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Comparison of the porphyrin patterns in patients with porphyria cutanea tarda in Czechoslovakia and Denmark
Inr.
J. Biochem.
Vol.
13. pp.
769
to 772,
002071
1981
lX/81/060769-04SO2.WO
Copyright 0 1981 Pergamon Press Ltd
Printed m Great Britain. All nghts reserved
COMPARISON OF THE PORPHYRIN PATTERNS IN PATIENTS WITH PORPHYRIA CUTANEA TARDA IN CZECHOSLOVAKIA AND DENMARK TORBEN K. WITH,* V. KORDAI: and JANE SERUP PEDERSEN Svendborg Hospital, Department of Clinical Chemistry and The First Medical Department of the Charles University. Prague, Czechoslovakia (Rrceiwd
6 Ocrober 1980)
Abstract-l. The incidence of porphyria cutanea tarda (PCT) has increased considerably in Denmark during the last decade (Table 1) but is much higher in Czechoslovakia than in Denmark. 2. We therefore made a detailed study of the urinary porphyrin excretion pattern in 10 cases of PCT from Copenhagen and 10 from Prague. 3. The results are presented (Table 2). They show no simple pattern. 4. Comparison with the type subdivision of Doss et al. (Table 3) and the important findings of Pitiol Aguade er al. show that such elaborate type division, involving considerable and time-consuming analytical work, belong to research and have limited value in clinical work.
INTRODUCTION Porphyria cutanea tarda (PCT) is today the most common type of porphyria. Earlier it was diagnosed less frequently and up to 1970 its incidence was considerably lower than that of acute intermittent porphyria (AIP) as illustrated by Table 1 presenting the number of new cases added to The Danish Porphyria Register (cf With, 1960, 1963, 1969a, 1973a-d). Since 1970 the incidence of PCT has increased in Denmark (cf Table 1). This disease is much more frequent in Czechoslovakia where at present more than 600 cases are under treatment by the First Medical Department of the Charles University of Prague. In comparison only 150 cases of PCT are registered in Denmark (cf Table 1). The increasing incidence of PCT is presumably due to dermatologists being increasingly aware of the diagnosis PCT combined with easier access to porphyrin analyses. but this can hardly be the whole explanation. Other factors such as increased alcohol consumption are presumably also active. Doss et a[. (1971) has proposed a subdivision of PCT in different types based on detailed analysis of the urinary porphyrin excretion. and when we got the opportunity to make a comparative study of the porphyrin excretion pattern in Czech and Danish PCT patients we used this opportunity to compare a patient material from a country with high incidence of PCT with one from a country with low incidence. MATERIALS AND
subjected to thin layer chromatography on silica gel with our modification of the system of Demole (1958tbenzene-ethylacetate-ethanol 90:20: 1.5 (by vol). The zones of the different porphyrins were scraped off the plate and eluted separately with chloroform-methanol (4: 1. v/v), evaporated, dissolved in suitable volumes of chloroform for spectrophotometric reading, and the extinctions at the Soret maxima compared after being corrected to the same volume for all the porphyrins. From the resulting corrected extinctions the relative amounts of the porphyrins were calculated (cf Rimington & With. 1973). Isomer analysis were performed by decarboxylation at atmospheric pressure (With, 1975) followed by lutidinewater paper chromatography (Eriksen. 1958).
RESULTS The results are presented in Table 2. The different porphyrin fractions are expressed as a percentage of the total porphyrin as found by summation of the fractions. The total urinary porphyrin concentrations could not be calculated because the analyses were based on talc adsorbates. All our patients showed domination of uroporphyrin making out between 50 and 80”, of the total porphyrin and the ratio Isomer I/Isomer III varied Table 1. The number of cases of different hepatic porphyrias added to the Danish Porphyria Register up to 1980 Type of porphyrias
METHODS
We selected10 patients with typical active PCT with high urinary porphyrin excretion from the Finsen Institute (Copenhagen) and 10 patients with typical active PCT with high excretion from Prague. The urines were adsorbed onto talc. the adsorbates esteritied (cf With, 1975b, 1976) and re-esterified to avoid partial esterification. and the concentrated chloroform extracts *This paper was presented in abbreviated form April 16th 1980 in a lecture by T. K. With at the Charles University, Prague. 769
PCT’ AIP’ VP3 HCP“
Before 1960 9 47+ 8 1
196c-1969 24 75 11 7
* To this comes 21 cases of sulphonal-trional acute porphyria. most of them before 1900. I Porphyria cutanea tarda. ’ Acute intermittent porphyria. 3 Variegate (or mixed) porphyria. 4 Hereditary coproporphyria.
197(t1979 109 30 10 3 induced
770
TORBENK. WITH et al.
Porphyria Table
TypeA,
cutanea
3. Types of chronic hepatic porphyria after Doss et al. (1971) 4~825~7~6
Type A2 4 > 8 > 7 > 5 > 6 8>4>7>5>6 Type B 8>7>4>5>6 Type C Type D 8 > 7 9 4(5,6) > 5(4,6) > 6(4,5) The figures signify the number of carboxyl groups-coproporphyrin having four. uro eight.
from 1:l to 9:l. i.e. marked domination of Isomer I. Heptacarboxylic porphyrins played a prominent part making out between 5 and 409/, of the total porphyrin. In seven of the Czech patients the percentage of hepta was, however, below the percentage of coproporphyrin (Doss’ type B) while the copro percentage was below that of hepta in all Danish cases. The ratio Isomer I/Isomer III for the hepta was between 1:20 and 3 : 7, i.e. marked domination of the III Isomer in all our 20 patients. The hexacarboxylic was only present as traces in the Czech cases but reached about 5”; in five of the Danish cases. The pentacarboxylic porphyrin was about 5”/b or below in most patients, except in two of the Czech cases where it reached 10 and 2556, in one exceeding the heptacarboxylic porphyrin. The III Isomer dominated in both the hexa and the penta, the ratio being 1:9-3:7. For the copro the percentage was higher in the Czech cases, making out l&25”/, in eight and 5% in two, as against below lLSp, in 8 of the Danish cases and 10% in two of them. In the copro, Isomer III dominated in all cases with ratios varying from 1: 1 to 1:20. It is noteworthy that isocoproporphyrin which often occurs in the feces in PCT-patients (Elder, 1972) was only present as traces in the urines studied. There was no significant difference in isomer ratios among the two groups. DlSCUSSlON
The only difference between the two groups of patients seems to be a tendency to a higher relative percentage of hepta and copro in the Czech patients and somewhat higher percentage of uro and hexa in the Danish patients. These differences could be due to the different chloroquine treatment used, as the Danish patients were treated with high intermittant doses while those from Prague were treated with low continuous doses (cf Kordai: et al., 1971). Our studies are fragmentary because they don’t include quantitative determination of total porphyrin and analysis of feces. A study of bigger materials, including patients in florid, moderate and latent clinical state and in different programmes of treatment, might disclose more consistent differences. Such studies would, however, be a time-consuming research project and our findings seem to show that results of such a project would hardly justify the labor involved. Our material can be used for evaluation of Doss et a/.‘~ (1971) subdivision of chronic hepatic porphyria in the types A,, Al, B, C and D according to their urinary porphyrin excretion pattern as summarized in Table 3. The types C and D show domination of uro followed by hepta while type B shows domination of
tarda
771
uro followed by copro. According to this seven of the Czech cases are type B while this type is not represented among the Danish cases. This difference may be due to the continuous chloroquine treatment used in the Czech cases. Piiiol Aguade et al. (1975) studied PCT (symptomatic porphyria) in Spain; 27 of his cases showed dominating uro, 21 showed dominating hepta, and eight showed dominating copro. These Spanish findings differ from both the Czech and Danish findings and from the types of Doss where uro always dominates over hepta: we have occasionally found patients in Denmark with hepta dominating, but in the Spanish material this was nearly as frequent as uro dominating over hepta. In the light of these findings the type subdivision of Doss seems too elaborate to be of practical clinical use. and the observations are at present somewhat confusing. This state of affairs is not surprising because the basic enzymatic disturbances, mainly in the urogen-decarboxylase enzyme. are believed to have a complex pathogenesis (Elder, 1977). Acknowledaemenrs-This paper was supported by a grant to Toiben K. With -from the Me&al Research Council of the Danish State (Grant No. 512-10.656) and by one to VBclav Kordaf from the Danish Ministry of Education. REFERENCES
DEMOLE E. (1958) Applications de microchromatographie d’adsorption sur couches minces. J. Chromat. 1, 24-35. p. 28 Legend to Fig. 2. Doss M.. MEINHOF W., LOCJK D., HENNING H., NAWROCKI P.. D~LLE W., STROHMEYERG. & FILIPPINI L. (1971) Porphyrins in liver and urine in AIP and chronic hepatic porphyrias. S. Afi. Med. J. 45, 5%54. ELDER G. H. (1972) Identification of a group of tetracarboxylate porphyrins. Eiochem. J. 126, 877-891. ELDER G. H. (1977) Porphyrin metabolism in PCT. Semin. Hemat.
24, 227-242.
ERIKSEN L. (1958) Paper chromatographic separation of the coproporphyrin isomers I and III. Stand. J. c/in. Lab. Inresr. 10, 319-322. KORDAE V., JIRSA M. & KALAB M. (1978) Chloroquine treatment of porphyria cutanea tarda. 2. k/in. Chemie Biochem. 16, 50. PI%OL AGUAD~ J., HERRERO C:. ALMEIDA J., SMITH S. G. & BELCHER R. V. (1975) Thin layer chromatography and counter-current analysis in porphyria. Br. J. Dermat. 93, 277-289.
RIMINGTON C. & WITH T. K. (1973) Porphyrin studies in congenital erythropoietic porphyria. Dan. med. Bull. 20, 5-12. WITH T. K. (1960) Porfyri i Danmark. Ugrskr. Lwg.. Copenhagen 122. 63-78. WITH T. K. (1963) Acute
intermittent Porphyria. Z. k/in. Chemie I, 134-143. WITH T. K. (1969a) Hereditary hepatic porphyrias. Acta med. stand. 186, 83-85. _ _ _ WITH T. K. (1969bl The oornhvrias in the light of Danish observations. Dan. med: 8~11.~16, 357-367. WITH T. K. (1973a) Akut intermitterende porfyri. Uyrskr. Lmg.. Copenhagen
WITH T. K. (1973b) hagen 135, 2450. WITH T. K. (1973~) Copenhuyen 135, WITH T. K. (1973d) Copenhagen
135, 2326-2328.
Variegat Heredit:er 2571. Porphyria
porfyri.
Uyeskr.
Lcrg., Copen-
koproporfyri.
Ugeskr.
Lwy..
cutanea
Uyeskr.
Lvy..
tarda.
135, 2691.
WITH T. K. (1975a)
Decarboxylation
of uroporphyrin
by
712
TOREIENK. WITH et al.
heating at atmospheric pressure. Biochem. J. 249-251. WITH T. K. (1975b) Clinical use of ester chromatography urine and feces. Dan. med. Bull 22, 74-80.
147, of
WITH T. K. (1976) A simplified system of clinical porphyrin analyses of urine and feces based on thin-layer chromatography. In Porphyrins in Human Diseases (Edited by Doss M.), pp. 492-499. Karger, Basel.
J. Biochem.
Vol.
13. pp.
769
to 772,
002071
1981
lX/81/060769-04SO2.WO
Copyright 0 1981 Pergamon Press Ltd
Printed m Great Britain. All nghts reserved
COMPARISON OF THE PORPHYRIN PATTERNS IN PATIENTS WITH PORPHYRIA CUTANEA TARDA IN CZECHOSLOVAKIA AND DENMARK TORBEN K. WITH,* V. KORDAI: and JANE SERUP PEDERSEN Svendborg Hospital, Department of Clinical Chemistry and The First Medical Department of the Charles University. Prague, Czechoslovakia (Rrceiwd
6 Ocrober 1980)
Abstract-l. The incidence of porphyria cutanea tarda (PCT) has increased considerably in Denmark during the last decade (Table 1) but is much higher in Czechoslovakia than in Denmark. 2. We therefore made a detailed study of the urinary porphyrin excretion pattern in 10 cases of PCT from Copenhagen and 10 from Prague. 3. The results are presented (Table 2). They show no simple pattern. 4. Comparison with the type subdivision of Doss et al. (Table 3) and the important findings of Pitiol Aguade er al. show that such elaborate type division, involving considerable and time-consuming analytical work, belong to research and have limited value in clinical work.
INTRODUCTION Porphyria cutanea tarda (PCT) is today the most common type of porphyria. Earlier it was diagnosed less frequently and up to 1970 its incidence was considerably lower than that of acute intermittent porphyria (AIP) as illustrated by Table 1 presenting the number of new cases added to The Danish Porphyria Register (cf With, 1960, 1963, 1969a, 1973a-d). Since 1970 the incidence of PCT has increased in Denmark (cf Table 1). This disease is much more frequent in Czechoslovakia where at present more than 600 cases are under treatment by the First Medical Department of the Charles University of Prague. In comparison only 150 cases of PCT are registered in Denmark (cf Table 1). The increasing incidence of PCT is presumably due to dermatologists being increasingly aware of the diagnosis PCT combined with easier access to porphyrin analyses. but this can hardly be the whole explanation. Other factors such as increased alcohol consumption are presumably also active. Doss et a[. (1971) has proposed a subdivision of PCT in different types based on detailed analysis of the urinary porphyrin excretion. and when we got the opportunity to make a comparative study of the porphyrin excretion pattern in Czech and Danish PCT patients we used this opportunity to compare a patient material from a country with high incidence of PCT with one from a country with low incidence. MATERIALS AND
subjected to thin layer chromatography on silica gel with our modification of the system of Demole (1958tbenzene-ethylacetate-ethanol 90:20: 1.5 (by vol). The zones of the different porphyrins were scraped off the plate and eluted separately with chloroform-methanol (4: 1. v/v), evaporated, dissolved in suitable volumes of chloroform for spectrophotometric reading, and the extinctions at the Soret maxima compared after being corrected to the same volume for all the porphyrins. From the resulting corrected extinctions the relative amounts of the porphyrins were calculated (cf Rimington & With. 1973). Isomer analysis were performed by decarboxylation at atmospheric pressure (With, 1975) followed by lutidinewater paper chromatography (Eriksen. 1958).
RESULTS The results are presented in Table 2. The different porphyrin fractions are expressed as a percentage of the total porphyrin as found by summation of the fractions. The total urinary porphyrin concentrations could not be calculated because the analyses were based on talc adsorbates. All our patients showed domination of uroporphyrin making out between 50 and 80”, of the total porphyrin and the ratio Isomer I/Isomer III varied Table 1. The number of cases of different hepatic porphyrias added to the Danish Porphyria Register up to 1980 Type of porphyrias
METHODS
We selected10 patients with typical active PCT with high urinary porphyrin excretion from the Finsen Institute (Copenhagen) and 10 patients with typical active PCT with high excretion from Prague. The urines were adsorbed onto talc. the adsorbates esteritied (cf With, 1975b, 1976) and re-esterified to avoid partial esterification. and the concentrated chloroform extracts *This paper was presented in abbreviated form April 16th 1980 in a lecture by T. K. With at the Charles University, Prague. 769
PCT’ AIP’ VP3 HCP“
Before 1960 9 47+ 8 1
196c-1969 24 75 11 7
* To this comes 21 cases of sulphonal-trional acute porphyria. most of them before 1900. I Porphyria cutanea tarda. ’ Acute intermittent porphyria. 3 Variegate (or mixed) porphyria. 4 Hereditary coproporphyria.
197(t1979 109 30 10 3 induced
770
TORBENK. WITH et al.
Porphyria Table
TypeA,
cutanea
3. Types of chronic hepatic porphyria after Doss et al. (1971) 4~825~7~6
Type A2 4 > 8 > 7 > 5 > 6 8>4>7>5>6 Type B 8>7>4>5>6 Type C Type D 8 > 7 9 4(5,6) > 5(4,6) > 6(4,5) The figures signify the number of carboxyl groups-coproporphyrin having four. uro eight.
from 1:l to 9:l. i.e. marked domination of Isomer I. Heptacarboxylic porphyrins played a prominent part making out between 5 and 409/, of the total porphyrin. In seven of the Czech patients the percentage of hepta was, however, below the percentage of coproporphyrin (Doss’ type B) while the copro percentage was below that of hepta in all Danish cases. The ratio Isomer I/Isomer III for the hepta was between 1:20 and 3 : 7, i.e. marked domination of the III Isomer in all our 20 patients. The hexacarboxylic was only present as traces in the Czech cases but reached about 5”; in five of the Danish cases. The pentacarboxylic porphyrin was about 5”/b or below in most patients, except in two of the Czech cases where it reached 10 and 2556, in one exceeding the heptacarboxylic porphyrin. The III Isomer dominated in both the hexa and the penta, the ratio being 1:9-3:7. For the copro the percentage was higher in the Czech cases, making out l&25”/, in eight and 5% in two, as against below lLSp, in 8 of the Danish cases and 10% in two of them. In the copro, Isomer III dominated in all cases with ratios varying from 1: 1 to 1:20. It is noteworthy that isocoproporphyrin which often occurs in the feces in PCT-patients (Elder, 1972) was only present as traces in the urines studied. There was no significant difference in isomer ratios among the two groups. DlSCUSSlON
The only difference between the two groups of patients seems to be a tendency to a higher relative percentage of hepta and copro in the Czech patients and somewhat higher percentage of uro and hexa in the Danish patients. These differences could be due to the different chloroquine treatment used, as the Danish patients were treated with high intermittant doses while those from Prague were treated with low continuous doses (cf Kordai: et al., 1971). Our studies are fragmentary because they don’t include quantitative determination of total porphyrin and analysis of feces. A study of bigger materials, including patients in florid, moderate and latent clinical state and in different programmes of treatment, might disclose more consistent differences. Such studies would, however, be a time-consuming research project and our findings seem to show that results of such a project would hardly justify the labor involved. Our material can be used for evaluation of Doss et a/.‘~ (1971) subdivision of chronic hepatic porphyria in the types A,, Al, B, C and D according to their urinary porphyrin excretion pattern as summarized in Table 3. The types C and D show domination of uro followed by hepta while type B shows domination of
tarda
771
uro followed by copro. According to this seven of the Czech cases are type B while this type is not represented among the Danish cases. This difference may be due to the continuous chloroquine treatment used in the Czech cases. Piiiol Aguade et al. (1975) studied PCT (symptomatic porphyria) in Spain; 27 of his cases showed dominating uro, 21 showed dominating hepta, and eight showed dominating copro. These Spanish findings differ from both the Czech and Danish findings and from the types of Doss where uro always dominates over hepta: we have occasionally found patients in Denmark with hepta dominating, but in the Spanish material this was nearly as frequent as uro dominating over hepta. In the light of these findings the type subdivision of Doss seems too elaborate to be of practical clinical use. and the observations are at present somewhat confusing. This state of affairs is not surprising because the basic enzymatic disturbances, mainly in the urogen-decarboxylase enzyme. are believed to have a complex pathogenesis (Elder, 1977). Acknowledaemenrs-This paper was supported by a grant to Toiben K. With -from the Me&al Research Council of the Danish State (Grant No. 512-10.656) and by one to VBclav Kordaf from the Danish Ministry of Education. REFERENCES
DEMOLE E. (1958) Applications de microchromatographie d’adsorption sur couches minces. J. Chromat. 1, 24-35. p. 28 Legend to Fig. 2. Doss M.. MEINHOF W., LOCJK D., HENNING H., NAWROCKI P.. D~LLE W., STROHMEYERG. & FILIPPINI L. (1971) Porphyrins in liver and urine in AIP and chronic hepatic porphyrias. S. Afi. Med. J. 45, 5%54. ELDER G. H. (1972) Identification of a group of tetracarboxylate porphyrins. Eiochem. J. 126, 877-891. ELDER G. H. (1977) Porphyrin metabolism in PCT. Semin. Hemat.
24, 227-242.
ERIKSEN L. (1958) Paper chromatographic separation of the coproporphyrin isomers I and III. Stand. J. c/in. Lab. Inresr. 10, 319-322. KORDAE V., JIRSA M. & KALAB M. (1978) Chloroquine treatment of porphyria cutanea tarda. 2. k/in. Chemie Biochem. 16, 50. PI%OL AGUAD~ J., HERRERO C:. ALMEIDA J., SMITH S. G. & BELCHER R. V. (1975) Thin layer chromatography and counter-current analysis in porphyria. Br. J. Dermat. 93, 277-289.
RIMINGTON C. & WITH T. K. (1973) Porphyrin studies in congenital erythropoietic porphyria. Dan. med. Bull. 20, 5-12. WITH T. K. (1960) Porfyri i Danmark. Ugrskr. Lwg.. Copenhagen 122. 63-78. WITH T. K. (1963) Acute
intermittent Porphyria. Z. k/in. Chemie I, 134-143. WITH T. K. (1969a) Hereditary hepatic porphyrias. Acta med. stand. 186, 83-85. _ _ _ WITH T. K. (1969bl The oornhvrias in the light of Danish observations. Dan. med: 8~11.~16, 357-367. WITH T. K. (1973a) Akut intermitterende porfyri. Uyrskr. Lmg.. Copenhagen
WITH T. K. (1973b) hagen 135, 2450. WITH T. K. (1973~) Copenhuyen 135, WITH T. K. (1973d) Copenhagen
135, 2326-2328.
Variegat Heredit:er 2571. Porphyria
porfyri.
Uyeskr.
Lcrg., Copen-
koproporfyri.
Ugeskr.
Lwy..
cutanea
Uyeskr.
Lvy..
tarda.
135, 2691.
WITH T. K. (1975a)
Decarboxylation
of uroporphyrin
by
712
TOREIENK. WITH et al.
heating at atmospheric pressure. Biochem. J. 249-251. WITH T. K. (1975b) Clinical use of ester chromatography urine and feces. Dan. med. Bull 22, 74-80.
147, of
WITH T. K. (1976) A simplified system of clinical porphyrin analyses of urine and feces based on thin-layer chromatography. In Porphyrins in Human Diseases (Edited by Doss M.), pp. 492-499. Karger, Basel.