THE NATURE OF THE SUGAR OF NORMAL URINE

I. THE PHENYLOSAZONES

BY MARK R. EVERETT AND FAY SHEPPARD

(From the Department of Biochemistry and Pharmacology, University oj Oklahoma Medical School, Oklahoma City)

(Received for publication, February 4, 1932)

Despite a general interest in the osazones of normal urine, surprisingly little chemical work has been done upon them, and no careful, critical estimate of such work is available.’ Hirsch1 (23) first noticed among the debris of the osazone test occasional irregular, thorn-apple-shaped crystals, soluble in alcohol and insoluble in water. Many others (17, 25, 28, 30, 36, 44, 45, 47, 49, 51, 53) have found crystals in the precipitate from every normal urine examined, and the frequency of the thorn-apple variety has been established (1, 6, 13, 25, 28, 30, 32).

As early as 1905 McEwen (32) made a clear distinction between the two kinds of crystals previously noted by Frank; namely, phenylglucosazone and the thorn-apple crystals, which McEwen described as “much smaller crystals, in the form of boat-shaped staves with centrally disposed radiating spicuke, like the seed-pod of Datura stramonium, and likened, save for their color, to crystals of ammonium urate.” Geelmuyden (15) gave the name “phys- iological sugar” to the non-glucose material from which the thorn-apple crrystals originated. In his Fig. f he pictured rosettes that were apparently phenylglucosazone, but after mechanical fracture were found to consist of thorn-apple (sword- like) crystals. This circumstance should have served as a warn- ing to microscopists who have been identifying such rosettes as phenylglucosazone, yet the finding has been generally ignored. While Geelmuyden said that the thorn-apple crystals were more reddish than phenylglucosazone, I&t (24) described the latter as the more deeply colored. Host’s results and descriptions lead

1 For general reviews consult Neubauer-Huppert (37) and Neuberg (38).

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432 Sugar of Normal Urine

us to believe that he was confused by the false phenylglucosazone of Geelmuyden.

Malmros’ (30) photographs of urine osazones may be ,roughly divided into the two types already mentioned. His Plate IV is a beautiful illustration of the effect of graded additions of glucose upon the crystal form, first shown by Frank (13). Here one may note the poorly formed crystals of the original urine, the inter- mediate thorn-apple types, and the ultimate sprays and sheaves of phenylglucosazone. At times Mahnros recrystallized his crude osazone precipitate from alcohol-water mixtures, getting broad needles, where none was visible originally.

Hassan (22) also fractionated the crude osazone precipitate by recrystallization. The preponderating physiological non-glucose component consisted of broad, flat, orange-yellow needles with gradually tapering ends, or blunt, spear-shaped points, tending to form irregular rosettes that resembled phenylmaltosazone. These crystals were soluble in hot water, 20 per cent alcohol, and alcohol-ether mixtures.

While a substance with the melting point of phenylglucosazone has occasionally been prepared from something in normal urine (2, 5, 19, 21, 22, 34, 36,46, 51, 55, 56), the usual result has been the production of rather impure crystals which have been variously designated as phenylosazones of glycuronic acid (5, 11, 16, 17, 21, 23, 49), isomaltose (3, 29, 46), maltose (30), pentose (14, 18, 25), or physiological sugar (15, 22, 24, 50). The osazones from urine previously hydrolyzed by Cammidge’s procedure are excellent examples of troublesome mixtures. Contrary to the findings of Cammidge (9) and Pekelharing and van Hoogenhuyze (47), Grimbert and Bernier (21), Smolenski (58), and Neuberg (39) made the osazone from normal urine and unhydrolyzed urine and sep- arated it into a high melting fraction and the water-soluble frac- tion of Cammidge. (See also Willcox (59).)

Realizing that a formation of mixed crystals (8, 20, 41) would account for the great confusion, we examined all original de- scriptions carefully and were surprised to find how many of these urinary osazones were obviously mixtures, with vague melting points and properties that attested the presence of gross impuri- ties. The original communications of Baisch (2, 3) and of Pavy and Siau (46), which have often been cited as proof of the presence

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M. R. Everett and F. Sheppard 433

of isomaltose in normal urine, display these facts. We believe with Salkowski (54) and Hassan (22) that most urinary phenylo- sazones were mixtures, whose physical properties were of little significance.

It seems curious to us that so little attention has been paid t.o the very suggestive, now almost forgotten work of Jai% (26) and Milrath (33). They found that 1-,phenylsemicarbazide separates as round, yellow, wart-like masses of needles from urine which has been heated with phenylhydrazine and acetic a.cid. Schulz (57) disposed of their findings by saying that the fear of confusion of glucose with urea is an exaggerated one, since human urine contains too little urea. This estimate of the possible danger from phenylsemicarbaaide may have misled many. At least, the only further experimentation was that of Percher (48), who noted the retarding effect of urea and of Liebig’s extract upon phenylglucosazone formation. In his experiments, whenever urea was added to glucose solution, the resulting osazone (actually much phenylsemicarbazide, or mixed crystals) redissolved upon heating the solution. Percher did not recognize this as phenyl- semicarbazide formation.

EXPERIMENTAL

In the course of our investigation of the sugar of normal urine we prepared several gm. of crystalline osazone from the night urines of dozens of normal persons. The method of preparation was essentially that of Hassan (22). 1 to 3 gm. of a mixture of 1 part of phenylhydrazine hydrochloride and 2 parts of sodium acetate were used for every 20 cc. of prepared urine, previously adjusted to pH 6.7. The mixture was heated in the boiling water bath for 1 hour, and allowed to cool in the bath for 12 to 15 hours. It was then centrifuged and the precipitate washed three times with ice water and then dissolved in 25 per cent ethyl alcohol solution. The solution was extracted three times with equal volumes of ether, and the latter was evaporated on the water bath. After adding an equal volume of water to the residual fluid, crystallization was allowed to take place in the refrigerator. The accumulated crystals were recrystallized from 3 per cent aqueous pyridine solution, the phenylhydrazine derivative sepa- rating as rosettes.

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434 Sugar of Normal Urine

The preparations used in the above procedure were either bone-black filtrates of urine, as recommended by Hassan, or partly purified extracts of urine made by evaporating the urine at 60” under reduced pressure, extracting the residue with absolute methyl alcohol, evaporating the extract under reduced pressure, dissolving this residue in distilled water, adding barium hydroxide solution to pH 7, and removing barium from the filtrate by means of sulfuric acid solution. By using only night samples of urine we expected to increase the yield of osazone from the uroketose (10) and to reduce the danger of alimentary mellituria to a minimum.

At first our products seemed to be phenylpentosazones, but we hesitated to regard them as such because of our inability to pre- pare the corresponding osazones from pure I-arabinose and d-xylose by the same process. Later we realized that continued recrystal- lization of the urinary osazones had raised their melting points to the vicinity of the corresponding semicarbazides. Thus, the first products from aqueous pyridine melted between 148-165’. After three or four recrystallizations, a dozen different prepara- tions melted between 160-168”. Our purest material, recrystal- lized six times, was optically inactive and melted at 170-172”. These temperatures were determined in the Roth apparatus. They were carefully checked against known materials and have been corrected. Mixing our material with Eastman’s pure phe- nylsemicarbazide result,ed in no change of melting point, while mixing it with pure phenylxylosazone caused the melting point t,o be greatly lowered.

The appearance of our derivative was microscopically identical with that of pure phenylsemicarbazide. Most authorities de- scribe the latter as plates or leaflets, but Holmgren’s description of his thorn-apple crystals from urine gives a much better picture of their actual appearance. He says that they were strange yellow crystals, arranged in regular balls which seemed to consist of needles, and yet when seen from the side, they were plate-like and had the form of lancet-like scales. After several recrystalliza- tions they became still more needle-like. When dry chloroform is used as a solvent, both pure phenylsemicarbazide and our urine derivative crystallize as perfectly white rectangular plates, with no rosettes. By Jaffe’s procedure we converted our substance into the corresponding nitroso derivative. This proved to be

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M. R. Everett and F. Sheppard 435

identical with that prepared from pure phenylsemicarbazide in every respect.

Micro analysis of our phenylhydrazine derivative by the Re- search Service Laboratories, New York, gave the following results.

4.985 mg. of substance, dried to constant weight in vacua, gave 10.210 mg. of COZ and 2.665 mg. of HzO; 4.899 mg. gave 1.191 cc. of N, at 26” and 758 mm.

C H N per cent pet cent per cent

Found................................... 55.85 5.98 27.67 Theory for phenylsemicarbazide.. . . . . . 55.60 6.00 27.81

I‘ “ phenylpentosazone........... 61.97 6.43 17.02

Urinary p-nitrophenylhydrazine and p-bromophenylhydrazine derivatives were also obtained, the former as orange-yellow needles, melting at 194-202”, and the latter as colorless, acicular needles, melting with decomposition at 226-228”. Pure p-nitrophenyl- semicarbazide melts at 211-212” and p-bromophenylsemicarbazide at 226” with decomposition. The further recrystallization of our p-nitrophenylsemicarbazide was not attempted because of the small amount prepared. We also experimented with or- and ,& naphthylhydrazines, m-nitrophenylhydrazine, methylphenylhy- drazine, diphenylhydrazine, and 2,4-dinitrophenylhydrazine, but these gave only oils or amorphous products not easily crystal- lized. Recognizable hydrazones could not be obtained by means of the hydrazines mentioned in this paper, either by classical methods, or by extraction of the mixture with ether, etc. Not only was this true for urine, but also for the concentrated, purified syrups which we have made from normal urine by a process soon to be published.

Jaffe (26) found that 2 per cent solutions of urea gave visible phenylsemicarbazide crystals, the yield being about 85 per cent of the theoretical in the course of 24 to 48 hours. He suspected the presence of phenylsemicarbazide in urinary osazones and was able to prepare an appreciable amount from normal dog urine. He was unable to form a definite conclusion about human urine.

We have secured crystals of discolored phenylsemicarbazide by the customary osazone procedure from artificial urines contain- ing 2.3 per cent urea. The artificial urine was prepared by mixing 1 cc. of each of the following with 2 cc. of 2.3 per cent potassium

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436 Sugar of Normal Urine

acid phosphat,e and 4 cc. of 0.4 per cent disodium urate *HzO. The mixture was adjusted to pH 6.5.

per cent per cent 2.0 alanine 21.0 magnesium chloride.6HzO 1.5 ammonium chloride 2.7 potassium chloride 4.4 “ sulfate 1.3 “ nitrate 8.0 calcium chloride.HzO 0.16 “ thiocyanate 0.15 “ lactate.5HzO 19.0 sodium chloride 0.3 p-cresol 1.3 “ hippurate 2.0 creatinine 46.0 urea

When the urea concentration was doubled the yield of crystals was greatly increased. When urea was omitted from the mixture these crystals were not found. The yield from the dilute mixture was uncertain and variable, but comparatively large amounts of phenylsemicarbazide, identified by its crystal form and melting point, could always be separated from the supernatant liquid by means of alcohol-ether extraction. This was also true for normal urine. The original crystals of the osazone precipitate are often very misleading, but they assume their characteristic shape after being recrystallized.

It is therefore obvious that l-phenylsemicarbazide will be pro- duced by the phenylhydrazine test from every normal urine. It is merely its massive crystallization, observed by Jaffd and Milrath, which requires longer heating. We have been able to show that urea and sodium hippurate, especially the former, in- crease the solubility of phenylsemicarbazide in water. Sodium chloride and ammonium sulfate, in larger concentrations, have an appreciable salting out effect, thus aiding the crystallization of phenylsemicarbazide, which has a tendency to form supersaturated solutions. It can easily be seen that the actual degree of crys- tallization of phenylsemicarbazide in t.he usual osazone test will be the result of the interplay of many factors. We wish to enumerate the following.

Concentration of U&e-As urine becomes concentrated phenyl- semicarbazide formation is hastened by the greater urea concen- tration. The increased concentration of salts will aid its crystal- lization. Several investigators (4, 13, 25, 42, 49) have noticed that the sensitivity of the osazone test is increased by concentrat- ing the urine.

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M. R. Everett and F. Sheppard 437

Acidity of Test Mixture-Certain modifications of von Jaksch’s t,est (27), such as Neumann’s (40), are said to be more sensitive (31), but they also employ a liberal amount of acid which, hasten- ing the production of cyanic acid from urea, produces larger yields of phenylsemicarbazide. Holmgren (25) showed that urines which had been made slightly alkaline gave no crystals, but he attributed this to the destruction of sugar by alkali.

Extent of Heating and Subsequent Cooling-Moritz (35) noticed that prolonged heating increased the sensitivity of the test. Since the heating period has been gradually lengthened from 15 minutes (27) to 2 hours (22) the errors from phenylsemicarbazide have been correspondingly increased.

Previous Formation of Cyanic Acid by Hydrolytic Procedures- We have been able to show very clearly that previous hydrolysis of artificial urine, or of dilute urea solutions, by Cammidge’s pro- cedure, results in a tremendously increased yield of phenylsemi- carbazide in the usual osazone test. We have assumed that the production of cyanate is the cause, since potassium cyanate solu- t,ions act just like Cammidge’s hydrolysate. The phenylsemi- carbazide separating from such test mixtures is well crystallized in the form of rosettes. It is quite obvious from these experi- ments that I-phenylsemicarbazide is one of the chief components of Cammidge’s osazone.

Cammidge stated that one peculiarity of his osazone was its marked solubility in 33 per cent sulfuric acid solution. Phenyl- semicarbazide is much more soluble in the latter than phenylglu- cosazone is. It is also quite soluble in chloroform, which accounts for Moritz’ observation (35). Its marked solubility in hot water may well account for the more soluble constituent of many osazone mixtures (12, 21, 22, 25, 30, 49).

In artificial urine mixtures and in phenylsemicarbazide solu- tions we have observed practically every form of osazone crystal described by others. By conducting the osazone test upon solu- tions containing both glucose and phenylsemicarbazide, we have found forms like the mixed crystals of Hassan (22), and have noted the growth of one of these substances fixed solidly upon a matrix of the other, as noted by several with normal urine (15, 22, 24, 30). We conclude that the physical form, composition, and melting point of osazones from normal urine, with its great urea

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43s Sugar of Normal Urine

content, are not trustworthy. While phenylglucosazone appears to form mixed crystals with phenylsemicarbazide under certain circumstances, it is nevertheless quite insoluble in the latter. Acid ammonium urate crystals, while similar to those of phenyl- semicarbazide in some respects, do not appear as mixed crystals and can scarcely be confused with either of the other substances.

After urine has been fermented for many hours (24, 30, 32), or after it has been allowed to undergo spontaneous ammoniacal fer- mentation (22, 30), the urea concentration, and hence the phenyl- semicarbazide formation, will be greatly reduced. Also we would expect Patein-Dufau filtrates of urine to yield little phenylsemi- carbazide, provided the urea has been properly precipitated. Therefore the osazones from such filtrates should be the purest and should have the sharpest melting points, if nothing other than phenylsemicarbazide is a disturbing factor. Actually, these osazones have had the least sharp melting points and were obvi- ously mixtures (5, 11, 12, 17, 21, 43). There are two reasons for this circumstance. First, some investigators have not made their precipitating mixtures definitely alkaline during the urea removal. It is true that Patein and Dufau’s directions (42) call merely for neutralization, or for the production of a slight acidity, but Gilbert and Baudouin (17) demonstrated many years ago that greater care must be exercised. Hence there must have been some urea present in certain of these preparations. After con- centration of the filtrate some phenylsemicarbazide would be formed. In other instances it appears that the urea has been carefully precipitated and we are forced to the conclusion that there is a second prolific source of error in the osazone test. Re- cently we have been fortunate enough to recognize the origin of this second interfering substance and will give details concerning it at another time. Suffice it to say that osazone preparations from Patein-Dufau filtrates of urine are far less trustworthy than usually supposed.

While we have called attention to these errors in the osazone test we also concede the frequent production of a high-melting phe- nylhydrazine derivative, probably phenylglucosazone, from some- thing in normal urine, but not necessarily from the reducing sugar. Our personal experience, during 5 years of experimentation, has

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M. R. Everett and F. Sheppard

been that the compounds of phenylhydrazine and the uroketose, if they exist at all, are very difficult to prepare. It is true that we have obtained both crystalline and amorphous derivatives of various hydrazines from purified urine preparations, but never with anything approaching the ease with which crystals may be obtained from normal urine itself. If there is so much difficulty in preparing a crystalline derivative from the more concentrated, much purer sugar syrups, how much more difficult will it be to get such substances from the original urine? The oft quoted work of Breul (7), based as it was upon the amount of the crude osazone precipitate made by lengthy heating in the presence of generous acidity, loses much of its significance. Breul’s values were merely fortuitous, since we now know that his precipitates must have contained much that was not osazone. (See also Raimann (52).) Whether the precipitate from normal urine contains any true osazone remains to be determined.

For clinical purposes the heating period in the phenylhydrazine test should not exceed an hour. Dilution of the urine with an equal volume of water, as recommended by Frank, and the use of Patein-Dufau reagent are good procedures, but t.his dilution re- sults in diminished sensitivity for glucose, and the use of mercuric salts is expensive. Probably the best practical means of differ- entiating phenylglucosazone from phenylsemicarbazide is to dis- solve the latter in hot water. For this purpose, conduct the osazone test in a centrifuge tube, examine the centrifuged residue not later than 12 hours after the heating, and confirm the presence of phenylglucosazone by adding to the residue a few cc. of water, warming, and stirring thoroughly on the water bath. Examine the centrifuged residue microscopically. If typical crystals still remain the test may be considered positive. To confirm the presence of phenylsemicarbazide in the water extract, add 5 volume of 95 per cent ethyl alcohol and extract the mixture with an equal volume of ether. Remove the ether solution and evap- orate it to dryness. Dissolve the residue in the smallest possible amount of 2 per cent warm hydrochloric acid solution. To this add saturated sodium nitrite solution, drop by drop, until the peculiar, characteristic crystals of the nitroso derivative can be distinguished with a microscope.

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440 Sugar of Normal Urine

SUMMARY

One of the principal components of the osazone precipitate of normal urine has been identified as I-phenylsemicarbazide. Its formation from urea and its subsequent crystallization have been studied in artificial urine. The effects of the concentration of urine, the acidity of the test mixture, the extent of heating, and the previous formation of cyanic acid by hydrolytic procedures have been considered. The presence of phenylsemicarbazide in osazone precipitates has necessitated a revaluation of previous microscopic and analytic findings. Cammidge’s test appears to involve the action of hydrochloric acid upon urea. A method is suggested for distinguishing between phenylglucosazone and phenylsemicar- bazide in the usual osazone test.

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Mark R. Everett and Fay SheppardPHENYLOSAZONES

NORMAL URINE: I. THE THE NATURE OF THE SUGAR OF

1932, 96:431-441.J. Biol. Chem. 

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