Experimental chloroleukemia in Sprague Dawley rats

MEHIVES

OF BIOCHEMISTKY

AND

Experimental

BIOPHYSICS

118,

58~389

Chloroleukemia

I. Isolation L. LEE,’ Department University

in Sprague

of Protoporphyrin

from

J. I?. RICHARDS of Biochemistry

of British Received

(1967)

Dawley

Rats

the Chloroma C. T. BEER,

ASD

and the Cancer Research Centre, Columbia, Vancouver, Canarlu August

22, 1966

A partition column chromatographic method on alumina for i he quantitative estimation of protoporphyrin ester by the gradient elution technique has been developed. It is rapid and suitable for the isolation of protoporphyrin, the principal porphyrin of experimental chloroma in Sprague Dawley rats.

Chloroma or chloroleukemia has been known in man for over 100 years (1). It is characterized by a leukemic blood picture and local tumors of a yellowish-green color in various sites within the body cavity. Under ultraviolet light, the tumor massesof the chloroma show a red fluorescence. The biochemical properties of the human chloroma have been studied by several workers in the last two decades (l-4). Due to the limiting amounts of clinical material available and the shortcomings of quantitative methods, conflicting data have been reported. Agner (3) suggested that the green color of t’he tumor was due t’o myeloperoxidase (formerly called verdoperoxidase), while Humble (1) and Durie (4) insisted that choleglobin was responsible for the color. Thomas (2) suggested that a protoporphyrin-protein complex was responsible for both t,he green color and the red fluorescence. However, Humble (1) could not find porphyrins in the tumor in spite of its red fluorescence. Chloroleukemia in the rat closely resembles that of the human (5). Chloroleukemia in the Sprague Dawley rat was initiated by the intravenous injection of actinium-227 (Cowden et al., S), and its first successful transplantation in weanling 1 Present University

address: of Hong

Department Kong, Tlong

of Pathology, Kong.

rats was reported by Zipf et al. (7). Morph0 logically, the tumor resembled t’he Shay chloroleukemia which developed in Wistar rats after the gastric instillation of 20-methylcholanthrene (8). Recently, Schultz et al. (9) have shown that the red fluorescence in the Shay chloroleukemia of the rat is due to a porphyrin that appears to be a dicarboxylic acid and which has the solubility and spectral charact,eristics of prot,oporphyrin. A green pigment’, slightly soluble in saline but insoluble in 65-70s alcohol, has been isolated from the chloroma and was shown t#o have a high myeloperoxidase activit’y with an absorption spectrum closely resembling that of myeloperoxidase. Schwartz (10) isolated protoporphyrin from the chloroma on a CaC03 column but his method suffers analytically due to the fact that not only a large amount of tumor has to be used for the analysis, but that also at least, thirteen different zones were observed on CaC03, representing porphyrins extracted from the chloroma tissue. In the present paper, a new and simple column chromatographic method on alumina is described for the isolation of pure protoporphyrin quantitatively, using a much smaller quantity of chloromatous tissue. MATERIALS Animals.

rats 585

were

AND

METHODS

Adult tumor-bearing Sprague Dawley used. (The chloroleukemic line in ollr

586

BEER,

LEE,

Tumor

AND

(5.0-7.0

RICHARDS

gm)

homogenized with 20 ml EtOAc/HOAc (4:l) at high speed for 10 minutes at 4”

t

Suspension centrifuged at 1lOOg for 10 minutes at 4”

Supernatant fraction

Precipitate repeated extraction with two lo-ml portions of EtOAc/HOAc and centrifuged

Precipitates

.&Discard

Combined supernatant 4-J fractions washed with 3% NaOAc (first washing contained 0.1 ml dilute I, solution)

Aqueous fraction (uroporphyrin)

Organic Et OAc/HOAc fraction repeated extraction with 5-ml portions of 3 N HCI

\f 3 N HCI extracts FIG. 1. Extraction

Organic fraction d of porphyrins

laboratory was initially transplanted into weanling Sprague Dawley rats but it has “changed” (“mutated”) so that it now can be transplanted into adult rats.) Radioactive glycine-2-W (1.5 X lo6 cpm) was injected intraperitoneally into chloroleukemic rats. Five hours after the injection the animals were killed by decapitation and the

Discard

from the tumor.

tumors were excised and used for the isolation of porphyrins. Ultraviolet spectroscopy. The absorption spectra of porphyrins in the visible and in the ultraviolet were measured with a Cary 11 recording spectrophotometer against reference cells containing the corresponding solvents.

ISOLATION

OF

MeasurenLent of radioactivity. Aliquots of samples were plated on platinum or aluminum planchettes and dried under an infrared lamp. The radioactivity was determined in a NIlclear Chicago windowless gas-flow counter. Exiraclion of porphyrins from the chlororna. The procedure used for the ext,raction of porphyrins from chloroma outlined in Fig. 1 is similar to that described by Schwartz and Wikoff (11) for the red blood cells. Since the vinyl side chains of protoporphyrin are ext.remely sensitive to light, all manipulations were carried out in dim light. The tumor was homogenized with a 4:l mixture of ethyl acetate and acet,ic acid. The protein-free organic extract was then washed free of acetic acid and buffered during washings with sodium acetate. (The addition of a trace of iodine in the conversion of any porphyrinogen present in t.he organic phase to porphyrins (12). Uroporphyriu is readily soluble in water and is found in the aqueous washes. The porphyrins remaining in the ethyl acetate-acetic acid solvent were then removed by repeated extractions with S-ml portions of 3 N TTCl until the HCl extract no longer showed the presence of porphyrins (absence of the Soret band). The ultraviolet data showed that the combined first, and second HCl extracts contained S-90(& of the total porphyrin extractable from the tumor, and these were used for the subsequent estimation of porphyrins in the tumor. The porphyrins may be identified and estimated as the free porphyrin, or after conversion into the ester. Free porphyrins are relatively urlstable, and in general practice, most workers prefer using the more stable esters. The esterificat.ion procedure llsed in the present work was essentially that of Falk (13). Column chromatography on alumina. ?jicholas (14) found that mixtures of chloroform and pet.roleum ether eluted the porphyrin esters from alumina in the order of proto-, copro-, and llroporphyrin. When the adsorptive power of the materials and the conditions of development were standardized, reproducible separations of porphyrin esters were obtained. It seems that the alumina column is a potentially useful tool for the qrlantitative estimation of porphyrin esters. Twent,y gm of alumina in an Erlenmeyer flask was partially deactivated by the addition of 2 ml of distilled water. The stoppered flask was shaken vigorously and, after standing overnight, the dry, free-flowing powder was suspended in 20 ml of ethylene dichloride-n-hexane mixture (1:4) and then slowly pollred into a glass column (1.8 X 30 cm) fitted with a cot,ton plug at the bottom. The aluminawas allowed to settle by gravity. The wall of the column was then washed twice with 2 ml of I he same solvent,. This aided in packing the top

587

PROTOPORPHYRIN

portion of the column more firmly and lessened the chance of the mechanical breaking of the column when samples were introduced. The surface of the column was protected by a layer of micro glass beads. RESULTS

Standavdization protoporphyrin

of method with authentic ester. Five hundred pg of

authentic protoporphyrin dimethyl ester (Sigma Chemical Company, Grade 1) in 1 ml of ethylene dichloride was diluted with 4 ml of n-hexane and then applied to the column. Although chloroform is the organic solvent generally used in porphyrin analysis, ethylene dichloride uras chosen in place of the former because: (1) it is a more stable solvent, (w) it does not need washing and drying before use, (3) it doesnot develop acidity, and (4) it does not contain phosgene and other impurities normally found in chloroform. The protoporphyrin dimethyl ester was eluted from the column with a linear gradient of ethylene dichloride and n-hexane (100 ml of a 1:4 mixture of ethylene dichloride and n-hexane in the mixing chamber, and 100 ml of a 4: 1 mixture of ethylene dichloride and n-hexane in the reservoir). The effluent was collected in 4.0ml fractions in a fraction collector. Spectra and optical densities were then measured in a Cary spectrophotometer. The elution pattern as shown in Fig. 2 gave two peaks (peaks a and b). The spectra of both peaks were porphyrin t,ype. Although effluents between fractions 25-32 absorbed around

Fraction FIG.

dimethyl

2. Chromatography ester on A1203.

number

of

protoporphyrin

400 mp t,heT did not fluoresce. Effluents beOween fractions 33 and 4S had spect’ra resembling those of pure protoporphyrin dimethyl ester and all of them fluoresced in ultraviolet light. This established that the main peak (peak b) was the protoporphyrin dimethyl ester. Sixty per cent of the protoporphyrin dimethyl ester applied to the column was recovered in this major peak (peak b). Fractions 46-54 forming the shoulder of the major peak also fluoresced and absorbed around 400 rnp. An examination of the column after chromatography revealed a wide zone of fluorescent material on top of the alumina. Re-chromatography of the material in fractions 35-38 on a new alumina column (Fig. 3) st’ill showed two peaks (peaks a’ and b’), but the shoulder between fractions 49 and 54 was absent. This indicated that the shoulder was probably due to impurities in the commercial protoporphyrin ester. As in the previous run, fractions 25-32 did not fluoresce. The recovery of protoporphyrin dimet,hyl ester in the effluent comprising the major peak (peak b’) was 75-80 % of the total amount applied to the column. The recovery of ultraviolet absorbing material between fractions 28 and 32 was 15-20 %. Since coproporphyrin might also be present in the tumor, it was necessary to check the chromatographic behavior of a sample of authentic coproporphyrin ester on the alumina column. It was found that coproporphyrin ester stayed at the top of the column if a linear ethylene dichloride nhexane gradient was used (same gradient as

for the elution of protoporphyrin.) However, if the column was then eluted with 50 ml of chloroform, the coproporphyrin ester moved rapidly down the column and could be recovered in the effluent in a yield of SO%. Isolation of protoporphyrin from the chlo?-oma.The porphyrins were extracted from the tumor and converted into the est’ers, and their radioactivities were determined. The crude esters were then chromatographed on alumina as described. The porphyrin ester from the chloroms was eluted from the column in a position corresponding to authentic protoporphyrin dimethyl ester (Fig. 4). Its absorption spectrum was also identical with t’hat of the pure dimethyl ester. There was only a very small amount of coproporphyrin in the tumor (about 1% of the protoporphyrin). The radioactivity of the porphyrin ester fractions from the column coincided with the optical density of the effluent’. A small radioactive peak (peak a) appeared between fractions 25 and 33. Although absorbing at 405 rnp, these fractions did not fluoresce in ultraviolet light. This secondary peak (peak a) was found in every chromatographic run of the porphyrin ester. However, its height varied from run to run, and it was suspectedthat it might be due to a photooxidation product of the protoporphyrin ester. In support of this suggestion, it was found that if the ester was prepared and chromatographed in the normal lighting of t’he laboratory, t’he peak (peak a’) was much greater than if the experiment was performed with another portion of the same

s .g

6

500

5

400

-is 2 4 E c% 3 ;

300” k 200

2 100

I 0 Fraction

FIG. 3. Re-chromatography on A1203.

IO

20

number

of fractions

35-38

30 Fraction

40 number

FIG. 4. Chromatography porphyrin

on alumina

in dim

50

of chloroma. light.

proto-

ISOLATION

Fraction FIG.

porphyrin laboratory.

3. Chromatography on alumina

OF

entirely (> 9s 5%) protoporphyrin. Free porphyrins have no known biological function, and they accumulate as by-products of the hemes. Under normal conditions only small amounts of free porphyrins occur in animal tissues. The presence of large amounts of protoporphyrin in the tumor is therefore an unusual finding. Whet,her these protoporphyrins are essential or incidental to the development of the leukemic condition is now under investigation.

number

in

of chloroma normal lighting

589

PROTOPORPHYRIN

protoof the

sample in near darkness (Fig. 5). The spectrum of the effluents comprising the nonfluorescent peak (peak a) differed from that of authentic prot,oporphyrin (fractions 34-43) in having the Soret band displaced to 405 rnp and two secondary peaks in the visible (instead of the normal Soret band at 407 rnp and four secondary peaks in the visible). However, the radioactivity per unit optical density of the material in peaks a and a’ (Figs. 4 and 5) was t,he same, irrespective of whether this experiment was done in bright, or dim light. This indicated that the small peak a was derived from protoporphyrin (or its ester) during manipulation, and its formation could be greatly reduced if care is taken to exclude light. It is therefore an artefact] of t,he method rather than of biochemical origin. DISCUSSION

The foregoing results indicate that the alumina column, as modified, is very suitable for the identification of protoporphyrin. It was easy t,o manipulate and gave consistent result,s in both the high recovery and t,he position of elubion. It was also found that the presence of traces of acid in the sample did not affect the elution pattern. By this method it has been shown t,hat the porphyrin present in the chloroma is almost

ACKNOWLEDGMENTS We wish to thank Dr. R. L. Noble for the use of the Cancer Research Centre laboratory facilities. This work was financed by grants from the Medical Research Council of Canada, and the National Cancer Institute of Canada. REFERENCES 1. HUMBLE, J. G., Quart. J. Med. 39, 299 (1946). 2. THOMAS, J., J. Bull. Sot. Chim. Riol. 20, 1058 (1938). 3. AGNER, K., Acta, Physiol. &and. 2 (Suppl. 8), 1 (1941). 4. DURIE, E. B., LEMBERG, R., AND SEAR, H. R., Med. J. Austr. 1, 397 (1950). 5. BESSIS, M., “Cytology of the Blood and Blood Forming Organs,” pp. 431-434. Grune and Stratton, New York (1956). 6. COIVDEN, R. N., AND MILLER, M., “Biological Quarterly Progress Report,” Mound Laboratory, Miamisburg, Ohio. Report No. MLM 989, June (1954). 7. ZIPF, R. E., CHILES, L., MILLER, M., AND KATCHMBN, B. J., J. Natl. Cancer Inst. 32, 669 (1959). 8. SH.~Y, H., GRUENSTEIN, M., MARX, 1~. E., AND LILLY, G., Cancer Res. 11, 29 (1951). 9. SCHULTZ, J., SHAY, H., AND GRUENSTEIN, M., Cancer Res. 14, 157 (1954). 10. SCHWARTZ, S., AND SCHULTZ, J., Cancer Res. 16, 565, (1956). 11. SCHWARTZ, S., AND WIKOFF, H. M., J. Biol. Chem. 194,563 (1952). 12. SCHWARTZ, S., ZIEVE, L., AND WATSON, C. J., J. Lab. C&n. Med. 37, 843 (1953). 13. FALK, J. E., AND DRESEL, E. I. B., Biochem. J. 63, 87 (1956). 14. NICHOLSS, R. E. H., Biochem. J. 48,309 (1951).