Preliminary examination of the influence of incubation time or cytosolic protein concentration on dihydropyrimidine dehydrogenase activity

ELSE’VIER

Clinica Chimica Acta 252 (1996) l-9

F’reliminary examination of the influence of inculbation time or cytosolic protein concentration on dihydropyrimidine dehydrogenase activity Tomonori Tateishi*, Hironori Nakura, Minoru Watanabe, Masami Tanaka, Toshio Kumai, Shinichi Kobayashi Department of Pharmacology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae. Kawasaki, Kanagawa, 216 Japan

Received 13 November 1995; revision received 22 January 1996; accepted 30 January 1996

Abstract

We examined the influence of incubation time or cytosolic protein concentration on the metabolite production of Sfluorouracil (SFU). Although the activity of dihydropyrimidine dehydragenase (DPDase) from rat liver is considered to be retained for up to 60 min, the production rate of 5FU metabolites was reduced, probably due to depletion of the substrate in 40 pmol/l and lower concentration of 5FU. Since the ratio of the metabolite production rate to the cytosolic protein became smaller in higher concentrations of cytosolic protein, the DPDase activity should be compared in the same concentration of cytosolic protein. The production rate of 5FU metabolites was considered to be linear up to 40 pmol/l 5FU incubated with 500 pg cytosolic protein. The rate of the metabolite production calculated by one-point sampling significantly correlated with the enzyme activity by the multi-point sampling method. Minimizing sampling points to determine the DPDase activity would save time and expense. Keywords: 5-Fluorouracil; Dihydropyrimide protein; Substrate concentration

dehydrogenase;

*Corresponding author. 0098-8981/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOOO9-8981(96)06315-2

Incubation

time; Cytosolic

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1. Introduction 5-Fluorouracil (SFU) has been considered the most active agent in the treatment of colorectal cancer [l]. Dihydropyrimidine dehydrogenase (DPDase, EC 1.3.1.2) is the initial and rate-limiting enzyme in 5FU catabolism [2]. Co-administering inhibitors of DPDase is intended to enhance the antitumor effect of 5FU [3,4]. On the other hand, deficiency of this enzyme activity induces lethal side-effects of 5FU [5,6]. These studies and case reports indicate that assessing this enzyme activity can be useful in predicting treatment or adverse effect of SFU. Although there have been many studies on the characteristics of DPDase, the assay method of this enzyme is different from study to study [7-lo]. In some studies the activities of DPDase were calculated from the slope of metabolites produced at various incubation times of up to 60 min [9,10]. However, there have been few studies to see how the incubation time or the enzyme concentration affect DPDase activity. The purpose of this study was to examine the influence of incubation time or cytosolic protein concentration on the metabolite production of 5FU and to establish an efficient assay method. 2. Materials and methods 2.1. Chemicals [6-3H]5-Fluorouracil (>99% radiochemical purity, 555 Bq/mmol) was purchased from Amersham Corp., Arlington Heights, IL, USA. Unlabelled 5FU was a generous gift from Kyowa Hakkou Kogyo Co., Ltd. (Tokyo, Japan). The radiolabeled drug was diluted with unlabeled 5FU to give appropriate specific activities. [6-3H]5-Fluorouracil was incubated at final drug concentrations ranging from 0.5 ,umol/l to 4 mmol/l and a final radioactivity concentration of 1 &i/sample (1.0 ml). All other chemicals were from Wako Chemicals (Osaka, Japan) and were of analytical grade. 2.2, Preparation of rat liver cytosol

Liver samples were obtained from three male Sprague-Dawley rats, aged 8 weeks and weighing 200-300 g. All procedures were carried out in accordance with the guiding principles for the care and use of laboratory animals approved by the Japanese Pharmacological Society. After cervical dislocation the livers were quickly removed and frozen in liquid nitrogen and stored at -80°C as the preparation for cytosol. The livers were minced and homogenized in 3 volumes of 35 mmol/l potassium phosphate (pH 7.4), 2.5 mmol/l magnesium chloride, 10 mmol/l 2-mercaptoethanol,

T. Tateishiet al. I Clinica ChimicaActa 252 (19%) l-9

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0.25 m#ol/l sucrose, 1 mmol/l benzamidine, 1 mmol/l aminoethylisothiouronium bromide and 5 mmol/l EDTA, using a glass homogenizer. The resulting homogenate was centrifuged at 9000 x g for 20 min at 4°C. Subseqjuently the supernatant was centrifuged at 100000 x g for 60 min at 4°C. The supernatant was removed and used as cytosols in the subsequent assay. Protein concentration of cytosolic fraction was determined by the method of Lowry et al. [ll]. 2.3. Incubation procedure Cytosolic incubations were carried out in a final volume of 1.0 ml with 35 mmol/l potassium phosphate (pH 7.4), 2.5 mmol/l magnesium chloride, 10 mmol/I 2-mercaptoethanol, 200 pmol/l NADPH, various concentrations of substrate and enzyme. DPDase activity was determined by measuring the metabolites of 5FU formed by reverse-phase HPLC. After a 3-min preincubation in the presence of 5FU, metabolism was initiated by the addition of cytosolic protein using open glass tubes in a shaking water ‘bath at 37°C. For the reaction mixture of 0.5 pmol/l and 4 ,umol/l 5FU, 1.50~1 were taken out at 5, 10 and 15 min and mixed with the same volume of 5% perchloric acid to quench the reaction. For the mixture of higher concentrations of 5FU, the same volume was taken out at 10, 20, 30,45 and 60 min. 2.4. Standard assay procedure and analytical method

The samples were centrifuged and subsequently filtered through a Columngard-LCR4 filter (Millipore Japan, Tokyo, Japan) prior to injecting HPLC. 5FU and its metabolites were separated by reverse-phase HPLC using two pumps (Applied Biosystems Japan, Tokyo, Japan), an AS-950 automatic sampler (JASCO, Tokyo, Japan), a 4.6 x 250 mm L-column ODS (Chemicals Inspection and Testing Institute, Tokyo, Japan), a 783 programmed absorbance detector (Applied Biosystems Japan, Tokyo, Japan), a D-2500 chromatointegrator (Hitachi, Tokyo, Japan) and a SF-212N fraction collector (JASCO, Tokyo, Japan). The absorbance of the eluate was monitored at 264 nm where the maximal absorbance was obtained for unlabeled 5FU in this HPLC system. The mobile phase was 20 mmol/l sodium phosphate (pH 3.5) at a flow rate of 0.5 ml/min. Following HPLC injection, the eluate was collected and counted in a LSC-1000 scintillation counter (Aloka, Tokyo, Japan). 2.5. Analytical method Vmax,app and L.app were estimated

Lineweaver-Burk

plots.

by linear regression analysis using

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3. Results 3. I. Typical radiochromatogram

The radiochromatogram obtained following incubation 5FU with rat liver cytosols is shown in Fig. 1.

of 400 pmol/l

3.2. E$ect of incubation time and cytosolic protein concentration on the production of metabolites

In 4 mmol/l and 400 pmol/l 5FU the generation of metabolites was linear for incubation times of up to 60 min (Fig. 2a,b). In 40 pmol/l and lower concentrations of 5FU the metabolite production was not increased proportionally to incubation times, especially in higher cytosolic protein concentrations (Fig. 2c,d,e). Although the metabolite production was linear for cytosolic protein concentration of up to 1000 pg per 1.0 ml sample, the ratio of the metabolite production rate to the cytosolic protein got smaller in higher concentrations of cytosolic protein (Fig. 3). The production rate of 5FU metabolites was considered to be linear up to 40 pmol/l incubated with 500 pg cytosolic protein per 1.0 ml sample (Fig. 4). 3.3. Correlation between one-point and multi-point sampling method

There was a significant correlation between the rate of the metabolite production calculated by one-point sampling at 20 min and the enzyme activity calculated by multi-point sampling (Fig. 5).

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Fig. 1. Radiochromatogram obtained for an incubate of 400 pmol/l 5-fluorouracil with rat liver cytosol(500 pg per 1 ml) and 1 $i [6-3H]5-fluorouracil. 5FU, DFU and FUPA are 5-fluorouracil, dihydrofluorouracil and fluoroureidopropionic acid, respectively.

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of rat liver cytosol and (a) 4 mmol/l, Fig. 2. Formation of 5FU metabolites after incubation with different concentrations (b) 400 pmol/l, (c) 40 pmol/l, (d) 4 pmol/l and (e) 0.5 pmol/l 5FU, as a function of the incubation time. Points are the means of data from three rats.

6o 1

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1

5FU

I I

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0

100

250

500

750

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Fig. 3. Formation of 5FU metabolites after incubation with different Muorouracil concentrations, as a function of the cytosolic protein concentration (compared with 100 pg/ml sample. Points are the means of data from three rats.

3.4. The apparent V,,,,, and K,,, The apparent V,,, obtained was 0.811 & 0.167 (mean f S.D., n = 3) nmol metabolites formed per min per mg cytosol protein and the apparent K, was 3.490 + 2.374 (mean f S.D., n = 3) pmol/l.

4. Discussion We have described the influence of the incubation time and the cytosolic protein concentration on the metabolite production of 5FU. Dihydrofluorouracil is produced from 5FU by DPDase and is subsequently metabolized to fluoroureidopropionic acid by dihydropyrimidinase [1,2]. Both enzymes are located in the cytosol [7]. There were two peaks before the 5FU peak in the radiochromatogram of Fig. 1. According to the previous studies [7,12], the earlier and later peaks are considered to be fluoroureidopropionic acid and dihydrofluorouracil, respectively. As fluoroureidopropionic acid is produced from dihydrofluorouracil [ 1,2], the sum of the radioactivity of these two metabolites reflects the activity of DPDase. In some studies the activities of DPDase were calculated from the slope of metabolites produced at various incubation times of up to 60 min [9,10]. In general, the enzyme activity can be lost gradually during incubation. However, there have been few studies of the influence of the incubation time or the enzyme concentration on the dihydropyrimidine dehydrogenase activity.

T Tateishiet al. I Clinica Chimica Acta 252 (19%) 1-9

cytosolicprotein

!

I loglo-'

log100

,

loglo'

loglo'

1

t

loglo'

log104

5FU (pmol/LI

Fig. 4. Formation of 5FU metabohtes after incubation with digerent concentrations of rat liver cytosol, as a function of the Muorouracil concentrations. Formation rate was calculated by metabolites generated at 5 min in 4 pmol/l and 0.5 pmol/l 5FU. Points are the meaurs of data from three rats.

The metabolite production of 5FU was linear for the incubation time in higher substrate concentrations, while it was not increased proportionally to-the incubation time, especially in higher cytosolic protein concent&tions incubated with 40 pmol/l and lower concentrations of 5FU. The

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Fig. 5. Relation between the rate of metabolite formation at 20 min and the slope calculated by metabolites generated at 10, 20, 30, 45 and 60 min in 4 mmol/l, 400 pmol/l, 40 pmol/l 5-fluorouracil with various cytosohc protein concentrations. Dotted lines show 95% confidence bands for the true mean of Y

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et al. I Clinica

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Acta 252 (19%)

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reduction in the slope of production rate might be related to a decrease in the enzyme activity due to the inhibition by metabolites produced and/or the depletion of 5FU in 40 pmol/l and lower concentrations. Since the metabolite production was linear in higher concentrations of 5FU and larger amounts of metabolites could be produced in those conditions, DPDase of rat liver was considered to maintain its activity up to 60 min and the depletion of substrate would contribute to the reduction in the production rate in this study. Although the metabolite production was linear for cytosolic protein concentration of up to 1000 pg/ml, the ratio of the metabolite production rate to the cytosolic protein became smaller in higher concentrations of cytosolic protein. These results suggested that the enzyme activity should be compared in the same concentration of cytosolic protein. In the previous studies [9,13] various concentrations of cytosolic protein were used to compare the DPDase activities. After the DPDase activity is corrected with the cytosolic protein the sample with the higher protein concentration might show lower activity than that with lower protein concentration even if they have equal activities at the same cytosolic protein concentration. As the rate of the metabolite production calculated by one-point sampling at 20 min significantly correlated with the enzyme activity calculated by the multi-point sampling method [9,10], the one-point sampling method would be as effective to measure DPDase activity as the multi-point sampling method. The production rate of 5FU metabolites was considered to be linear for up to 40 pmol/l incubated with 500 pg cytosolic protein. Therefore, DPDase activity can be compared in 40 pmol/l 5FU with 500 pug cytosolic protein incubated for 20 min. In the one-point sampling method we can shorten the incubation time for the reaction mixture and the running time for HPLC as well as reducing the final volume of the reaction mixture. In summary, we examined the influence of incubation time or cytosolic protein concentration on the metabolite production of 5FU. The production rate of 5FU metabolites was reduced, probably due to the depletion of 5FU in lower concentrations with higher cytosolic protein. In addition, DPDase activity should be compared in the same concentration of cytosolic protein. The one-point sampling method was considered efficient in determining the DPDase activity. Minimizing sampling points will save time and expense and will serve as a useful tool for determining DPDase activity.

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References [l] Kijhne-Wiimpner C-H, Schmoll H-J, Harstrick A, Rustum YM. Chemotherapeutic strategies in metastatic colorectal cancer: an overview of current clinical trials. Semin Oncol 1992;19(S3):105-125. [2] Pinedo HM, Peters GF. Fluorouracil: biochemistry and pharmacology. J Clin Oncol 1988;6:1653-1664. [3] Rustum YM, Cao S, Spector T. 5-Ethynyluracil (776C85) is a potent modulator of thmetherapeutic activity of 5-fluorouracil. Proc Am Assoc Cancer Res 1993;34:283. [4] Baccanari DP, Davis ST, Knick VC et al. 5-Ethynyluracil (776C85): a potent mlodulator of the pharmacokinetics and antitumor efficacy of 5-fluorouracil. Proc N,atl Acad Sci USA 1993;90:11064-11068. [S] Tuchman M, Stoeckeler JS, Kiand DT, O’Dea RF, Ramnaraine ML, Mirkin BL. Familial pyrimidinemia and pyrimidinuria associated with severe fluorouracil toxicity. N Engl J Med 1985;313:245-249. [6] Dtasio RB, Beavers TL, Carpenter JT. Familial deficiency of dihydropyrimidine dehydrogenase. Biochemical basis for familial pyrimidinemia and severe 5fluorouracil-induced toxicity. J Clin Invest 1988;81:47-51. [7] Naguib FNM, el Kouni MH, Cha S. Enzymes of uracil catabolism in normal and ne:oplastic human tissues. Cancer Res 1985;45:5405-5412. [S] Tuchman M, Ramnaraine MLR, O’Dea RF. Effects of uridine and thymidine on the degradation of 5-fluorouracil, uracil, and thymine by rat liver dihydropyrimidine dehydrogenase. Cancer Res 1985;45:5553-5556. [9] Lu Z, Zhang R, Diasio RB. Dihydropyrimidine dehydrogenase activity in human peripheral blood mononuclear cells and liver: population characteristics, newly identified deficient patients, and clinical implication in 5-fluorouracil chemotherapy. Cancer Res 1993;53:5433-5438. [lo] Lu Z, Zhang R, Diasio RB. Comparison of dihydropyrimidine dehydrogenase from human, rat, pig and cow liver. Biochem Pharmacol 1993;46:945-952. [11] Lowry OH, Rosebrough NJ, Farr AL et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. [12] Sommadossi J-P, Gewirtz DA, Diasio RB, Aubert C, Cano J-P, Goldman ID. Rapid ca.tabolism of 5-fluorouracil in freshly isolated rat hepatocytes as analyzed by high performance liquid chromatography. J Biol Chem 1982;257:8171-8176. [13] Etienne MC, Lagrange JL, Dassonville 0 et al. Population study of dihydropyrimidine dehydrogenase in cancer patients. J Clin Oncol 1994;12:2248-2253.