Studies on Cis Urocanic Acid*

Vol. 50, No. 6

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

1968 by The Williams & Wilkins Co.

Printed in U.S.A.

STUDIES ON CLS IJROCANIC ACID* J. 11. ANGLIN, Ja., PHD. AND W. H. BATTEN

In epidermis, metabolism of histidine terminates with the formation of urocanic acid (RCA) (1). UCA accounts for about 80% of the LW absorbancy of acid soluble extracts of

Dowex-1 is delayed, but ci.s RCA is invariably eluted before the trans isomer. In addition, urine contains many UV absorbing substances which in-

terfere with spectrophotometric detection. For these reasons labeled cis RCA was added to the

guinea pig epidermis (2). It has also been shown urine before ion exchange chromatography to facilthat only the trans isomer of UCA is present in itate recognition of fractions containing this sub-

Radioactive fractions from the anion normal epidermis, but following exposure of stance. exchanger contained appreciably more TJV abskin to IJV a mixture of the cis and trans sorbancy than that associated with the labelled isomers is formed (3, 4). In a study employ- cis RCA added to the urine aliquot. These fractions ing RCA, Kuroguchi et al. investigated the were pooled and adjusted to pH 3.0 for absorption effect of rabbit liver homogenates on cis RCA on Dowex-50-H (cation exchanger). When eluted with 7.0% NaCl in a gradient scheme the fractions and found no decrease in 3 hours, while a little containing radioactivity were collected. degradation was found after 24 hours (5). This Lyophilization of fractions collected from the report is concerned with disposition of cis UCA cation exchanger yielded a product with a low concentration of cis UCA in NaCI. The ialt was reby mammals. moved from the cis UCA by dilution to 3% NaCl and reabsorption on the same cation exchanger. MATERIALS

dehyde and 2-C'4 malonic acid. It had a specific

This was followed by washing with water and elution with 0.5N NaOH. Fractions 20—30 contained radioactivity and were neutralized with acetic acid. When reabsorbed on Dowex-1-acetate and eluted with 0.04N acetic acid as described above, cis RCA was obtained free from NaCl. Thin layer chromatography. Materials to be in-

activity of 1.4 ic/eM.

vestigated by thin layer chromatography were

Trans UCA. Trans UCA was purchasedt or synthesized in this laboratory by the method of Pyman (6) or Anatol (7). Labeled ( C'4) trans RCA was preparedi: by a modification of the latter method, that is by condensation of imidozole 4-al-

Cis UCA. A mixture of cis and trans isomers of RCA was prepared by irradiation of aqueous solutions of 7.0 emoles trans UCA, PH 8.0, with 6.7 X 10' Einsteins of 280—320 mj light. The cis isomer was isolated from the irradiation mixture by ion-

applied to commercial silica gel chromatographic sheets It and developed in small chromatographic chambers by the ascending technique. The solvents

exchange procedures described below. Animals. White male guinea pigs weighing 300— 700 gm or rats weighing 250—350 gm. were used in the experiments involving laboratory animals.

employed were: n-butanol/acetic acid/H,O (60/ 25/15), n-propanol/ammonia (sp gr. 0.90/H2O (60/30/10), n-butanol/ethylacetate/acetic acid! 11,0 (1/1/1/1), n-butanol/acetic acid/11,O (2/1/1), all prepared in volume ratios. Chromatograms were developed either unidirectionally, adjacent to authentic materials, or in 2 dimensions using different solvents. Alkaline diazo reagent (8) was

METHODS

employed for chemical detection of imidazoles and autoradiography was utilized for localizing radioactivity.

Ion-exchange chromatography. Anion-exchange chromatography employing Dowex-1-acetate in a gradient elution scheme as described previously (8) effectively separates the cis and trans isomers

The RCA recovered from urine by application of the ion-exchange procedures, was further punfled for JR Spectrum determination by thin layer

of UCA. If urine or other solutions containing relatively large amounts of other anions are chromatographed, elution of the UCA isomers from * From the Department of Dermatology, TJniversity of Oklahoma Medical Center, Oklahoma

chromatography. The material was applied to

Merck TLC plates and developed in the propanol/

ammonia system described above. When the

chromatograms were examined under 2537 mg light the UV absorbancy seen was at the R, expected for cis RCA. Silica gel from the RV absorbing area on the plate was removed and collected. Elution of cis RCA from the collected

City, Oklahoma.

This work was supported by IJSPHS, National

Institutes of Health grants AM 09161 and AM 05278.

Accepted for publication December 29, 1967. t Calbiochem, Los Angeles, California.

t Nuclear Research Chemicals, Inc., Orlando,

II Eastman Chromagram sheet, type K 301R2,

Florida. § "Sanitron" Model FS 20, 80 W, Atlantic Ultraviolet Co., Inc., Long Island, N.Y.

Eastman Kodak, Rochester, N.Y. TLç-plates

463

Silica Gel F254, E. Merck. Darmstadt, Bninkman Instruments, Westbury, N.Y.

464

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

was used for all radioisotope determinations. Meas-

ured aliquots of organ extracts or urine were applied to filter paper, dried and positioned within the counting chamber. The count rate was read from the rate meter. The counting efficiency was

•

30% with a precision of

Homogenates. A motor driven, all glass tissue grinderJ was used to prepare the homogenates from shaved rat or guinea pig epidermis collected

by the scraping technique (0). The tube was

cooled during the grinding operation to reduce heating by friction. Approximately 30% suspensions were prepared in physiological saline. A simi-

lar procedure was employed in preparation of liver and kidney homogenates. RESULTS

A

e

Fia. 1. Autoradiogram print of C'4 labeled cis

IJCA. (A) starting cis IJCA in 1% NaC1 (B)

radioactivity in urine eluted from anion exchanger after oral administration of 014 labeled cis UCA

to a rat. (C) radioactivity from a 15% guinea pig epidermal homogenate incubated with C' cis UCA for 4 bours at 22° in 1% NaCl. (D) Same as (C) except incubated in 0.15 M phosphate buffer pH 6.8. Solvent: Propanol/ammonia/H20 (60/30/10).

silica gel with water and lyophilization of the eluent yielded the cis UCA recovered from urine that

Incubation of 011 Cis UCA with Organ Homogenates. Investigation of the chromatographic behavior of the radioactivity recovered from homogenates of skin, liver and kidney when incubated with labeled cis UCA at 22° for 24 hours gave no indication that any new radioactive substance had been formed either by the homogenate or by contaminating bac-

teria. It was established that the chromato-

graphic solvent systems which were employed would resolve cis and trans UCA, imidazole propionic acid and glutamic acid, substances likely to be formed by enzymatic modification of cis UCA. Figure 1 is an autoradiogram of radioactivity recovered from urine or homogenates and compared with that of cis UCA. Intraperitoneal injection. Cis UCA, having 270,000 cpm (0.46 emoles) was dissolved in physiological saline (pH 7.0) and injected intrapcritoneally into a white male rat weighing 280

was incorporated into a KBr pellet for Infra-red gm. The urine collected in the next 4 hours (IR) Spectrum determination. Autoradiography. Thin layer chromatograms contained 70% of the radioactivity injected. In with spots of the starting cis UCA and radioactiv- the interval of the next 20 hours 19% was ity recovered from urine or from incubation of ci.s UCA with organ homogenates were developed in the manner described. After air drying, the chro-

matograms were placed in contact with photographic film and indexed. Following exposure to approximately 1 >< 106 atomic disintegrations (2—10

days) the film was removed and developed in the usual manner. The location of darkening of the photographic emulsion by radioactivity was compared with that of the color produced by chemical

recovered in the urine making a total of 89% recovered within 24 hours. Table I lists the data obtained in several similar experiments.

Investigation of the chromatographic prop-

erties of the radioactivity in the urine by anion-exchange showed it to be similar to the

cis UCA which was injected. When further characterized by thin layer chromatography spray reagents for imidazoles. using a variety of solvent systems, the reAnalytical Procedures. A Gary Model 14 Spec- covered urinary radioactivity had the same trophotometer was used for determination of the molar extinction coefficient (18,000 at 265 m/z) employed for spectrophotometric quantitation of UCA. A model JR-b Beckman JR spectrophotom-

mobilities as cis UCA.

Following intraperitoneal injection of trans UCA 98,000 cpm (0.35 molc) the urine col-

eter was employed for determination of IR lected during the next 24 hours contained no despectra in KBr pellets. A model 880 Vanguard chromatogram scanner ¶ Kontes Glass Company, Vineland, New Jersey.

465

CIS UROCANIC ACID

tectable radioactivity. Apparently the labeled trans TJCA was degraded by urocanase and was not excreted by the kidney. Feeding experiments. Administration of 300,000 cpm (0.63 emoles) C14-tagged cis TJCA

TABLE II Urinary excretion of C14 labeled cis UCA administered orall CPM X 101

by stomach tube to a 300 gm. white male rat Animal w• yielded 75% of the count in the urine within four hours and an additional 25% in the urine within the next 20 hours. This accounts for all of the ingested count. Investigation of the uri- Rat 300 gm nary radioactivity by means of ion-exchange and thin layer chromatography indicated that Rat 300 gm this product was cis TJCA. Table II lists the data obtained in this and two other similar experiments. Rat 330 gm Irradiation in vivo. The cis UCA content in the urine collected during 24 hours following whole body irradiation of a normal 22 year old

Ad ministered

Found in urine 0—4 hr.

0—8 hr.

0—24 hr.

226

255

301

92 (37%)

—

17

—

0—48 hr.

300

301 (75%) (85%) (100%) (100%)

250

100

155

200

(62%) (80%)

(17%)

77

89

(77%) (89%)

year old white male the urine was collected for

white male exposed to 3 minimal erythema 48 hours. The cis isomer administered was 90% doses of UV was evaluated. The maximum urinary cis UCA content was determined employing a modification of the isotope dilution pro-

cedure wherein the 265 m absorbancy and

accounted for in the urine by application of the ion-exchange techniques described above.

Infrared spectra. Comparison of the JR

spectrum of cis UCA recovered from urine count rate of labeled cis 1IJCA added to a ali- after purification on both anion and cation quot of the urine was compared with that iso- exchangers showed absorption maxima at frelated. The data indicated that less than 1 X quencies not observed in the spectrum of au10 moles cis UCA was contained in the 24 thentic cis UCA. When it was further purihour specimen.

Oral administration. Following oral administration of 3 mg. non-labeled cis UCA to a 45

TABLE I Urinary excretion of G' labeled cis UCA injected intraperitoneally CPM X 10-' Animal

Wt. Admin-

Found in urine

istered

0-4 hr. 0—s hr. 0—24 hr. 0-48 hr.

Rat

280 gm

270

190

Rat

350 gm

1740

Rat

300 gm

210

Rat

300 gm

32

600

241 241

authentic cis UCA showed that absorption maxima were present at the same frequencies. DISCUSSION

The initial experiments employing labeled cis IJCA utilized homogenates prepared from epidermis, liver and kidney. The enzymatic changes of cis UCA by organ homogenates considered most likely were, isomerization to trans UCA, reduction to imidazole propionic

acid, or conjugation with an amino acid or sugar derivative. There was no indication that any new C15 labeled product had been formed.

21.4 66%

Guinea 500 gm Pig

228

70% 84

fied by thin layer chromatography comparison of the JR spectra of this material with that of

The possibility that cis UCA is modified in vivo was investigated by intraperitoneal injection of labeled cis UCA. Determination of the radioactivity in urine showed that, within experimental error, all of the label was excreted within 24 to 48 hours. Separation of the radio-.

activity from urine by both anion and cation exchange and determination of the mobility of the radioactivity in thin layer chromatographic systems indicated that all of the label was asso-

THE JOUENAL OF INVESTIGATIVE DEEMATOLOGY

466

ciated with unchanged cis UCA. The same resuits were obtained when cis UCA was administered by mouth, confirming that the liver does not modify this pbotoproduct. To investigate the urinary excretion of cis UCA formed in situ, a normal adult male was irradiated with DV and the urine was collected for 24 hours. Addition of labeled ins UCA to an aliquot of the urine followed by separation of the cis UCA and subtraction of DV absorbancy attributable to the exogenous UCA left essentially no DV absorbancy to be attributed to cis UCA transported from the epidermis to the urine. In addition, it was found that about the same increment of DV absorbancy was seen

3. Cis UCA formed in viva by DV irradiation of epidermis was not found in the urine within 24 hours after irradiation. Thus, cis UCA formed in situ by DV irradiation does

hours preceding DV irradiation.

5. Kuroguchi, Y., Fukui, Y., Nakagawa, T. and Yamamoto, I.: A note on cis urocanic acid.

not normally enter the systemic circulation. REFERENCES 1. Schwarz, E.: Abban von histidin zu urocaninsSure in der epidermis. Biochem. z, 334: 415, 1961.

2. Tabachnick, J.: The free amino acids, ammonia, urocanic acid and nucleic acid content of nor-

mal albino guinea pig epidermis. J. Invest. Derm.,32: 563, 1959.

3. Anglin, J. H., Jr., Bever, A. T., Everett, M. A.

and Lamb, J. H.: Ultraviolet light induced alterations in urocanic acid in vivo. Biochem.

Biophys. Acta, 53: 40S, 1961.

9.: Cis- and trans-urocaninsäure als by application of identical separation pro- 4. Pascher, bestandtile des stratum corneum. Arch. Klin. cedures to urine of the subject from the 24 Exp. Dermat., 214: 234, 1962. 5UMMARY

1. Experiments in vitro with skin, liver or kidney homogenates indicated that cis UCA was not metabolized.

2. When administered orally or intraperi-

Jap. J. Pharmacol., 6: 147, 1957.

6. Pyman, F. L.: A new synthesis of histamine (4 (5)-p Amino ethylglyoxahne). J. Chem. Soc., 1911 Part 1: 668.

7. Anatol, Jesus: French Patent Number 1,254,992, Chem. Abs., 57: 5930, 1962.

S. Anghn, J. 11., Jr. and Eyerett, M. A.: Photodimerization of urocanic acid in viva and in

toncally cis IJCA was neither degraded nor con-

vitro. Biochem. Biophys. Acta, 88: 492, 1964.

changed.

sulfhydryl. J. Invest. Derm., 35: 239, 1960.

H., Knox, J. M. and Griffin, A. C.: Sepajugated but was excreted by the kidney un- 9. Ogura, ration of epidermis for the study of epidermal

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

24. Wynn, C. H. and Iqbal, M.: Isolation of rat

skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.

human epidermis, p. 511, The Epidermis

Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-

No warranty is given about the accuracy of the copy.

Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.

Cell Biol., 27: 603, 1965.

27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-

C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.

30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.

31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-

lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.

32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.

33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.

34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:

Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.

43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.

44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.

45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.

46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,

Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by

Res., 33: 176, 1964.