THE NATURE OF THE SUGAR IN FOUR CASES OF PENTOSURIA.*

BY ISIDOR GREENWALD.

(From the Littauer Pneumonia Research Fund, New York University, New York.)

(Received for publication, May 17, 1930.)

Ever since the discovery of the first case of pentosuria by Salkowski and Jastrowitz (1) the nature of the pentose excreted has excited considerable interest. The first definite report was that of Neuberg (2) who stated that he had isolated i-arabinose. Because of this report and also because of the apparent optical inactivity of the pentose-containing urine, it has generally been believed that Garabinose is present in all cases. However, more recent studies indicate that the apparent, optical inactivity of the urine may have been due to the low concentration of the sugar and that, at least in a number of cases (3-lo), the sugar was actu- ally dextrorotatory,

In fact, only Neuberg, Cammidge and Howard (II), and possi- bly Aron (12) have isolated from the urine any derivatives of i-arabinose. All of the other reported attempts at the isolation of the pentose or of its derivatives have indicated that the sugar was probably d-xyloketose (S-10, 13). This identification was made quite certain by Levene and La Forge (9).

The present paper is a report of the isolation from the urine of four different individuals of an osazone and a p-bromophenyl- hydrazone having properties identical with those described by Levene and La Forge. A more extended study was made with the material obtained from the first of these cases.. This included the liberation of the sugar from the p-bromophenylhydrazone, de- termination of its optical activity, and attempts at crystallization. The latter were unsuccessful as were, also, all attempts at the

* Bided by a grant from the American Association for the Advancement of Science.

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2 Nature of Sugar in Pentosuria

separation of the sugar from the urine without the use of the expensive p-bromophenylhydraeine.

All four individuals are Jews. One is a woman; the others, males. Case 1 has already been reported by Margolis (14). For Case 2, I am indebted to Dr. Herman Sharlit and for Cases 3 and 4 to Dr. Herman 0. Mosenthal.

In view of the association of pentosuria with migraine by Margolis, it is interesting to observe that our Case 2 had periodic headaches between the ages of 8 and 18 and that these had dis- appeared shortly before the presence of sugar in the urine had been discovered, 3 years ago. Although they were questioned in re- gard to this, there is no history of headache in either of the other two individuals. They are now 40 and 20 years old respectively and the condition has been known to exist for 18 months in Case 3 and for 6 months in Case 4.

The ratio of sugar to nitrogen in the urine of all four cases was about the same, 1: 3 or 4. The osazones, prepared in the usual manner, melted at 157-160’. When recrystallized with an equal weight of Il-xylosazone, the melting point, rose to 195-202”. The optical properties of the osazone wcrc determined in the first case.

In spite of several attempts, it was not found possible to obtain p-bromophcnylhydraaone from the urintc of Case 1 by following the directions given by Levene and La Forge. This may have been due to the prcscncc in the urine of foreign substances due to the ingestion of various sedatives by the patient. Several modi- fications of the method were tried and one was finally adopted. This was also employed upon the urine of the three other individ- uals. It is possible that the original method of Levene and La Forge might have been successful with these urine samples. It was not tried because only limited quantities of the urine were available.

The method depends upon the removal of phosphate ion and most of the sulfate and chloride ions with neutral lead acetate, precipitation of the sugar with basic lead acetate and sodium hydroxide (Plimmer and Skelton (15)), decomposition with car- bon dioxide (Levene and La Forge), removal of most of the re- maining nitrogenous constituents with HgS04 and Ba(OH)% (modified from West, Scharles, and Peterson (16)), concentration by means of alcohol, and final separation as the hydrazone.

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I. Greenwald 3

10 liters of the urine are treated with a concentrated solution of neutral lead acetate until no more precipitate is produced. After standing about an hour, most of the liquid is decanted and filtered through a large Buchner filter. The precipitate is then trans- ferred to the filter and sucked nearly dry. The suction is then discontinued and about a liter of water is placed in the funnel. This is allowed to stand overnight. Most of the water filters through, washing the precipitate. The suction is then again applied and the precipitate is pressed dry with the aid of a sheet of rubber, as described by Gortner (17). The filtrate is cooled to 5” in a freezing mixture and, while being stirred vigorously, a liter of a 25 per cent solution of basic lead acetate (Goulard’s extract) is added. The cooling and stirring are continued while 40 per cent sodium hydroxide is added from a pipette or burette. About 100 cc. are required. Portions of the mixture are removed and filtered. The addition of the alkali is continued until further addition to the clear filtrate gives no precipitate. An excess of alkali must be avoided. At the correct point, the filtrate will give no precipitate upon the addition of a drop of sodium hydroxide solution and no color or only a trace upon the addition of thymol- phthalein. The cold mixture is filtered upon two large Buchncr funnels and pressed dry, a sheet of rubber again being usxl. This %lt,rat,ion requires about 4 hours, The precipitate is suspended in about 4000 cc. of water, preferably wil;h the aid of a mechanical stirrer. About 200 gm. of solid carbon dioxide are then added and the stirring is continued. The decomposition of the precipitate is rapid and its completion is announced by the sudden foaming of the mixture. This can easily be controlled by the addition of a few drops of caprylic alcohol. The mixture is then acid to litmus. The stirring is continued for from 30 to 45 minutes longer and the mixture is then filtered on a large Buchner funnel. This material filters rapidly. The precipitate is pressed dry and is then sus- pended in about 2500 cc. of water and again stirred with carbon dioxide. After 30 minutes, or longer, the mixture is filtered and pressed dry. The filtrates are separately treated with HzS and are filtered, in order, upon the same %uted paper which is then washed with a little water. The filtrates are evaporated in vucuo to a volume of about 3000 cc. The nitrogen content is then de- termined upon 1 cc. portions, with 0.01 N acid and alkali. Enough

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Nature of Sugar in Pentosuria

of a 30 per cent solution of HgS04 in 10 per cent H&J04 to combine with all the nitrogen found, upon the assumption that each gm.- equivalent of nitrogen requires 1 mol of HgSOa, is then added and the mixture is cooled by the addition of ice. A hot concentrated solution of Ba(OH)z is then added until the mixture is just neutral to litmus. It is then filtered, and the precipitate washed and pressed dry. The small amount of mercury in the filtrate is re- moved with HzS and t,he filtrate is evaporated in vacua to about 75 cc. It is then slowly run into 1500 cc. of absolute alcohol, with constant stirring. After the addition of 150 cc. of anhy- drous ether, the precipitate settles rapidly. It is filtered off and washed with a little absolute alcohol. The filtrate is again eva- porated in vacua to a volume of 35 or 40 cc. and the precipitation with alcohol is repeated. Only a small amount of precipitate is now formed. This is filtered out, the volume of the filtrate is measured, and 1 cc. is diluted to 10 or 25 cc. for a sugar determina- tion by Sumner’s method (18). The remainder is evaporated in vacua to about 75 cc. and is then transferred to a glass evapo- rating dish. p-Bromophenylhydrazine in amount equal in weight to the indica,ted “glucose” content of the liquid is added and the mixture is stirred until it is all dissolved. Crystallization may begin in a few minutes bul; it) is usually absent. The dish is placed in a vacuum desiccator over NaOH for 1 or 2 days. Henlircg is unnecessary, 13y that time, most of the alcohol has evaporated and a red oil which may or may not contain crystals is left. The dish is filled with cracked ice and water is slowly added. As the oil is mixed with the ice water, it sets to a mass of crystals. This is stirred to secure thorough mixture and, after an hour or two at 0”, is filtered and washed with ice water. The crystals are then ex- tracted with alcohol-free ether until the filtrate is colorless. They are dried in a vacuum desiccator and recrystallized by solution in warm alcohol and precipitation with ice water. Extraction of the recrystallized material with ether generally removes a little more coloring matter. The yield is about 0.5 gm. of hydrazone for each gm. of “sugar” in the original urine. This is less than was ob- tained by Levene and La Forge.

The material obtained from Case 1 was identified by its melting point, decomposition point, and optical rotation. The ot.her preparations of the hydrazone were identified merely by the melt-

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I. Greenwald 5

ing and decomposition points, which remained unchanged when the material was mixed with an equal quantity from Case 1. The data are summarized in Table I and the following text.

E$ect of Bromine upon the Urine Pentose.

Case I.-A solution of the partly purified sugar, before the treatment with alcohol, contained 3.68 mg. per cc. After treat- ment with bromine for 2 days, it contained 3.23 mg. per cc.

Case .%?.-A solution of the partly purified sugar, after the treat- ment with alcohol, contained 10.4 mg. per cc. After treatment with bromine for 1 day, it contained 10.0 mg. per cc.

TABLE I.

Data Concerning the Excretion and Properties of Pentose in Urine.

Case No. Nitrogen.

qm.

1 87.6t 2 72.4 3 40.6$ 4 16.38

-

Pentox?.*

____

CJm.

29.2 17.8 13.1 4.6

From urine Mixed with me,ting ’ 2-xy10syA0ne

point. meltmg point.

“C. T*

157-160 20&202 154-157 108-200 161-163 lw=188 156158 202

- s-Bromophenylhydrazone.

Melting point.

Decomposi- tion point.

--

“C.

127-128 X3&132 130-131 1%~&132

____-

“(;‘.

165

165 164 165

-

* The rr!duciug action was determined by Sumner’s method (18) and the apparent glucose content calculated,

t See Margolis (14) for variations in the ratio of nitrogen to pentose excretion.

$4600 cc. g 2600 cc.

Optical Activity of Phenylosaxone.

0.200 gm. of osazone from Case ‘L was dissolved in a mixture of 4 cc. of pyridine and 6 cc. of methyl alcohol. Read in a 1 dm. tube, [01] in 10 minutes was 0.25”; after 48 hours, 0.60”.

Optical Activity of p-Bromophenylhydraxone.

0.500 gm. of the hydrazone from Case 1 was dissolved in 5 cc. of alcohol. Read in a 1 dm. tube, [CX] in 10 minutes was -1.87”; after 24 hours, +2.43”.

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6 Nature of Sugar in Pentosuria

Decomposition of p-Bromophenylhydrazone.

7.2 gm. of the hydrazone from Case 1 were decomposed with benzaldehyde according to the method described by Levene and La Forge. After extraction with ether, the aqueous solution was evaporated in vacua and then diluted to 100 cc. In a 1 dm. tube, [a] = 1.18”. Since the calculated pentose content was 3.39 gm., [a]: = 34.8”. Levene and La Forge give 33.15” but state that this value is to be regarded as a minimum.

The reducing action of the solution was determined by various methods. 1 mg. of the pentose was found to be the equivalent of 1.25 mg. of glucose by Sumner’s method, 1.22 by Benedict’s copper method (19), 1.45 mg. of glucose by Benedict and Osterberg’s sodium hydroxide-picrate method (20), and 1.18 by their sodium carbonate-picrate method (21).

SUMMARY.

The phenylosazone and the p-bromophenylhydrazone of the sugar have been isolated from the urine of four cases of pento- suria. Their properties are identical with those described by Levene and La Forge and indicate the sugar to be d-xyloketose.

The method of preparation of the p-bromophcnylhydrazone has been modified.

BIBLIOGRAPHY.

1. Salkowski, E., and Jastrowitz, M., C&r. wbed. Il’issensch., 30,337 (1892). 2. Neuberg, C., Ber. &em. Ges., 33,2243 (1900). 3. Blumenthal, F., Dezctsch. Klin., 3, 305 (1902). 4. Rosenfeld, F., Med. Klin., a,1041 (1906). 5. Schriler L., Berl. klin. Woch., 47, 1322 (1910). 6. Luzzatto, R., Beitr. them. Physiol. u. Path., 6,87 (1905). 7. Klercker, K. O., Deutsch. Arch. klin. Med., 108,277 (1912). 8. Zerner, E., and Waituch, R., Mona&h. Chem., 34, 1639 (1913). 9. Levene, P. A., andLa Forge, F. B., J. BiaZ. Chem., 18,319 (1914).

10. Hiller, A., J. BioZ. Chem., 30,129 (1917). 11. Cammidge, P. J., andHoward, H. A. H., Brit. Med. J., 2,777 (1920). 12. Aron, H., Monatschr. Kinderheilk., 12,177 (1913). 13. Elliott, J. H., and Raper, H. S., J. BioZ. Chem., 11,215 (1912). 14. Margolis, J. I., Am. J. Med. SC., 177,348 (1929); J. Am. Med. Assn., 93,

173 (1929). 15. Plimmer, R. H. A., and Skelton, R. F., Biochem. J., 8,641 (1914). 16. West, E. S., Scharles, F. H., and Peterson, V. L., J. BioZ. Chem., 82,

137 (1929).

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17. Gortner, R. A., J. Am. Chem. SOL, 36,1967 (1914). 18. Sumner, J. B., J. Biol. Chem., 66,393 (1925). 19. Benedict, S. R., J. Biol. Chem., 64,207 (1925). 40. Benedict, S. R., and Osterberg, E., J. Biol. Chem., 48, 51 (1921). 21. Benedict, S. R., and Osterberg, E., J. Biol. Chem., 34,195 (1918).

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Isidor GreenwaldCASES OF PENTOSURIA

THE NATURE OF THE SUGAR IN FOUR

1930, 88:1-7.J. Biol. Chem. 

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