Clinical Science (1970) 39, 147-158.
T H E P A T T E R N O F P O R P H Y R I N ISOMER
ACCUMULATION A N D EXCRETION IN
SYMPTOMATIC P O R P H Y R I A
E. DOWDLE, P. GOLDSWAIN, NORMA SPONG
AND
L. EALES
Department of Medicine, University of Cape Town
(Received 29 December 1969)
SUMMARY
1 . Thin-layer chromatography of porphyrin methyl esters provides a useful and
rapid technique for resolving mixtures of porphyrins from biological samples.
2. Application of this technique to urinary, faecal and hepatic porphyrins from
patients with symptomatic porphyria revealed five porphyrins of interest which could
be identified by mass spectrometry as the methyl esters of porphyrins with 8-, 7-,
6-, 5- and 4-carboxyl groups. Millimolar extinction coefficients at the Soret absorption
maxima for these porphyrin methyl esters in chloroform solution were measured.
3. A consistent pattern of urinary porphyrin excretion in symptomatic porphyria
was seen with 8-, 7-, 6-, 5- and 4-carboxyl porphyrins constituting approximately
60%, 25%, 3%, 3% and 9% of the total respectively. Uroporphyrin (73%), heptacarboxylic porphyrin (26%) and hexa-carboxylic porphyrin (I %) were the only
porphyrins detected in the liver of one patient.
4. Isomer analysis of excreted and hepatic porphyrins revealed that uroporphyrin
was approximately35% isomer I11,hepta- and hexa-carboxylic porphyrins almost all
isomer I11 and penta-carboxylic and coproporphyrin approximately 50% isomer 111.
5. To explain these findings it is suggested that there are two metabolic pathways
for the handling of 6-aminolaevulic acid (ALA) in the liver.
Symptomatic porphyria (porphyria cutanea tarda) is a well defined syndrome characterized
clinically by increased mechanical fragility, blistering, pigmentation and superficial ulceration
of the sun-exposed skin. A history of alcohol abuse and evidence of chronic parenchymatous
liver disease can usually be elicited. The serum iron concentration is frequently raised and
siderosis of the liver of varying degree is invariably present. Unlike inherited forms of hepatic
porphyria, a positive family history of the disorder is exceptional and acute attacks are
unknown.
The disease is distinguished biochemically by a characteristic pattern of haem precursor
Correspondence: Dr E. Dowdle, Department of Medicine, University of Cape Town Medical School,
Observatory, Cape, South Africa.
147
148
E. Dowdle et al.
excretion and accumulation. Urinary porphyrin excretion is considerably elevated with that
fraction measured by solvent extraction procedures as ‘uroporphyrin’ predominating. The
faecal porphyrin concentration is only moderately elevated if at all. Hepatic accumulation of
porphyrins is invariable and may in fact be the only manifestation of the disease (Lundwall,
personal communication). Excessive urinary excretion of 6-aminolaevulic acid (ALA) or
porphobilinogen (PBG) is not a feature of symptomatic porphyria.
The recent development of thin-layer chromatographic techniques for the separation of
porphyrin mixtures (Grosser, Sweeney & Eales, 1967; DOSS,1967) has contributed a very useful
analytical procedure to the study of human porphyria. Use of these techniques affords a means
of separating excreted porphyrins that is less laborious than conventional methods and
superior to solvent extraction techniques in the degree of resolution obtained. In this paper
we report on the pattern of porphyrin and porphyrin isomer excretion in human symptomatic
porphyria as observed when thin-layer chromatography of esterified porphyrins is used as the
major initial separation procedure.
MATERIALS A N D METHODS
Clinical
Twelve patients with symptomatic porphyria were studied. The relevant clinical and biochemical details are summarized in Table 1 . Patient J.M. presented with symptoms due to an
incidental carcinoma of the stomach and liver tissue for porphyrin analysis was obtained at
laparotomy. In his case it was therefore possible to analyse urine, liver and stool samples
collected during the same 24-h period. The fragment of liver obtained was macroscopically
free of metastatic deposits.
Urine samples were collected as pooled 24-h specimens without added preservative and
were processed within 48 h of collection.
Experimental
Extraction of urinary porphyrins as the methyl esters. The pH of each urine sample was
adjusted to between 3.0 and 4-0 with glacial acetic acid; and approximately I/lOth volume of
powdered talc was added. The slurry was stirred for 1 h, after which the talc and adsorbed
porphyrins were harvested by filtration with suction. This procedure was repeated until an
acetic acid: ether:amyl alcohol (1 :1 :1) extract of the filtrate no longer fluoresced under
ultra-violet light. The talc was then washed with 1% (v/v) aqueous acetic acid and water and
thoroughly dried in a vacuum dessicator over silica gel.
The adsorbed porphyrins were eluted and esterified in one step by suspending the talc in
methanol:H,SO, (20: 1) and allowing the suspension to stand overnight in the dark. The
suspension was then filtered and the talc washed with methanol:H,SO, (40:l) until it no
longer fluoresced. The pooled filtrate and washing were adjusted to pH 4.0 with saturated
sodium acetate and the porphyrin methyl esters were extracted into chloroform. The chloroform solution of methyl esters was washed four times with distilled water, and taken to dryness
under reduced pressure.
Extraction of faecalporphyrins as their methylesters. A sample of faeces (about 5 g) was dried
in Y ~ C U Oand the residue pulverized with a glass rod. The powder was suspended in approximately 50 ml of methanol: H,S04 (20: 1) and esterification was allowed to proceed overnight
M
M
M
M
M
M
M
M
M
M
M
M
R.A.
C.N.
R.L.
J.T.
B.M.
P.T.
E.M.
G.M.
E.C.
J.J.
G.O.
J.M.
43
42
50
45
52
46
43
36
60
45
63
51
Age
Liver
disease?
+
+
+
+
+
+
+
+
+
+
+
+
Alcohol
abuse*
+
+
+
+
+
0
+
+
+
+
+
+
163
165
49
1.9
2.5
1.2
4.4
1.4
2.0
3.7
1.2
3.3
4.1
291
521
257
371
538
2240
10249
3289
1607
3545
103
31
-
138
71
94
28
13
101
56
38
39
+
-
+
+
+
+
+
+
Livers
biopsy
5’
3
%
%
5
tomy
s.
3
a
3
G
3
3
g.
s2
33’
5
2
4
Diabetes. Chronic
pancreatitis.
Carcinoma of the
stomach Iaparo-
Diabetes.Chronic
pancreatitis.
Tuberculous
lymphadenitis
Remarks
Clearly palpable, lhn hepatomegaly; raised S.G.O.T. ; abnormal flocculation tests; elevated serum bilirubin, raised serum globulin, bromsulphthalein retention or liver biopsy.
3 The following methods were used without modification:
Urinary ALA and PBG-Mauzerall & Granick (1956).
Urinary uroporphyrin and coproporphyrin-Rimington & Sveinsson (1950).
Faecal coproporphyrin and protoporphyrin-Holti et al. (1958).
§ Liver biopsy: + = confirmation; - = not done.
132
153
93
37
153
0.7
0.1
0.6
1.1
3.3
1.8
4.8
5.9
3417
5056
6908
5011
217
1144
837
824
656
131
640
6740
2680
3434
1.0
0.8
0.8
105
110
110
‘Coproporphyrin’ ALA PBG Copro- ProtoOlgl1)
(mg/l)
bglg dry wt)
Faecal
porphyrint
3.6
2.8
1.5
‘Uroporphyrin’
Olgm
* Based on patient’s own assessment of his drinking habits.
t Liver disease judged to be present on the basis of two or more of the following criteria:
Sex
Subject
Urinary haem precursorst
TABLE1. Details of twelve patients with symptomatic porphyria
150
E. Dowdle et al.
in the dark. The suspension was then filtered, and the residue extracted repeatedly with
methanol: H,S04 (40: 1) until no further red fluorescence could be detected in the filtrate. The
pooled filtrate and washings were adjusted to pH 4.0 with saturated sodium acetate and the porphyrin methyl esters estracted into chloroform, washed and dried as in the case of the urine.
Extraction of porphyrins from liver as their methyl esters. A portion of liver (2.6 g) from J.M.
was minced finely and freeze-dried in vacuo. The dried tissue was pulverized and the porphyrins
esterified and extracted into chloroform, as for the faecal samples.
PuriJcation of the porphyrin methyl esters. The dried methyl esters were taken up in a small
volume of chloroform and applied as streaks 2 cm from the edge of 20 x 20 cm glass plates
coated with a 0.5 mm layer of silica gel without binder. The thin-layer plates were developed
in a continuous elution apparatus (Shandon Scientific Co., London, England) with benzene:
ethyl acetate: methanol (85 :13:2) until inspection under ultraviolet light revealed that the
components of the mixture had been separated satisfactorily. The bands were identified by
reference to pure coproporphyrin and uroporphyrin methyl ester markers.
The five main bands (see Results section) were removed quantitatively from the thin-layer
plates with a small suction device and the porphyrin methyl esters eluted from the silica gel
with chloroform: methanol (90: 10). The chloroform: methanol solution was taken to dryness
under a stream of nitrogen and the methyl ester residue redissolved in a measured volume of
redistilled, washed chloroform for spectrophotometric quantitation.
Precise chemical identification of the porphyrin methyl esters required purer compounds
that were obtained in the following manner. The dried methyl ester residue was hydrolysed
and freed from traces of contaminating porphyrins by descending paper chromatography of
the free acid on Whatman No. 3 filter paper using 2:6 lutidine: water (5: 3.5) as the solvent
system in an atmosphere of ammonia. The developed chromatogram was dried and the porphyrin recovered by immersing the rolled porphyrin-containing paper spot in methanol:
H,S04 (20: 1) for 24 h. The resulting ester was extracted into chloroform, dried, rechromatographed on thin-layer plates to remove traces of free porphyrin and recrystallized twice from
chloroform :methanol as described by Falk (I 964).
Paper chromatography of porphyrin free acids, Purified porphyrin methyl ester crystals were
hydrolysed by dissolving in a small volume of 7.5 N HCl and standing overnight. The HCl
was evaporated in vacuo and the resulting porphyrin free acids were separated by ascending
chromatography for 7-8 h with 2:6 1utidine:water (5:3.5) in an atmosphere of ammonia
(Eriksen, 1953).
Paper electrophoresis. The free porphyrins obtained by hydrolysis of the methyl esters were
taken up in a minimum volume of 7.0 N ammonia and applied as a line near the cathode end
of a 20 x 10 cm strip of Whatman No. 3 filter paper that had been moistened with 0.04 M
Na,CO, containing
M EDTA and equilibrated by pre-electrophoresis for 15 min. A
potential difference of 10 V/cm was applied until inspection under ultraviolet light revealed
adequate resolution. Authentic uroporphyrin and coproporphyrin markers were electrophoresed simultaneously.
Decarboxylation of porphyrin methyl esters. Chloroform solutions of the purified methyl
esters were evaporated to dryness in 10 ml Carius tubes and hydrolysed by the addition of
0.2 ml of 7.5 N HC1 for approximately 1 h. The HC1 was then adjusted to 0-3 N by the addition
of 4.8 ml water. The Carius tubes were evacuated, sealed and heated in an oven at 180" for
3 h, by which time decarboxylation to coproporphyrin was complete. The Carius tubes were
151
Porphyrin excretion in symptomatic porphyria
opened, their contents adjusted to pH 4.0 with saturated sodium acetate, and the coproporphyrin extracted into ether for further processing.
The isomer composition of the purijied porphyrin methyl esters was determined by decarboxylation of the methyl ester to coproporphyrin free acid as described above. The coproporphyrins I and I11 in the reaction mixtures from the decarboxylation were separated by chromatography on paper and determined by spectrofluorophotometry of the eluted coproporphyrin
isomer spots (Sweeney & Eales, 1964).
Spectrophotometry. The absorption spectra of the porphyrin methyl esters were recorded
automatically using a Beckman DK 2A ratio recording spectrophotometer. For quantitative
work and for more precise definition of the absorption maxima, chloroform solutions of the
methyl esters or hydrochloric acid solutions of the free acids were examined in a Zeiss PMQ I1
spectrophotometer at a slit width of 0.02 mm. The wavelength scale was calibrated against the
486.1 nm and the 656.3 nm emission bands of a hydrogen lamp.
Millimolar extinction coefficients (E&
for porphyrin methyl esters other than coproporphyrin and uroporphyrin methyl ester were determined by decarboxylation of the porphyrin
ester contained in a measured volume of a chloroform solution whose extinction at the Soret
maximum was known. The extinction of the resulting hydrochloric acid solution of coproporphyrin free acid was determined at the Soret peak and the millimolar concentration of methyl
ester in the original chloroform solution was calculated assuming a stoichiometricyield of free
coproporphyrin from the methyl ester and an cmMfor coproporphyrin in 0-1 N HC1 of 489
(Falk, 1964).
Mass spectrometry. Mass spectra of the porphyrin methyl esters were recorded on an MS9
double-focussing instrument (A.E.I. Ltd.) by the direct injection technique at an operating
temperature of approximately 250".
RESULTS
Technical
The mixture of porphyrin methyl esters from the urine could be separated into five main
fractions by thin-layer chromatography. The fastest of these fractions moved with coproporphyrin methyl ester and the slowest with uroporphyrin methyl ester. The three intermediate
bands showed chromatographic mobilities appropriate for penta-, hexa- and hepta-carboxylic
porphyrin methyl esters. Similarly, paper chromatography and paper electrophoresis of the
hydrolysed purified methyl esters identified five porphyrins, one of which behaved as coproporphyrin, one as uroporphyrin and three of which had intermediate mobilities. The pentacarboxylic porphyrin ran as two spots with 2:6 1utidine:water on paper. This proved, on
further investigation to be due to the ability of this system to resolve the I and I11 isomers of
penta-carboxylic porphyrin.
Mass spectrometry of the purified material from the five methyl ester bands showed in each
case a clearly defined, major molecular ion (M') peak with a confirming metastable peak.
Molecular ion peaks for the five methyl esters gave integral molecular weights in order of
decreasing chromatographic mobility of 710,768,826,884 and 942, so confirming their identity
as the methyl esters of coproporphyrin, penta-, hexa- and hepta-carboxylic porphyrin and
uroporphyrin respectively. Minor metal complex peaks were visible at M+ 53 and M+ 61
mass units in the case of the 5-, 6-, 7- and 8-carboxyl methyl esters and at M + + 54 and M+ + 62
+
+
E. Dowdle et al.
152
mass units in the case of coproporphyrin methyl ester. The cleavage of the propionate ester
side chains at the bond p to the nucleus was shown in the fragmentation patterns by the successive loss of seventy-three mass units for each propionate residue until the last, which involved
the loss of seventy-four units. A similar observation was made by Jackson, Kenner, Smith,
Aplin, Budzikiewicz & Djerassi (1965).
Spectrophotometry of the purified methyl esters in chloroform solution showed Soret
maxima at 400, 403, 404, 405 and 406 nm with millimolar extinction coefficients of 181, 198,
203, 206 and 216 for the 4-, 5-, 6- 7 and 8-carboxyl porphyrins respectively. These data are
summarized in Table 2 and compared with similar data obtained by other workers.
TABLE
2. Soret maxima (A) and millimolar extinction coefficients (emM)for
porphyrin methyl esters in chloroform.
Present study
Porphyrin methyl ester
A
Emiv
(nm)
Uroporphyrin
Hepta-carboxylic porphyrin
Hexa-carboxylic porphyrin
Penta-carboxylic porphyrin
Coproporphyrin
406
405
404
403
400
216
206
203
198
181
Doss (1969)
I
&mM
(nm)
405.5
404.0
402.5
401.0
399.5
216
207
198
189
180
Falk (1964)
A
&mM
(nm)
406
215
-
-
-
-
400
180
Although thin-layer chromatography of the crude porphyrin methyl ester mixtures gave
clearly separated bands, mass spectrometry of the individual methyl esters without further
purification showed slight contamination with faster moving components, as evidenced by
the presence of peaks at fifty-eight mass units, or integral multiples thereof, less than the M f
peak. The uroporphyrin methyl ester fraction (M’ = 942), for example, showed minor peaks
(from 1 to 2% of the M + peak) at m/e values of 884, 826, 768 and 710. The procedure described in the Methods section for obtaining pure material was necessary to obtain ‘clean’ mass
spectra. This phenomenon of chromatographic ‘entrainment’ is particularly troublesome with
porphyrins and has been described by other workers (Bogorad & Marks, 1960; Grinstein,
Schwartz 4% Watson, 1945).
Experimental
When the five main porphyrin methyl ester bands were eluted from the thin-layer plates and
the relative amounts of each measured, the results summarized in Table 3 were obtained.
Uroporphyrin methyl ester invariably predominated in the mixture of methyl esters separated
from the urine, constituting between 51 % and 76% of those measured. This was followed by
the methyl esters of hepta-carboxylic porphyrin (18-32%) and coproporphyrin (2-12%).
Penta-carboxylic and hexa-carboxylic methyl esters were present in approximately the same
amount and constituted from 1 to 4%. The methyl esters of the faecal porphyrins isolated
from J.M. showed an entirely different pattern. Considering only those porphyrins with four
or more carboxyl groups, copro-, hepta- and hexa-carboxylic porphyrin methyl ester predominated and uroporphyrin methyl ester contributed only 6% to the total. The methyl esters
Porphyrin excretion in symptomatic porphyria
153
from the liver showed only uroporphyrin and hepta-carboxylic porphyrin methyl esters with a
very faint trace of hexa-carboxylic methyl ester. Coproporphyrin and penta-carboxylic porphyrin methyl esters could not be detected. These results are consistent with observations that
TABLE3. Relative proportions of porphyrins with 4 to 8 carboxyl
groups isolated from patients with symptomatic porphyria. Values
expressed as percentages of the total
Subject
Sample
R.A.
C.N.
R.L.
J.T.
B.M.
P.T.
E.M.
G.M.
E.C.
J.J.
G.O.
Urine
J.M.
Urine
Faeces
Liver
9,
Number of carboxyl groups on porphyrin
8765458
51
62
65
56
72
59
60
62
76
62
21
30
24
21
32
18
28
30
29
20
23
3
4
2
3
3
4
2
2
2
1
2
3
3
3
3
2
2
1
2
3
2
2
10
12
9
8
52
6
73
33
24
26
6
17
1
5
4
-
4
47
-
I
4
10
6
4
2
9
TABLE
4. Isomer composition of porphyrins with 4 to 8 carboxyl
groups isolated from patients with symptomaticporphyria.
Subject
Sample
R.A.
C.N.
R.L.
J.T.
B.M.
P.T.
E.M.
G.M.
E.C.
J.J.
Urine
19
G.O.
J.M.
Urine
Faeces
Liver
Number of carboxyl groups on porphyrin
81654(Isomer 111expressed as % of the total)
42
24
30
35
33
28
32
30
34
27
97
82
86
93
88
84
99
92
94
85
93
85
86
84
91
87
88
96
74
95
85
40
47
31
100
100
100
100
100
100
40
56
51
40
38
54
29
55
26
45
72
44
50
53
62
63
64
44
59
66
56
58
37
52
52
50
154
E. Dowdle et al.
we have made previously on fragments of liver obtained by needle biopsy from eight patients
with symptomatic porphyria. When the porphyrins contained in these pieces of liver were
extracted and chromatographed, inspection under ultraviolet light invariably showed the predominant presence of uroporphyrin and hepta-carboxylic porphyrin, the occasional presence
of traces of hexa-carboxylic porphyrin and the absence of penta-carboxylic porphyrin or
coproporphyrin.
Analysis of the isomer composition of the porphyrins revealed only the series I and I11
isomers (Table 4). In the urine uroporphyrin was approximately 30% isomer 111, hepta- and
hexa-carboxylic porphyrin approximately 90% isomer 111, while coproporphyrin and pentacarboxylic porphyrin were approximately 50% isomer 111. The faecal porphyrins isolated from
J.M. showed a similar preponderance of series I isomer in the uroporphyrin fraction, the heptaand hexa-carboxylic porphyrins were virtually all series I11 isomers and coproporphyrin and
penta-carboxylic porphyrin were approximately half series I and half series I11 isomer. The
liver porphyrin from J.M. showed a similar pattern of isomer composition, uroporphyrin
being 37% isomer I11 while the 7- and 6-carboxyl porphyrins were all isomer 111.
DISCUSSION
Thin-layer chromatography of a crude mixture of methyl esters of porphyrins isolated from
biological materials provides a rapid technique for detecting the nature and relative abundance
of main porphyrins of interest in human porphyria. While this technique is adequate for most
diagnostic purposes it should be noted that entrainment of minor components does occur (as
shown by mass spectrometry) and more rigorous procedures are required before any particular
porphyrin methyl ester can be judged pure. Such procedures would obviously be necessary if
valid conclusions were to be drawn from experiments with radioactive porphyrin precursors.
The occurrence of porphyrins other than coproporphyrin and uroporphyrin in the urine of
patients with porphyria has been known for many years (Nicholas, 1951;Grinstein et al., 1945;
McSwiney, Nicholas & Prunty, 1950; Canivet & Rimington, 1953) and various names such as
‘Waldenstrom porphyrin’, ‘pseudo-uroporphyrin’, ‘208-porphyrin’ and ‘phyria porphyrin’
were given to these. Technical procedures available at the time afforded poor resolution of
porphyrins with similar characteristics so that many of these were, in fact, mixtures. Paper
chromatography and electrophoresis gave far better resolution than had been achieved by
solvent extraction procedures and enabled workers to distinguish other porphyrins in addition
to coproporphyrin and uroporphyrin. These techniques have been extensively reviewed by
Falk (1964).
Chu & Chu (1959) separated the urinary porphyrins from a patient with symptomatic porphyria by column chromatography of the methyl esters on Hyflo Super-Cel. They, too, noted
the presence of five main constituents and reported the absorption spectra, paper chromatographic characteristics and pH-dependent fluorescence of these. The excreted porphyrins were
compared with the products of partial decarboxylation of uroporphyrin and on the basis of
similarity the five main porphyrins in symptomatic porphyria were assigned 8-, 7-, 6-, 5- and
4-carboxyl groups.
Lockwood & Davies (1962) observed five main porphyrins on electrophoresis of the porphyrin free acids isolated from the urine of patients with acute porphyria and porphyria cutanea
tarda. They tentatively identified these as 8-, 7-, 6-, 5- and 4-carboxyl porphyrins but, on
Porphyrin excretion in symptomatic porphyria
155
subjecting the eluted fractions to chromatography on paper with a lutidine :water solvent
system, the electrophoretically homogeneous bands yielded multiple spots. This phenomenon
was attributed to contamination by adjoining fractions.
The addition of mass spectrometry to the available techniques for the identification of
porphyrin methyl esters in the studies reported here leaves little doubt of the identity of the
five main porphyrins excreted in symptomatic porphyria.
The millimolar extinction coefficients that we obtained for the 7-, 6- and 5-carboxyl porphyrins differ somewhat from those obtained by Doss (1969). Since Doss obtained millimolar
extinction coefficients by interpolation between those for uroporphyrin and coproporphyrin,
while we have used a more direct procedure, we have preferred to use our own values.
In all cases uroporphyrin and hepta-carboxylic porphyrin accounted for 80-90% of the
excreted urinary porphyrins and for virtually 100% of the porphyrin that accumulated in the
liver in the case of J.M. Essentially similar findings have been reported by Chu & Chu (1967).
The smaller relative amount of uroporphyrin in the faeces from J.M. can be explained by the
known preference for urinary excretion of uroporphyrin while coproporphyrin is preferentially eliminated via the bile (Rimington, 1963). Since it is known that faecal porphyrin excretion in symptomatic porphyria is quantitatively relatively minor (Eales, 1961), one can
draw the general conclusion that the characteristic biochemical abnormality of symptomatic
porphyria is an accumulation and excessive excretion of uroporphyrin and hepta-carboxylic
porphyrin.
If, as seems probable from the available evidence (Dowdle, Mustard & Eales, 1967; Levere,
1966; Zail & Joubert, 1968), hepatic 6-aminolaevulic acid synthetase activity is increased in
symptomatic porphyria, and an exogenous load of ALA is preferentially excreted as uroporphyrin in symptomatic porphyria (Dowdle et al., 1968), one must ask why is it that the endogenous ALA overload in this disease leads to excessive synthesis of uroporphyrin and heptacarboxylic porphyrin, without other haem precursors participating in the abnormal excretory
pattern? An obvious explanation would be to postulate a defect in the decarboxylation of
hepta-carboxylic porphyrinogen, but experiments with [I4C]ALA have indicated that haem
biosynthesis is normal in symptomatic porphyria and, moreover, that the rate of incorporation of radioactivity into urinary coproporphyrin is unimpaired in symptomatic porphyria
(Dowdle et al., 1968 and unpublished observations). It seems more probable that the liver disease
which accompanies this syndrome operates in such a manner as to create a separate metabolic
pool of uroporphyrinogen and hepta-carboxylic porphyrinogen with limited access to the
decarboxylation steps that normally serve haem biosynthesis. It may, for example, be that
highly carboxylated porphyrinogens, by virtue of their polarity and poor lipid solubility, are
more critically dependent upon healthy intracellular membrane-bound organelles for their
transport and metabolic disposal. This however, is conjecture and further work is needed to
frame a sounder explanation.
Measurement of the isomer composition of the urinary porphyrins gave consistent results
similar to those reported by Chu & Chu (1967), with uroporphyrin showing a slight preponderance of isomer I, hepta- and hexa-carboxylic porphyrin being almost all isomer 111and pentacarboxylic porphyrin and coproporphyrin being approximately half isomer I and half isomer
111.
In view of these differences it is quite clearly unacceptable to conceive of the decarboxylation
of uroporphyrinogen to coproporphyrinogen as proceeding under the influence of decarboxy-
156
E. Dowdle et al.
lases with similar kinetics for the I and I11 isomers, and generating a sequence of intermediately
carboxylated porphyrinogen pools from which porphyrins are derived by oxidation without
isomer discrimination. If this were so, all of the porphyrins would have identical isomer
compositions. Since the isomer compositions of the faecal, hepatic and urinary porphyrins
isolated from J.M. were similar, one cannot explain the enrichment of urinary hepta- and
hexa-carboxylic porphyrin with I11 isomer by postulating preferential biliary excretion or
hepatic accumulation of the series I isomers. Although there have been no definitive studies in
mammalian systems, the work of Hoare & Heath (1958) with acetone-dried Rhodopseudomonas
spheroides would indicate that isomerases for individual porphyrinogens do not exist and that
the final isomer type is determined during the conversion of porphobilinogen to uroporphyrinogen. It is unlikely, therefore, that the observed differences in isomer composition reflect
different equilibrium constants for individual porphyrinogen isomerases.
Two other explanations have occurred to us. Firstly, it may be that the decarboxylation of
uroporphyrinogen, hepta-carboxylic porphyrinogen and hexa-carboxylic porphyrinogen
proceed with different kinetics for the series I and I11 isomers. If, for example uroporphyrinogen I11 were rapidly converted to hepta-carboxylic porphyrinogen I11 and subsequent decarboxylation was slow, while the decarboxylation of uroporphyrinogen I were slow and that of
hepta- and hexa-carboxylic porphyrinogen were rapid, the observed accumulation of isomer
types might be expected.
Secondly, it may be that there are two functionally distinct metabolic paths that ALA may
follow in the liver, the one generating porphyrinogen pools that equilibrate only to a limited
extent with those generated by the other. Since, as argued earlier, the existence of such a second,
‘isolated’ pathway would also explain the accumulation of uroporphyrin and hepta-carboxylic
porphyrin in symptomatic porphyria, we prefer this explanation and propose the tentative
model of hepatic porphyrin metabolism depicted diagrammatically in Fig. 1.
In this model we suggest that under normal circumstances ALA is converted via PBG,
uroporphyrinogen I and 111, and the intermediately carboxylated porphyrinogens I and 111, to
a mixture of coproporphyrinogens I and I11 (pathway A in Fig. 1). This pathway has a limited
capacity to handle ALA, in the first instance at the stage of the further utilization of coproporphyrinogen and in the second instance at the stage of the conversion of ALA to PBG.
Under conditions of mild ALA overload such as occur in hepatic cirrhosis (Levere, 1967),
coproporphyrinogen accumulates and coproporphyrinuria alone results. When, however,
endogenous ALA overproduction becomes more pronounced in symptomatic porphyria, the
second rate-limiting step is exceeded and the excess ALA is now handled by a second pathway
(B in Fig. 1) in which it is converted to PBG and a mixture of uroporphyrinogens I and 111.
In this pathway there is no decarboxylase for uroporphyrinogen I or hexa-carboxylic porphyrinogen I11 and the decarboxylation of uroporphyrinogen I11 and hepta-carboxylic porphyrinogen I11 are rate-limiting. The net result is an accumulation of uroporphyrin I
and 111 (the former predominating since I11 can be decarboxylated) and hepta-carboxylic
porphyrin I11 with traces of hexa-carboxylic porphyrin 111. If this model has validity, pentacarboxylic porphyrin and coproporphyrin in symptomatic porphyria would be derived from
pathway A, whereas porphyrins with 8-, 7- and 6-carboxyl groups would be derived from
pathway B.
While the model as proposed in Fig. 1 shows two functionally distinct pathways for the
disposal of ALA, our observations could similarly be used to support a model in which there
157
Porphyrin excretion in symptomatic porphyria
were two functionally distinct pathways for the metabolic disposal of PBG. Further studies
are at present underway to examine this possibility.
I
1
1
7 1
7 m
1
1
6 1
6 I11
7LrI
6
1III
1
1
5 1
5 m
1
coprdgen I
I
Coprdgen III
1
Hoem
FIG.1. Model proposed to explain the pattern of abnormal hepatic porphyrin metabolism
encountered in symptomatic porphyria. ALA = 6-aminolaevulic acid. PBG = porphobilinogen.
Uro’gen = uroporphyrinogen Copro’gen = coproporphyrinogen. A and B are postulated
alternative metabolic pathways. See text for further details. The numerals 7, 6, 5 refer to heptahexa- and penta-carboxylic porphyrinogen respectively.
ACKNOWLEDGMENTS
This work was supported by Grant PHS AM-03997 from the National Institute of Arthritis
and Metabolic Diseases, Public Health Service, United States of America. It formed part of the
programme of the CSIR/UCT Renal-Metabolic Research Group which is supported at the
University of Cape Town by the South African Council for Scientific and Industrial Research,
the South African Atomic Energy Board and the University of Cape Town Staff Research
Fund.
Dr S. Eggers of the National Chemical Research Laboratories of the Council for Scientific
and Industrial Research kindly recorded the mass spectra of the porphyrin methyl esters.
The authors acknowledge with gratitude the interest and suggestions of Professor A Neuberger, Dr G. Tait and Dr A. Gorchein of the Department of Chemical Pathology, St Mary’s
Hospital, London.
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