NOTES

The Journal of Biochemistry, Vol. 49, No.1, 1961

The Quantitative Determination of Uridine Diphosphoglucuronic Acid*

By MINORU KAWADA

(From the Department of Biochemistry, Faculty of Medicine, University of Tokyo, Tokyo)

(Received for publication, September 5, 1960)

For the quantitative determination of assay on a large amount of sample, and the uridine diphosphoglucuronic acid (UDPGA) the method of Levvy and Storey (1) is now being used (2), which measures the rate of o-aminophenyl glucuronide synthesis by a mouse liver slice, but this method is both troublesome and time-consuming. In 1957, Strominger et al. (3) reported that the amount of UDPGA could be estimated through the formation of phenolphthalein glucuronide, which was determined from the decrease in optical density at 540 mp after incubation of phenolphthalein, UDPGA, and guinea pig microsomes. This method is more convenient, but it was found necessary to centrifuge off micro somes after color development and moreover not applicable for samples which contained a large amount of protein. TCA and Somo gyi method (4) were not suitable for deprote inization in this case, as phenolphthalein was absorbed by precipitated proteins. The author's experiments indicated that it is more favorable to carry out simultaneous deproteinization and extraction of free phenol phthalein by treatment with chloroform and the chloroform layer then reextracted with

glycine buffer. This method did not need FIG. I. Calibration curve centrifugation and at the same time yellowish Condition was the same as described in the color originating from proteins or microsomes text with the exception that the reaction mixture did not interfere in the course of the assay. did not contain potassium saccharate and contained 0.0927ƒÊmole of phenolphthalein instead of 0.1 This method could also be applied to the mole.•\•›•\ ƒÊ amount of microsomes 0.3ml. (300mg. wet * The following abbreviations are used in this liver) •\• •\ amount of microsomes 0.2ml. (200mg. wet paper:TCA, trichloroacetic acid; UDP-glucose, uri liver) dine diphosphoglucose; UDPGA, uridine diphospho __ amount of microsomes 0.1ml. (100mg. wet glucuronic acid. liver) Notes 79

accuracy was increased in this method by 0.1M phosphate buffer (pH 7.5), 0.1ml. of

the pH value of the sample was kept con microsomes obtained from 100 mg. of guinea stant 10.6 for phothometric determination. pig wet liver, and the sample, was made the The calibration curve is shown in Fig. 1. total volume to 1-2ml. After incubation at

Microsomes obtained from 100mg. of wet 37•Ž for one hour, 3ml. of chloroform was

liver would be adequate for this assay, be added, the mixture was shaken, and 2ml. of

cause the excess of microsomes would cause chloroform layer was removed to another

the adsorption of phenolphthalein and its too tube after 30minutes. Three ml. of 1M gly

small amount would decrease the enzyme cine buffer (pH 10.6) was added to the chloro

activity of glucuronide formation. In one form solution, the mixture was shaken, and

hour incubation almost all the UDPGA added the optical density (A) of the water layer was

was conjugated with phenolphthalein. measured at 550mƒÊ. As a blank, the optical

This method was not influenced by the density (B) was measured with the same mix

coexistence of a large amount of UDP-glucose, ture without incubation. The amount of

glucuronic acid 1-phosphate, or glucuronic UDPGA was calculated by the following acid. Addition of potassium saccharate, which equation: is a specific inhibitor of ƒÀ-glucuronidase, was B-A/B•~(amount of phenolphthalein added, able to eliminate the interference by the co existence of phenolphthalein monoglucuronide mole)=(amount of UDPGA, ƒÊmole) ƒÊ

in this procedure. The recovery test of The author wishes to express his gratitude to UDPGA is shown in Table I. The recovery Prof. N. Shimazono and Dr. Y. Mano for their

ratio was 110per cent and the standard de helpful advices and continuous encouragement.

TABLE I Recovery Test

viation was 12.8per cent. If the sample did not contain protein, the standard deviation REFERENCES was 2.5per cent. (1) Levvy, G. H., and Storey, I. D. E., Biochem. J., The following condition seemed to be the 44, 295 (1949) most suitable for determination of UDPGA. (2) Dutton, G. J., Biochem. J., 71, 141 (1959) The reaction mixture containing phenolphtha (3) Strominger, J. L., Maxwell, E. S., Axelrod, J., lein (0.1ƒÊmole) dissolved in 0.3ml. of 0.001M and Kalekar, H. M., J. Biol. Chem., 224, 79 (1957) sodium hydroxide, 0.1ml. of 0.1M MgCl2, 0.1 (4) Somogyi, M., J. Biol. Chem., 160, 69 (1945) ml. of 0.001M potassium saccharate, 0.2ml. of