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glycoluril). (8,. 9) as the chromogen. From The Burnsides Research Laboratory, University of Illinois at Urbana-Champaign,. Urbana, Ill. 61801, and The Nuclear.
ChemkjaI Basis of the CarbamidodiacetylMicromethodfor Estima’tionof Urea,Citrulline,andCarbamylDerivatives Michael

P. Veniamin

and Catherine

Vakirtzi-Lemonias

The chemical

pathway of the carbamidodiacetyl colorimetric assay was investigated. The experimental variables that were studied include reaction temperature, heating time, and the ratio of the mineral acid mixture to the reactants. Evidence is presented establishing the involvement of either 7or 8-methyl, or 7,8.d imethyltetrahyd roimidazo(4,5-d)imidazole-2,5-d iones as chromogens, all three being equally acceptable.

T long

colorimetric assay has been the method of choice in many clinical laboratories for the determination of urea, citrulline, and carbamyl derivatives (1, 2). Convenient reagent kits (3), as well as a mechanized adaptation (4), have been described for reportedly simple and rapid application of the assay to the analysis of biologic fluids. The addition of either semidine (5) or thiosemicarbazide (6) to the reaction mixture has been the latest modification. In addition, biologically important adducts of a-diketo compounds with the guanine residues of viral RNA have been described (7). The observed cancerostatic activity of the a-diketo compounds, an activity which may thus be linked with a specific modification of nucleic acids, has focused unusual attention on the mechanism of the carbamidodiacetyl reaction. In this communication we describe the simulation of critical stages of the colorimetric assay as a means of elucidating the overall reaction scheme. Both starting materials and potential intermediates have been subjected to the standardized conditions (2) of the assay (the acid mixture is H2S04 : HPO4: 1120, 1:3:4; Fe3, 0.2 mmol/liter; 100#{176}C) and to a series of milder treatments. A computer program has been written for the study of the kinetics of two of these intermediate stages. This experimentation has led to the proposal of an overall reaction scheme, which essentially involves either 7- or 8-methyl or 7,8-dimethyltetrahydroimidazo (4,5-d)imidazole-2,5-diones (the trivial name for the parent compound: glycoluril) (8, 9) as the chromogen. HE

CARBAMIDODIACETYL

From The Burnsides Research Laboratory, University of Illinois at Urbana-Champaign, Urbana, Ill. 61801, and The Nuclear Research Center “Democritus,” Division of Biochemistry and Experimental Medicine, Aghia Paraskevi, Attica, Greece. Received Aug. 7, 1969; accepted Sept. 10, 1969.

Materials

and Methods

Computer

and Program

A computer program was developed for the computation of rate constants and characterization of the molecularity of the reaction. The program was written in FORTRAN IV for the IBM 360 Model 75 computer. A listing of the source deck of the program together with a sample run has been deposited in the Perkin-Elmer Program Exchange Library. Analytical

Methodology

The carbamidodiacetyl assay was run as previously reported (2) with occasional variation of reaction temperature, heating time, and ratio of acid mixture to reactants. The a-diketones and the ureas used were purchased from Eastman Organic Chemicals, and the kinetic studies were conducted with a constant temperature water bath (Lapine Scientific Co.). Elementary microanalyses were done at the Microanalytical Laboratory, Department of Chemistry, University of Illinois at Urbana-Champaign. Detection

of Chromogen

To detect any potential pigment precursor, we conducted the assay under the following conditions: A mixture of 223 mol of urea, 3 mol of 1phenyl-1 ,2-propanedione, and 5.2 mmol HCl (0.5 ml ACS grade reagent) was brought to 4.5-ml final volume with absolute ethanol. Time course of the reaction was studied at 50#{176}C. Figure 1 shows that as the a-diketone (X,,, = 228 nm) disappears, the pigment (Xrnax = 550 nm) develops, until a common plateau is reached corresponding to a steady state of chrornogen concentration. The initial pattern reappears beyond the plateau.

CLINICAL CHEMISTRY, Vol. 16, No. 1, 1970 3

#{149} STANDARDIZEDCONDITIONS AFTER 48 HOUR EXPOSURE IN DIRECT NEON LIGHT

1.00

#{176}

#{149}0.2x ABSORBANCYAT 228 mu o ABSORBANCYAT 550 mp

2.25

0.8

2.00 1.75 U)

O.6

w 1.50 C.,

z

4

1.25

0

0

4

40.4

1.00 0.75

0.2

0.50 0.25

25

50 REACTION

75 TIME

100

125

0.00 025

IN MINUTES

p MOLES

Fig. 1. Simulation of the carbamidodiacetyl

microassay under mild conditions of temperature (50#{176}C) and acid concentration. Solid circles show the time course of the disappearance of 1.phenyl.1,2-propanedione. Outline circles indicate the time course of the appearance of the pigment

Glycolurils as Chromogens Glycoluril derivatives (formula B, Fig. 5) were synthesized (10). Elementary analysis and spectral data (8, 11) were used to confirm the identity of each glycoluril. The various glycolurils were subjected to conditions analogous to the standardized conditions of the assay (2); the procedure, as exemplified by the treatment of 3a,6a-dimethylglycoluril, was as follows: Ten to 15 mg of 3a,6a-dimethylglycoluril were refluxed in 100 ml of absolute ethanol. The solution was filtered and the optical density was measured at 278 nm. The glycoluril content of the solution was then measured by use of the value E = 294, which was determined in a separate experiment. Several aliquots, each containing 0.5 to 1.5 ml of the above solution, were added to 5 ml of the acid mixture and 0.5 ml of water. The volume in the colorimeter tubes was brought to 7 ml with absolute ethanol. The original procedure was subsequently followed (2). Figure 2 shows the linearity of molar response in pigment production by the glycoluril, with and without exposure to light. The variation in pig-

4 CLINICAL CHEMISTRY, Vol. 16, No. 1, 1970

0.50

0.75

1.00

i25

OF 3a,6a-DIMETHYLGLYCOLURIL

Fig. 2. Standard curves obtained for 3a,6a.dimethylglycoluril under: (a) the standardized conditions described in the text (solid circles); (b) after additional 48-hr exposure to direct neon light (outline circles). Absorbancies were read at 478 nm

ment production in Table 1.

with various

Time Course of Pigment

glycolurils

is shown

Development

The time course of the conversion of 3a,6adimethyiglycoluril (Xmax = 256 rim) to the yellow pigment (Xmax = 478 nm) is shown in Fig. 3. The experiment was conducted under the standardized conditions (2) of the assay, except that the temperature was lowered to 50#{176}C, and equal volumes

Table 1.

Variation

Glycoluril

of Pigment Production

chromogen

3a-Phenyl.6a-methyl 3a-Phenyl-6a-methyl- derivative of N,N’.dimethylurea 3a,6a.Dimethyl 3a.Methyl- derivative of N,N’.dimethylurea 3a,6a.Diphenylglycoluril (parent compound) o

No pigment

production.

Pigment

X,,,

550 450 478 497

(nm)

2.25

#{149} ABSORBANCY

AT 256mg

150

o ABSORBANCY AT 478mp 2.OC

#{149} ABSORBANCYAT 290mp o ABSORBANCY AT 264mu

1.25 1.75

1.00

1.50 U) Lii C.)

U)

C)

z

z

1.25

4 U)

o.75

4

0

U) 0 U)

U,

1.00

4

U)

4

0.50 0.75

0.50

0.25

0.25 0.00 0.00

10

20 30 40 50 60 REACTION TIME IN HOURS

Fig. 3. Time course of conversion of 3a,6a.dimethyl. glycoluril (Xmox = 256 nm) into the pigment (X0,00 = 478 nm) at 50#{176}C

of the alcoholic glycoluril solution the acid mixture were used.

(10 mg/ml)

and

Acid Hydrolysis of Glycolurils This experiment (Fig. 4) is presented to demonstrate conditions under which hydrolysis of the glycoluril, and not pigment development, prevails. Ninety-two pmol of 3a-methyl-6a-phenylglycoluril were dissolved in 100 ml N,N ‘-dimethylformamide. At zero time 36.4 mmol (3.5 ml) HCI (ACS reagent) was added and the time course of the hydrolysis of the glycoluril to the corresponding 4,5-dihydroxy-2-imidazolidinone (11, 12) (formula A, Fig. 5) was followed at 20#{176}C. Neither pigment nor hydantoin (13) was produced. Computation

25

50

75

REACTION TIME IN MINUTES

of Rate Constants

A computer program has been written optimized estimation of rate constants for sults plotted in Figs. 3 and 4. As in Fig. 3a,6a-dimethylglycoluril is converted to the pigment (Xmx = 478 rim) with a half-life of 10 sec and a rate constant of 2.51 X 106

for the the re3, the yellow 2.75 X sec’.

Fig. 4. Time course of acid hydrolysis of 3a.methyl-6aphenylglycoluril at 20#{176}C. Solid circles show the disappearance of 3a-methyl-6a-phenylglycoluril (Xmox = 290 nm). Outline circles show the appearance of 4-methyl5-phenyl-4,5.dihydroxy-2-imidazolone (Xm = 264 nm).

On the other hand, as in Fig. 4, the 3a-methyl-6aphenyiglycoluril is hydrolyzed with a half-life of 3.1 X 10’ see and a rate constant of 2.23 X 10 sec .

Discussion

The existence of glycolurils as the chromogens of the colorimetric assay is flO surprise, since they have long been prepared in high yields by means of carbamidodiacetyl reaction, though under milder reaction conditions (10, 14). The reaction stops in Step 1 of the scheme (Fig. 5) only in the benzilN,N ‘-dimethylurea system. This phenomenon has also been observed in the alkaline condensation of the same reactants (11) and must not be confused with the negative colorimetric reaction given by benzil and other diketo compounds that are devoid of enolizable ct-carbon hydrogens. The latter case not only demonstrates the requirements of acarbon hydrogens, but also suggests a possible analogy to the chemistry of bile pigment production, which sequentially involves active methylene and methenyl bridges between the heterocyclie rings (15).

CLINICAL CHEMISTRY, Vol. 16, No. 1, 1970 5

STEP I

OH

OH

CH_C_C_Rr+RI.NH_C_NH_RH+CH,_C. II

II

II

0

0

0

C-R1 I

H

R -NN-R

STEP 2

(A)

STEP

(B)

(A)

a

R CH3 R-NH--C-NH-R II 0

+

niH4

O=C

H

,NC-N

(B)

I

N-C-N7

3

H’ F

e ,O5h)) pigment > [corynoid (2)]

Fig. 5. Proposed

reaction

Photolysis

Products

scheme of the carbamidodiacetyl

Although the acid hydrolysis rate constant of the glycoluril and the pigment-formation rate constant appear to differ by a factor of 100, no attempt should be made to correlate the two systems. They differ in reaction temperature, heating time, solvent system, and acid concentration, and they merely represent two extremes: total hydrolysis of glycoluril and total conversion of glycoluril to pigment. The irreversible formation of the pigment in the actual assay will probably cause continuous shifting of the equilibrium to the right (Fig. 5). The addition of either semidine (5) or thiosemicarbazide (6) to the reaction mixture does not produce the expected orange color (Emax = 478 nm), but rather a more stable pink color (semidine E,, = 545 nm, thiosemicarbazide Ema,, = 535 nm). In such a case, semidine or thiosemicarbazide is the final electron acceptor of the reaction scheme. Preliminary results indicate that the same reaction scheme applies to the carbamidodiacetyl assay conducted under strongly alkaline conditions (10% NaOH) and to the alkaline guanidinodiacetyl reaction (Voges-Proskauer reaction) (16).

References

3. Veniamin,

6 CLINICAL CHEMISTRY, Vol. 16, No. 1, 1970

M. P., Ed.,

tis Press, Athens, Greece,

“Methodi

Klinikis

Chimias,”

Anaplio-

1967, p 41.

4. Walton, H. M., Automation. Advan. ClAN. CHEM., 2, 301 (1959). 5. Hunninghake, D., and Grisolia, A., A sensitive and convenient, micromethod for estimation of urea, citrulline, and carbamy! derivatives. Anal. Biochem. 16, 200 (1966).

6. Mather, A., and Roland, D., The automated bazide-diacetyl monoxime method for plasma urea. 15, 393 (1969).

thiosemicarCLIN.

CHEM.

7. Shapiro, R., Cohen, B. I., Shieuey, S., and Maurer, H., On the reaction of guanine with glyoxal, pyruvaldehyde, and kethoxal and the structure of acylguanines. A new synthesis of N’-acylguanines Biochemistry 8,238 (1969). 8. Nematollahi, J., and Ketcham, R., Imidazoimidazole. I. The reaction of ureas with glyoxal. Tetrahydroimidazo (4,5-d)imidazole-2,5-diones. J. Org. Chem. 28, 2378 (1963). 9. Dunnavant, W. R., and James, F. L., Molecular rearrangements. I. The base-catalyzed condensation of benzil with urea. J. Amer. Chem. Soc. 78, 273 (1965). 10. Williams, J. W., Antivesicant compounds and clothing impregnated therewith. U.S. Patent 2,649,389 (1953). 11. Neville, R. G., Formation of 1,3-dimethyl-5,5-diphenylhydantoin and related reactions. J. Org. C/tern. 30, 2179 (1965).

12. Vail, S. L., Barker, R. H., and Mennit, P. G., Formation and identification of cis- and trans-dihydroxyimidazolidinones from ureas and glyoxal. J. Org. Chern. 30, 2179 (1965). 13. Henze, H. II., and Speer, H. J., Identification of organic compounds through conversion into hydantoins. J. Amer. C/tern. Soc. 64, 522 (1942). 14. Biltz, H., Zur Kenntnis der Diureine. Chem. Ber. 40, 4806 (1907). 15. Gray,

W. l{., The carhamido diacetyl reaction: A test for citrulline. Biochem. J. 33,902 (1939). 2. Fenton, P. F., and Vakirtzi-Lemonias, C., Urea formation in nephrectomized mice. Amer. J. Phyiol. 198, 1272 (1960). 1. Fearon,

microassay

C.

H.,

Kulczycka,

A.,

and

Nicholson,

D.

chemistry of bile pigments. Part IV. Spectrophotometric tion of bile pigments. J. Chent. Soc. 1961,2276. 16. Eddy, B. P., The Voges-Proskauer reaction cance: A review. J. Appl. Bacteriol. 24,27(1961).

C.,

The

titra-

and its signifi-