Hereditary coproporphyria in Germany: clinical-biochemical studies in 53 patients

Clinical Biochemistry, Vol. 33, No. 6, 465– 473, 2000 Copyright © 2000 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/00/$–see front matter

PII S0009-9120(00)00159-4

Hereditary Coproporphyria in Germany: ClinicalBiochemical Studies in 53 Patients ALEXANDRA KU¨HNEL, ULRICH GROSS, and MANFRED O. DOSS Abteilung fu¨r Klinische Biochemie, Universita¨tsklinikum Marburg, Marburg, Germany Objectives: To describe the biochemical and clinical features in hereditary coproporphyria (HCP). Design and method: Within the last 20 years, we investigated 53 patients (male:female ⫽ 1:2.5; age ⫽ 8 – 86 years) suffering from HCP. We describe the characteristic levels of urine, and fecal porphyrins and their precursors in hereditary coproporphyria and present the clinical features. Especially, we measured the coproporphyrin isomers I and III. Results and conclusion: The group of hereditary coproporphyria patients exhibited a significantly higher (p ⬍ 0.0001) excretion of urinary porphyrin precursors, ␦-aminolevulinic acid (median ⫽ 84 ␮mol/24 h) and porphobilinogen (median ⫽ 39 ␮mol/24 h), as compared to controls (␦-aminolevulinic acid: 22 ␮mol/24 h, porphobilinogen: 3 ␮mol/24 h; median, n ⫽ 20). The median of coproporphyrin in urine (1315 nmol/24 h) and feces (1855 nmol/g) were enhanced 12and 168-fold, as compared to healthy subjects (urinary coproporphyrin: 106 nmol/24 h, fecal coproporphyrin: 11 nmol/g; median, n ⫽ 20). During therapy on one female patient, with IV application of heme arginate, a considerable decline of porphyrin precursors and porphyrin excretion was observed. The examination of urinary and fecal coproporphyrin isomers I and III revealed an excessive elevation of the coproporphyrin isomer III of 87% in urine and 94% in feces, respectively (normal: urinary isomer III ⫽ 69 – 83% and fecal isomer III ⫽ 25– 40%). In feces the increase of isomer III caused an inversion of the physiologic coproporphyrin isomer III:I ratio that could be recognized in all various stages in hereditary coproporphyria and in children. Acute attacks of hereditary coproporphyria are accompanied by an acute polysymptomatic clinical syndrome, and this is associated with high levels of urinary porphyrin precursors. On review of our patients, the highest percentage had abdominal pain (89%), followed by neurologic (33%), psychiatric (28%), cardiovascular (25%), and skin symptoms (14%). Copyright © 2000 The Canadian Society of Clinical Chemists

KEY WORDS: acute hepatic porphyria; hereditary coproporphyria; coproporphyrinogen oxidase; fecal isomer ratio; neurologic manifestation; heme arginate.

Introduction ereditary coproporphyria (HCP) has been observed in Germany since 1978 as the third most common acute hepatic porphyria in Europe. It is an autosomal dominant inherited disorder characterized by an abnormal hepatic and renal excretion of

H

Correspondence: Manfred O. Doss, Abteilung fu¨r Klinische Biochemie, Klinikum der Philipps-Universita¨t, Deutschhausstra␤e 171⁄2, D-35037 Marburg (Lahn), Germany. E-mail: [email protected] Received March 23, 2000; revised July 14, 2000; accepted July 17, 2000. CLINICAL BIOCHEMISTRY, VOLUME 33, AUGUST 2000

coproporphyrin and by a deficient activity of the mitochondrial enzyme coproporphyrinogen oxidase (1–3). In the majority of cases the enzyme activity is reduced to nearly 50% in heterozygotes; in very rare cases of HCP, presumed to be homozygous for the coproporphyrinogen III oxidase defect, it is about 2% (4 – 8). Clinical attacks are characterized by an acute polysymptomatic syndrome with abdominal, cardiovascular, neurologic, and psychiatric symptoms (1, 2,9). Additionally in nearly 20% of cases HCP may be associated with photosensitivity (10). Secondary factors (drugs, most commonly barbiturates, estrogens, sulfonamides, and hormonal oral contraceptives; also alcohol, caloric deprivation, infection, endocrine factors, or stress) can lead to manifestation of HCP in susceptible individuals (11–13). Thus an early detection of the metabolic disorder is important in the prevention of attacks, so that patients can be advised to avoid the precipitating factors. Enzyme tests are often technically difficult and require tissues such as cultured fibroblasts, lymphocytes, or liver biopsy material and, therefore, they are rarely used (14). Genetic analysis produces a precise diagnosis in some of the porphyrias provided the patient has one of the previously described point mutations, but this technique is currently confined to a few research laboratories (14). For this reason the diagnosis of an acute HCP is based on increased excretion of urinary ␦-aminolevulinic acid (ALA) and porphobilinogen (PBG) and a markedly elevation of coproporphyrin in both feces and urine (8,15,16). Diagnosis of HCP in carriers of the defective gene, who are in the clinically latent phase; however, is not always possible by the conventional biochemical analyses and, in this case, the determination of fecal coproporphyrin isomers I and III has become a helpful complementary diagnostic method (17). Treatment of HCP includes the omission of precipitating factors and regulatory treatment by glucose or IV administration of heme (18,19). Both glucose and heme compounds as hemin and heme arginate have shown to normalise the production of excess heme precursors, as well as to lead to a clinical improvement (19,20 –23). Early diagnosis and a well-timed administration of carbohydrates 465

¨ HNEL, GROSS AND DOSS KU

together with a withdrawal of dangerous drugs and alcohol cannot be overemphasised for an uncomplicated course of porphyria. The aim of the study was to show the characteristic biochemical and clinical features obtained from 53 patients suffering from HCP. We specifically wanted to evaluate the diagnostic relevance of fecal coproporphyrin isomer III in patients as well as in asymptomatic gene carriers. Materials and methods PATIENTS Investigations were carried out over a period of 20 years on 53 patients suffering from HCP (37 females and 16 males, age: 8 – 86 years). Most of the patients are not related to each other with exception of two couples and two families. One family with a 10-yearold girl was described previously (24). The second family we described in context with the case report of a 29-year-old female patient, suffering since 1969 from recurrent abdominal complaints. Regarding to the various stages of HCP 6 of the 53 patients are in the acute phase of the porphyria disease process, whereas 40 of them are in the subclinical stage, which means that patients have had previous acute attacks but are not experiencing symptoms at present. The remaining seven patients are described by the term latent meaning that the subjects have inherited the enzyme deficiency but have never displayed clinical features of the disease. The latter were investigated in the context of family studies. In all patients the diagnosis of HCP was established by analyses of urinary porphyrin precursors (ALA and PBG) and of coproporphyrin in urine and feces. Statistical evaluation was performed by Mann– Whitney U-test. A group of 20 healthy subjects (11 females and 9 males, age: 25–53 years) were examined for comparison. Their recruitment occurred by using random selection. Procedures involving human subjects were performed in accordance with the Helsinki declaration revised in 1983, and informed consent was obtained from all subjects before their inclusion in the study. DETERMINATION

OF PORPHYRIN PRECURSORS

ALA and PBG in urine were assayed with the method of Mauzerall and Granick (25). In this method, ALA and PBG are adsorbed on the ion exchange resins: the upper, anionic column adsorbs PBG, and the lower, cationic column the ALA. The urine sample, which is applied to the upper column, runs through both resins. Urea is removed from the columns with water. Then the columns are separated, and ALA and PBG are eluted. ALA is converted to a pyrrole. The monopyrrols form colored derivatives with p-dimethylaminobenzaldehyde in acid solution (Ehrlich’s Reagent), which are measured spectrophotometrically (26,27). 466

DETERMINATION

OF URINARY AND FECAL PORPHYRINS

First the urine and feces samples were lyophilized and, subsequently, they were incubated overnight with methanol-sulfuric acid (95:5, v/v). The esterification mixture was extracted with chloroform washed twice with distilled water and subsequently dried with sodium sulfate. The mixture was filtered, and chloroform was removed under vacuum. Dried porphyrin methyl esters were redissolved in chloroform and separated via silica gel thin-layer chromatography. The sheets were prerun with chloroformmethanol (120:20, v/v), and samples were separated in petroleum ether (40 – 60°C)-diethyl ether (3:1, v/v). When the front had reached the edge, sheets were run in benzene-ethyl acetate-methanol (85: 13.5:1.5, by volume). The different porphyrin methyl esters were eluted from the sheets with chloroform and subsequently quantified spectrophotometrically (26). HIGH-PERFORMANCE

LIQUID CHROMATOGRAPHY

ANALYSIS OF COPROPORPHYRIN ISOMERS

I

AND

(HPLC) III

The separation and the quantification of urinary and fecal coproporphyrin isomers I and III were performed as free acids by reversed-phase HPLC (Table 1). Separation was carried out on a Lichrospher RP 8 column (250 ⫻ 4 mm, 5 ␮m, Merck, West Point, PA, USA) by using a gradient elution between a buffered salt solution containing acetonitril and between methanol. Porphyrins were detected spectrophotometrically by a UV/VIS detector (Merck/ Hitachi L-4250) and spectrofluorometrically (RF 535 Shimadzu®, Columbia, MD, USA). For the identification of the peaks standards from Paesel and Lorei (Hanau, Germany) were used with mesoporphyrin as an internal standard. The isomer proportions were calculated from peak area ratios from the spectrofluorometric diagram. Results BIOCHEMICAL

INVESTIGATIONS

The medians and the ranges of the urinary and fecal heme precursors from 53 patients suffering from HCP are shown in Table 2. Total porphyrin excretions were significantly increased about 15and 33-fold, and ranged widely from 332 to 35585 nmol/24 h in urine and from 541 to 61317 nmol/g in feces (control group: urine ⫽ 135 (85–157) nmol/24 h, feces ⫽ 65 (46 –209) nmol/g; median (Xmin–Xmax)). In urine this elevation could be explained by a significant rise of the porphyrin precursors, ALA and PBG up to 4- and 13-fold, uroporphyrin up to 13-fold, and coproporphyrin up to 12-fold, respectively. In feces, especially the excessive rise of coproporphyrin about 170-fold is obviously accompanied by a small but not significant elevation of protoporphyrin. The mean proportions of the urinary and fecal coproporphyrin isomers III and I are CLINICAL BIOCHEMISTRY, VOLUME 33, AUGUST 2000

HEREDITARY COPROPORPHYRIA IN GERMANY

TABLE 1 The Chromatographic and Detection Conditions for the Analyses of the Coproporphyrin Isomers I and III Injection volume Separation column Pre-column Solvent

50-␮L sample volume by manual injection LiChrospher威 100 RP-8 (5 ␮m), 250 ⴱ 4 mm (Merck, Darmstadt) LiChrospher威 100 RP-8 (5 ␮m), 4 ⴱ 4 mm (Merck) A ⫽ 10% acetonitrile (v/v) in 1 M ammonium acetate, pH 5.5 C ⫽ methanol LiChrosolve (Merck) Step-wise gradient 0 30 46 50 51 60 100 35 10 5 0 0 0.6 mL/min variable 29 °C 59 min Ex: 405 nm, Em: 620 nm 406 nm

Gradient Time (min) Solvent composition A (%) Flow rate Pressure Temperature Stop time Fluorometric detection UV-Detection

presented in Table 3. Physiologically type III isomer predominates the type I isomer in urine (isomer III ⫽ 69 – 83%; isomer I ⫽ 17–31%), whereas the reverse is true in bile (isomer III ⫽ 25– 40%; isomer I ⫽ 65–75%). An excessive rise of coproporphyrin isomer III, up to 87 ⫾ 4% in urine and to 94 ⫾ 2% in feces [x˜ ⫾ SD, p ⬍ 0.001], was observed in patients suffering from HCP. In feces the amount of isomer III exceeded that of isomer I. This leads to an inversion of the physiologic fecal coproporphyrin isomer III:I ratio, which is normally ⬍1 (Table 3). The chromatograms of urinary and fecal coproporphyrin isomers I and III from a patient with HCP and a healthy person were shown in Figure 1. PHASES

OF HEREDITARY COPROPORPHYRIA

The pathobiochemical criteria for the phase differentiation are based on clinical and biochemical investigations of the 53 HCP patients. Table 4 shows, that especially in the acute phase of the porphyria disease process the excretion of urinary and fecal heme precursors were increased. Thus, we could recognize a 36- and a 89-fold enhancement of porphyrin precursors ALA and PBG, respectively. Urinary uroporphyrin, coproporphyrin, and total porphyrins were elevated 89-, 222-, and 133-fold, respectively, compared to healthy subjects. However

patients in the subclinical or in the latent phase of HCP showed, if at all, only a slight enhancement of both ALA, PBG as well as of urinary uroporphyrin and coproporphyrin (Table 4). In feces, the median of coproporphyrin and protoporphyrin were enhanced 129- and 3-fold in the acute phase, respectively, compared with the median of the control group. In the subclinical phase the coproporphyrin excretion amount to 1850 nmol/g, whereas patients in the latent stage showed an excretion only up to 525 nmol/g. Protoporphyrin was within the normal range (Table 5). The examination of urinary and fecal coproporphyrin isomer III showed no significant difference between patients in the acute and patients in the nonacute phase of the porphyria disease process. CLINICAL

ASSESSMENT

As expected during acute manifestation 89% had abdominal pain, 33% had neurologic symptoms, 28% had psychiatric symptoms, and 25% had cardiovascular symptoms. Skin photosensitivity was observed in five cases with HCP. Additionally the correlation of common incidence of abdominal pain and neurologic symptoms was calculated to prove their causal coexistence. In 69% of our patients, abdominal pains occurred without neurologic complaints and 27%

TABLE 2 Urinary and Fecal Heme Precursors of 53 Patients with HCP Compared with Healthy Controls Patients (n ⫽ 53) Excretion Urinary

Fecal

Metabolites ␦-Aminolevulinic acid (␮mol/24 h) Porphobilinogen (␮mol/24 h) Uroporphyrin (nmol/24 h) Coproporphyrin (nmol/24 h) Total Porphyrins (nmol/24 h) Coproporphyrin (nmol/g dw) Protoporphyrin (nmol/g dw) Total Porphyrins (nmol/g dw)

Controls (n ⫽ 20)

Median (Xmin–Xmax) 84 (52–1102) 39 (12–370) 225 (41–27307) 1315 (150–29176) 1991 (332–35585) 1855 (96–60458) 73 (41–165) 2130 (541–61317)

22 (7–45) 3 (2–5) 17 (12–26) 106 (61–119) 135 (85–157) 11 (8–35) 43 (30–139) 65 (46–209)

p ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 0.94 ⬍0.001

dw ⫽ dry weight. CLINICAL BIOCHEMISTRY, VOLUME 33, AUGUST 2000

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¨ HNEL, GROSS AND DOSS KU

TABLE 3 Isomer Composition Expressed as Percent of Total Coproporphyrin (Median ⫾ SD) Excretion Urinary

Fecal

Metabolites Copro isomer I Copro isomer III Ratio of Copro isomers III:I Copro isomer I Copro isomer III Ratio of Copro isomers III:I

Patients (n ⫽ 53)

Controls (n ⫽ 20)

13 ⫾ 4% 87 ⫾ 4% 6.7

24 ⫾ 4% 76 ⫾ 4% 3.2

6 ⫾ 2% 94 ⫾ 2% 15.7

70 ⫾ 3% 30 ⫾ 3% 0.4

showed abdominal and neurologic manifestations. Only 4% of the patients had neurologic symptoms alone. SPECIAL

OBSERVATIONS

A young woman, born 1969, suffering since 1990 from recurrent abdominal complaints. A hiatus hernia and a reflux esophagitis were established gastroscopically, and they were first treated with Antra and Propulsin. In addition, an obviously increased urinary porphyrin excretion could be recognized and

the diagnosis of an acute hepatic porphyria was taken into consideration. Therefore, differential diagnostic analyses were made which showed the characteristic excretion pattern of HCP in a subclinical phase of the porphyria disease process (urine: ALA ⫽ 7 ␮mol/24 h, PBG ⫽ 9 ␮mol/24 h, uroporphyrin ⫽ 20 nmol/24 h, coproporphyrin ⫽ 262 nmol/24 h; feces: coproporphyrin ⫽ 391 nmol/g, coproporphyrin isomer I ⫽ 11%, coproporphyrin isomer III ⫽ 89%, protoporphyrin ⫽ 34 nmol/g). With the exception of a moderate increase of PBG, the porphyrin precursors were in the physiologic ranges. The excretion of coproporphyrin III in urine and feces was still increased. One month later, the young woman had a viral infection and a general urticarial exanthem, which was treated with an injection of Dimetindene. Dimetindene is a porphyrinogen drug and together with the virus infection it induced an acute attack accompanied by sudden abdominal pain. Thus, the patient suffered from severe complaints, muscular asthenia, paresthesia, and tachycardia, and received Pethidine IV and Propanolol. Increased urinary excretion of ALA and PBG and an increase in fecal coproporphyrin confirmed the diagnosis of an acute manifestation. Under long-wave ultravioletlight the feces exhibited a red fluorescence contain-

Figure 1 — HPLC separation of urinary and fecal coproporphyrin isomers I and III (percentage of total coproporphyrins) from a healthy person and a patient with hereditary coproporphyria. Chromatograms of the fluorescence detector. copro ⫽ Coproporphyrin; dw ⫽ dry weight. a) Normal. Feces: copro I ⫽ 3 nmol ⫻ g⫺1 dw, copro III ⫽ 1,2 nmol ⫻ g⫺1 dw, copro I:III ⫽ 71:29%. Urine: copro I ⫽ 30 nmol ⫻ 24 h⫺1, copro III ⫽ 98 nmol ⫻ 24 h⫺1, copro I:III ⫽ 23:77%. b) Hereditary coproporphyria. Feces: copro I ⫽ 150 nmol ⫻ g⫺1 dw, copro III ⫽ 2133 nmol ⫻ g⫺1 dw, copro I:III ⫽ 7:93%. Urine: copro I ⫽ 35 nmol ⫻ 24 h⫺1, copro III ⫽ 161 nmol ⫻ 24 h⫺1, copro I:III ⫽ 18:82%. 468

CLINICAL BIOCHEMISTRY, VOLUME 33, AUGUST 2000

HEREDITARY COPROPORPHYRIA IN GERMANY

TABLE 4 Urinary Heme Precursors of 53 Patients in Phases of Hereditary Coproporphyria Acute Phase (n ⫽ 6) Urinary Excretion ␦-Aminolevulinic acid (␮mol/24 h) Porphobilinogen (␮mol/24 h) Uroporphyrin (nmol/24 h) Coproporphyrin (nmol/24 h) Copro isomer III [%] Total porphyrins (nmol/24 h)

Subclinical Phase (n ⫽ 40)

Latent Phase (n ⫽ 7)

Median 802 267 5377 11370 83 29905

ing an extremely high concentration of coproporphyrin more than 5 mg/g dry wt. After therapeutic glucose infusion a considerable decline of urinary ALA, PBG, and porphyrins between 82% to 99%, and of fecal porphyrins between 38% to 50% was observed (11). Since 1994 the patient reported intermittent abdominal symptoms. However, excretion of urinary and fecal heme precursors was only slightly increased. Thus the complaints could not be correlated to the porphyria disease process. They may have been caused by the reflux esophagitis or may be understood in a psychosomatic way. The patient tended to abuse analgetics during the last years (Pethidine, Propanolol, and Tilidin). In the summer of 1997, she suffered again from severe acute abdominal and neurologic symptoms and her excretion pattern shows the characteristic signs of an acute attack (Table 6). Consequently, she was treated by IV application of heme arginate. The clinical condition improved associated by a decrease in the excretion of heme precursors down to roughly normal values. For prophylactic reasons, an interval treatment by IV application of heme arginate, one ampulle per month (3 mg heme arginate/kg body weight per month) has been continued for 1 year (Table 6). In Table 7 the biochemical examinations of the whole family are shown. Her father, brother 1, and her sister showed an elevation of uroporphyrin and coproporphyrin in urine and of coproporphyrin in feces with dominance of coproporphyrin isomer III. In view of the porphyrin precursors only a slight increase of ALA could be found. With the exception of brother 1 PBG was within normal ranges. Thus,

Normal

46 7 101 856 85 1179

53 4 115 800 76 1097

⬍49 ⬍8 ⬍29 ⬍119 ⬍70 ⬍224

the father, brother 1, and the sister were found to be heterozygous gene carriers for the coproporphyrinogen oxidase defect in the clinically latent phase. Her mother and brother 2 showed no sign of HCP. Discussion PATHOBIOCHEMICAL

DIAGNOSIS

Diagnosis of overt porphyria requires metabolite studies. Enzyme assays have only a minor part to play in the routine diagnosis and management of the porphyrias (14,28). The presence of an enzyme defect, most of the time, does not necessarily mean the patient will have clinical manifestations of the associated porphyria. Decreased enzyme activity reflects only that the patient carries an enzyme mutation. As clinical experience shows, only in a minority of genetically affected family members does the mutation lead to disease expression. In establishing the diagnosis of porphyria under clinical circumstances one has to start with metabolite studies. In agreement with other investigations (10,29 –32) an increased excretion of urinary porphyrin precursors and a marked elevation of coproporphyrin isomer III in both feces and urine are characteristic findings in patients in the acute phase of the porphyria disease process. Early diagnosis of HCP is difficult, however, since coproporphyric patients in the subclinical or latent phase only show a slight-to-moderate increase of metabolite excretion (33). Accordingly the 47 patients in the nonacute stages of the hereditary disease process showed no enhancement of porphyrin precursors and only a small increase of urinary

TABLE 5 Fecal Heme Precursors of 53 Patients in Phases of Hereditary Coproporphyria

Fecal Excretion Coproporphyrin (nmol/g dw) Copro isomer III (%) Protoporphyrin (nmol/g dw) Total porphyrins (nmol/g dw)

Acute Phase (n ⫽ 6)

Subclinical Phase (n ⫽ 40)

Latent Phase (n ⫽ 7)

Median

Median

Median

Normal

4768 93 371 5508

1850 93 98 1730

525 94 43 694

⬍37 ⬍40 ⬍151 ⬍224

dw ⫽ dry weight. CLINICAL BIOCHEMISTRY, VOLUME 33, AUGUST 2000

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¨ HNEL, GROSS AND DOSS KU

TABLE 6 Urinary and Fecal Excretion of Heme Precursors by the Patient Receiving Heme Arginate During the Acute Phase of HCP

Metabolites

Acute Phase (1997)

After 8 Months

After 10 Months

Normal

␦-Aminolevulinic acid (␮mol/24 h) Porphobilinogen (␮mol/24 h) Uroporphyrin (nmol/24 h) Coproporphyrin (nmol/24 h) Total porphyrins (nmol/24 h) Coproporphyrin (nmol/g dw) Protoporphyrin (nmol/g dw) Total porphyrins (nmol/g dw)

12320 12672 187 1310 1931 11880 406 12607

62.6 10.2 87.8 267 455 29.3 2.8 289

34.4 8.0 74.6 591 689 140 21.5 164

⬍49 ⬍8 ⬍29 ⬍119 ⬍165 ⬍37 ⬍151 ⬍224

Excretion Urinary

Fecal

Intervall Treatment with Heme Arginate

dw ⫽ dry weight.

and fecal total porphyrins, compared with the median of the normal range (Tables 4 and 5). In this case, the determination of coproporphyrin isomers I and III is a helpful complementary diagnostic method.

coproporphyrin isomer III:I ratio. Recently, in three families the gene carriers of the coproporphyrinogen oxidase defect could be recognized by their characteristic fecal isomer inversion. In three persons the diagnoses were confirmed by molecular studies.

ISOMER

CLINICAL

ANALYSIS FOR DETECTION OF GENE CARRIERS

The diagnostic value of the fecal coproporphyrin isomers I and III was first described by Sieg and Doss (34). Thus, in HCP the most important clinical diagnostic aspect is the fact that in patients the disease can be detected by the inversion of their fecal coproporphyrin isomers III:I ratio, independent of the clinical phase exists (Table 5). Analyses of urinary coproporphyrin isomers with dominance of coproporphyrin isomer III could also contribute to the diagnosis of HCP but in cases of normal excretion of total urinary coproporphyrin the isomer distribution showed inadequate diagnostic sensitivity (17). The importance of isomer analyses is evident from the case study of the 29-year-old female patient. Her father, brother 1, and her sister are latent gene carriers although their heme precursors were only slightly increased or even in the normal ranges. Although they showed high values of coproporphyrin in urine and feces the unambiguous HCP diagnosis was confirmed by the inversion of the fecal

EXPRESSION

All the four types of hepatic porphyria present clinically with an identical abdominal-neurologic syndrome. Manifestations of acute life-threatening attacks include autonomic neuropathy with severe abdominal pain, vomiting, constipation, hypertension, tachycardia, and bladder dysfunction. Other manifestations are peripheral neuropathy causing motor weakness and paralysis and mental symptoms that occur without detectable morphologic anomalies in the brain (8). The clinical manifestations are accompanied by an abnormal excretion and endogenous accumulation of porphyrin precursors in urine and porphyrins in feces and urine. Even 22 years ago Brodie et al. (1977) investigated 111 patients with HCP, whereby 35% present in acute manifestation (1). Regarding to the clinical symptoms, 80% had abdominal pain, 34% vomiting, 29% solar sensitivity, 23% neurologic involvement, 23% psychiatric symptoms, and 20% severe constipation. Only two fatalities have been published, both from

TABLE 7 Urinary and Fecal Excretion of Heme Precursors of the 29-Year-Old Female Patient and Her Family Excretion Urinary

Fecal

Metabolites

Father

Mother

Patient

Brother 1

Brother 2

Sister

Normal

␦-Aminolevulinic acid (␮mol/24 h) Porphobilinogen (␮mol/24 h) Uroporphyrin (nmol/24 h) Coproporphyrin (nmol/24 h) Ratio of Copro isomers I:III (%) Total porphyrins (nmol/24 h) Coproporphyrin (nmol/g dw) Ratio of Copro isomers I:III (%) Protoporphyrin (nmol/g dw) Total porphyrins (nmol/g dw)

52 3 341 625 32:68 1146 525 10:90 43 603

41 2 8 116 31:69 143 12 65:35 30 45

580 511 749 18130 11:89 22395 6676 6:94 185 7283

108 15 300 1901 18:82 2611 4982 5:95 92 5386

40 1 4 115 44:56 141 24 64:36 149 183

56 4 298 625 32:68 1097 608 6:94 46 694

⬍49 ⬍8 ⬍29 ⬍119 24:76 ⬍165 ⬍37 70:30 ⬍151 ⬍224

dw ⫽ dry weight. 470

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respiratory failure. There was a female preponderance of cases in attack of 2.5:1 and in the latent case of 1.5:1, suggesting hormonal provocation in the uncovering of the disease. How these changes are triggered and the pathogenesis of the neuronal dysfunction that produces the symptoms are little understood. In the literature different hypothesis are discussed (8,35–37). Two hypotheses: (1) the possible neurotoxicity of ALA and PBG; or porphyrin production and accumulation in neuronal tissues; and (2) a decrease in heme levels in the nervous system and/or in the liver, which could lead to an inability of cells in the nervous system to synthesise adequate amounts of key hemoproteins, causing a deficiency in the ATP supply and thus neuronal dysfunction are discussed for example by Bonkovsky (1993) (35). Corresponding data from porphobilinogen-deaminase-deficient mice are presented by Lindberg et al. (1999) (38). The present evidence suggests that multiple mechanisms interact in causing the varied symptoms, including ALA interaction with GABA receptors, altered tryptophan metabolism, and possibly heme depletion in nerve cells (39). Regarding our investigations, a correlation between the excessive excretion of neurotoxic porphyrin precursors and the occurrence of clinical symptoms could be recognized. This result confirms the possibility that the porphyrin precursors may act as pharmacological agents in this disease, compounding the effects of heme deficiency. Probably oxygen intermediates produced by porphyrins being exposed to light play an important role in skin lesions. In case of delayed diagnosis severe complications appear. Thus, in two female patients (50 and 25 years old) the diagnosis of HCP was fist made after beginning or after long-timed tetraparese. The 50year-old women with the clinical diagnosis Landry paralysis and Guillain-Barre´ syndrome died because of a secondary infection after tetraplegia, ileus and respiratory insufficiency, tachycardia and hypokalemia (29). The second case a 28-year-old patient had been ill for the last 12 years. She had been treated in 14 different hospitals more than 20 times, where gastroenterologic, neurologic-psychiatric, gynecologic, and urologic diagnoses were made. These were followed by therapeutic measures including nine operations. Sudden cardiac-rhythm irregularities led to a decreased blood pressure, which could not be corrected by a temporary pacemaker. She died because of a respiratory insufficiency with cardiac paralysis (33). In both cases, a variety of porphyrinogenic drugs such as analgetics (i.e., pyrazolone), barbiturates, and sulfonamides were given. The 28year-old patient especially was treated with primidone for over 5 years, because of epilepsy. In both cases the diagnosis of porphyria was established by high excretion of urinary ALA and PBG and coproporphyrin III in urine and feces. Analyses of coproporphyrinogenoxidase in the 28-year-old woman showed that the activity was reduced by 48% in the CLINICAL BIOCHEMISTRY, VOLUME 33, AUGUST 2000

liver and 40% in the kidney compared to controls (30). TREATMENT

AND PROPHYLAXIS OF HEREDITARY

COPROPORPHYRIA

After the treatment with heme arginate the laboratory and the clinical effects of the application were remarkable (19 –22). The results reported by Schoenfeld et al. (23) showed, that after four infusions of heme arginate over 4 consecutive days, within 48 h after the first infusion the massively increased urinary excretion of ALA, PBG, and coproporphyrin returned to normal values. These results are comparable with our examinations. Table 6 shows that prophylactic treatment with heme arginate can stabilize the latent phase of HCP successfully and could prevent a renewed manifestation. MOLECULAR

BIOLOGY

The identification of the gene abnormality in HCP families is now possible since the cDNA and the gene of human coproporphyrinogenoxidase have been isolated and sequenced. Isolation of cDNAs for mouse and human coproporphyrinogenoxidase revealed a single gene, which in humans is located on chromosome band 3q12 (40,41). To date, 16 mutations in the heterozygous case of the coproporphyrinogenoxidase gene have been described (6,7,24,40 – 48). Also, homozygous HCP (49,50) and one child with compound heterozygous HCP have been reported in the literature (24). Four other cases of homozygous coproporphyrinogen oxidase deficiency have been reported with childhood onset of severe jaundice, hemolytic anemia, and hepatosplenomegaly (51,52). However, in addition to elevations in urinary and fecal coproporphyrin excretion, these patients displayed marked increases in excretion of harderoporphyrin. Lymphocyte coproporphyrinogen oxidase had abnormal kinetic properties in three of these cases (53) and a marked decrease of activity (43). In this kindred, the transmission of the disease was autosomal recessive (43). All mutations in the coproporphyrinogen oxidase gene, including two for the homoallelic form of HCP and harderoporphyria, are different, confirming the genetic heterogeneity of HCP. Due to this heterogeneity a molecular diagnosis is methodically difficult. However, DNA analysis could be helpful to detect asymptomatic gene carriers with normal excretion patterns, and consequently to improve the prevention of the acute clinical expression of these porphyrias. But molecular studies do not establish that the actual clinical findings are due to the acute hepatic porphyria. In establishing the diagnosis under clinical circumstances one has to start with metabolite studies: high levels of urinary porphyrin precursors and of porphyrins in urine and feces. Therefore the analysis of heme precursors and especially of coproporphyrin isomer III in feces is still the method of choice in clinical diagnosis. 471

¨ HNEL, GROSS AND DOSS KU

Conclusion Summarizing the main points of our study on hereditary coproporphyria, we conclude that: 1) Diagnosis and differential diagnosis of HCP are based on analysis of urinary porphyrin precursors (ALA and porphobilinogen) and of coproporphyrin in urine and feces. 2) The inversion of the fecal coproporphyrin isomers III:I ratio could be the decisive factor to identify patients in the subclinical (n ⫽ 40) and in the latent (n ⫽ 7) stage of hereditary coproporphyria. 3) Clinically, hereditary coproporphyria is similar to acute intermittent porphyria, characterized by acute abdominal and neurologic symptoms. In addition and in contrast to acute intermittent porphyria skin symptoms will appear in about one third of cases compatibly to variegate porphyria in the middle and north Europe. 4) In patients with severe acute neurovisceral manifestation of hereditary coproporphyria the treatment of choice is administration of heme arginate and should be started immediately.

9. 10. 11.

12.

13. 14. 15.

Acknowledgement

16.

The authors thank Thomas Dietze (Department of Medical Biometrics and Medical Information of the Philipp University, Marburg, Germany) for statistical analysis of the data. The study was supported by the German Research Association (Grant GR 1363/2-2) and by the Hans-FischerGesellschaft (Munich, Germany).

17.

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