Britishjoumal of Dermatology (i975) 935 291.

The use of thin layer chromatography in the separation of free porphyrins and porphyrin methyl esters S.G.SMITH Department of Medical Biochemistry, Welsh National School of Medicine, Heath, Cardiff Accepted for publication 28 February 1975

SUMMARY

Thin layer chromatographic techniques for the separation and subsequent quantification of free porphyrins, coproporphyrin isomers and the methyl esters of porphyrins are described including a 2-dimensional system. The relevant RFs for a wide range of porphyrins are given. These methods have been in use in Cardiff and Barcelona for a number of years and were used in the survey of cases described by Pifiol et al. (1975) which resulted in the discovery of the new porphyria, hepato-erythrocytic porphyria.

The thin layer chromatography (TLC) techniques to be described were used in the survey of porphyrin excretion patterns from various porphyries in Barcelona during the period 1971-1974 (Pifiol et al., 1975). They have also been widely used routinely and for research purposes in Cardiff for several years. The technique used for free porphyrins is based on the lutidine system described by Eriksen (1958) who used paper chromatography. Two methods of separation of porphyrin methyl esters are described. Method (A) is similar to that described by Doss (1967) except that it is based on toluene in place of benzene. Method (B) is derived from the system used by Chu, Green & Chu (1951) for paper chromatography of the porphyrin esters. Methods (A) & (B) may be used sequentially in a 2-dimensional system which further improves separation. The method described for the separation of coproporphyrin isomers uses the lutidine system of Jensen (1963) applied to cellulose coated TLC plates in place of silica gel coated plates. Extraction and quantification of the separated porphyrins and porphyrin esters are also described. MATERIALS AND METHODS

The porphyrins used as standards were taken from the laboratory stock with the exception of bis291

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S.G.Smith

hydroxy deuteroporphyrin, kindly supplied by Professor Sano, Kyoto University, and isocoproporphyrin kindly supplied by Dr G.Elder, Welsh National School of Medicine, Cardiff. Three types of silica gel plates were used: (a) Merck (Kieselgel 60), (b) plates prepared on a Shandon plate spreader, (c) plates prepared on a Desaga spreader. The laboratory-prepared plates utilized silica gel layers of 0-2 mm thickness. All plates were 20 X 20 cm. The tanks used were of dimensions 30 x 9 x 22 cm. Separation of free porphyrins using silica gel plates

A well shaken solution of 50 ml distilled water in 100 ml 2:6 lutidine was poured into the tank which contained two small beakers of 30% v/v o-88o ammonia solution in water. The tank was Uned with two pieces of thick filter paper about 60 x 100 cm soaked in the 30% ammonia solution. The tank was put into a larger plastic tank containing water at about 40° C until the temperature of the chromatographic tank reached 22-24° C. It was then ready for use. The free porphyrins to be chromatographed were dissolved in a few drops of 2N NH4OH and applied to the plate by micropipette. The time taken for a 10 cm run was about 2 h. Separation of porphyrin methyl esters using silica gel plates

The methyl esters of the porphyrins were prepared by dissolving the free porphyrins in 5% cone. H2SO4 in methanol and allowing them to stand in the dark at room temperature for 24 h. An equal volume of distilled water was added to the esterification mixture and the porphyrin esters extracted with chloroform. The chloroform phase was separated and washed successively with 2% ammonia solution (v/v, to remove unesterified material) and distilled water. The resultant solution vvas evaporated to dryness on a rotary film evaporator. System {A) A well mixed solution containing 80 ml of toluene, 15 ml of ethyl acetate and 3 ml of ethanol was poured into the tank and allowed to equilibrate for 10 min. System (B) A well mixed solution of 60 ml of chloroform, 20 ml of redistilled kerosene and 4 ml of ethanol was poured into the tank and allowed to equilibrate for 30 min. The porphyrin esters to be applied to the TLC plates in systems (A) & (B) were dissolved in small quantities of CH CI3 and applied by micropipette. The tanks should be freshly loaded with solvent each day. A 2-dimensional separation could be achieved by removing the developed plate from System (A), drying in hot air, and placing in the System (B) tank at 90° to the axis of the first run. The running time for both Systems (A) & (B) was 30-40 min each. Separation of coproporphyrin isomers using cellulose plates

The tank was loaded with a well mixed solution of 30 ml of distilled water in 80 ml of 2:6 lutidine. Two small beakers containing SG o 880 ammonia solution were placed in the bottom of the tank. The tank was allowed to equilibrate overnight and was then ready for use. The coproporphyrin mixture was dissolved in a small volume of 2N NH4OH and applied to the plate. An excellent separation of the isomers was achieved in 4-6 h. Quantification

The porphyrin bands were detected by their red fluorescence under u.v. light and identified by comparison with concurrently run standards. Each band was scraped off the plate and put into a

Thin layer chromatography for separation of porphyrins

293

stoppered centrifuge tube. The free porphyrins were eluted by first shaking with 3 ml of 20% acetic acid in ether, followed by a shaking with i ml of 5% HCl. After centrifuging, the fluorescent aqueous layer was transferred to a i cm path width microcell and read in the spectrophotometer. The porphyrin methyl esters were eluted in a similar way with 2 ml chloroform. Usually, one extraction was sufficient, but the elution procedure was repeated with heavily loaded bands using 2 ml of 50% CH CI3 in ethanol. Relevant Soret band wavelengths, extinction coefficients and corrections for background absorption were described by Rimington (1958) and by Falk (1964). An assessment ofthe percentage of each porphyrin in a mixture, sufficiently accurate for routine purposes, may be made by a simple comparison ofthe Soret band absorptions. All the porphyrins listed in Tables i, 2 and 3 have been reported in human tissues or excreta with the exception of the hydroxylated porphyrins (2) and (3), and coproporphyrins H and IV. The following RF values were recorded using systems (A) & (B) (Table i) (Merck plates).

TABLE I. RF values recorded using systems (A) & (B) (Merck plates) System (A) System (B) (155 mm run) (132 mm run)

Porphyrin Methyl ester Haematoporphyrin Bis-hydroxy deuteroporphyrin Hydroxyethyl, ethyl deuteroporphyrin Uroporphyrin Heptacarboxyl porphyrin Hexa carboxyl porphyrin Penta carboxyl porphyrin Isocoproporphyrin Coproporphyrin I Coproporphyrin IH Harderoporphyrin Isoharderoporphyrin Deuteroporphyrin Protoporphyrin

(I) (2)

(3) (4) (5) (6) (7) (8) (9)

013 014 028

018

0-21

043

025

0-47

0 29 035 039

0-55 0-57

022

0-39 051

0-45

058

0-58

(II)

046 0-51

(12)

052

(13) (14)

059

067

062

0-68

(10)

062 062

RESULTS

The separation of the porphyrin methyl esters from an extract from the urine of a patient with hepato-erythrocytic porphyria (Piriol et al., 1975) is shown in Fig. i and shows a typical separation using the 2-dimensional system. Practical notes

The commercial Merck silica gel plates give sharper separations and allow the apphcation of smaller spots or bands to the plates. Silica gel plates made in the laboratory using the Shandon or Desaga processes give softer layered plates from which porphyrins may be extracted more easily than from Merck plates. The fluorescence of porphyrin esters after single runs in system B is more intense than after the system A development. It may therefore be preferred if photography is anticipated.

S.G.Smith

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TABLE 2. RF values for various free porphyrins using the lutidine system with silica gel plates Free porphyrin

RF value

Uroporphyrin Heptacarboxyl porphyrin Hexacarboxyl porphyrin Pentacarboxyl porphyrin Coproporphyrin I Coproporphyrin HI Harderoporphyrin I soharderoporphyrin Protoporphyrin \ Deuteroporphyrin J

0-0

o 05-0-15 O 20

0-25 0-27 032 038 0-47

System (A) is similar to that of Doss (1967) except that it gives a greater separation of hydroxylated porphyrin esters. These porphyrins may be further identified by the acetylation procedure of Barrett (1959)5 acetylation ofthe hydroxyl group(s) causing an increase in the RF value ofthe porphyrin. This system also largely avoids the health hazard ofthe benzene system of Doss, toluene being nearly ten times less toxic (Patty, 1963). A clear unequivocal separation of the coproporphyrin isomers is best achieved by first separating coproporphyrin from other porphyrins in an extract. This may be done by using the first described system (for free porphyrins) preparatively. The extracted coproporphyrins may then be separated using the isomer system. The use ofthe lutidine system for free porphyrins may be readily apphed to extracts of porphyrin mixtures prior to methylation. It does not separate 5, 6, 7 and 8 carboxyl porphyrins as well as the

i

0.

System (B)

- 2 nd run

FIGURE I. 2-dimensional separation of porphyrin esters from the urine extract from a patient with hepato-erythrocytic porphyria. (See Table I for numerical key.)

Thin layer chromatography for separation of porphyrins

295

TABLE 3. Typical RF values for the separation of the coproporphyrin isomers after a 4 h 30 min run on Merck cellulose plates (010 mm thickness) using the lutidine system described for the separation of isomers Porphyrin

RF value (4^ h run)

Coproporphyrin I Coproporphyrins III and IV Coproporphyrin II

063 050 o 43

ester systems, but allows separation of protoporphyrin and barderoporphyrins before metbylation, a process wbich causes some losses of tbese porphyrins. CONCLUSION

In order to arrive at the correct diagnosis of a porphyric, it is necessary to study the patient clinically and biochemically. A separation, identification and quantification of the porpbyrins from urine, faeces and blood may be required, together witb a coproporphyrin isomer analysis. In some cases it may be necessary to establish the isomer distribution of the bepta-, hexa- and penta-carboxyl porphyrins. Tbis may be achieved by decarboxylation of the acetic acid side chains of these porphyrins (Falk, 1964) and subsequent coproporphyrin isomer analysis. Tbe TLC systems described enable tbe relevant porphyrin separations to be carried out. Tbe use of these systems resulted in the discovery of a new type of porphyria, hepato-erythrocytic porphyrin (Pinol et al., 1975). REFERENCES BARRETT, J. (1959) Detection of hydroxyl groups in porphyrins and chlorins. Nature, 183, 1185. CHU, T . C , GREEN, A. A. & CHU, E . J . H . (195 I) Paper chromatography of methyl esters of porphyrins. Journal of Biological Chemistry, 190, 643.

Doss, M. (1967) Quantitative separation of porphyrins and protohaemin as methyl esters by TLC. Journal of Chromatography, 30, 265.

ERIKSEN, L . (1958) Paper chromatographic separation of the coproporphyrin isomers I & III. Scandinavian Journal of Clinical and Laboratory Investigation, 10, 319. FALK, J.E. (1964) Porphyrins and Metalloporphyrins. Elsevier, Amsterdam. JENSEN, J. (1963) Separation of the coproporphyrin isomers I and III by ThC. Journal of Chromatography, 10, 236. PATTY, F . A . (1963) Industrial Hygiene and Toxicology, vol. II. Interscience. PINOL-AGUAD^, J. HERRERO, J., ALMEIDA, J., SMITH, S.G. & BELCHER, R . V . (1975) Thin layer chromatography

and counter current analysis in porphyrias. British Journal of Dermatology, 93, 277. RIMINGTON, C . (1958) Quantitative determination of porphobilinogen and porphyrins in urine and faeces. Broadsheet no. 21 (New Series). The Association of Clinical Pathologists.

The use of thin layer chromatography in the separation of free porphyrins and porphyrin methyl esters.

Thin layer chromatographic techniques for the separation and subsequent quantification of free porphyrins, coproporphyrin isomers and the methyl ester...
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