urine sucrose determination by GLC (r Å 0.92). The assay range of 0.03–2.10 mmol/liter is suitable for the quantitation of serum sucrose following iv administration and for the quantitation of urine sucrose at basal levels and following the consumption of an oral test dose of sucrose. This method was used to analyze urine samples from a group of human subjects who consumed 20 g of sucrose for the assessment of gastroduodenal permeability. This convenient assay provides for the rapid and specific estimation of sucrose and has the potential to be used in a variety of manual, semiautomated, or automated formats. q 1997 Academic Press, Inc.

Much of the effort toward the development of methods for the quantitative determination of sucrose has been directed toward applications in the area of plant physiology and the food sciences. However, there has also been a steady interest in the measurement of sucrose in animal tissues and body fluids for the purposes of investigating the excretion of dietary sucrose in health and disease (1, 2) and evaluating sucrose uptake and metabolism in the gastrointestinal tract (3). Sucrose has also been used as a marker for the estimation of extracellular fluid volume (4), and the measurement

of serum sucrose following an intravenous dose continues to be used to investigate this parameter in children and adults (5). The newest application of sucrose determination in humans involves the measurement of urinary excretion following the administration of an oral test dose of the sugar (6). Sucrose challenge tests of this sort appear to be sensitive and specific indicators of increased gastroduodenal permeability in animals and humans where sucrose excretion has been shown to increase in proportion to the severity of epithelial damage (7). Increased permeability of orally administered sucrose has been observed in subjects with gastric ulcers (7), alcohol- (8, 9) and nonsteroidal anti-inflammatory drug-mediated gastropathies (6), and celiac disease (10). Much of the current enthusiasm for the sucrose challenge test is due to the possibility that it might constitute a noninvasive, inexpensive alternative to endoscopy as a means for assessing integrity of the GI tract. There are three general categories of analytical methods for the determination of sucrose in mammalian body fluids. Colorimetric methods using fructoseselective modifications of the anthrone reaction (11) have the required sensitivity and specificity for serum determinations, but the harsh reaction conditions preclude the development of automated analyses. The chromatographic techniques like GLC (12) and HPLC with amperometric detection (13) also provide the required sensitivity and specificity, but both techniques are labor intensive and require expertise and equipment that is often not readily available. The enzymatic sucrose assays have the potential to provide the required specificity and sensitivity in a format that is especially suited to high-volume manual or automated testing on a routine basis. The most widely used enzymatic sucrose assay utilizes a coupled assay system in which invertase hydrolyzes sucrose to fructose and glucose and one or more additional enzymes that transform the glucose by reactions that result in a propor103

0003-2697/97 $25.00 Copyright q 1997 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tional change in the absorbance or oxygen tension of the reaction mixture (14, 15). One drawback of using a glucose-dependent secondary reaction for sucrose determination is the potential for interference by glucose in samples that contain both sugars. This problem can be especially significant in human serum or urine where the molar ratio of glucose to sucrose may be high and variable. While it is possible to minimize the effects of glucose interference by sample pretreatment or the use of blank reagents, these approaches make the assay more difficult to automate and may reduce assay precision at low sucrose concentrations. One way to retain the specificity of the invertasecatalyzed primary reaction and avoid interference by glucose is to use the invertase reaction along with a fructose-specific indicator reaction. Fructose dehydrogenase (FDH)1 from Gluconobacter has already been shown to be useful for the determination of fructose in aqueous buffers (16) and in human seminal fluid (17). The present paper describes the combination of invertase and FDH for the quantitation of sucrose according to the following reactions: Invertase

D-Fructose

Sucrose

/ D-Glucose

[1]

Reagent Preparation Citric phosphate buffer (CPB) at pH 4.5 consisted of 0.05 M citric acid, 0.09 M dibasic sodium phosphate in distilled water. A stock solution of FDH was prepared at 125 U/ml in an enzyme diluent that consisted of 1% Triton X-100 and 1 mmol/liter 2-mercaptoethanol in CPB. This stock was stored at 0207C in small aliquots until used in the assay. Invertase was prepared in CPB at a concentration of 1000 U/ml and stored in the same manner. Under these conditions, both enzymes were stable for at least 6 months. MTT was prepared in CPB at a concentration of 0.6 mg/ml and stored at 47C, where it was stable for at least 6 months. PMS was prepared at a concentration of 2.4 mg/ml just prior to use. A working reagent sufficient to carry out 17 sucrose assays was prepared by mixing 2.8 ml CPB, 400 ml enzyme diluent, 120 ml PMS solution, and 40 ml FDH solution. A 1.7-ml aliquot of this mixture was combined with 170 ml of invertase solution to prepare a test reagent that reacted with both sucrose and fructose. A second 1.7-ml aliquot was mixed with 170 ml CPB to prepare a ‘‘blank’’ reagent that reacted only with fructose. The two reagents were used in concert to provide a specific measure of the sucrose concentration in fluids that contained both sugars.

Fructose dehydrogenase

D-Fructose

/ MTT

Sample Collection and Preservation

Phenazine methosulfate

5-Keto-D-fructose / MTT formazan.

[2]

These reactions, which proceed with an increase in the absorbance at 570 nm, are carried out in a microtiter plate format and provide for a rapid, automatable analysis of sucrose in unextracted samples of serum and urine. The assay covers the range of serum sucrose concentrations of interest when sucrose is used as a marker for extracellular fluid volume and is well suited to the determination of urinary sucrose excretion in sucrose challenge tests. MATERIALS AND METHODS

Enzymes and Chemicals All of the chemicals used were of the highest purity available and were purchased from Sigma Chemical Co. (St. Louis, MO). The FDH (EC 1.1.99.11) was from Gluconobacter sp. and had a specific activity of 29 U/ mg protein. The invertase (EC 3.2.1.26) was from baker’s yeast and had a specific activity of 852 U/mg protein. Other key components of the assay included phenazine methosulfate (PMS) and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT). 1

Abbreviations used: CPB, citric phosphate buffer, pH 4.5; FDH, fructose dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; PMS, phenazine methosulfate.

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Urine samples from human subjects were obtained from several clinical studies in which intestinal permeability to lactulose, mannitol, and xylose was assessed by the measurement of urinary sugar excretion using gas–liquid chromatography. The original studies had been approved by the Institutional Review Board of Loyola University Medical Center, and informed consent was obtained from all of the participants. The samples assayed in the present study were either randomly voided specimens or aliquots of 5-h collections following the consumption of an oral test dose that contained sucrose. In the latter case, subjects fasted overnight, emptied their bladders, drank 8 oz of water containing sucrose (20 g), xylose (5 g), lactulose (7.5 g), and mannitol (2 g), and collected all urine passed over the next 5 h. Urine samples were preserved with sodium fluoride (0.4 mg/ml urine). Urine volumes were recorded, and the samples were stored at 0207C. On the day of assay, the samples were thawed, adjusted to pH 4.5 with acetic acid, and centrifuged at 10,000g for 5 min. The resulting supernatants were used for the assay without any further treatment. Serum samples, prepared by centrifuging clotted whole blood, were stored at 0207C and assayed directly. Sucrose Assay Assays were performed in flat-bottom 96-well microtiter plates. The absorbance at 570 nm was monitored

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every 3 min over a 60-min incubation period at 377C using a microplate reader (Ceres 900 Scanning Autoreader and Micro Plate Workstation, Bio-Tek Instruments, Inc., Winooski, VT). The plates were shaken at a moderate rate for 3 s prior to each reading. Twenty microliters of urine or serum was added to a well and the assay was initiated by the addition of 100 ml of either the test or the blank reagent. Sucrose and fructose standards were prepared by mixing stock solutions of known concentrations with urine or serum obtained from a fasting subject. A series of sugar standards was analyzed in duplicate along with each batch of unknowns. The difference between the absorbance changes observed with the ‘‘test reagent’’ (which reacts with fructose, sucrose, and background reductants) and the ‘‘blank reagent’’ (which reacted with all of the aforementioned except sucrose) was taken to represent the absorbance change due to the sucrose in the sample. Evaluation of the Performance Characteristics of the Assay The effects of increasing amounts of FDH and invertase on the reaction rate for fructose and sucrose were tested by varying each enzyme’s concentration while holding the other conditions constant. The effects of potential urine preservatives and of potential crossreacting sugars on the coupled assay for sucrose were tested by adding these compounds to the standard assay system. The overall imprecision of the assay was assessed by measuring aliquots of a pooled urine sample in quadruplicate on three separate occasions. The minimum detectable sucrose concentration was calculated as the minimum concentration that gave an initial rate or a net absorbance change that was two standard deviations above that of a urine sample that contained no sucrose. Comparison of the Enzymatic Assay with Gas–Liquid Chromatography Aliquots of samples from 19 subjects were assayed by the enzymatic method and by gas–liquid chromatography (12). For GLC analysis, 400 ml of urine was mixed with an internal standard solution containing inositol and phenyglucoside, applied to a 2-ml bed of DEAESephadex, and eluted with 5 ml of deionized water. The eluent was evaporated to dryness, the residue was dissolved in 200 ml of pyridine containing 25 mg/ml hydroxylamine, and the mixture was heated at 707C for 60 min. Trimethylsilyl derivatives of the nonreducing sugars and sugar oximes were prepared by mixing 100 ml of the pyridine extract with an equal volume of trimethylsilylimidazole and heating for 30 min at 707C. The silyl derivatives were chromatographed on a 2 mm 1 6 ft column of 3% SE-30 with temperature programming from 200 to 2607C at 207C/min. Detection was by

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flame ionization. A series of sucrose standards in human urine was processed and analyzed exactly as for the unknown samples. Unknown concentrations were determined by comparing the peak height ratios (sucrose/phenylglucoside) to those of the standards. Under the conditions described above phenylglucoside and sucrose had retention times of 5.8 and 8.3 min, respectively. The urine sucrose concentrations of the samples used in the method comparison study ranged from 0.05 to 1.34 mmol/liter (mean, 0.57 mmol/liter; median, 0.47 mmol/liter). Determination of Urine Sucrose Concentrations before and after Sucrose Consumption by Human Subjects Urine samples were collected from subjects just prior to consumption of a test solution containing 20 g of sucrose and for 5 h following the dose. The subjects were 12 prospective bone marrow transplant recipients (1 male, 11 females) who were free from severe gastrointestinal disease, hemodynamic compromise, and respiratory failure. RESULTS AND DISCUSSION

Optimization of the Assay The FDH-catalyzed oxidation of fructose was carried out under the assay conditions described by Nakashima et al. (17). The similarity of the pH optima of FDH and invertase suggested that citric phosphate buffer at pH 4.5 would be appropriate for both enzymes. Studies using CPB at pH values ranging from 3.5 to 6.5 showed that values between 4.5 and 5.0 provided maximum rates for the coupled enzyme system (data not shown). Under the standard assay conditions, the initial rate of FDH-catalyzed fructose oxidation increased with the FDH concentration over the range of 0.18–2.00 U/ml of reagent (Fig. 1). While these results showed that FDH concentrations of 2.0 U/ml (or even higher) would provide higher initial rates, a concentration of 1.4 U/ml was selected for routine use in the interest of economy. At this FDH concentration, the initial rate of the reaction with sucrose as the substrate reached a plateau at an invertase concentration of about 90 U/liter (Fig. 1, inset). Based on these results, 90 U/liter was selected as the invertase concentration for future analyses. Reaction Rates and Standard Curves On the average, the net absorbance change for a mixture of saline and the complete test reagent was 5 mAU/h. A mixture of blank urine (obtained from a fasted human) and the complete test reagent (invertase and FDH) resulted in an absorbance change of approximately 27 mAU/h. When the same blank urine was incubated with a reagent that lacked invertase, a change of 26 mAU/h was observed. The small difference

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between these two rates indicated that the sucrose concentration of the blank urine sample was negligible. When both invertase and FDH were omitted from the reagent, the absorbance change was about 18 mAU/h. These results indicated that the blank urine contained a fructose concentration of about 0.13 mmol/liter of fructose, as well as substances other than sucrose and fructose that reduced MTT under the standard assay conditions. For this reason, the blank reagent was added to the protocol for the assay of sucrose in human urine. This blank reagent, which was identical to the test reagent except that it contained no invertase, produced an absorbance change proportional to the sum of the concentration of fructose and the nonspecific reductants in the sample. The difference between the absorbance changes with the blank and the test reagents was therefore proportional to the invertase-releasable fructose concentration of the sample. Examples of the reaction of urine-based sucrose standards with the test reagent are shown in Fig. 2. Standards containing increasing sucrose concentrations

FIG. 1. The effects of FDH and invertase concentrations on the reaction rate. Urine samples containing different amounts of fructose were assayed under standard assay conditions except that the FDH concentration was varied between 0.08 and 2.00 U/ml. The initial reaction rates were calculated from the absorbance changes over the first 12 min of incubation. Inset: A urine sample containing 4.25 mmol/liter sucrose was assayed under standard conditions (an FDH concentrations of 1.40 U/ml) except that the invertase concentration was varied from 0.9 to 293 U/ml. Initial reactions rates were determined as described above.

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FIG. 2. Reaction of sucrose with the enzymatic reagent under the standard assay conditions. A blank urine sample from a fasting subject and sucrose standards prepared in blank urine were incubated for 60 min at 377C with a test reagent containing 1.4 U/ml FDH, 90 U/ml invertase, and the other components described under Materials and Methods. The absorbance at 570 nm was recorded at 3-min intervals using an automated microplate reader. Samples were analyzed in duplicate and progress curves were constructed from the means of the 21 readings for each well.

showed proportional increases in initial rates over a wide range of sucrose concentrations. The 60-min net absorbance change showed a similar relationship, but over a more limited concentration range. These results suggested that, depending on the analytical range desired, either initial rate or net absorbance change could serve as the basis for a quantitative analysis. Dose–response curves for the determination of sucrose in urine and serum using the blanked reaction scheme are presented in Fig. 3. The initial rates and net changes for the urine standards were approximately linear up to a sucrose concentration of 1.0 mmol/ liter. The low standard errors of the composite curves, which represent the average of results from four different batches of reagents prepared over a period of 6 weeks, indicated good assay-to-assay reproducibility for the estimation of the standard curve. The shape of the urine standard curve suggests that a linear interpolation would suffice for calculating unknown values that are less than 1.0 mmol/liter, but that a point-topoint interpolation should be used for calculating higher values. The reagents developed for the urine assay were also applicable to the direct determination of sucrose in human serum (Fig. 3B), where linear relationships for both rate and net change were observed for sucrose concentrations between 0.1 and 1.0 mmol/liter. Unfortunately, the sensitivity of the assay in its current state is not sufficient for the analysis of basal levels of sucrose in sera of subjects with normal gastroduodenal

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FIG. 3. Standard curves for the determination of sucrose in urine and serum. (A) Urine sucrose standards were analyzed in duplicate under the standard assay conditions using four batches of reagents that were prepared over a 6-week period. The average absorbance change for the first 12 min (representing the initial reaction rate) and the average change over a 60-min period are plotted (bars represent the standard errors for the four assays). (B) Serum sucrose standards were assayed in duplicate under the same conditions used for the urine assay. The error bars represent the pooled within-assay standard errors as calculated by ANOVA.

permeability where concentrations are typically õ0.03 mmol/liter even after the consumption of an oral sucrose load (E. Holmes, unpublished observation). However, the serum standard curve does cover the appropriate range for the measurement of serum sucrose when this sugar is used for the estimation of extracellular fluid volume (5). Interference by Preservatives, Sugars, and Other Urine Constituents Some of the preservatives that might be used to prevent microbial growth in urine specimens were tested for their effect on the assay. The following compounds did not interfere when present in the urine at or below the stated concentration: streptomycin, 35 mg/ml; vancomycin, 25 mg/ml; thymol, 1 mg/ml; sodium azide, 0.1% (w/v); sodium fluoride, 2.3 mg/ml. Sodium fluoride at a concentration of 0.4 mg/ml was routinely used for the preservation of timed urine collections from subjects undergoing the sucrose challenge test. The specificity of the assay for sucrose was investigated by analyzing urine samples that were supplemented with other sugars. Fructose reacted with the test reagent to the same extent as and at a rate similar to that of sucrose. Thus, the blanking scheme described above was used to quantitate sucrose in fluids that contained both sugars. None of the following sugars interfered with the sucrose assay when present in urine at a concentration of 3.3 mmol/liter: mannitol, galactose, rhamnose, glucose, xylose, lactulose, lactose, or cellobiose. As anticipated by the substrate specificity of invertase, both raffinose and stachyose reacted with the test reagent. Raffinose produced 25% and stachyose

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11% of the absorbance of an equimolar concentration of sucrose. While both of these sugars may be present in small amounts in the diet, the absorption and urinary excretion of raffinose are low compared to those of sucrose (1) and the absorption and excretion of stachyose would be expected to be even lower. Therefore, neither sugar should be a significant source of interference in fasting patients who consume a test dose of sucrose. In the course of assaying urinary sucrose in samples from more than 90 subjects (most of whom were patients receiving multiple prescription drugs and/or dietary supplements), seven cases of positive interference were observed. In all cases, the interference was due to a sucrose- and fructose-independent reduction of MTT as supported by the observation that an increase in absorbance with time was observed with a reagent formulation that lacked both invertase and FDH. While the interfering substance(s) have not been identified, interference was easily distinguished from the absorbance changes produced by physiological or pathological concentrations of sucrose (Fig. 4). Five of the seven cases of interference showed a high initial absorbance with no further increase during the incubation period (Fig. 4, curve A). The other two cases showed a high initial rate of reaction with a plateau of the net absorbance within the first 6 min of incubation (curves B and C). The shape of the absorbance versus time curves in these two cases differed from those observed for sucrose. Furthermore, the initial reaction rates greatly exceeded those observed for sucrose even when the urine sucrose concentration was extremely high (curves D and E). Since urine fructose concentrations exceeding 35 mmol/liter are not likely to be encountered even after the administration of oral sucrose

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tained by GLC (x) gave the following results by linear regression analysis: slope Å 1.16 { 0.11; intercept Å 0.18 { 0.08 mmol/liter; r Å 0.93 and Syx Å 0.21 mmol/ liter; n Å 19. The slope of the regression line was not significantly different from 1, nor was the intercept significantly different from 0, indicating an absence of constant and proportional bias. The correlation coefficient indicated a good agreement between the two methods of analysis. Urine Sucrose before and after an Oral Sucrose Dose

FIG. 4. Assay interference by unknown reductants in human urine samples. Urine samples containing interfering substances (A–C) and urine sucrose standards at 36 (D) and 7 mmol/liter (E) were assayed with the test reagent under standard assay conditions. The most common type of interference was characterized by a high initial absorbance (A). A second type of interference was recognized by a high initial rate and a plateau of the absorbance within the first 12 min (B and C). Both types of interference could be differentiated from the absorbance changes due to physiological or pathological concentrations of sucrose.

The enzymatic assay was used to determine urine sucrose concentrations in human subjects before and after a 20-g oral sucrose dose (Fig. 5). The mean urine sucrose was 0.18 mmol/liter just before the dose and 0.41 mmol/liter in the 5-h collections following the dose. Note that all of the pre- and postdose results for this group of subjects were less than 1 mmol/liter, placing them within the linear range of the urine assay. The difference between the pre- and postdose means was significant (P Å 0.013, paired t). The average 5-h excre-

to persons with a severe abnormality in gastroduodenal permeability (7–9), subject-specific, ‘‘no enzyme’’ blanks are not required to detect positive interference. Precision and Sensitivity The overall imprecision (run-to-run plus within-run) of the urine sucrose assay was evaluated using both the initial reaction rate (over the first 12 min) and the 60-min net absorbance change as the basis for quantitation. At a urine sucrose concentration of 0.32 mmol/ liter, the overall and within-assay coefficients of variation were 9.7 and 7.5%, respectively, for the initial rate assay and 7.5 and 6.7%, respectively, for the 60-min assay. The minimum detectable urine sucrose concentrations by the initial rate and the 60-min assays were 0.06 and 0.03 mmol/liter, respectively. In view of these results, the 60-min assay was selected for routine use with the standard reagent formulation. The results in Fig. 1 suggest that, when necessary, increased sensitivity and precision can be obtained at shorter incubation times by increasing the FDH concentration of the test reagent. In its present form, the new sucrose assay has a measuring range, a minimum sensitivity, and a level of imprecision similar to those of a glucose-directed assay that was recently developed for the measurement of urinary sucrose in rodents (18). Method Comparison A comparison of urine sucrose results determined with the enzymatic sucrose assay (y) with those ob-

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FIG. 5. Urine sucrose concentration and 5-h excretion following an oral sucrose dose. Urine sucrose was measured in random urine samples collected just prior to a 20-g oral sucrose dose and in the samples collected over the next 5 h (the asterisks indicate that the plotted point represents two observations). The average postdose urine sucrose concentration (mean { SE) of 0.41 { 0.08 mmol/liter was significantly increased over the predose value of 0.18 { 0.04 mmol/liter (P Å 0.002). The total 5-h sucrose excretions for each subject in mg/5 h are shown to the right of the point marking each subject’s postdose sucrose concentration. Six of the subjects had results that exceeded the upper limit of 42 mg/5 h that was recently reported by Vogelsang et al. (10) for a group of ostensibly healthy control subjects.

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tion in this group was (mean { SE) 38 { 8 mg/5 h, which represents 0.19 { 0.04% of the administered dose. Six of these subjects exceeded the upper limit of 42 mg/5 h that was recently observed in a reference sample of healthy subjects who consumed a 20-g oral sucrose dose (10). However, increases of the order observed in the subjects in the present study represent only slight increases in gastroduodenal permeability. By comparison, chronic alcoholics with liver disease had urinary sucrose excretions that were increased 20fold over those of control subjects (9). In summary, I have presented a coupled enzymatic assay which provides for the measurement of sucrose in serum and urine. One advantage of the new method is that it gives satisfactory results for serum and urine samples without the need for deproteinization or extraction of the samples. A second advantage is that the FDH indicator reaction generates reducing equivalents that can be detected using a variety of different acceptors and a number of different analytical methods. These advantages and the microscale of the present procedure provide the simplicity and economy required for routine testing in situations where sucrose is used as a probe for gastroduodenal permeability or extracellular fluid volume. ACKNOWLEDGMENTS The author is grateful to Ms. Fe Balauag for her technical assistance, to Ms. Decarlo Sykes for help in preparing the manuscript, and to Dr. Mark A. Jandreski for reviewing and critiquing the manuscript.

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2. Woodruff, G. G. (1958) J. Pediatr. 52, 66–72. 3. Bond, J. H., Currier, B. E., Buchwald, H., and Levitt, M. D. (1980) Gastroenterology 78, 444–447. 4. Keith, N. M., and Power, M. H. (1937) Am. J. Physiol. 120, 203– 210. 5. Bauer, K., Versmold, H., Pro¨lss, A., DeGraaf, S. S. N., Meeuwsen-Van DerRoset, W. P., and Zijlstra, W. G. (1990) Pediatr. Res. 27, 256–259. 6. Meddings, J. B., Sutherland, L. R., Byles, N. I., and Wallace, J. L. (1993) Gastroenterology 104, 1619–1626. 7. Sutherland, L. R., Verhoef, M., Wallace, J. L., Van Rosendaal, G., Crutcher, R., and Meddings, J. B. (1994) Lancet 343, 998– 1000. 8. Keshavarzian, A., Fields, J. Z., Vaeth, J., and Holmes, E. W. (1994) Am. J. Gastroenterol. 89, 2205–2211. 9. Holmes, E. W., Patel, M., Pethkar, S., Adelman, K., Iber, F., and Keshavarzian, A. (1996) Alcoholism Clin. Exp. Res. 20, (Suppl. 2), 127A. 10. Vogelsang, H., Oberhuber, G., and Wyatt, J. (1996) Gastroenterology 111, 73–77. 11. Lal Somani, B., Khanade, J., and Sinha, R. (1987) Anal. Biochem. 167, 327–330. 12. Toba, T., and Adachi, S. (1977) J. Chromatogr. 135, 411–417. 13. Frias, J., Price, K. R., Fenwick, G. R., Hedley, C. L., Sorensen, H., and Vidal-Valverde, C. (1996) J. Chromatogr. A 719, 213– 219. 14. Bergmeyer, H. U., and Bernt, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.), 2nd ed., Vol. 3, pp. 1176–1179, Verlag Chemie, Weinheim. 15. Outlaw, W. H., Jr., and Tarczynski, M. C. (1986) in Methods of Enzymatic Analysis (Bergmeyer, H. U., Bergmeyer, J., and Graß1, M., Eds.), 3rd ed., Vol. VI, pp. 96–103, Verlag Chemie, Weinheim. 16. Ameyama, M., Shinagawa, E., Matsushita, K., and Adachi, O. (1981) J. Bacteriol. 145, 814–823. 17. Nakashima, K., Takei, H., Adachi, O., Shinagawa, E., and Ameyama, M. (1985) Clin. Chim. Acta 151, 307–310. 18. Davies, N. M., Corrigan, B. W., and Jamali, F. (1995) Pharm. Res. 12, 1733–1736.

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