J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from J. clin. Path., 1972, 25, 1013-1033

The porphyrias: a review

G. H. ELDER, C. H. GRAY, AND D. C. NICHOLSON From the Department of Chemical Pathology, King's College Hospital Medical School, Denmark Hill, London

The porphyrias are disorders of the biosynthesis of Chemistry and Biochemistry of the Porphyrins protohaem, the ferrous iron complex of proto- porphyrin IX, in which characteristic clinical The general pathway of biosynthesis of haem for features are accompanied by specific patterns of haem protein formation is well known except for porphyrin and porphyrin precursor overproduction, the details of very early stages and the later stages accumulation, and excretion, each pattern defining in uroporphyrinogen and coproporphyrinogen syn- a particular form of porphyria. thesis. Until the formation of protoporphyrin IX All those forms of porphyria in which there is this pathway involves not the porphyrins but overproduction of porphyrins have one clinical porphyrinogens, the hexahydro derivatives of feature in common-sensitivity of the skin to porphyrins. Succinyl co-enzyme A, derived from sunlight-although the nature of the lesions pro- acetate via the Kreb's cycle and a-oxoglutarate, is duced differs between diseases. The photosensitivity condensed by the enzyme 5-aminolaevulinic acid

is due to the photodynamic action ofthe porphyrins synthetase (ALA-S) with glycine, pyridoxal phos- by copyright. that accumulate in the skin when the plasma porphy- phate participating, to form a-amino-fl-ketoadipic rin concentration is increased (Rimington, Magnus, acid which rapidly loses CO2 non-enzymically to Ryan, and Cripps, 1967) and which probably act as form ALA. Under the influence of the sulphydryl sensitizers for singlet-oxygen-mediated destructive enzyme ALA dehydratase, two molecules of ALA processes, for example, the peroxidation of lipids in condense to form one molecule of the monopyrrole the membranes of lysosomes (Allison, Magnus, and PBG with the structure of 2-aminomethyl-3- Young, 1966; Magnus, 1972). carboxymethyl-4-carboxyethyl pyrrole (Fig. la). The other main clinical feature ofthe porphyrias- The enzyme uroporphyrinogen synthetase (Bogorad, neurological lesions typically causing severe abdom- 1958) causes four molecules of porphobilinogen to inal pain, peripheral neuropathy, and often mental condense to give uroporphyrinogen I in which http://jcp.bmj.com/ disturbance, and frequently precipitated by drugs 4-carboxymethyl and 4-carboxyethyl groups are such as the barbiturates-is associated with in- arranged alternately around the hexahydroporphin creased excretion of the porphyrin precursors, ring. When uroporphyrinogen synthetase acts in the porphobilinogen (PBG) and 5-aminolaevulinic acid presence of uroporphyrinogen III co-synthetase, (ALA) and does not occur in those forms of which may be a separate enzyme (Bogorad, 1963), porphyria in which excretion of these precursors is or, as in mammalian liver, part of a complex con- always normal. taining the synthetase (Bogorad, 1958), uropor- on September 23, 2021 by guest. Protected In some patients only the biochemical features phyrinogen III is formed; in this the carboxymethyl are apparent. Such clinically latent porphyria may andcarboxyethylgroups attachedtopyrrole ringDare occur either as a phase in an episodic illness or as the reversed (Fig. Ib). Two otherisomericporphyrinogens only manifestation throughout life. Proper treatment are theoretically possible but are not found in nature. of patients with porphyria depends upon accurate In haem-synthesizing tissues, four carboxymethyl diagnosis, which in turn depends entirely upon the groups are presumed to be successively decarboxy- accurate interpretation of proper laboratory in- lated to methyl groups to give hepta-, hexa-, and vestigations and proper enquiry into the family pentacarboxyl porphyrinogens and finally the history. Discussion of the prevention and manage- tetracarboxyl coproporphyrinogens I and III. Copro- ment of porphyria is beyond the scope of this review porphyrinogen I is not further metabolized but and is summarized by Goldberg (1971). coproporphyrinogen III undergoes a dehydrogena- tion-decarboxylation reaction convertingthecarboxy- Received for publication 19 October 1972. ethyl groups on rings A and B to vinyl groups 1013 J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

1014 G. H. Elder, C. H. Gray, and D. C. Nicholson

(c0011 COOH (LY(CINF (H2 Fig. la The biosynthetic CH2 P A haem IN2NCH2 COOH ALA CH, [1 pathway of formation synthetase H. N (H COOH -co, CH2 ALA dehydratase L (formation ofporphobilinogen) 1OOCCCH2 C(i2 COCoA O=(('H ('12COOH C=O N CHH2 sH SUCCINYL COENZYMF A a AMINO KETOADIPI(' AC(' ) CH, NH, PORPHOBILINOGFN Transient) NH,

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Fig. b The biosynthetic pathway of haemn formation (conversion ofporphobilinogeni to porphy!rins and haem) on September 23, 2021 by guest. Protected yielding protoporphyrinogen which is readily de- unknown and there may well be a separate pool of hydrogenated to provide protoporphyrin (Sano and this compound not directly formed via the Kreb's Granick, 1961). In the presence of iron and the cycle. Secondly, the mechanism of formation of enzyme ferrochelatase (Labbe, Hubbard, and uroporphyrinogens I and III from porphobilinogen Caughey, 1963), this is converted to haem for haem remains obscure although numerous theories have protein synthesis (Figure lb). At all stages in the been proposed (Llambias and Batlle, 1970). There biosynthetic pathway the porphyrinogens are is need for this to be established as there are almost readily converted to the corresponding porphyrins certainly enzymic deficiencies of this pathway other which, with the exception of protoporphyrin, than that of uroporphyrinogen III co-synthetase in cannot be further metabolized. congenital erythropoietic porphyria. Thus there is There are several points of this pathway that are evidence that abnormality or deficiency of uropor- in need of further elucidation. The precise source of phyrinogen synthetase itself may be the primary succinyl coenzyme A used for ALA synthesis is abnormality in acute intermittent porphyria (Strand, J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1015 Felsher, Redeker, and Marver, 1970; Miyagi, R NI Cardinal, Bossenmaier, and Watson, 1971). The H third area which requires elucidation, particularly in C mammalian liver, is the decarboxylation of uropor- phyrinogen III to coproporphyrinogen III which is usually accepted as occurring sequentially. Recently r7, Dowdle, Goldswain, Spong, and Eales (1970), HC (H studying that form of porphyria known as sympto- matic porphyria, attribute a paradoxical distribution of isomers I and III in conversion of uroporphy- rinogen through hepta- and pentacarboxyl porphy- rinogens to two distinct metabolic pathways in the P p liver. In one, ALA is converted sequentially to haem via PBG, uroporphyrinogens I and III, copropor- Fig. 2 New porphyrins recently isolatedfromfaeces phyrinogen III and protoporphyrinogen, while in (see text). the other, which operates under conditions of ALA Isocoproporphyrin (IR = CH2 * CH3) overload, uroporphyrinogen III gives rise only to Deethylisocoproporphyrin (11 R = -H) hexa- and heptacarboxyl porphyrinogens which Hydroxyisocoproporphyrin (IIIR = -CH(OH)CH3) accumulate and are excreted as the respective Dehydroisocoproporphyrin (IVR = -CH= CH2) porphyrins. Isotope experiments have suggested that In I, II, III, IV, R' = CH2COOH (Elder, 1971, 1972) there may be two distinct metabolic pools of uro- Acrylic analogue ofcoproporphyrin-III isomer (VR = -CH=CH2 R' = CH3) porphyrinogen (Goldswain, Dowdle, Spong, and (French, Nicholson, and Rimington, 1970) Eales, 1970). The situation of the acrylic group shown here in More recently, detailed studies of urinary and position 2 of structure V still requires final proof. faecal and biliary porphyrins have shown that in one form of porphyria-variegate porphyria-there is by copyright. excretion of a peptide conjugate of a porphyrin Changeux, and Jacob, 1963), thus inhibiting its (Rimington, Lockwood, and Belcher, 1968). In activity. End product repression usually involves symptomatic cutaneous hepatic porphyria a vinyl combination oftheendproductwithan 'aporepressor' tricarboxyethyl carboxymethyl trimethyl porphyrin molecule synthesized by the activity of a regulator occurs (Elder, 1972) (Fig. 2, IV) which is excreted in gene (Jacob and Monod, 1961). Normally the the bile with a hydroxy derivative in which the aporepressor molecule combined with the end pro- elements of water have been added to the vinyl duct acting as a co-repressor represses the activity of group and is partially converted to ethyl and hydryl an operator gene directing the synthesis of the

(deutero) derivatives by intestinal microorganisms m-RNA controlling the synthesis of the enzyme. If http://jcp.bmj.com/ (Elder, 1972). It is not known whether this reflects the end product is deficient, or has to compete for the exaggeration of a normal pathway by an enzyme binding sites with compounds which can act as por- block in symptomatic cutaneous hepatic porphyria phyrinogenic agents, the aporepressor molecule no or the presence of a unique metabolic pathway in longer represses the operator gene which leads to this condition. increased synthesis and induction of the enzyme thus Aminolaevulinic acid synthetase (ALA-S) is the providing a feedback control of the biosynthetic rate-limiting enzyme in the biosynthesis of haem There is that inhibi- pathway. evidence end-product on September 23, 2021 by guest. Protected (Granick and Urata, 1963) and normally its amount tion and repression of ALA synthetase by the end and activity are adjusted so precisely that only traces product haem takes place in Rhodopseudomonas of haem precursors are present in the normal spheroides (Burnham and Lascelles, 1963) and re- organism. In most forms of porphyria, the activity pression by haem occurs in rabbit reticulocytes and in of the enzyme is increased (Dowdle, Mustard, embryo chicken liver cell cultures (Granick, 1966) Spong, and Eales, 1967). This may result from in- (Fig. 3). The action of compounds which cause creased substrate availability or by diminution of experimental porphyria is outside the scope of this end product inhibition or repression. In the first review but has been surveyed by Tschudy and mechanism an increased formation of succinyl CoA Bonkowski (1972). or of glycine might activate the enzyme but there is no evidence that this plays a significant part in Classification and Nomenclature increasing ALA synthetase activity in the porphyrias. In end product inhibition, the end product changes There have been numerous classifications of the the allosteric conformation of the enzyme (Monod, porphyrias based on consideration of clinical and J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

1016 G. H. Elder, C. H. Gray, and D. C. Nicholson

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Fig. 3 Possible mechanisms for the control ofhaemoglobin synthesis. by copyright. biochemical features and patterns of inheritance In the erythropoietic porphyrias the metabolic (Gunther, 1911; Waldenstrom, 1937; Watson, 1951; abnormality is believed to be confined to the bone Goldberg and Rimington, 1962; Conference Dis- marrow and in the hepatic porphyrias to the liver. cussion, 1963; Gray, 1970; Tschudy, 1969; Marver In recent years it has become apparent that in ery- and Schmid, 1972). The system of classification thropoietic protoporphyria, which was first identified followed in this review is shown in Table I. The by Magnus, Jarrett, Prankerd, and Rimington in porphyrias are usually divided into two main groups 1961, porphyrins are produced in excess in both according to the site of accumulation and, by in- erythropoietic and hepatic cells (Gray, Kulczycka, http://jcp.bmj.com/ ference, of overproduction of haem precursors Nicholson, Magnus, and Rimington, 1964), and within the body (Schmid, Schwartz, and Watson, Scholnick, Marver, and Schmid (1971) have pro- 1954; Watson, Lowry, Schmid, Hawkinson, and posed that the condition should be renamed cry- Schwartz, 1951). throhepatic protoporphyria. Overproduction of porphyrins in erythropoietic tissues leads to accumulation of porphyrins within Erythropoietic porphyrias can thus erythrocytes. Erythropoietic on September 23, 2021 by guest. Protected 1 Congenital erythropoietic porphyria 2 Erythropoietic coproporphyria be distinguished from the hepatic porphyrias by measurement of erythrocyte porphyrin levels, for in Erythrohepatfc the purely hepatic porphyrias overproduction of I Protoporphyria porphyrins is not accompanied by an increase in Hepatic erythrocyte porphyrin levels. 1 Hepaticporphyriasinherited as autosomal dominants i Acuteintermittentporphyria There is general agreement that the three forms of ii Variegate porphyria hepatic porphyria-acute intermittent and variegte iii Hereditarycoproporphyria 2 Symptomaticcutaneoushepaticporphyria(symptomaticporphyria) porphyria and hereditary coproporphyria-which i Those with no familyhistory ofthe diaease are transmitted by separate autosomal dominant (a) associated with alcoholism, liver disease, iron overload, and 1967) are oestrogen therapy genes (Waldenstrom Haeger-Aronsen, (b) due to hexachlorobenzene poisoning distinct disease entities: but there is less agreement ii Those with family history of the disease about the classification of the remaining patients 3 Cutaneous porphyria due to hepatic tumours with hepatic porphyria. The majority of these have Table I Classification ofthe porphyrias certain clinical and biochemical features in common J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1017 which differentiate them from the other forms of for the purely cutaneous 'middle-aged alcoholic' hepatic porphyria and, although aetiologically type of porphyria. In 1957, Waldenstrom proposed diverse, have been classified as a single group: that 'porphyria cutanea tarda' was in fact a mixed symptomatic cutaneous hepatic porphyria (sympto- group of at least two diseases and modified his matic porphyria). Only two patients with acquired classification accordingly. The patients with both porphyria due to overproduction of porphyrins cutaneous lesions and acute attacks were re-classified within an hepatoma have been described and will be as 'porphyria cutanea tarda hereditaria' or, to take discussed separately (Tio, Leijnse, Jarrett, and into account the characteristic faecal abnormality, Rimington, 1957; Thompson, Nicholson, Farnan, 'protocoproporphyria'. This group of patients is Whitmore, and Williams, 1970). now classified under variegate porphyria. The No system for the classification of the hepatic remaining patients in whom the diseaseappearedtobe porphyrias is satisfactory. Some patients cannot be acquired later in life were classified as 'porphyria classified even after extensive investigation (Watson, cutanea tarda symptomatica'. In this category 1960; Pifiol-Aguade, Castells, Indacochea, and Waldenstr6m placed both the European and Rodes, 1969) and a more satisfactory classification American patients with alcoholic liver disease-and will have to await greater understanding of the the occasional non-alcoholic patient-and the Bantu fundamental abnormalities in this group of dis- porphyria of South Africa, a purely cutaneous form orders. of non-familial porphyria associated with liver The use of the term 'porphyria cutanea tarda' in disease, malnutrition, and alcoholism common respect of a specific cutaneous porphyria of slow or amongst the Bantu peoples. Subsequently porphyrin late onset has caused much confusion. Waldenstr6m excretion patterns of variegate porphyria and introduced the term to describe a group of patients 'porphyria cutanea tarda symptomatica' were shown developing a cutaneous form of porphyria after to be quite distinct, largely as the result of extensive puberty (Waldenstrom, 1937), clearly differentiated comparisons in South Africa (Eales, Levey, and from congenital erythropoietic porphyria which was Sweeney, 1966b). Thus the existence of a purely present from birth and from acute intermittent cutaneous form of hepatic porphyria that could be by copyright. porphyria in which cutaneous symptoms do not distinguished both from the erythropoietic porphyrias occur. Although Waldenstrbm recognized that and from variegate porphyria and hereditary copro- attacks of abdominal pain were occasionally a porphyria became established. This condition is feature of 'porphyria cutanea tarda', subjects with referred to as 'symptomatic cutaneous hepatic cutaneous symptoms were never found amongst porphyria' (or 'symptomatic porphyria') (Eales, families with typical acute intermittent porphyria. 1963; Eales and Dowdle, 1968). The term 'porphyria Subsequent reports established that 'porphyria cutanea tarda' without qualification has been used cutanea tarda' was predominantly a disease of men for the same condition, especially in the United over the age of 40, who frequently gave a history of States (Taddeini and Watson, 1968; Tschudy, 1969), http://jcp.bmj.com/ alcoholism and showed evidence of liver dysfunction but should be avoided since it has also been used to and that both attacks of acute porphyria and a family describe variegate porphyria. history of porphyria were uncommon in this group (Szodoray and Sumegi, 1944; Brunsting, Mason, and Aldrich, 1951; Brunsting, 1954). However, there were The Erythropoietic Porphyrias also accounts of a less common cutaneous porphyria

in which both photosensitivity and acute attacks CONGENITAL ERYTHROPOIETIC PORPHYRIA on September 23, 2021 by guest. Protected occurred (Gray, Rimington, and Thomson, 1948; This disease (Gunther's disease) (Gunther, 1911; Watson, 1951 ; MacGregor,Nicholas, and Rimington, 1922) is a very rare condition characterized by onset 1952; Rimington, 1952; Calvert and Rimington, at or soon after birth of variable, but usually severe 1953; Discombe and Treip, 1953; Wells and Riming- and mutilating, skin lesions due to photosensitivity ton, 1953; Holti, Rimington, Tate, and Thomas, and often accompanied by haemolytic anaemia 1958). In general these patients were younger than (Gray and Neuberger, 1950; Goldberg, 1966; the former group and in some there was evidence of Taddeini and Watson, 1968). porphyria in other members of the family although Porphyrins of isomer series I accumulate within there was no difference between the skin lesions in the body often discolouring the bones, teeth, and the two groups. In Europe these patients were also skin and are excreted in large amounts in the urine referred to as cases of 'porphyria cutanea tarda' and faeces. Urine from patients with congenital (Rimington, 1952; 1958a) though in the United erythropoietic porphyria not only contains uropor- States Watson (1954) described them as 'mixed phyrin I and coproporphyrin with eight and four porphyria' and reserved 'porphyria cutanea tarda' carboxyl groups respectively but also small quantities J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

1018 G. H. Elder, C. H. Gray, and D. C. Nicholson of at least one other octacarboxyl porphyrin, perhaps protoporphyrin in the erythrocytes and is distin- uroporphyrin III as well as porphyrins containing guished from Gunther's disease in which the ery- seven, six, and five carboxyl groups (Rimington and throcytes contain predominantly uroporphyrin. Miles, 1951). The faeces contain large amounts of Chapel, Stewart, and Webster (1972) and Cripps coproporphyrin I. and MacEachern (1971) showed that only vari- The underlying enzyme defect which is apparently able percentages of the red cells in erythropoietic restricted to the erythroid cells (Schmid, Schwartz, protoporphyria fluoresced in ultraviolet light, and and Sundberg, 1955; Watson, 1968) is a deficiency ClarkandNicholson(1971)foundtheexcessporphyrin of uroporphyrinogen III cosynthetase relative to to be in the young rather than the old cells. Faecal uroporphyrinogen synthetase. This leads to the protoporphyrin and coproporphyrin are increased, formation of a greater than normal proportion of especially the former. In contrast to the other forms uroporphyrinogen I from porphobilinogen, while of cutaneous porphyria the urinary porphyrins are the overall production of series IHI porphyrinogens usually normal, and, unlike variegate porphyria and is normal or may even be increased (Taddeini and acute intermittent porphyria, there is no excessive Watson, 1968). Uroporphyrinogen I cannot be used urinary excretion of porphyrin precursors. for haem synthesis and is deposited in the tissuesand The source of the excess porphyrins in erythro- converted to uroporphyrin I, partly excreted in the hepatic protoporphyria was originally assumed to urine and partly converted to coproporphyrinogen I be mainly the bone marrow and the condition was and coproporphyrin I for excretion mainly but not called erythropoietic protoporphyria (Rimington entirely in the faeces. The abnormality is inherited et al, 1967). However, the incorporation of 15N as an autosomal recessive gene (Marver and Schmid, glycine (Gray et al, 1964) and of 14C ALA (Nichol- 1972). Abnormalities of porphyrin metabolism son, Cowger, Kalivas, Thompson, and Gray, 1973) (Heilmeyer, Clotten, Kerp, Merker, Parra, and into erythrocytes, plasma and faecal porphyrins, Wetzel, 1963) and a slightly decreased co-synthetase and stercobilin showed a pattern of labelling in red cells and cultured fibroblasts (Romeo, Glen, suggesting disturbances of porphyrin biosynthesis in and Levin, 1970; Romeo, Kaback, and Levin, 1970) both the liver and erythropoietic tissues and that theby copyright. have been described in clinically normal hetero- early peak of labelling of bile pigment was increased, zygotes. probably due to an increased turnover of non- Heilmeyer and Clotten (1964) have described a erythropoietic haems. Moreover, Miyagi (1967) has mild form of cutaneous photosensitivity associated shown increased ALA synthetase activity in both with an increase in erythrocyte coproporphyrin but erythroid and hepatic tissue. Scholnick et al (1971) no further patients with this condition have been concluded that in erythropoietic protoporphyria encountered. some of the excess hepatic protoporphyrin refluxes into the blood contributing by uptake from the

ERYTHROHEPATIC PROTOPORPHYRIA plasma to erythrocyte protoporphyrin. They have http://jcp.bmj.com/ This condition, although an uncommon cause of therefore renamed the condition 'erythrohepatic photosensitivity (Prentice, Goldberg, and Thompson, porphyria'. A different interpretation of labelling 1969), is the commonest form ofcutaneous porphyria experiments was made by Schwartz, Johnson, associated with increased erythrocyte porphyrin. Stevenson, Anderson, Edmondson, and Fusaro The condition is inherited as an autosomal dominant (1971) who consider both faecal and erythrocyte gene (Waldenstrom and Haeger-Aronsen, 1967) and porphyrins to be mainly of erythropoietic origin, is usually associated with mild photosensitivity the latter pool being complex and the secondary on September 23, 2021 by guest. Protected (Rimington et al, 1967) usually beginning in child- source of early labelled bile pigment. hood. Exposure to sunlight is rapidly followed by a The enzyme block in erythrohepatic porphyria tingling or burning sensation, irritation, erythema, responsible for the increased synthesis of ALA urticaria, sometimes associated with oedema but synthetase is not known with certainty, but is rarely with blisters; the lesions usually heal without probably a deficient synthesis from protoporphyrin severe scarring (Rimington et al, 1967). Gallstones of haem required for a specific haem protein im- are a complication (Cripps and Scheuer, 1965). The portant in the feedback mechanism. This may affect condition was regarded as benign but in recent years both the liver and the bone marrow. The excess a number of patients have been reported who have protoporphyrin in the liver is then partially excreted died of liver failure (Barnes, Hurworth, and Miller, in the bile and faeces and partly transferred via the 1968; Donaldson, McCall, Magnus, Simpson, blood plasma to the circulating red cells which may Caldwell, and Hargreaves, 1971). take up protoporphyrin passively, thus augmenting Erythrohepatic protoporphyria is characterized the protoporphyrin accumulating because of the biochemically by the presence of excessive free defect in the erythropoietic tissue. J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1019 The Hepatic Porphyrias (Nielsen and Thorn, 1965). The hyponatraemia is usually associated with vomiting and inadequate HEPATIC PORPHYRIAS OF AUTOSOMAL DOM- salt and water intake, but renal factors (Eales et al, INANT INHERITANCE 1971) and inappropriate secretion of antidiuretic hormone, perhaps due to a hypothalamic lesion The acuteporphyric attack (Hellman, Tschudy, and Bartter, 1962; Nielsen and Acute intermittent porphyria, variegate porphyria, Thorn, 1965; Perlroth, Tschudy, Marver, Berard, and hereditary coproporphyria have one important Ziegel, Rechcigl, and Collins, 1966), may also be clinical feature in common: patients with any one important in some patients. Additional evidence for of these disorders may develop clinically identical involvement of the hypothalamus comes from attacks of acute porphyria. Main clinical features reports of failure of growth hormone (Perlroth, which may occur separately or in combination are Tschudy, Waxman, and Odell, 1967) and ACTH severe colicky abdominal pain, vomiting and con- release in response to appropriate stimuli (Waxman, stipation, a neuropathy which is mainly motor in Berk, Schalch, and Tschudy, 1969). type but often associated with severe pain in the Biochemically the attacks are characterized by extremities, and psychiatric disturbances; respiratory increased excretion of the porphyrin precursor, PBG, paralysis is not uncommon. All these features and to a lesser extent ALA. They do not occur in probably have a neurological basis (Goldberg, 1959; those types of porphyria in which the excretion of Ridley, 1969). Attacks are frequently precipitated PBG is always within normal limits. by drugs (Table II), especially barbiturates, but may The three hereditary hepatic porphyrias can be disinguished on the basis of their clinical and biochemical features. Antipyrine Isopropyl dipyrone Barbiturates Methyldopa Chlordiazepoxide Meprobamate Acute intermittenttporphyria Chlorpropamide Oestrogens (natural and synthetic) Diazepam Patients with this condition suffer only variable and Dichioral phenazone Pentazocine (Fortral) periodic acute attacks of the type described above, by copyright. Ergot preparations Progestogens there being an associated overproduction of por- Glutethemide Sedormid Griseofulvin Succinimides phobilinogen (Goldberg, 1959; Stein and Tschudy, Hydantoins Sulpha drugs 1970). Skin lesions due to photosensitivity do not occur. In some the disease may be clinically latent Table If Some of the mnore important drugs known or reported to precipitate attacks of acute intermittent throughout life, while in others the frequency and porphyria severity of attacks varies widely. The conventional tests of liver function are usually normal in acute intermittent porphyria, except for be provoked by fasting, alcohol, infections, or occur retention of bromsulphthalein (BSP) (Stein and http://jcp.bmj.com/ spontaneously (Eales, 1971). They occur most Tschudy, 1970) due to decreased excretion of BSP commonly between the second and fourth decades by the liver cell (Stein, Bloomer, Berk, Corcoran, and and are commoner in women. In both sexes they are Tschudy, 1970). The presence of this defect as well as rare before puberty; in some women their onset may an increase in serum protein-bound iodine (PBI) and, be related to a particular phase of the menstrual in females, thyroxine-binding globulin (Hollander, cycle. Acute attacks during pregnancy are un- Scott, Tschudy, Perlroth, Waxman, and Sterling,

common but there is an increased incidence during 1967) without hyperthyroidism has led to the on September 23, 2021 by guest. Protected the postpartum period (Stein and Tschudy, 1970). suggestion that an 'oestrogen effect' operates in this The severity of the attacks is variable and slow disease (Stein et al, 1970). recovery from the neuropathy may entail prolonged Other metabolic abnormalities that have been morbidity (S0rensen and With, 1971). The overall described include raised plasma iron, hypercholes- mortality in variegate porphyria has recently been terolaemia (Stein and Tschudy, 1970) with hyper-p- estimated as about 20% (Eales, 1971), while in a lipoproteinaemia (Lees, Song, Levere, and Kappas, recent series of 46 patients with acute intermittent 1970), and abnormalities of glucose metabolism porphyria (Stein and Tschudy, 1970) two males and (Stein and Tschudy, 1970). two females died. The characteristic biochemical abnormality of Disturbances of salt and water metabolism are both the manifest and the latent disease is a per- common during the acute attack (Eales, Dowdle, and sistently increased excretion of PBG (Waldenstr6m, Sweeney, 1971). Hyponatraemia, often accompanied 1937; Watson and Schwartz, 1941) and ALA in the by hypovolaemia, is the commonest disturbance, urine, the level of PBG exceeding that of ALA but hypokalaemia and hypomagnesaemia may occur (Haeger-Aronsen, 1958; Waldenstrom and Haeger- J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

1020 G. H. Elder, C. H. Gray, and D. C. Nicholson Aronsen, 1963). Although the amount of PBG 1958) and from the United States (Watson, 1960). excreted may reach a peak during acute attacks the As might be expected from the clinical features, increase persists during remission in the majority of overproduction and excretion of both porphyrin patients (Taddeini and Watson, 1968) but the level precursors and porphyrins are found in variegate varies widely from patient to patient and from day porphyria. The most characteristic biochemical to day and does not correlate with the likelihood or abnormality is a marked increase in the concentra- severity of an acute attack (Ackner, Cooper, Gray, tion of porphyrin in the faeces with the levels of Kelly, and Nicholson, 1961). It is probable that the protoporphyrin exceeding those of coproporphyrin excretion of PBG is only rarely within normal limits (Waldenstrom, 1957; Barnes, 1958; Holti et al, 1958; in subjects carrying the gene for acute intermittent Dean and Barnes, 1959; Eales, Dowdle, Saunders, porphyria, for Wetterberg (1967) found that the and Sweeney, 1963; Eales et al, 1966b). In the urine of 55% of 197 siblings of patients contained majority of South African variegate porphyrics, the abnormally increased amounts of PBG, which is faecal porphyrin concentration, although greatly close to the theoretical gene distribution of 50%. increased in all phases of the disease including However, normal excretion of precursors has been latency (total ether-soluble porphyrin > 500 ug/g reported in a few patients during the latent phase of dry wt in 92% of patients, Eales et al, 1966b), is acute intermittent porphyria (With, 1961; 1963) and particularly high during acute attacks (Eales, such subjects have been shown to carry the gene by Dowdle, Levey, and Sweeney, 1966a). The concen- loading tests with ALA and appropriate enzyme tration of ether-insoluble porphyrin in the faeces is estimations (Meyer, Strand, Doss, Reese, and also increased (Watson, 1960; Sweeney, 1963; Marver, 1972). Rimington et al, 1968; Eales, Grosser, and Sano, Although the condition is essentially one of over- 1971). Rimington et al (1968) found that much of production of porphyrin precursors rather than of this fraction consists of porphyrin-peptide con- porphyrins, there are probably small increases in jugates ('X porphyrins') and suggested that the porphyrin excretion. Interpretation of urinary excretion of increased amounts of these conjugates porphyrin analyses is difficult owing to the large in the faeces, and in the urine during acute attacks,by copyright. amounts of uroporphyrin formed in the urine by is a prominent biochemical feature of variegate non-enzymic polymerization of PBG, but there may porphyria. In the past this hydrophilic material may be some increase in coproporphyrin III excretion at have been described mistakenly as uroporphyrin. least during acute attacks (Waldenstrom and Haeger- The porphyrins of bile are similarly abnormal Aronsen, 1963). Faecal protoporphyrin and copro- (Smith, Belcher, Mahler, and Yudkin, 1968; porphyrin concentrations are often normal but may Belcher, Smith, and Mahler, 1969). be slightly increased in some patients (Waldenstrom During the acute attack the concentration of PBG and Haeger-Aronsen, 1963; Wetterberg, Haeger- in the urine is increased, often to levels similar to

Aronsen, and Stathers, 1968). More frequently there those of acute intermittent porphyria, and there is an http://jcp.bmj.com/ are small increases in the ether-insoluble faecal increase in urinary coproporphyrin III excretion. porphyrin fraction which may contain both uro- However, in contrast to acute intermittent porphyria, porphyrin (Watson, 1960; Taddeini and Watson, during remission the amount of PBG excreted in the 1968; Rimington et al, 1968) and small amounts of urine falls rapidly over a few weeks, usually returning porphyrin-peptide conjugates (Rimington et al, to normal within a few weeks (Eales et al, 1966a). 1968; Moore, Thompson, and Goldberg, 1972). Urinary coproporphyrin III concentration is variable remain increased remission but may during (Eales on September 23, 2021 by guest. Protected Variegateporphyria et al, 1963). The level of coproporphyrin always In this form of porphyria, which is particularly exceeds that of uroporphyrin except occasionally common in the white population of South Africa, during the acute attack when a large amount of both acute attacks and cutaneous lesions occur uroporphyrin is formed by non-enzymic polymer- though not necessarily in the same patient at the ization of porphobilinogen. same time. In a series of 133 South African patients It is possible that each form of dominantly 50% presented with an acute attack, accompanied inherited hepatic porphyria although clinically and by cutaneous lesions, 34% had cutaneous lesions biochemically distinct is genetically heterogeneous. alone, while in 15 % an acute attack occurred alone Thus With (1969) has emphasized small differences (Eales, 1963). The incidence of variegate porphyria that occur between the same disease in different in the white population of South Africa has been families and has suggested that the condition seen estimated as 3 per 1000 (Dean, 1963). Elsewhere the in each family is the expression of a genotype disease is much rarer but similar patients have been peculiar to that family. In this respect it is interesting described in Europe (Waldenstrom, 1957; Holti et al, to note the remarkable uniformity of the South J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1021 African patients with variegate porphyria when Hunter, and Rechcigl reported marked increase in compared with those from other parts of the world, the activity of ALA-S, the rate-limiting enzyme of since all the South African patients are believed to hepatic protohaem synthesis, in the liver of a patient be descended from one of the original free burghers who had died from acute intermittent porphyria. who married at Cape Town in 1688 (Dean, 1963). This finding, since confirmed in other patients using Some points of difference reported in families from liver tissue obtained by open surgical or needle outside South Africa, not all of which are likely to biopsy (Nakao, Wada, Kitamura, Uono, and Urata, be due to environmental factors, are the larger 1966; Dowdle, Mustard, and Eales, 1967; Masuya, number of latent porphyrics, the lower incidence of 1969; Strand et al, 1970), provided the first direct cutaneous manifestations, and the greater variability evidence in support of the suggestion of Watson, of faecal porphyrin levels (Watson, 1960; Hamn- Runge, Taddeini, Bossenmaier, and Cardinal (1964) strom, Haeger-Aronsen, Waldenstr6m, Hysing, and that acute intermittent porphyria is an 'overpro- Molander, 1967; Cochrane and Goldberg, 1968; duction' disease in which excessive amounts of Rimington et al, 1968). In particular Rimington and porphyrin precursors are produced due to an in- his coworkers have reported a number of British herited defect in the regulation of ALA-S activity. patients in whom impaired hepato-biliary function An increase in the activity of this enzyme in the liver during the acute attack led to diversion of porphy- has since been found in variegate porphyria (Dowdle rins from the bile to the urine (Gray et al, 1948; Mac- et al, 1967; Strand et al, 1970) and in hereditary Gregor et al, 1952; Wells and Rimington, 1953). coproporphyria (Kaufman and Marver, 1970; Such reciprocity ofporphyrin excretion is apparently McIntyre, Pearson, Allan, Craske, West, Moore, not a feature of the disease in South Africa (Eales, Paxton, Beattie, and Goldberg, 1971.) 1963) or the United States (Watson, 1960) except An increase in ALA-S activity in the liver in these when complicated by intercurrent cholestatic conditions could be either a primary effect of the jaundice (Eales et al, 1966b). The existence of these genetic lesion or could be secondary to interruption

differences suggests that the biochemical diagnostic ofthe feedback control of ALA-S activity by a partial by copyright. criteria established by study of the plentiful South block in haem synthesis, by increased catabolism of African patients may not always be applicable haem to bilirubin or other catabolites (Landaw, elsewhere. Callahan, and Schmid, 1970), or by increased in- corporation into haemoproteins. Hereditary coproporphyria In recent years, evidence has accumulated that, This is probably the least common of the genetic at least in acute intermittent porphyria, the increase hepatic porphyrias, although accurate assessment in ALA-S activity is not the primary defect in haem of its incidence is difficult since it is frequently biosynthesis in these conditions. Thus if over- asymptomatic (Watson, Schwartz, Schulze,Jacobsen, production of ALA in the liver were the only and Zagaria, 1949; Berger and Goldberg, 1955; lesion in haem biosynthesis one would expect the http://jcp.bmj.com/ Goldberg, Rimington, and Lochhead, 1967; Haeger- pattern of porphyrin excretion to be the same in Aronsen, Stathers, and Swahn, 1968; Lomholt and each disease and to resemble that produced by With, 1969). administering ALA to a normal person (Berlin, The condition is characterized by the excretion of Neuberger, and Scott, 1956) whereas in fact the differ- large amounts of coproporphyrin III, mainly in the ent forms of porphyria are characterized by specific faeces. Porphobilinogen and ALA excretion is inherited patterns of porphyrin excretion (Dowdle normal except during an acute attack, which is the et al, 1968). Since it is unlikely that two separate on September 23, 2021 by guest. Protected most frequent clinical feature. Photosensitivity is inherited lesions are present in each type ofporphyria uncommon and in four patients, in which it has been -one leading to an increase in the activity of reported has been accompanied by hepatic in- ALA-S and one determining the consequent ex- sufficiency (Goldberg et al, 1967; Connon and cretion pattern-Kaufman and Marver (1970) have Turkington, 1968; Hunter, Khan, Hope, Beattie, proposed that the primary inherited defect is a par- Beveridge, Smith, and Goldberg, 1971). tial block in the biosynthesis of haem at a different, characteristic step for each disease. The increase in THE NATURE OF THE METABOLIC ABNORMALITY ALA-S activity would then come about through the IN THE INHERITED HEPATIC PORPHYRIAS feedback control mechanism operating to increase Current theories of the pathogenesis of the hepatic the synthesis of intermediates to maintain normal porphyrias depend on the concept that increased levels of hepatic haem in the face of such a block. production of ALA, the first committed precursor Strand et al (1970) and Miyagi et al (1971) have of protohaem, in the liver is an underlying abnor- recently reported thattheactivityofuroporphyrinogen mality. In 1965, Tschudy, Perlroth, Marver, Collins, synthetase in the liver is decreased in patients with J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

1022 G. H. Elder, C. H. Gray, and D. C. Nicholson acute intermittent porphyria, an observation which A number of clinical observations suggest that neatly explains the observed excretion of large steroid hormones are important in the pathogenesis amounts of PBG and ALA, but not porphyrins, in of acute attacks in the inherited hepatic porphyrias. this condition, and Meyer et al (1972) have shown Thus acute attacks are commoner in females, are that the activity of erythrocyte uroporphyrinogen rare before puberty, and in some patients their onset synthetase and the ability to convert an exogenous may be related to a particular phase of the menstrual load of ALA to porphyrins are both decreased in cycle. The administration of oestrogens and/or patients carrying the gene for acute intermittent progestogens increases the excretion of PBG and porphyria whether or not it is clinically manifest. ALA (Welland et al, 1964) and may provoke acute Strand, Manning, and Marver (1971) have predicted attacks in some patients with acute intermittent that enzyme defects at different sites may similarly porphyria (Welland et al, 1964; Wetterburg, 1964) underlie hereditary coproporphyria and variegate and variegate porphyria (McKenzie and Acharya, porphyria. 1972). On the other hand inhibition of ovulation by Indirect evidence from measurement of bilirubin oral contraceptives may prevent acute attacks in production rates suggests that hepatic haem turn- those patients who have cyclical attacks related to over is normal in acute intermittent porphyria the menstrual cycle (Perlroth et al, 1966). Although (Dowdle et al, 1968; Bloomer, Berk, Bonkowsky, oestrogens are weak inducers of ALA-S in chick Stein, Berlin, and Tschudy, 1971; Jones, Bloomer, embryo liver cell cultures and in whole animals and Berlin, 1971) and in variegate porphyria (Dowdle (Granick and Kappas, 1967; Tschudy, Waxman, et al, 1968) although it is probable that the methods and Collins, 1967), the ALA-S in the chick embryo used were insufficiently sensitive to detect small liver is induced by oral contraceptives due to the alterations in bilirubin production rates. progestational component (Rifkind, Gillette, Song, There is some evidence that the activity of ALA-S and Kappas, 1970). is further increased during acute attacks. The There is also some evidence that the metabolism in tends to be of endogenous steroids may be abnormal in some excretion of PBG and ALA the urine by copyright. higher during acute attacks in all three forms of patients with acute intermittent porphyria. Thus porphyria (Taddeini and Watson, 1968). Goldberg, Moore, Beattie, Hall, McCallum, and Many of the drugs that provoke acute attacks Grant (1969) have shown that the excretion of (Table II) induce hepatic ALA-S and are metabolized various 17-oxosteroids may be increased and that in the liver by haem-containing microsomal enzyme one of these, dehydroepiandrosterone, and its systems (Granick, 1966; Marver and Schmid, 1968). sulphate conjugate produce a significant elevation The induction of ALA-S is normally short-lived and of hepatic ALA-S when injected intraperitoneally appropriate to the provision of the extra haemo- into rats. Gillette, Bradlow, Gallagher, and Kappas protein particularly cytochrome P450 required for (1970) have shown that in some patients metabolism metabolism of the inducer, presumably since any of exogenously administered androgens is diverted http://jcp.bmj.com/ further increase in haem levels represses further from the 5a-H pathway towards the 5/3-H pathway, synthesis of the enzyme. It may be that in patients thus producing metabolites which are known to be with inherited hepatic porphyria, regulation of potent inducers of ALA-S (Granick and Kappas, ALA-S induction fails due to an inability to increase 1967). haem levels sufficiently in the presence of a partial If the fundamental inherited defects in these block in haem synthesis; thus they 'respond to the conditions lie in the pathway of haem biosynthesis administration of such drugs with a sustained the abnormality of haem metabolism must be on September 23, 2021 by guest. Protected increase in ALA-S activity and hence massive directly responsible for the neurological disturbance overproduction of the intermediates preceding the characterized morphologically by axonal degenera- block in the pathway. tion (Ridley, 1969) that underlies the clinical features Carbohydrate loading and starvation are known of the acute attack. In the past the demonstration to influence the excretion of PBG and ALA and the that ALA and PBG are pharmacologically inert activity of hepatic ALA-S in animals with experi- (Goldberg, Paton and Thompson, 1954) has led to mental porphyria (Rose, Hellman, and Tschudy, suggestions that there is a deficiency of a substance 1961), and also affect the amounts of these precursors required to protect nerve function, or of pyridoxine excreted by patients with porphyria. Thus increased (Ridley, 1969), or that the abnormality of porphyrin dietary carbohydrate decreases the excretion of precursor metabolism is merely a spectacular side PBG and ALA in acute intermittent and variegate effect of a more fundamental metabolic disturbance porphyria (Welland, Hellman, Gaddis, Collins, (Neuberger, 1968) possibly involving oxidation of Hunter, and Tschudy, 1964). Conversely starvation NADH (Tschudy and Bonkowski, 1972). Recently may provoke acute attacks. Becker, Viljoen, and Kramer (1971) have shown J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1023 that ALA inhibits brain tissue ATPase and have lesions include erosions, crusts, and scabs which suggested that in the acute phase of porphyria ALA finally heal with scar formation. Sclerodermatous is taken up by nerve tissue and causes paralysis of changes may occur and milia are frequent. Both conduction by inhibition of the Na+-K+-dependent hirsutism and pigmentation, particularly of the face, ATPase, while Feldman, Levere, Lieberman, Car- are common and may be the only clinical features. dinal, and Watson (1971) have reported that PBG There are no diagnostic differences between the skin in concentrations similar to those found in the lesions of symptomatic porphyria and the other cerebrospinal fluid during acute attacks, and one of hepatic porphyrias accompanied by cutaneous its non-enzymically produced condensation products symptoms. porphobilin, produce presynaptic neuromuscular Acute attacks of porphyria with abdominal pain inhibition. Although the concentration of PBG in and neurological complications do not occur and in the cerebrospinal fluid is about one quarter that in this respect the reaction to drugs, suchasbarbiturates, plasma (Bonkowsky, Tschudy, Collins, Doherty, is normal (Taddeini and Watson, 1968). Bossenmaier, Cardinal, and Watson, 1971), there The incidence of diabetes mellitus is increased in is some doubt as to whether ALA crosses the symptomatic porphyria (Brunsting, 1954; Eales and blood-brain barrier in more than trace amounts. Dowdle, 1968) and an association with syphilis Nothing is known of the consequences of disordered (Berman and Bielicky, 1956; Gheorghui and Forsea, haem metabolism within nervous tissue. 1968) and connective tissue disorders (Hetherington, Jetton, and Knox, 1970; Rimington, Sears, and SYMPTOMATIC CUTANEOUS HEPATIC P OR- Eales, 1972) has been noted. PHYRIA (SYMPTOMATIC PORPHYRIA) Biochemically the condition is characterized by a Almost all the patients with hepatic porphyria who marked increase in the urinary excretion of uro- do not have one of the three forms of hepatic porphyrin usually to between 1 0 and 10-0 mg/day, porphyria described above have a purely cutaneous with a smaller increase in the coproporphyrin form of porphyria-symptomatic porphyria-in fraction, whereas the concentrations of PBG and, which skin lesions indistinguishable from those of usually, ALA in the urine are normal (Eales et al, by copyright. variegate porphyria (Eales, 1963; Magnus, 1972) 1966b; Taddeini and Watson, 1968). Numerous are accompanied by a characteristic abnormality of detailed analyses of the urinary porphyrins, em- porphyrin metabolism. This is the commonest form ploying chromatographic separation of individual of cutaneous porphyria encountered in the United porphyrins, have confirmed that uroporphyrin is the Kingdom. main porphyrin excreted in the urine, but large With rare exceptions there is no evidence of quantities of heptacarboxylic porphyrin with smaller inheritance, and this form of porphyria has been but increased amounts of porphyrins with six, five regarded as acquired (Waldenstrom, 1957; Goldberg and four carboxyl groups are also-present (Sweeney, and Rimington, 1962), although possibly only by 1963; Nacht, San Martin de Viale, and Grinstein, http://jcp.bmj.com/ genetically predisposed individuals (Waldenstrom 1970; Dowdle et al, 1970; Doss, Meinhof, Look, and Haeger-Aronsen, 1967; Taddeini and Watson, Henning, Nawrocki,D6lle, Strohmeyer, andFilippini, 1968). The condition has been reported in association 1971b). with liver disease (particularly when due to alcohol), Alterations in faecal porphyrin concentration are with oestrogen therapy, and with an outbreak of less striking, although probably as much porphyrin hexachlorobenzene poisoning in Turkey (Cam and is excreted by this route as in the urine (Sweeney,

Nigogosyan, 1963). 1963; Herbert, 1966). The faeces usually contain on September 23, 2021 by guest. Protected In Europe and N. America the majority of increased amounts of both ether-soluble and ether- patients are men between the ages of 40 and 60 insoluble porphyrins (Eales et al, 1966b; Taddeini (Ippen, 1959) but the age and sex incidence depends and Watson, 1968; Eales et al, 1971; Moore, Thomp- on the underlying aetiology. The onset of the con- son, and Goldberg, 1972). The total ether-soluble dition is insidious and spontaneous remission may porphyrins are not increased to the same extent as in occur. The skin lesions, which are the most striking variegate porphyria and may occasionally be within clinical feature of symptomatic porphyria, occur normal limits. Most of the increase is due to an mainly in areas of the skin exposed to sunlight, particularly the face and the backs of the hands and increase in the coproporphyrin fraction which forearms. Increased fragility of the skin in response frequently exceeds the level of protoporphyrin. to trivial mechanical or thermal trauma is usually However, much of the 'coproporphyrin fraction' is more prominent than photosensitivity. The acute not coproporphyrin but a mixture of tetracarboxylic skin lesions are erythema, vesicles and bullae, which porphyrins-isocoproporphyrin, de-ethylisocopro- may be haemorrhagic or become infected. Later porphyrin, and hydroxyisocoproporphyrin (respec- J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

1024 G. H. Elder, C. H. Gray, and D. C. Nicholson tively I, II, and 111, Fig. 2) of which the first two are FACTORS ASSOCIATED WITH SYMPTOMATIC probably formed from dehydroisocoproporphyrin CUTANEOUS HEPATIC PORPHYRIA (IV, Fig. 2), which is a prominent component of the bile in symptomatic porphyria (Elder, 1971, 1972, Liver disease and unpublished observations), by intestinal micro- Liver damage frequently occurs in symptomatic organisms. Small amounts of penta-, hexa-, and porphyria even in the absence of known causative heptacarboxylate porphyrins are also usually present factors such as alcoholism or hexachlorobenzene in this fraction. poisoning (Taddeini and Watson, 1968). Liver The concentration of ether-insoluble porphyrin function tests, particularly the bromsulphthalein in the faeces is frequently increased (Watson, 1960 retention time, show moderate, not usually severe, Sweeney, 1963; Herbert, 1966; Eales et al, 1971; impairment of liver function (Waldenstrom and Elder, 1971; Magnus and Wood, 1971; Moore et al, Haeger-Aronsen, 1960). Hepatomegaly is common, 1972). When this fraction is estimated by methods the most frequent histological findings, apart from using urea-Triton X100-extraction (see below) siderosis, being fatty change, periportal fibrosis (Rimington et al, 1968) the quantities of porphyrin sometimes with round cell infiltration, and cirrhosis, found may be as great as in variegate porphyria especially in those patients with a long clinical (Eales et al, 1971; Magnus and Wood, 1971; history (Uys and Eales, 1963; Lundvall and Weinfeld, Moore et al, 1972). Examination of the composition 1968). The electron microscopic findings have been of the porphyrin fraction extracted by urea-Triton described by Jean, Lambertenghi, and Ranzi has shown that heptacarboxylic porphyrin and uro- (1968) and by Timme (1971). porphyrin (Elder, 1971; Magnus and Wood, 1971) Alcohol rather than porphyrin-peptide conjugates (Moore Alcoholism is a frequent but not invariable factor et al, 1972) are the main ether-insoluble porphyrins in the development of liver disease in this condition. excreted in this condition confirming earlier studies Withdrawal of alcohol may lead to clinical and in which other methods of extraction were biochemical improvement especially if there is im-by copyright. used (Watson, 1960); Sweeney, 1963; Herbert, proved nutrition. Nevertheless symptomatic por- 1966). phyria is uncommon in alcoholics suggesting that The isomer type of the porphyrins excreted in alcohol may unmask an inherited predisposition to symptomatic porphyria is variable. Uroporphyrin this condition (Waldenstr6m and Haeger-Aronsen, is about 70% type I, coproporphyrin and penta- 1967; Taddeini and Watson, 1968; Benard, Gajdos, carboxylic porphyrin are about 50% type I, while and Gajdos-Tbrok, 1958). hepta- and hexacarboxylic porphyrins are mainly type III (Nacht et al, 1970; Dowdle et al, Iron metabolism 1970). Hepatic siderosis (increased stainable haemosiderin In addition to excreting excess uroporphyrin and iron) is very common but not invariable in sympto- http://jcp.bmj.com/ heptacarboxylic porphyrin, patients with sympto- matic porphyria (Turnbull, 1971). Nevertheless matic porphyria accumulate large amounts of these severe iron overload is uncommon and the condition porphyrins within the liver so that liver biopsy is only rarely associated with haemochromatosis samples viewed directly in ultraviolet light show an (Sauer, Funk, and Finch, 1966). Some hepatic intense red fluorescence. Increased amounts of siderosis with liver cell damage and cirrhosis is porphyrin within the liver cell may persist after common among heavy drinkers of beers and wines otherwise complete clinical and biochemical re- with high iron content, eg, home-brewed Bantu on September 23, 2021 by guest. Protected mission (Lundvall and Enerback, 1969) and might beers (Saunders, Williams, and Levey, 1963) and the precede the onset of symptoms (Doss, Look, and red wine of Brescia (Perman, 1967). Symptomatic Henning, 1971a). porphyria is unusually common amongst drinkers The large amount of porphyrin stored in the liver of these beverages but in the Bantu malnutrition underlies the unique response of patients with this may also be an important factor. Increased iron condition to chloroquine administration, which absorption and mild to moderate hepatic siderosis produces a transient febrile reaction accompanied are not infrequent inpatientswith cirrhosis (Williams, by massive uroporphyrinuria and biochemical Williams, Scheuer, Pitcher, Loiseau, and Sherlock, evidence of liver cell damage (Sweeney, Saunders, 1967) and may also be due to enhancement of iron Dowdle, and Eales, 1965; Felsher and Redeker, absorption by alcohol itself. Although the cause and 1966). Chloroquine forms a complex with porphyrins role of the increased iron stores in some patients and an abnormally high intracellular concentration with symptomatic porphyria is not clear, depletion may account for its hepatotoxic action in these of storage iron by repeated venesection (Ippen, 1961; patients (Scholnick and Marver, 1968). Epstein and Redeker, 1968; Lundvall and Weinfeld, J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1025 1968) or by long-term desferrioxamine treatment cularly when more specific assays are used (Kaufman (Wohler, 1964) produces a clinical remission in the and Marver, 1970; Strand et al, 1970). The activity majority of patients which is reversed by replenish- of the enzyme apparently depends on the stage of ment of iron stores. activity of the condition, for Shanley et al (1969) showed that it is increased by alcohol ingestion and Oestrogens Moore et al (1972) have reported that remission There is a significantly increased incidence of induced by venesection is accompanied by a decrease symptomatic porphyria in patients taking oestrogens, in activity. eg, men with prostatic carcinoma or women treated In this condition, as in the inherited hepatic for carcinoma of the breast or postmenopausal porphyrias, the increase in ALA production is symptoms (Warin, 1963; Copeman, Cripps, and possibly secondary to a partial block in haem Summerly, 1966; Felsher and Redeker, 1966; synthesis which determines the characteristic por- Zimmerman, McMillin, and Watson, 1966; Vail, phyrin excretion pattern. Although the nature of this 1967). The condition (symptomatic porphyria) is fundamental disturbance is unknown, the similarity rare in women taking oral contraceptives. The rarity between the porphyrin excretion patterns of most of symptomatic porphyria as a complication of patients with this condition suggest that it is common oestrogen therapy, the absence of abnormalities of to all aetiological groups. At present it is not clear porphyrin excretion in most patients taking similar why acute attacks and porphobilinogenuria do not amounts of oestrogens (Theologides, Kennedy, and occur. Watson, 1964; Roenigk and Gottlob, 1970), and the One factor that the majority, if not all, these lack ofevidence of a dose-related effect suggests that, patients have in common is disturbed liver function as with alcohol, some underlying predisposition is and it is possible that ultrastructural damage within present. the hepatocyte leads to disruption of the normal rigid compartmentalization of haem synthesis. As a Heredity consequence, oxidation of porphyrinogens to Most patients with symptomatic porphyria have no porphyrins may be enhanced (Heikel, Lockwood, by copyright. family history of porphyria and porphyrin excretion and Rimington, 1958; Rimington, 1963) or alter- is usually normal in other members of their families native metabolic pathways that are quantitatively (Hickman, Saunders, and Eales, 1967; Waldenstrom insignificant under normal conditions may become and Haeger-Aronsen, 1967; Taddeini and Watson, activated (Dowdle et al, 1970; Elder, 1972). The fact 1968) although a few patients have been described that symptomatic porphyria is a rare complication with the clinical and biochemical syndrome and of several common forms of liver disease has led to evidence oflatent or manifest symptomatic porphyria the suggestion that some patients with this condition in their families (Waldenstrom and Haeger-Aronsen, have an inherited predisposition to develop the

1967; Taddeini and Watson, 1968). There is a high disease in response to liver injury (Waldenstrom and http://jcp.bmj.com/ incidence of alcoholism and evidence of liver dys- Haeger-Aronsen, 1967; Taddeini and Watson, 1968), function in these patients and it is likely that liver while in others, notably those poisoned with hexa- damage may unmask porphyria in these families. chlorobenzene, the disease may be truly acquired. Although iron is probably not a primary aetiolo- NATURE OF THE METABOLIC ABNORMALITY gical agent (Kalivas, Pathak, and Fitzpatrick, 1969; There is some evidence that, as in the inherited Shanley, Zail, and Joubert, 1970) it appears to

hepatic porphyrias, there is endogenous overpro- enhance the excessive excretion of porphyrin both on September 23, 2021 by guest. Protected duction of ALA by the liver in symptomatic in symptomatic porphyria and in hexachlorobenzene porphyria. Kaufman and Marver (1970) have poisoning in rats (Taljaard, Shanley, and Joubert, pointed out that if as seems probable there is no 1971) without affecting the pattern of porphyrins increase in haem synthesis in this condition (Dowdle excreted. et al, 1968) the excessive amounts of porphyrins excreted do not require a great increase in ALA PORPHYRIN-PRODUCING HEPATIC TUMOURS production and that the necessary increase in ALA-S Two patients with a purely cutaneous form of por- activity may be difficult to detect. An increase in phyria due to overproduction of porphyrin by a hepatic ALA-S activity has been detected in many tumour surrounded by normal liver tissue have been patients with this condition (Dowdle et al, 1967; described. In one the tumour was a benign hepatic Zail and Joubert, 1968; Shanley, Zail, and Joubert, adenoma and the porphyria disappeared after its 1969; Moore, Turnbull, Bernardo, Beattie, Magnus, removal (Tio et al, 1957); the other was due to a and Goldberg, 1972) but in others there is no increase malignant primary hepatoma in an otherwise normal (Zail and Joubert, 1968; Shanley et al, 1969) parti- liver (Thompson et al, 1970). The porphyrin ex- J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

l1026 G. H. Elder, C. H. Gray, and D. C. Nicholsont

cretion pattern in both these patients differed from NORMAL URINARY, FAECAL, AND ERYTHROCYTE that usually found in symptomatic porphyria and PORPHYRINS they probably constitute a separate form of acquired The normal values are summarized in Table IV; all cutaneous hepatic porphyria that must be con- values refer to total porphyrin, ie, including that sidered in the differential diagnosis of symptomatic formed from porphyrinogen by oxidation during porphyria. Such a distinction is tentative and as analysis. Values for urine are presented in ,ug/day, more patients are described some may be found in- for faeces in ,tg/g of dry weight of faeces, and distinguishable from symptomatic porphyria. Indeed erythrocyte porphyrins in jug/100 ml of packed red it is mandatory that all patients with symptomatic cells: plasma normally contains no measurable porphyria should be carefully examined for the porphyrin. presence of an hepatic tumour. Urine Laboratory Investigation of the Porphyrias Urine contains small amounts of PBG, ALA, and

Table III shows the abnormalities which may be found in the various forms of porphyria and makes Porphyrin Normal Value clear the importance of examining urine, faeces, and Urine erythrocytes if a correct diagnosis is to be established. Porphobilinogen <1-0 mg/day I Mauzerall and Aminolaevulinicacid <2 5 mg/day fGranick (1956) Coproporphyrin 0-160 Ag/day PRESERVATION OF SAMPLES Uroporphyrin 5-30 Mg/day Porphobilinogen is unstable in urine particularly Faeces (4g/g dry stool) under acidic conditions; if specimens cannot be Coproporphyrin 0-20 estimated immediately after collection, thepH should Protoporphyrin 0-76 be adjusted to neutrality and the sample stored at Ether-insoluble porphyrin 0-21' - 200 for up to one month (Bossenmaier and Erythrocytes (jAg/100 mlpacked cells) are Coproporphyrin 0-4 by copyright. Cardinal, 1968). Urinary porphyrins also stable Protoporphyrin 4-52 under these conditions. If faecal porphyrins cannot be estimated within a few hours of collection, the Table IV Normal values for urinary, faecal, and faeces may be preserved for at least a month at erythrocyte porphyrins1 -20°. Blood for porphyrin analysis should not be 'Values quoted by Rimington (1971) except where otherwise stated. collected unless can be carried out im- 2Reported Values for ether-insoluble porphyrins are variable. The analysis ,normal' range shown here is derived from a study of only 13 subjects mediately. (Elder, unpublished observations). http://jcp.bmj.com/ Blood Urine Faeces Isomtter Type (I or 111) Erythtropoietic Congenital erythropoietic Erythrocyte porphyrin PBG and ALA normal Coproporphyrin increased. I (Gunther's disease) increased (mainly Uroporphyrin much increased Some increase in protopor- uroporphyrin) Coproporphyrin increased phyrin and uroporphyrin. Erythrohepatic protoporphyria Erythrocyte porphyrin Normal usually Protoporphyrin increased. III increased (mainly Coproporphyrin sometimes protoporphyrin) increased Hepatic on September 23, 2021 by guest. Protected Acute intermittent porphyria Normal PBG and ALA increased Normal Uroporphyrin may be present from non-enzymatic condensation ofPBG. Hereditary coproporphyria Variable. PBG and ALA may Increase in coproporphyrin III be increased. Coproporphyrin increased. Symptomatic porphyria Normal PBG and ALA normal. Large Normal to moderate increase Complex (see increase in porphyrins of 'copro' and protopor- text) (mainly uroporphyrin). phyrin with'copro' > proto. Often increased ether- insoluble porphyrin (see text). Variegate porphyria Normal PBG and ALA increased, but Large increase in proto- and Mainly llI may be normal during coproporphyrin with proto remission. Porphyrins may > copro. 'X' peptidoporphyrin be increasedwith the increased. copro > uro. Table III Porphyrins and porphyrin precursors occurring in blood, urine, andfaeces in the porphyrias J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1027 coproporphyrin, together with traces of uropor- Plasma phyrin and hepta-, hexa-, and pentacarboxylic Coproporphyrin cannot be detected in normal plasma porphyrins. The ratio of coproporphyrin I to III is but protoporphyrin is just detectable in amounts variable, although coproporphyrin I usually pre- which cannot exceed 2-3 ,ug/dl (Masuya, 1969). dominates. METHODS OF DETERMINATION Faeces Coproporphyrin of normal faeces is also predom- PBG andALA inantly isomer type I. Comparison of the amounts The widely used screening tests for PBG in which of porphyrin excreted in the bile and in the faeces equal volumes of urine and modified Ehrlich's suggests that the dicarboxylic porphyrins of normal reagent (paradimethylaminobenzaldehyde in HCI) faeces are mainly of exogenous origin arising from give a red colour are unreliable because of the the activity of microorganisms in the gastrointestinal presence of interfering substances (Table VI), tract (Table V) (England, Cotton, and French, 1962; French and Thonger, 1966). Protoporphyrin,whether Pyrrolic compounds Urobilinogen Phylloerythrinogen 1 Endogenous Pyrrole mono- and di-carboxylic acids which with (a) From the products of intermediary metabolism excreted in the Indole paradimethyl- bile Indoxyl aminobenzalde- 5:6 Dihydroxyindole (melanogen) hyde also give a 2 Exogenous red colour (a) From precursors degraded to porphyrins by intestinal micro- Unidentified urinary substances after organisms administration of: (i) from chlorophyll Levomepromazine -infood Cascara sagrada bark extract (ii) from haem and haemoproteins Sedormid -in food Methyl red I which become red -haemorrhage from walls of alimentary tract by copyright. -desquamation of cells from walls of alimen- Pyridium (phenazopyridinium chloride) > in the acid ofthe tary tract Urosein J reagent (b) Synthesis by intestinal microorganisms from simple precursors (c) Preformed porphyrins in food Urea .1 which both inhibits colour due to por- V phobilinogen and Table Origin offaecalporphyrins produces a yellow colour Bilirubin which produces a of endogenous or of exogenous origin, is further green colour changed by intestinal microorganisms so that Tryptophan which produces an a orange brown normal faeces contain variable mixture of proto-, colour meso-, deutero-, and pemptoporphyrins (French, http://jcp.bmj.com/ England, Lines, and Thonger, 1964). In addition, Table VI Substances which may affect the detection of small quantities of uroporphyrin (Aziz and Watson, urinary porphobilinogen by the Ehrlich's reagent 1969) and porphyrin-peptide conjugates ('X por- phyrins') (Rimington et al, 1968) may be present. and substances, eg, urea, which inhibit colour Normal bile and meconium contain small amounts formation. They are also limited in sensitivity, only of other porphyrins including an acrylic analogue of reliably detecting concentrations ofPBG above about

coproporphyrin (V, Fig. 2) (French and Thonger, 10 mg/litre. Separation and distinction of the Ehrlich on September 23, 2021 by guest. Protected 1966; French, Nicholson, and Rimington, 1970) reagent complexes of porphobilinogen and uro- but this has not been detected in normal faeces, bilinogen may be affected by extraction of the latter perhaps because of its reduction to coproporphyrin into chloroform after neutralization of acid present by microorganisms. in the reagent with sodium acetate. It is essential to allow development of colour for 1-5 minutes before Erythrocytes making this neutralization and extraction and a Normal erythrocytes contain only small amounts of mixture of benzyl and amyl alcohols is a better protoporphyrin and coproporphyrin; the latter is extractant than chloroform (Rimington, 1958b). predominantly of isomer type I (Koskelo and The test is strongly positive in almost every patient Toivonen, 1968). It is possible that porphyrins are presenting acute symptoms but its sensitivity is in highest amount in the younger cells as in erythro- inadequate for reliable detection of the condition in hepatic protoporphyria (Clark and Nicholson, 1971). patients without clinical symptoms. All positive Uroporphyrin is not detectable in normal erythro- tests should be confirmed by quantitative estimation cytes. of ALA and PBG using a method by which por- J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

1028 G. H. Elder, C. H. Gray, and D. C. Nicholson phobilinogen is isolated by ion exchange chromato- hydrochloric acid. Some uroporphyrin, especially graphy before determination with Ehrlich's reagent. the I isomer, remains in the proteinaceous precipitate Such a method is that of Mauzerall and Granick formed from the cells during analysis and may be (1956) or some modification of it (eg, Grabecki, extracted into 10% ammonia. The two aqueous Haduch, and Urbanowicz, 1967; Doss and Schmidt, uroporphyrin fractions are then united for spectro- 1971). A convenient kit for these determinations is photometry or spectrofluorimetry (Schwartz et al, obtainable from the Biorad Company (New York). 1960). These methods have the added convenience of per- Ether-insoluble porphyrins in faeces are best mitting the simultaneous estimation of ALA by estimated by the method of Rimington et al (1968) condensation with acetyl-acetone to give an Ehrlich- in which 45 % (w/v) urea containing 4% (v/v) Triton reacting substance which may be similarly estimated. X-100 is used as extractant. It is important to ensure that ether-soluble porphyrins have been wholly Porphyrins removed by exhaustive extraction with ether-acetic Porphyrins, especially protoporphyrins, are unstable acid before this method is applied to the faecal and all measurements must be rapidly carried out in residue. Even so the ether-insoluble porphyrin subdued light as soon as possible after samples are fraction obtained in this way from both normal and obtained. It is especially important that all solvents porphyric faeces is complex and may yet contain be peroxide-free. ether-soluble porphyrins, which have escaped ex- Quantitive estimation of porphyrins is time- traction by adsorption on solid faecal matter as well consuming. Their intense red fluorescence in near as true ether-insoluble porphyrins such as uropor- ultraviolet (Wood's) light, however, provides a phyrin and other polycarboxylic porphyrins, por- sensitive method of detection and this forms the phyrin-peptide conjugates ('X porphyrin'), and basis of widely used screening tests for increased possibly other porphyrin conjugates. For this reason porphyrin concentrations in urine, faeces, and the ether-insoluble porphyrins extracted by urea- erythrocytes. Suitable tests for faeces and urine have Triton solution should not be referred to as 'X por- been described by Rimington (1958b) and their use phyrin' or porphyrin-peptide conjugates until this by copyright. is in the diagnosis of the porphyrias is discussed by confirmed by additional techniques such as reaction Eales et al (1966b). Similar techniques may be used with 14C-labelled dinitrofluorobenzene (Rimington for the screening of whole blood (Rimington and et al, 1968) or electrophoresis (Rimington et al, 1968; Cripps, 1965) but in erythropoietic protoporphyria Magnus and Wood, 1971). Before this can be done a similar technique may be used for the screening of all Triton X-100 must be removed from the sample whole blood but it is more satisfactory to determine (Rimington et al, 1968) since its presence affects the the percentage of fluorocytes by examination of the chromatographic and electrophoretic mobility of fresh red cells in ultraviolet microscopy using light porphyrins. from an iodine quartz lamp (Chapel et al, 1972). In all methods depending on solvent extraction, http://jcp.bmj.com/ The plasma should also be examined for fluorescence porphyrins are divided into fractions according to in ultraviolet light. Positive screening tests and tests their solubility properties but are not identified, and where the interpretation is in doubt should always it is well recognized that the fractions designated be confirmed by quantitative estimations, which protoporphyrin, coproporphyrin, and uroporphyrin should also be used for investigation of the families may contain other porphyrins as well. For this reason ofpatients with porphyria. in recent years increasing use has been made of

Most methods for the quantitative determination methods in which porphyrins are separated byelectro- on September 23, 2021 by guest. Protected of porphyrins in urine, faeces, and erythrocytes phoresis (Lockwood and Davies, 1962; Magnus and depend upon preliminary extraction and fraction- Wood, 1971) or by thin-layer chromatography after ation by solvent partition followed by spectro- extraction and usually methyl esterification (Doss, photometric or fluorimetric determination (Schwartz, 1970). The individual porphyrins are then estimated Berg, Bossenmaier, and Dinsmore, 1960). either spectrophotometrically or fluorimetrically after The methods most widely used in Britain for the elution from the plates or by fluorescence scanning estimation of porphyrins in urine and ether-soluble in situ (Doss, Ulshofer, and Philipp-Domiston, porphyrins in faeces and erythrocytes have been 1971c). The sensitivity of these methods which have described in detail in a recent Association of Clinical been used, particularly for urine, is increased if the Pathologists Broadsheet (Rimington, 1971). Much porphyrins are converted to their zinc chelates before erythrocyte uroporphyrin may be recovered from separation (Doss, 1971). the aqueous phases left after fractionation oferythro- cyte copro- and protoporphyrins, by absorption on DETERMINATION OF ISOMER TYPE alumina and subsequent extraction into 1-5 M In certain circumstances, determination of isomer J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

The porphyrias: a review 1029 type of porphyrin present may be necessary. This mittent porphyria by intravenous infusions of hematin. Proc. nat. Acad. Sci. (Wash.), 68, 2725-2729. may be carried out in the case of coproporphyrins Bossenmaier, I., and Cardinal, R. (1968). Stability of 8-aminolevulinic by chromatography in a lutidine:water:ammonia acid and porphobilinogen in urine under varying conditions. Clin. Chem., 14, 610-614. system (Eriksen, 1958), or in the case of uropor- Brunsting, L. A. (1954). Observations on porphyria cutanea tarda. phyrins by decarboxylation to coproporphyrins Arch. Derm. Syph. (Chic.), 70,551-564. same way Brunsting, L. A., Mason, H. L., and Aldrich, R. A.(1951). Adult form which may then be characterized in the of chronic porphyria with cutaneous manifestations: report (Edmundson and Schwartz, 1953). of 17 additional cases. J. Amer. med. Ass., 146, 1207-1212. Burnham, B. F., and Lascelles, J. (1963). Control of porphyrin Future Considerations biosynthesis through a negative feedback mechanism. Biochem. J., 87, 462-472. seen are an Calvert, R. J., and Rimington, C. (1953). Porphyria cutanea tarda in It will be that the porphyrias interesting relapse: a case report. Brit. med. J., 2, 1131-1134. group of metabolic diseases. The diversity of their Cam, C., and Nigogosyan, G. (1963). Acquired toxic porphyria clinical manifestations and biochemical features has cutanea tarda due to hexachlorobenzene: a report of 348 cases caused by this fungicide. J. Amer. med. Ass., 183, 88-91. stimulated much useful collaboration between Chapel,T.A., Stewart, R. H., and Webster, S.B. (1972). Erythropoietic physicians, pathologists, and biochemists. Extensive protoporphyria: Report of a case successfully treated with carotene. Arch. Derm., 105, 572-574. fundamental knowledge of porphyrin biochemistry Clark, K. G. A., and Nicholson, D. C. (1971). Erythrocyte proto- and metabolism has accrued from this. Probably the porphyrin and iron uptake in erythropoietic protoporphyria. Clin. Sci., 41, 363-370. most important problems worthy of further study Cochrane, A. L., and Goldberg, A. (1968). A study of faecal porphyrin include the nature of the central nervous system in- levels in a large family. Ann. hum. Genet., 32, 195-206. volvement in the inherited hepatic porphyrins, the Conference Discussion (1963). S. Afr. J. Lab. clin. Med., 9, 301-302. Connon, J. J., and Turkington, V. (1968). Hereditary coproporphyria. mode of action of the porphyrinogenic drugs, and the Lancet, 2, 263-264. mechanism of the photosensitization in porphyria. Copeman, P. W. M., Cripps, D. J., and Summerly, R. (1966). Cutaneous porphyria and oestrogens. Brit. med. J., 1, 461 463. There is need also for refinement of the methods Cripps, D. J., and MacEachern, W. N. (1971). Hepatic and erythro- whereby the porphyrias are diagnosed and differ- poietic protoporphyria: delta-aminolevulinic acid synthetase, fluorescence and microfluorospectrophotometric study. Arch. entiated. Path., 91, 497-505. D. and P. J. (1965). Hepatobiliary changes in References Cripps, J., Scheuer, by copyright. Ackner, B., Cooper, J. E., Gray, C. H., Kelly, M., and Nicholson, erythropoietic protoporphyria. Arch. Path., 80. 500-508. D. C. (1961). Excretion of porphobilinogen and 8-aminolae- Dean, G. (1963). The prevalence of the porphyrias. S. Afr. J. Lab. clin. vulinic acid in acute porphyria. Lancet, 1, 1256-1260. Med., 9, 145-151. Allison, A. C., Magnus, I. A., and Young, M. R. (1966). Role of Dean, G., and Barnes, H. D. (1959). Porphyria in Sweden and South lysosomes and of cell membranes in photosensitization. Africa. S. Afr. med. J., 33, 246-253. Nature (Lond.), 209, 874-878. Discombe, G., and Treip, C. S. (1953). Cutaneous manifestations of Aziz, M. A., and Watson, C. J. (1969). An analysis of the porphyrins porphyria. Brit. med. J., 2, 1134-1136. of normal and cirrhotic human liver and normal bile. Clin. Donaldson, E. M., McCall, A. J., Magnus, I. A., Simpson, J. R., chim. Acta, 26, 525-531. Caldwell, R. A., and Hargreaves, T. H. (1971). Erythropoietic Barnes, H. D. (1958). Porphyria in South Africa: the faecal excretion protoporphyria: two deaths from hepatic cirrhosis. Brit. J. of porphyrin. S. Afr. med. J., 32, 680-683. Derm., 84, 14-24. Barnes, H1. D., Hurworth, E., and Millef, J. H. D. (1968). Erythro- Doss, M. (1970). Formation of zinc chelates from porphyrin methyl esters for spectrophotometric analysis. Z. klin. Chem., 8,

poietic porphyrin hepatitis. J. clin. Path., 21, 157-159. http://jcp.bmj.com/ Becker, D., Viljoen, D., and Kramer, S. (1971). The inhibition of red 208-2 11. cell and brain ATPase by 8-aminolaevulinic acid. Biochim. Doss, M. (1971). Conversion of porphyrin methyl esters to zinc and biophys. Acta (Amst.), 225, 26-34. copper chelates for spectrophotometric analysis. Analyt. Belcher, R. V., Smith, S. G., and Mahler, R. (1969). Biliary protein- Biochem., 39, 7-14. bound porphyrins in porphyria variegata. Clin. chim. Acta, 25, Doss, M., Look, D., and Henning, H. (1971a). Chronic hepatic 45-52. porphyria in chronic aggressive hepatitis. Klin. Wschr.. 49, B_nard, H., Gajdos, A., and Gajdos-Torok, M. (1958). Porphyries: 52-54. H., Nawrocki, P., Etude Clinique et Biologique. Bailliere, Paris. Doss, M., Meinhof, W., Look, D., Henning, Dolle, W., Strohmeyer, G., and Filippini, L. (1971b). Porphyrins Berger, H., and Goldberg, A. (1955). Hereditary coproporphyria. Brit. med. J., 2, 85-88. in liver and urine in acute intermittent porphyria and chronic Berman, J., and Bielicky, K. (1956). Einige aussere Faktoren in der hepatic porphyria. S. Afr. J. Lab. clin. Med., 17, 50-54. on September 23, 2021 by guest. Protected Atiologie der porphyria cutanea tarda und des Diabetes mellitus Doss, M., and Schmidt, A. (1971). Quantitative Bestimmung von mit besonderer Bzrucksichtigung der syphylitischen Infektion 8-Aminolavulinsiune und Porphobilinogen im Urin mit und ihrer Behandlung. Dermatologica (Basel), 113, 78-87. Ionenaustauschch romatographie-Fertigsaulen. Z. klin. Chem., Berlin, N. I., Neuberger, A., and Scott, J. S. (1956). The m:tabolism of 9, 99-102. d-aminolaevulinic acid. Biochem. J., 64, 90-100. Doss, M., Ulshofer, B., and Phillip-Domiston, W. K. (1971c). Bloomer, J. R., Berk, P. D., Bonkowsky, H. L., Stein, J. A., Berlin, Quantitative thin layer chromatography of porphyrins by N. I., and Tschudy, D. P. (1971). Blood volume and bilirubin in situ fluorescence measurements. J. Chromat., 63, 113-120. production in acute intermittent porphyria. New Engl. J. Med., Dowdle, E., Goldswain, P., Spong, N., and Eales, L. (1970). The pattern 284,17-20. of porphyrin isomer accumulation and excretion in sympto- Bogorad, L. (1958). The enzymatic synthesis of porphyrins from matic porphyria. Clin. Sci., 39, 147-158. E. B., Mustard, P., and Eales, L. (1967). 8-Aminolaevulinic porphobilinogen. I. Uroporphyrin I. It. Uroporphyrin IHI. III. Dowdle, Uroporphyrinogens as intermediates. J. biol. Chem., 233, acid synthetase activity in normal and porphyric human 501-509, 510-515, and 516-519. livers. S. Afr. med. J., 41, 1093-1096. Bogorad, L. (1963). Enzymatic mechanisms in porphyrin synthesis: Dowdle, E., Mustard, P., Spong, N., and Eales, L. (1968). The possible enzymatic blocks in porphyrias. Ann. N. Y. Acad. Sci., metabolism of [5-"'C] 8-aminolaevulic acid in normal and 104,676-688. porphyric subjects. Clin. Sci., 34, 233-251. Bonkowsky, H. L., Tschudy, D. P., Collins, A., Doherty, J., Bossen- Eales, L. (1963). Porphyria as seen in Cape Town: a survey of 250 maier, I., Cardinal, R., and Watson, C. J. (1971). Repression patients and some recent studies. S. Afr. J. Lab. clin. Med.; 9, of the overproduction of porphyrin precursors in acute inter- 151-162. J Clin Pathol: first published as 10.1136/jcp.25.12.1013 on 1 December 1972. Downloaded from

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