MICROANALYSIS OF GLUCURONIDE GLUCURONIC ACID AS APPLIED TO /3-GLUCURONIDASE AND

GLUCURONIC ACID STUDIES*

BY WILLIAM H. FISHMAN AND SIDNEY GREEN

(From the Cancer Research and Cancer Control Unit, and the Department of Biochemistry and Nutrition, Tufts College Medical School, and the Cancer

Research Laboratory, the New England Center Hospital, Boston, Massachusetts)

(Received for publication, December 28, 1954)

In recent years, interest in the metabolism of glucuronic acid has grown. Contributions have ranged from studies of its biosynthesis and conjugation in surviving tissues and exper,imental animals to its utilization in man (l-lo). In all spheres, especially the latter, progress has been hampered by the lack of an analytical method by which glucuronides could be differen- tiated from free glucuronic acid in their mixtures.

The naphthoresorcinol reaction (11, 12) is widely used in metabolic studies, but is positive for both free and conjugated glucuronic acid. The principle of the proposed procedure depends on the selective oxidation of free glucuronic acid and other oxidizable interfering substances before the reaction with naphthoresorcinol. It has been possible also to improve the analytical performance of this latter reaction so that valid figures have been obtained for the small amounts of glucuronides present in human blood. Under special circumstances, both free and conjugated acid can be determined, making possible a number of important applications of the method, examples of which are presented.

Method

Principle-Glucuronic acid is eliminated from a mixture of the acid and glucuronides when the free aldehyde group is oxidized by hypoiodite at pH 10.1, forming saccharic acid which is insensitive to the naphthoresor- cinol glucuronic acid reaction. Glucuronides are not affected by this treatment, since the aldehyde group is protected at carbon 1 by its linkage to the aglycone. Subsequently, the strongly acid conditions of the naph- thoresorcinol test liberate glucuronic acid from its conjugate, giving rise to a violet-colored pigment, the amount, of which is measured photocolori- metrically. An estimate of the unconjugated glucuronic acid is obtained

* Aided by a grant-in-aid from the Damon Runyon Memorial Fund for Cancer Research, by a fellowship from the Corn Products Refining Company, Argo, Illinois, and by an Institutional Grant of the American Cancer Society, New York. Pre- sented before the 127th meeting of the American Chemical Society at Cincinnati, March 31,1955.

527

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528 P-GLUCURONIDASE AND GLUCURONIC ACID

by computing the difference in the values obtained for glucuronic acid before and after the oxidation procedure. This estimate is valid only in the absence of interfering amounts of hexoses.

Reagents- Toluene (Merck reagent). Ethyl alcohol, 95 per cent. Bu$er, pH 10.1. 8.4 gm. of sodium bicarbonate (Merck reagent) and

36.0 gm. of sodium carbonate (anhydrous, Merck reagent) are dissolved in a liter of distilled water.

0.1 N iodine in potassium iodide solution. It is stored (glass-stoppered amber-colored bottle) out of direct sunlight.

Sodium bisuljite solution (1.0 M). 10.4 gm. of sodium bisulfite (Merck reagent) are dissolved in 100 ml. of distilled water. A fresh solution is made up every 2 weeks.

6 N sulfuric acid. This solution may be prepared by diluting 1 volume of 18 N sulfuric acid with 2 volumes of water.

18 N sulfuric acid. 1 volume of concentrated sulfuric acid (Merck re- agent) is poured into 1 volume of distilled Hz0 and allowed to cool to room temperature before use.

Aqueous naphthoresorcinol solution, 0.4 per cent. A fresh solution is used for each day’s determinations. It is essential to pulverize the naph- thoresorcinol (Schwarz Laboratories, Inc., New York) with a mortar and pestle. It is necessary to shake the powdered naphthoresorcinol-water mixture in a glass-stoppered, amber-colored mixing cylinder for 10 min- utes either by hand or with a rapid shaking apparatus. The filtered solu- tion is protected from sunlight.

Apparatus-A Pyrex constant temperature bath, 16 inches in diameter, that includes a relay box, mercury thermocouple, stirrer, 500 watt immer- sion heater, and adjustable shelf manufactured by the Precision Scientific Company, Chicago, was used. Oil (Blandol, Howe and French, Inc., Boston) was substituted for water to maintain a constant immersion level of the tubes throughout the heating period. It was necessary to insulate the bath to avoid excessive heat loss and to install a 1000 watt auxiliary heater to shorten the time required to bring the temperature of the oil bath up to 100”. It is advisable to place the bath in a ventilating hood or underneath an exhaust vent.

Evelyn photoelectric calorimeter. Procedure-5 ml. of solution are pipetted into an Erlenmeyer flask (50

ml. capacity) containing 2.05 ml. of carbonate buffer. 1.5 ml. of iodine solution are added, shaken gently, and the flask is stoppered and allowed to stand in the dark for 30 minutes. At the end of this time, 0.15 ml. of sodium bisulfite solution is added, the flask agitated, and an addition made

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W. H. FISHMAN AND S. GREEN 529

of 0.3 ml. of 6 N sulfuric acid. Any residual iodine coloration can be re- moved by 1 additional drop of bisulfite solution. The flask is shaken to remove the excess CO2 from the solution. The subsequent determination of glucuronic acid by the naphthoresorcinol reaction on this mixture yields a value for glucuronide glucuronic acid.

To obtain a figure for total glucuronic acid, another 5 ml. of solution is pipetted into a solution containing iodine, bisulfite, and HzS04 pre- pared in the amounts and in the sequence used before. This mixture is ready for the next step without the 30 minute interval.

Duplicate samples of 4 ml. aliquots are then pipetted into non-protein nitrogen type boiling tubes1 with a ground glass mouth (purchased from the Macalaster Bicknell Company, Cambridge, Massachusetts). To each are added 2 ml. of 0.4 per cent naphthoresorcinol and 2 ml. of 18 N sulfuric acid (13). The contents of the tubes are mixed well, and the tubes are arranged, unstoppered, in a suitable metal rack and placed in the oil bath at 100” for 90 minutes. The oil level should be about 2 inches from the top of the tubes. The rack of tubes is then immersed in a container of cold water to which Alconox in the prescribed amount has been added. After cooling, 10 ml. of 95 per cent alcohol (14) are added to each tube, the tubes are shaken to dissolve the pigment, and 8 ml.’ of toluene (13) are added. Ground glass stoppers are inserted and the tubes vigorously shaken 100 times to extract the violet pigment into the toluene phase. The aqueous layer is removed completely by suction with a tube drawn out to a capillary attached to an aspirator and collecting bottle. The toluene extracts are then transferred carefully into Evelyn tubes.

All the above operations should be performed away from direct sunlight, since the pigment is sensitive to sunlight. After allowing the extracts to stand 5 minutes in the dark to permit them to clear, the optical density of each tube is measured in the Evelyn calorimeter at 565 rnp with the 6 ml. well, set to zero optical density with a toluene blank. Duplicate readings should agree within 2 galvanometer divisions. The reagent blank, which consists of 5 ml. of water, should not read below 85 per cent transmittance. It is also desirable to include a standard with each day’s determinations.

Cal&a&n Curve-5 ml. of solutions that contain 1.25, 2.5, 5, 10, and 16 y of glucuronic acid2 per ml., respectively, are pipetted into Erlen- meyer flasks that already contain the previously stated amounts of buffer, iodine, bisulfite, and acid (final volume, 9 ml.). 4 ml. a&pots of this mix-

1 The washing procedure for these tubes includes an alcohol rinse after use, then soaking in chromic acid solution. The tubes should be marked in lead pencil only on the ground glass spot.

* Glucuronolactone serves equally well as a standard, provided the data are cor- rected to glucuronic acid. Both pure glucuronic acid and glucuronolactone were generously provided by Dr. E. R. Weidlein and Dr. N. E. Arts.

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530 P-GLUCURONIDASE AND GLUCURONIC ACID

ture, run in duplicate, are then pipetted for the naphthoresorcinol reaction. The concentration of glucuronic acid in the 4 ml. aliquot is plotted against optical density to yield a straight line which is readily reproducible. A new curve should be run whenever a new batch of naphthoresorcinol or sulfuric acid is used.

Calculation-The average optical density of the duplicate readings is substituted in the standard calibration curve to yield the concentration of glucuronide glucuronic acid in 4 ml. The free glucuronic acid value is then obtained by subtracting the figure for glucuronide glucuronic acid from the total glucuronic acid value.

Solution of Glucuronides- 9

y of glucuronide glucuronic acid X a = y glucuronide glucuronic acid in 5 ml. ana-

lyzed Serum (1: 10 Dilution in Protein-Free Filtrate)-

y glucuronide glucuronic acid 1000

X i X f X 100 = mg. glucuronide glucuronic acid per

100 ml. serum Urine (Diluted 1:60)-

y glucuronide glucuronic acid * 1000

X i X s X 100 = mg. per 100 ml. urine

EXPERIMENTAL

Measurement of Glucuronides and Glucuronic Acid in Aqueous Mixtures of Pure Compounds-Mixtures were made of glucuronides with glucuronic acid in varying proportions. The results of the analysis of 10 such mix- tures are given in Table I. Agreement between expected and found amounts of glucuronides is reasonably good, even in mixtures favoring free glucuronic acid by a factor of 8 to 18. The standard percentage devia- tion from theoretical recovery of glucuronides in this series was f3.3 per cent. The method is sensitive to glucuronic acid in a concentration of 0.50 y per ml. or 2 y in the test system. It has been our practice to work with concentrations of glucuronides or of glucuronic acid which yield values for optical density somewhere in the middle of the calibration curve.

Determination of Serum Glucuronides-2 ml. of serum are pipetted into 16 ml. of distilled water in an Erlenmeyer flask. The mixture is depro- teinized by the addition of 1 ml. each of 10 per cent sodium tungstate and 0.66 N sulfuric acid, followed by filtration 10 minutes later. Two 5 ml. aliquots are taken for analysis as described above. Table II lists values for serum glucuronides obtained on healthy subjects and on ill patients.

The choice of serum rather than whole blood as the material for study represents a current trend, since it eliminates from the system glucuronic acid-rich leucocytes (15, 16).

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W. H. FISHMAN AND S. GREEN 531

The range of serum glucuronides is from 0.6 to 1.35 mg. per cent in healthy subjects, with occasional elevations in patients.

Previous workers (17) have introduced an empirical correction factor for blood glucose designed particularly for the study of diabetic serum glucuronic acid, which was considered to be abnormally high in most cases. However, the data obtained with the present method indicate clearly that serum glucuronides are within the normal range in diabetic patients. The non-glucuronide substances reacting in the naphthoresor-

TABLE I

Determination of Glucuronides and Glucuronic Acid in Mixtures of Known

The results are in microgram 1s

“rs- No.

T Total glum-&c acid I Free glucuronic acid Glucuronide* glucuronic acid

Expected Found Expected Found Expected Found

1 9.68 9.68 0.48 0.20 9.20 9.48 2 9.60 9.55 0.90 1.22 8.70 8.33 3 9.66 9.78 1.90 1.95 7.76 7.83 4 9.64 9.85 2.85 3.49 6.79 6.36 5 9.62 9.78 3.80 4.04 5.82 5.74 6 9.60 9.90 4.75 5.17 4.85 4.73 7 9.58 9.68 5.70 5.85 3.88 3.83 8 9.56 9.90 6.65 6.86 2.91 3.04 9 9.52 9.50 8.55 8.54 0.97 0.96

10 9.52 10.24 9.03 9.68 0.49 0.56

Composition

per ml.

* The glucuronides of menthol, phenolphthalein, and pregnanediol in a ratio of 2: 1: 1 were dissolved in water to provide the stock solution of glucuronide, which was analyzed for glucuronide glucuronic acid. Mixtures ‘of stock’solution with a so- lution of glucuronic acid were prepared in the proportions stated.

cinol test in diabetic blood thus may be attributed to glucose. Studies on glucuronide formation in diabetic patients now become possible.

Determination of Urinary Glucuronides-Ordinary urines are diluted 1:50 with distilled water, and 5 ml. aliquots in duplicate are analyzed. Glucuronide-rich urines may require a 1: 500 dilution. Some data on 24 hour urine collections on normal subjects appear in Table II.

Course of Glucuronide Formation in Humans-By utilizing the techniques described for measuring glucuronides, the study of the rate of conjugation of exogenous glucuronidogenic substances becomes possible. Fig. 1 illus- trates the changes observed in the blood and urine of several normal sub- jects after the oral ingestion of menthol.

The advantages of the present analytical method over previous ones are

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532 P-GLUCURONIDASE AND GLUCURONIC ACID

(1) that isolation of the urinary menthol glucuronide is not necessary be- fore it can be determined and (2) that the small amounts present in blood serum can be measured. It is now possible to correlate rates of appearance

TABLE II

Values for Serum and Urinary Glucuronides

The results are in mg. per cent for serum glucuronides; in mg. per 24 hours for urinary glucuronides.

Subject No. Serum glucuronides Subject No. Serum glucuronides

Normal, healthy men

1 1.01,1.12,0.85,0.90 3 0.79,0.79,0.90,0.79 2 0.79, 1.24, 0.73 4 1.00, 1.35, 0.62, 1.1

Diabetic subjects

1.7 1.2 1.9 1.4 1.1 1.3 1.2

8 1.2 9 1.2

10 1.0 11 1.0 12 1.2 13 1.0

Prostatic cancer patients

1 1.6, 1.6,0.9, 1.2 6 1.7, 1.6, 1.4 2 1.6, 1.4 7 1.2 3 1.4,1.4,1.4,1.0 8 1.0 4 1.1,0.8, 1.1 9 0.8 5 2.1, 6.8, 3.0, 0.9, 4.2

1 2

Normal, healthy men

Urinaryglucuronides

324, 356, 362 400, 450, 387, 463

of the glucuronide in blood and urine and to compute the percentage con- version of the substance administered to its glucuronide without its pre- vious isolation. For menthol, this amounts to 62 to 66 per cent of the dose administered.

Assay of p-Glucuronidase Activity-Since, in vitro, p-glucuronidase cata- lyzes the hydrolysis of glucuronides, yielding the products glucuronic acid and the aglycone, the present techniques offer a means for assaying p-glu-

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W. H. FISHMAN AND S. GREEN 533

x

I\

MG. % SERUM E.L.H. 2 \ W.W.M.

x x-x !

I

I \ “\ a

I MG./HR. URINE 1 t200

n, 1

1 64%

MG. % SERUM

SO.

n ; z x

1 MG./HR. URINE

JL t’“” n 66%

1’1

I -L I I

I o-20 HOURS o

I I I -1 IO 20 30 40

GLUGURONIDES

FIG. 1

020 tl

PHENOLPHTHALEIN PHENOLPHTHALEIN

GLUCURONIC ACID GLUCURONIC ACID

I I I I I I

I23456 I23456 HOURS HOURS FIG. 2 FIG. 2

0

X

FIG. 1. Alterations in blood serum and urinary glucuronides following the inges- tion of menthol. All subjects except S. G. (1 gm.) received orally 1.5 gm. of men- thol in aqueous suspension at 16 hours. Specimens of venous blood were taken at 16, 17, 19, 21, and 23 hours and the serum analyzed for glucuronide glucuronic acid. Urine collections were made at the intervals of 0 to 16, 16 to 19, 19 to 23, and 23 to 40 hours and were similarly analyzed. The conversion of administered menthol to the increment in urinary glucuronides has been computed in per cent.

FIG. 2. &Glucuronidase-catalyzed hydrolysis of phenolphthalein glucuronic acid. The system contained 0.5 ml. of 0.01 M phenolphthalein glucuronic acid, 4 ml. of 0.1 M acetate buffer, pH 4.5, and 0.5 ml. of purified liver p-glucuronidase. In Curve A, the activity of the added enzyme solution was 500 units (al), and, in Curve B, 58 units per 0.5 ml. Such digests incubated at 38” in a water bath were removed at the inter- vals indicated and immersed in boiling water for 1 minute. Duplicate samples of 1 ml. each were taken for phenolphthalein measurement (21), and two 1 ml. aliquots were individually acidified with 0.1 ml. of 0.6 N H2S04 solution, made up to 5 ml. with Hz.O, and then extracted three times with 5 ml. of ethyl acetate. Analyses were performed on the aqueous phase for total and glucuronide glucuronic acid, from which data figures for free glucuronic acid were computed. The phenolphthalein and glucuronic acid liberated are expressed in micromoles per ml. of digest.

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534 &CLUCURONID.~~E AND GLUCURONIC ACID

curonidase activity. Accordingly, a test was made of this idea by utiliz- ing pure phenolphthalein glucuronide as the substrate and highly purified calf liver p-glucuronidase as the enzyme. The results are presented in Fig.

DISAPPEARANCE

0 450 1350 2250 &GLUCURONIDASE UNITS

FIG. 3

SERUM

---GLUCURONIDES

4 GLUCURONIC ACID

HOURS

FIG. 4

FIG. 3. Appearance of free glucuronic acid and disappearance of glucuronide glucuronic acid upon incubating a new glucuronide with @-glucuronidase. The system contained 85 y (glucuronide glucuronic acid) of an ethyl acetate-extractable product from a testosterone-liver slice incubation, in 3 ml. of aqueous solution plus 5 ml. of HzO, 1 ml. of M acetate buffer (pH 4.5), and 1 ml. of purified liver ,T-glucuroni- dase. Three digests containing increasing amounts of enzyme as indicated in the figure were incubated for 4 hours at 37”. After deproteinization with 1 ml. of 10 per cent ZnSOl plus 2.5 ml. of 0.5 N NaOH and 0.5 ml. of H,O, the supernatant fluids were analyzed for free and glucuronide glucuronic acid.

FIG. 4. Fluctuations of serum glucuronides and “glucuronic” acid in two human subjects observed after the ingestion of 5 gm. of glucuronolactone. The glucuronic acid figures were obtained by correcting the figures for oxidizable naphthoresorcinol- positive material in serum, expressed as glucuronic acid by the zero time value, it being assumed that the increment in serum was due to the glucuronolactone admin- istered.

Measurements were made of the glucuronic acid liberated rather than of the disappearance of substrate so that these figures could be compared directly with values for phenolphthalein liberated. The good agreement found indicates that /Sglucuronidase can be assayed by measuring glu- curonic acid liberation by a more specific procedure than the determination of increase in reducing power employed previously. Moreover, phenol- phthalein liberation has provided an independent yardstick with which to measure the performance of the present method for determining glucuronic

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W. H. FISHMAN AND S. GREEN 535

acid. It is expected that this assay will find application in the use of substrates whose aglycone moiety cannot be conveniently measured or when the enzyme acts on mixtures of glucuronides.

Identi$cation of /3-Glucuronides-fi-Glucuronidase has been employed as an analytical agent to identify P-glucuronides. However, as a rule, no evidence for the liberation of free glucuronic acid is given, the identification of the aglycone alone being made after enzymic hydrolysis. On the other hand, instances may occur in which the amounts of material available are so small that identification of the aglycone by isolation and chemical char- acterization is not possible after P-glucuronidase hydrolysis. Thus, the use of present analytical techniques for establishing the presence of a new /3-glucuronide formed after incubating testosterone with surviving rat liver slices8 is illustrated in Fig. 3.

Utilization of Glucuronolactone by Human Subjects--It *was not possible to distinguish between glucuronic acid and its conjugates by the methods used in previous studies. Information bearing on the question of the uti- lization of glucuronolactone in the formation of glucuronides is therefore lacking. Fig. 4 presents a demonstration of the fluctuations in serum

glucuronides following the oral ingestion by humans of glucuronolactone. The increase in oxidizable naphthoresorcinol-positive material above the control level at zero time is ascribed to the blood level of the substance administered. These same conditions can be employed in studies of glu- curonic acid utilization.

DISCUSSION

The use of alkaline iodine solutions for mild oxidation of aldoses was

first described by Romj;n (18) in 1897 and has been widely used since (19). The conditions employed for glucuronic acid oxidation in this laboratory are essentially the same as those Levy and Doisy (20) used for quantitative oxidation of glucose. The oxidation procedure is efficient (see Table II), the conditions being adequate for the complete oxidation of at least 30 times the amount of glucuronic acid employed in these experiments. The product, saccharic acid, gives a negative naphthoresorcinol test. Glu- curonides are completely resistant to this oxidation and can be measured successfully by the naphthoresorcinol color reaction after the oxidation step.

In order to obtain accurate data for the low levels of blood serum glu- curonides, it was necessary to improve 2- to 3-fold the sensitivity and performance of the existing naphthoresorcinol reaction. This was accom- plished by careful testing of conditions of heating, mineral acid and naph- thoresorcinol concentration, and of the technique of pigment extraction.

* Fishman, W. H., and Sie, H.-G., Federation Proc., 14,211 (1955).

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536 &GLUCURONIDASE AND GLUCURONIC ACID

Accuracy and reproducibility depended not only on good laboratory tech- nique but also on the maintenance of even heating (oil bath at 100°) and on the close adherence to details of preparing the naphthoresorcinol solu- tion. The standard curve was reproduced many times without exception, and duplicate transmission readings were as a rule within 1 per cent of each other in all the analyses.

The specificity of the procedure has also been studied. Whereas, with the use of the naphthoresorcinol reaction under conditions of lower acidity, glucuronic and galacturonic acids were the only materials which produced pigment to any important degree, it was found that hexoses and ascorbic acid registered with the present sensitive procedure. This interference is of no consequence in the determination of glucuronides, since it is elimin- ated by the oxidation step. However, this interference is important in interpreting the significance of naphthoresorcinol-positive oxidizable ma- terial in blood and urine. Thus far, no reports have appeared which identify free glucuronic acid as a component of normal blood. Our re- peated attempts in this direction with the sensitive techniques of paper chromatography have been negative. Consequently, no significance with regard to glucuronic acid is now attached to this fraction in blood and urine except in cases in which glucuronic acid or the lactone is administered.

Confidence in the reliability and accuracy of the method is based both on satisfactory recovery of glucuronide glucuronic acid from mixtures with free acid and on the good agreement in the figures for phenolphthalein and glucuronic acid liberated in the enzymic hydrolysis of phenolphthalein glucuronic acid.

Examples of problems which can now be approached experimentally with the aid of the present method have been described to illustrate its potential applications and require no further discussion.

SUMMARY

1. Details are given of a procedure for determining glucuronides in the presence of glucuronic acid, which is based on the prior oxidation of the acid and other interfering aldehydic substances with alkaline hypoiodite solution, the remaining glucuronides being analyzed by the naphthoresor- cinol color reaction.

2. Satisfactory recovery is obtained of glucuronides from their mixtures with glucuronic acid. The sensitivity of the method is 2 y of glucuronide glucuronic acid and the standard deviation in the neighborhood of 3 per cent.

3. The procedure may be utilized for the assay of @-glucuronidase activity when the amount of the liberated aglycone is not readily determined.

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W. H. FISHMAN AND S. GREEN 537

4. The identification of p-glucuronides is made more certain by the new method, applied to /3-glucuronidase hydrolysates.

5. The determination of the small amounts of glucuronides circulating in the blood is feasible, making possible time studies of the rates of conjuga- tion and excretion of administered glucuronidogenic substances (e.g. men- thol).

6. The course of absorption and excretion of administered glucuronolac- tone can be followed, and any alteration in the amount of excreted glu- curonides can be easily measured.

BIBLIOGRAPHY

1. Mosbach, E. H., and King, C. G., J. Biol. Chem., 186, 491 (1950). 2. Storey, I. D. E., Biochem. J., 47, 212 (1950). 3. Bidder, T. G., J. Am. Chem. Sot., 74,1616 (1952). 4. Packham, M. A., and Butler, G. C., J. Biol. Chem., 194, 349 (1952). 5. Doerschuk, A. P., J. Biol. Chem., 196,855 (1952). 6. Dutton, G. J., and Storey, I. D. E., Biochem. J., 67,275 (1954). 7. Fishman, W. H., Smith, M., Thompson, D. B., Bonner, C. D., Kasdon, S. C., and

Homburger, F., J. Clin. Invest., 30, 685 (1951). 8. Sudhof, H., Z. physiol. Chem., 290, 72 (1952). 9. Hollmann, S., and Wille, E., 2. physiol. Chem., 290,91 (1952).

10. Fretwurst, F., and Ahlhelm, H. A., Arch. exp. Path. u. Pharmakol., 217, 382 (1953).

11. Tollens, B., and Rorive, B., Ber. them. Ges., 41, 1783 (1908). 12. Mandel, J. A., and Neuberg, C., Biochem. Z., 13, 148 (1908). 13. Neuberg, C., and KobeI, M., B&he-m.Z., 243,435 (1931). 14. Maughan, G. B., Evelyn, K. A., and Browne, J. S. L., J. BioZ. Chem., 128, 567

(1938). 15. Deichmann, W. B., and Dierker, M., J. BioZ. Chem., 163,753 (1946). 16. Follette, J. H., Valentine, W. N., Hardin, E. B., and Lawrence, J. S., J. Lab. and

CZin. Med., 43, 134 (1954). 17. Saltzman, A., Caraway, W. T., and Beck, I. A., Metabolism, 3,ll (1954). 18. Romjin, G., 2. anal. Chem., 38,349 (1897). 19. Pigman, W. W., and Goep, R. M., Chemistry of the carbohydrates, New York

(1948). 20. Levy, M., and Doisy, E. A., J. BioZ. Chem., 77,733 (1928). 21. Fishman, W. H., Springer, B., and Brunetti, R., J. BioZ. Chem., 173,449 (1948).

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William H. Fishman and Sidney GreenACID STUDIES

-GLUCURONIDASE AND GLUCURONICβGLUCURONIC ACID AS APPLIED TO

MICROANALYSIS OF GLUCURONIDE

1955, 215:527-537.J. Biol. Chem. 

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