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Skin porphyrin assay in porphyria
55
Clinica Chimica Acto, 83 (1978) 55-59 @ Elsevier/North-Holland Biomedical Press
CCA 9024
SKIN PORPHYRIN ASSAY IN PORPHYRIA
L. MALINA
a, VI. MILLER
b and LA. MAGNUS
cv*
a Dermatology Department, Faculty of Medicine and Hygiene, Charles University, Prague (Czechoslovakia), b Znstitute of Biochemistry and Pharmacology, Charles University, Prague (Czechoslovakia), c Department of Photobiology, Institute of Dermatology, Homerton Grove, London E9 6BX (U.K.) (Received
July 8th, 1977)
Summary The analysis of skin porphyrins in biopsies in patients with porphyria cutanea tarda has been achieved by high pressure liquid chromatography. Porphyrins were determined as their methyl ester copper-chelates and 2-, 4-, 5-, 6-, 7- and Scarboxylic porphyrins distinguished and quantified.
Introduction Some reported methods for the assay of porphyrins in the skin of porphyric humans or animals are defective in quantifying and have been capable, at most, of measuring only three of these pigments. Furthermore values by different teams, being at the border of sensitivity of the methods used, are not comparable. We here report the assay of skin porphyrins using high pressure liquid chromatography (HLPC). Materials and methods Preliminary work was completed with excretions and skin from rats and mice treated with porphyrinogenic drugs. The rats received 0.5% hexachlorobenzene with their food pellets for four months, and the mice received 2.5% griseofulvin in their standard diet for 5 days prior to sacrifice. The patients with porphyria cutanea tarda (PCT) were males, aged 38 to 64 years with a characteristic history of increased fragility to minor mechanical trauma, photosensitivity and a typical clinical picture of a bullous and erosive eruption mostly of the backs of hands. The diagnosis was confirmed by a char-
* To whom reprint requests and correrpondence should be addrewed.
56
acteristic urinary excretion of porphyrin, predominantly of uro- and 7-carboxylic porphyrin. Thin-layer chromatography of the urine, by standard methods [ l] showed the usual concentration pattern: 8-COOH > ‘7-COOH > 4-COOH > 6-COOH > 5-COON; the 24-h excretion of urinary eoproporphyrin was 130-540 pg, of uroporphyrin 730-4680 pg. Skin samples (190-280 mg) were kept at 20°C until analysis. Fatty tissue was then removed and the skin scissored and homogenized. For simplicity the next 2 stages, porphyrin extraction and esterification, were combined by mixing the homogenate with 5% methanolie sulphuric acid; subsequent work was completed in subdued light. The optimal conditions for complete extraction and esterification were investigated and found to be 37°C for 12 h. The supernatant obtained by centrifugation was diluted with chloroform and washed once with 1 M sodium carbonate solution and twice with redistilled water in a separatory funnel. The chloroform solution, dried by filtration, was evaporated and the residual porphyrin esters were redissolved in a measured quantity of dry chloroform. For the HLPC analyses of the highly concentrated porphyrins from animals, aliquots of the ester solution were applied, in the Varian 8500 liquid chromatograph, to a 0.2 cm diameter, 25 cm column of MicroPak-CN with a bound alkyl nitrile phase, Porphyrin esters were eluted in the isocratic mode with ~-heptane/ethyl acetate (60 : 40, v/v) and the individu~ fractions were detected by absorptivity at 400 nm. Modified procedures were necessary for the analyses of mixtures containing the low concentrations of porphyrins necessarily obtained from patient’s skin biopsies. With these fluorescence was unstable and the HPLC fractions showed double instead of single peaks. That these phenomena resulted from Cu-complex formation was apparent from spectrophotometry which revealed the typical absorbance maxima [2] and from HPLC by the characteristic shorter retention time of the Cu-complexes. Possible sources of copper ions are metal parts of the HPLC instrument, reagents used in the assay or laboratory glassware, but attempts to reduce this complication were unsuccessful. Accordingly, we deliberately converted free esters to Cu-complexes for determination of the porphyrins as Cu-chelates. This was achieved by dissofving the dried porphyrin esters in 0.1% aqueous copper acetate in methanolic chloroform, storing the mixture at room temperature for 8 h and then evaporating the methanolic chloroform. The dried porphyrin chelates were then redissolved in a measured volume of chloroform which effectively separated the excess copper acetate reagent from the porphyrin complexes. Identification of porphyrins by HPLC was effected by comparison of retention times with those of previously separated complexed and uncomplexecl standard solutions of proto-, copro- and uroporphyrin methyl esters (Sigma Co., U.S.A.). The concentration of porphyrin methyl ester-Cu-chelates was determined from the under-peak areas of the chromatogram. A trace, cut and weigh procedure, was used and the qu~tities of porphyrins were calculated on the basis of calibration of copro- and uroporphyrin standard solutions prepared freshly for every batch of analyses. The concentration of the porphyrin standard solutions was determined spectrophotometrically using the absorbance maxima and molar extinction coefficients of Falk [ 33 and Doss [2]. The amounts of intermediate porphyrins were determined with coefficients calcu-
57 TABLE I CONCENTRATION PHYRIA CUTANEA Patient NO.
OF PORPHYRIN TARDA
Weight of the biopsy specimen (B)
WITH POR-
Skin porphyrins @g/g wet weight)
0.260 0.213 0.196 0.228 0.110 0.210
5 2 3 4 5 6
IN SKIN BIOPSY SPECIMENS FROM PATIENTS
Uroporphyrin
7-COOH
6-COOH
5615.0 134.1 86.5 -
231.2 42.3 -
37.0 -
38.2 -
72.5 -
47.0 -
110.0 -
620.8
348.0
87.9
41.9
5COOH
Coproporphyrin
Protoporphyrin
70.6 -
Traces 54.7 52.9 257.7 -
Traces 43.8
33.8
lated by interpolation from calibration curves of absorbance against porphyrin concentration. That the porphyrins, as measured by HPLC, were indeed Cucomplexes, was confirmed by atomic absorption spectrometry using a JarrellAsh 82520 instrument. Results Figs. l-3 illustrate various stages of Cu-chelation in samples of methyl esters of skin porphyrin extract. Fig. 1 shows spontaneous Cu-chelation after 3 days
a
A
7
t
I,
2I
36
4h
5
time (mins)
6
7I
1
2
3
4
5
6
7
time (mins)
Fig. 1. HPL chromatogram of skin porphyrin extract. The specimen has been esterified and allowed to remain 3 days on the laboratory bench. AXTOWat left of time scale. Injection point. The subsequent 2 high peakn are due to the “solvent front”. The numbered paakr refer to the carboxylic groups of the unchelate? porphyrins, I.e. 4- refers to coproporphyrin. 8- to uroporphyrin. 2- to protoporphyrin (not shown on thb diagram). The other figures. viz. b. 6.. 7-. refer to porphyrin with these numbers of carhoxylic groups. The peab labelled “X” immediately to the left of each numbered peak refer to porphyrin thelated with metal. Fig. 2. The result 2 h after deliberate chelation of methyl ester porphyrin extract with 0.1% of copper acetate.
58
a 2
7
I2 6
,
1
1 t
I,L_
2
3
4
time
5
(mins)
6
P
1
1
7 t
I
A
1
1
I
I
2
3
4
5
6
7
timc(mins)
Fig. 3. The complete chelation of the previous sample (Fig. 2) after 8 h. Fig. 4. Typical methyl ester Cu-chelate porphyrin HPL chromatogram
of PCT skin sample.
standing on the bench, Fig. 2 shows deliberate but incomplete Cu-complexing 2 h after the addition of 0.1% copper acetate and Fig. 3 the same after 8 h. Fig. 4 shows the results found in the skin extract of patient No. 1 (Table I) and is a typical methyl ester Cu-chelate porphyrin chromatogram of a PCT skin sample. Table I shows the skin porphyrin concentration in our group of PCT patients. It can be seen that not only 2-, 4-, 7- and 8-COOH porphyrin are present in porphyric skin, as previously reported [4] but even that other so-called “intermediary” porphyrins with 5- and 6-carboxylic groups. The range of concentration of skin porphyrin was wide, viz. O-1000 ng per g wet weight. Preliminary indications suggest that urinary excretion and skin porphyrin concentration may perhaps be correlated positively, but further conjecture seems unwarranted until much more data are available. Recovery experiments gave an average yield of 50.8%. The range was 20.4-85.6s in 6 duplicate experiments in which uroporphyrin was added to normal skin specimens previously shown to be porphyrin free. Discussion For the study of human skin porphyrins, a micromethod permitting estimation in skin biopsy specimens of no more than 300 mg is necessary. The method must thus measure quantities of the order of rig/g wet tissue. Earlier methods [ 4-61 do not fulfil this criterion, being less specific and achieving, in some instances, relatively poor resolution of the various porphyrins. We have, therefore, focused our attention on the more recent analytical method, HPLC, to solve these difficulties [ 7-91.
59
Skin porphyrin concentration is seemingly not of direct diagnostic help in the clinical management of a patient, however it is likely to be of importance in our understanding the basis of skin photosensitivity in the porphyrias. The analytical method here described assays whole skin porphyrins and is suitable for routine clinical studies. It could certainly be modified for special studies such as those on separated epidermis and dermis. The procedure should therefore be suitable also for detailed studies relative to photodynamic action of porphyrins in different types of porphyria, for the investigation of the deposition, persistence or turnover and metabolism of skin porphyrins in patients with either erythropoietic or hepatic porphyrias. Morphological data from studies of the skin in porphyria give some indirect evidence for the primary localisation of porphyrins in the endothelium of the cutaneous vasculature [lo]. There is also similar indirect evidence that porphyrins may be present in epidermal cells and the dermis [ 111. If these observations could be placed on a firm basis with quantitative studies, the origin of skin photosensitive reactions after irradiation with appropriate wavelengths could be better explained on the basis of the photodynamic action of porphyrin. Acknowledgements Acknowledgements are due for helpful criticism to Bill Lockwood and Dr. Dennis Nicholson, also to the Wellcome Trust for financial assistance to one of us (L.M.). References 1 2 3 4 5 6 7 8 9 10 11
Doss, M. (1970) Z. Klin. Chem. KIin. Biochem. 8.197-207 Doss, M. (1971) Anal. Bbchem. 39.7-14 FaIk, J.E. (1964) Porphyrins and MetaUoporphyrins, Elsevier. Amsterdam Miura. T., Magnus. LA.. Jones, K. and Doyle, M. (1975) Dermatologica 151, 80-88 Pathak, M.A. and Burnett, J.W. (1964) J. Invest. Dermatol. 43,119-120.421427 Van Gog, H. and Schothorst. A.A. (1973) J. Invest. Dermatol. 61.4245 Gray, C.H.. Lim, C.K. and Nicholson, D.C. (1976) in High Pressure Liquid Chromatography in CIinical Chemistry (Dixon, P.F. et al., eds.), PP. 79-85, Academic Press, London Gray, C.H., Lim. C.K. and Nicholson, D.C. (1977) CIin. Chim. Acta 77,167-178 Carbon. R.E. and Dolphin, D. (1976) in High Pressure Liquid Chromatography in Clinical Chemistry (Dixon, P.F. et al., eds.), PP. 87-96, Academic Press, London Gschnait, F., Wolff, K. and Konrad, K. (1975) Br. J. Dermatol. 92. 546-567 (N.B.: first author cited in jourhal as F.G. Schnait) Baart de k Faffle, H. (1976) Erythropoietic protoporphyria. Thesis, Oosthoek, Scheltema and Holkema, Utrecht
Clinica Chimica Acto, 83 (1978) 55-59 @ Elsevier/North-Holland Biomedical Press
CCA 9024
SKIN PORPHYRIN ASSAY IN PORPHYRIA
L. MALINA
a, VI. MILLER
b and LA. MAGNUS
cv*
a Dermatology Department, Faculty of Medicine and Hygiene, Charles University, Prague (Czechoslovakia), b Znstitute of Biochemistry and Pharmacology, Charles University, Prague (Czechoslovakia), c Department of Photobiology, Institute of Dermatology, Homerton Grove, London E9 6BX (U.K.) (Received
July 8th, 1977)
Summary The analysis of skin porphyrins in biopsies in patients with porphyria cutanea tarda has been achieved by high pressure liquid chromatography. Porphyrins were determined as their methyl ester copper-chelates and 2-, 4-, 5-, 6-, 7- and Scarboxylic porphyrins distinguished and quantified.
Introduction Some reported methods for the assay of porphyrins in the skin of porphyric humans or animals are defective in quantifying and have been capable, at most, of measuring only three of these pigments. Furthermore values by different teams, being at the border of sensitivity of the methods used, are not comparable. We here report the assay of skin porphyrins using high pressure liquid chromatography (HLPC). Materials and methods Preliminary work was completed with excretions and skin from rats and mice treated with porphyrinogenic drugs. The rats received 0.5% hexachlorobenzene with their food pellets for four months, and the mice received 2.5% griseofulvin in their standard diet for 5 days prior to sacrifice. The patients with porphyria cutanea tarda (PCT) were males, aged 38 to 64 years with a characteristic history of increased fragility to minor mechanical trauma, photosensitivity and a typical clinical picture of a bullous and erosive eruption mostly of the backs of hands. The diagnosis was confirmed by a char-
* To whom reprint requests and correrpondence should be addrewed.
56
acteristic urinary excretion of porphyrin, predominantly of uro- and 7-carboxylic porphyrin. Thin-layer chromatography of the urine, by standard methods [ l] showed the usual concentration pattern: 8-COOH > ‘7-COOH > 4-COOH > 6-COOH > 5-COON; the 24-h excretion of urinary eoproporphyrin was 130-540 pg, of uroporphyrin 730-4680 pg. Skin samples (190-280 mg) were kept at 20°C until analysis. Fatty tissue was then removed and the skin scissored and homogenized. For simplicity the next 2 stages, porphyrin extraction and esterification, were combined by mixing the homogenate with 5% methanolie sulphuric acid; subsequent work was completed in subdued light. The optimal conditions for complete extraction and esterification were investigated and found to be 37°C for 12 h. The supernatant obtained by centrifugation was diluted with chloroform and washed once with 1 M sodium carbonate solution and twice with redistilled water in a separatory funnel. The chloroform solution, dried by filtration, was evaporated and the residual porphyrin esters were redissolved in a measured quantity of dry chloroform. For the HLPC analyses of the highly concentrated porphyrins from animals, aliquots of the ester solution were applied, in the Varian 8500 liquid chromatograph, to a 0.2 cm diameter, 25 cm column of MicroPak-CN with a bound alkyl nitrile phase, Porphyrin esters were eluted in the isocratic mode with ~-heptane/ethyl acetate (60 : 40, v/v) and the individu~ fractions were detected by absorptivity at 400 nm. Modified procedures were necessary for the analyses of mixtures containing the low concentrations of porphyrins necessarily obtained from patient’s skin biopsies. With these fluorescence was unstable and the HPLC fractions showed double instead of single peaks. That these phenomena resulted from Cu-complex formation was apparent from spectrophotometry which revealed the typical absorbance maxima [2] and from HPLC by the characteristic shorter retention time of the Cu-complexes. Possible sources of copper ions are metal parts of the HPLC instrument, reagents used in the assay or laboratory glassware, but attempts to reduce this complication were unsuccessful. Accordingly, we deliberately converted free esters to Cu-complexes for determination of the porphyrins as Cu-chelates. This was achieved by dissofving the dried porphyrin esters in 0.1% aqueous copper acetate in methanolic chloroform, storing the mixture at room temperature for 8 h and then evaporating the methanolic chloroform. The dried porphyrin chelates were then redissolved in a measured volume of chloroform which effectively separated the excess copper acetate reagent from the porphyrin complexes. Identification of porphyrins by HPLC was effected by comparison of retention times with those of previously separated complexed and uncomplexecl standard solutions of proto-, copro- and uroporphyrin methyl esters (Sigma Co., U.S.A.). The concentration of porphyrin methyl ester-Cu-chelates was determined from the under-peak areas of the chromatogram. A trace, cut and weigh procedure, was used and the qu~tities of porphyrins were calculated on the basis of calibration of copro- and uroporphyrin standard solutions prepared freshly for every batch of analyses. The concentration of the porphyrin standard solutions was determined spectrophotometrically using the absorbance maxima and molar extinction coefficients of Falk [ 33 and Doss [2]. The amounts of intermediate porphyrins were determined with coefficients calcu-
57 TABLE I CONCENTRATION PHYRIA CUTANEA Patient NO.
OF PORPHYRIN TARDA
Weight of the biopsy specimen (B)
WITH POR-
Skin porphyrins @g/g wet weight)
0.260 0.213 0.196 0.228 0.110 0.210
5 2 3 4 5 6
IN SKIN BIOPSY SPECIMENS FROM PATIENTS
Uroporphyrin
7-COOH
6-COOH
5615.0 134.1 86.5 -
231.2 42.3 -
37.0 -
38.2 -
72.5 -
47.0 -
110.0 -
620.8
348.0
87.9
41.9
5COOH
Coproporphyrin
Protoporphyrin
70.6 -
Traces 54.7 52.9 257.7 -
Traces 43.8
33.8
lated by interpolation from calibration curves of absorbance against porphyrin concentration. That the porphyrins, as measured by HPLC, were indeed Cucomplexes, was confirmed by atomic absorption spectrometry using a JarrellAsh 82520 instrument. Results Figs. l-3 illustrate various stages of Cu-chelation in samples of methyl esters of skin porphyrin extract. Fig. 1 shows spontaneous Cu-chelation after 3 days
a
A
7
t
I,
2I
36
4h
5
time (mins)
6
7I
1
2
3
4
5
6
7
time (mins)
Fig. 1. HPL chromatogram of skin porphyrin extract. The specimen has been esterified and allowed to remain 3 days on the laboratory bench. AXTOWat left of time scale. Injection point. The subsequent 2 high peakn are due to the “solvent front”. The numbered paakr refer to the carboxylic groups of the unchelate? porphyrins, I.e. 4- refers to coproporphyrin. 8- to uroporphyrin. 2- to protoporphyrin (not shown on thb diagram). The other figures. viz. b. 6.. 7-. refer to porphyrin with these numbers of carhoxylic groups. The peab labelled “X” immediately to the left of each numbered peak refer to porphyrin thelated with metal. Fig. 2. The result 2 h after deliberate chelation of methyl ester porphyrin extract with 0.1% of copper acetate.
58
a 2
7
I2 6
,
1
1 t
I,L_
2
3
4
time
5
(mins)
6
P
1
1
7 t
I
A
1
1
I
I
2
3
4
5
6
7
timc(mins)
Fig. 3. The complete chelation of the previous sample (Fig. 2) after 8 h. Fig. 4. Typical methyl ester Cu-chelate porphyrin HPL chromatogram
of PCT skin sample.
standing on the bench, Fig. 2 shows deliberate but incomplete Cu-complexing 2 h after the addition of 0.1% copper acetate and Fig. 3 the same after 8 h. Fig. 4 shows the results found in the skin extract of patient No. 1 (Table I) and is a typical methyl ester Cu-chelate porphyrin chromatogram of a PCT skin sample. Table I shows the skin porphyrin concentration in our group of PCT patients. It can be seen that not only 2-, 4-, 7- and 8-COOH porphyrin are present in porphyric skin, as previously reported [4] but even that other so-called “intermediary” porphyrins with 5- and 6-carboxylic groups. The range of concentration of skin porphyrin was wide, viz. O-1000 ng per g wet weight. Preliminary indications suggest that urinary excretion and skin porphyrin concentration may perhaps be correlated positively, but further conjecture seems unwarranted until much more data are available. Recovery experiments gave an average yield of 50.8%. The range was 20.4-85.6s in 6 duplicate experiments in which uroporphyrin was added to normal skin specimens previously shown to be porphyrin free. Discussion For the study of human skin porphyrins, a micromethod permitting estimation in skin biopsy specimens of no more than 300 mg is necessary. The method must thus measure quantities of the order of rig/g wet tissue. Earlier methods [ 4-61 do not fulfil this criterion, being less specific and achieving, in some instances, relatively poor resolution of the various porphyrins. We have, therefore, focused our attention on the more recent analytical method, HPLC, to solve these difficulties [ 7-91.
59
Skin porphyrin concentration is seemingly not of direct diagnostic help in the clinical management of a patient, however it is likely to be of importance in our understanding the basis of skin photosensitivity in the porphyrias. The analytical method here described assays whole skin porphyrins and is suitable for routine clinical studies. It could certainly be modified for special studies such as those on separated epidermis and dermis. The procedure should therefore be suitable also for detailed studies relative to photodynamic action of porphyrins in different types of porphyria, for the investigation of the deposition, persistence or turnover and metabolism of skin porphyrins in patients with either erythropoietic or hepatic porphyrias. Morphological data from studies of the skin in porphyria give some indirect evidence for the primary localisation of porphyrins in the endothelium of the cutaneous vasculature [lo]. There is also similar indirect evidence that porphyrins may be present in epidermal cells and the dermis [ 111. If these observations could be placed on a firm basis with quantitative studies, the origin of skin photosensitive reactions after irradiation with appropriate wavelengths could be better explained on the basis of the photodynamic action of porphyrin. Acknowledgements Acknowledgements are due for helpful criticism to Bill Lockwood and Dr. Dennis Nicholson, also to the Wellcome Trust for financial assistance to one of us (L.M.). References 1 2 3 4 5 6 7 8 9 10 11
Doss, M. (1970) Z. Klin. Chem. KIin. Biochem. 8.197-207 Doss, M. (1971) Anal. Bbchem. 39.7-14 FaIk, J.E. (1964) Porphyrins and MetaUoporphyrins, Elsevier. Amsterdam Miura. T., Magnus. LA.. Jones, K. and Doyle, M. (1975) Dermatologica 151, 80-88 Pathak, M.A. and Burnett, J.W. (1964) J. Invest. Dermatol. 43,119-120.421427 Van Gog, H. and Schothorst. A.A. (1973) J. Invest. Dermatol. 61.4245 Gray, C.H.. Lim, C.K. and Nicholson, D.C. (1976) in High Pressure Liquid Chromatography in CIinical Chemistry (Dixon, P.F. et al., eds.), PP. 79-85, Academic Press, London Gray, C.H., Lim. C.K. and Nicholson, D.C. (1977) CIin. Chim. Acta 77,167-178 Carbon. R.E. and Dolphin, D. (1976) in High Pressure Liquid Chromatography in Clinical Chemistry (Dixon, P.F. et al., eds.), PP. 87-96, Academic Press, London Gschnait, F., Wolff, K. and Konrad, K. (1975) Br. J. Dermatol. 92. 546-567 (N.B.: first author cited in jourhal as F.G. Schnait) Baart de k Faffle, H. (1976) Erythropoietic protoporphyria. Thesis, Oosthoek, Scheltema and Holkema, Utrecht