Original Article
Bile Porphyrin Analysis in the Evaluation of Variegate Porphyria
N Engl J Med 1991; 324:1408-1411May 16, 1991
- Abstract
- Abstract
Background.
Variegate porphyria is a genetic disorder of porphyrin metabolism in which patients may have both neurologic dysfunction and photocutaneous lesions. Biochemical confirmation of the diagnosis can be difficult, particularly in patients without neurologic dysfunction at the time of testing. The demonstration of increased fecal excretion of porphyrin is frequently used for this purpose, but levels may be normal. Since elevated fecal porphyrin levels in variegate porphyria are presumably a consequence of increased biliary excretion, we evaluated whether analysis of porphyrins in bile distinguishes better between patients with variegate porphyria and controls.
Methods.
Bile samples were collected by duodenal aspiration from 10 patients with proved variegate porphyria who had no neurologic symptoms when they were studied and 17 control subjects. Bile and fecal porphyrin levels were measured fluorometrically.
Results.
The mean total porphyrin concentration in bile in the patients with variegate porphyria was significantly higher than that in the controls (67.8 vs. 0.71 μmol per liter; P<0.00002). There was more than a ninefold difference between the highest level in any control subject and the lowest level in any patient with variegate porphyria. The mean fecal porphyrin level in the patients with variegate porphyria also differed significantly from that in the controls (0.79 vs. 0.14 μmol per gram of dry weight; P<0.007), but four patients had levels within the control range.
Conclusions.
The concentration of porphyrin in bile is higher in patients with variegate porphyria than in controls, and the difference is greater than that for fecal porphyrin. Bile porphyrin measurements may be helpful in the evaluation of asymptomatic patients suspected of having variegate porphyria. (N Engl J Med 1991; 324:1408–11.)
Media in This Article
Figure 1
Protoporphyrinogen Metabolism in Variegate Porphyria.Figure 2
Total Porphyrin Levels in Bile Samples from Control Subjects, Patients with Protoporphyria (PP); and Patients with Variegate Porphyria (VP).Article Activity
- Article
VARIEGATE porphyria is an autosomal dominant disorder characterized clinically by attacks of neurologic dysfunction, photocutaneous lesions, or both. The symptoms that occur during attacks, chiefly abdominal pain, are identical to those of acute intermittent porphyria.1 2 3 4 5 Skin lesions may be present when patients are not having attacks of neurologic dysfunction, but they were present in less than half of the affected persons in some studies.2 Moreover, the skin lesions may be mild and may not be mentioned by the patient.
During acute attacks, the urinary abnormalities of variegate porphyria are indistinguishable from those of acute intermittent porphyria, with marked elevations in the excretion of porphobilinogen and δ-aminolevulinic acid. Between attacks, urinary excretion of these substances may be normal. The excretion of protoporphyrin in the feces may be increased, not only during acute attacks but also between them; therefore, fecal protoporphyrin levels have been measured in asymptomatic persons suspected of having variegate porphyria.6 , 7 The increase in fecal excretion of protoporphyrin reflects the underlying enzyme defect, which is a deficiency of protoporphyrinogen oxidase activity. This enzyme catalyzes the conversion of protoporphyrinogen to protoporphyrin in mitochondria,8 9 10 11 and deficiency of this enzyme is thought to result in excessive accumulation and excretion of protoporphyrinogen. The nonenzymatic oxidation of protoporphyrinogen to protoporphyrin subsequently leads to increased levels of protoporphyrin in bile and the feces (Fig. 1Figure 1
Protoporphyrinogen Metabolism in Variegate Porphyria.).Unfortunately, food and bacterial metabolism in the intestine contribute to the porphyrin content of feces12 , 133 and so may obscure the difference in levels between normal persons and patients with variegate porphyria. To overcome this shortcoming, we measured porphyrin levels in bile. We found considerably greater differences in bile levels than in fecal levels in the two groups.
Methods
Subjects
The study protocol was approved by the Committee on Human Investigation at the University of Minnesota. All the subjects gave informed consent to participate in the study. There were 17 control subjects, 10 patients with variegate porphyrin, and 6 patients with protoporphyria. None of the controls had biochemical or clinical evidence of porphyria. They included 12 men and 5 women, ranging in age from 18 to 68 years (mean, 49); 7 were normal, healthy subjects, 7 had hyperlipidemia, and 3 had recurrent abdominal pain for which no cause had been found. There were nine women and one man with variegate porphyria, ranging in age from 24 to 65 years (mean, 43). All 10 patients with variegate porphyria had had one or more episodes of neurologic dysfunction characterized by abdominal pain associated with increased urinary excretion of δ-amino-levulinic acid and porphobilinogen. Nine of the 10 had a history of bullous skin lesions in areas exposed to the sun. At the time of the study, none had neurologic dysfunction, but six had increased skin fragility with erosions and bullae. We also studied six patients with protoporphyria: two women and four men, ranging in age from 18 to 34 years (mean, 27). These patients all had typical photosensitivity and elevated erythrocyte protoporphyrin levels. None had clinical signs or symptoms of liver disease, except for a mild increase in the serum alanine aminotransferase level in one patient (58 U per liter; upper limit of normal, 43). These patients served as positive controls, since patients with protoporphyria, including three of these six patients, have been found to have elevated bile porphyrin concentrations.14
Sample Collection
Bile was collected by duodenal aspiration. After topical lidocaine was applied to the pharynx, each subject swallowed a polyvinyl tube weighted with a mercury bag and capped by a metal aspiration tip. The tip passed into the duodenum to the level of the ampulla of Vater. The location was determined by aspiration of duodenal contents by gravity drainage. A rise in the pH to 7 or more associated with bilious return was used to confirm that the tip of the tube was at the level of the ampulla of Vater. Gallbladder contraction was stimulated by the intravenous administration of cholecystokinin octapeptide (sincalide [Kinevac], Squibb, Princeton, N.J.) at a dose of 20 ng per kilogram of body weight given over a 10-minute period.15 Bile-enriched fluid was collected for 15 minutes on ice and transferred to a sealed tube flushed with nitrogen. All subjects provided blood samples. Stool samples were provided by all the patients with variegate porphyria and eight of the controls (five of the normal, healthy subjects and the three subjects with unexplained abdominal pain).
Biochemical Measurements
The samples of bile were analyzed within one to two hours after collection. Total bile porphyrin levels were measured fluorometrically before and after oxidation of the bile sample with chloranil. The bile samples were diluted in glacial acetic acid/95 percent ethanol/0.6 N hydrochloric acid (1:1:2, vol/vol/vol) with and without chloranil in a concentration of 0.1 mg per milliliter until the solution was colorless. The fluorescence emission spectrum of each sample was scanned from 500 to 700 nm, with excitation at 405 nm. The total bile porphyrin level was defined as the sum of the porphyrin moieties. Chloranil was found in control studies not to enhance the fluorescence of porphyrins. The increase in fluorescence intensity of the bile sample after treatment with chloranil was due to the oxidation of porphyrinogens to porphyrins. The level of porphyrinogen was therefore determined as the difference between the nonoxidized and the oxidized sample. Coproporphyrin I (Sigma Diagnostics, St. Louis) was used as the standard for the bile porphyrin assay. Total bilirubin was measured by a modification of the method of Jendrassik and Gróf.16
High-performance liquid chromatography was used to determine the porphyrin profile in the samples of bile from each subject.17 The samples were diluted in a solution of 1.5 M citric acid, 0.1 M oxalic acid, and 10 percent acetic acid. The solution was mixed and filtered through a 0.45-μm nylon filter. A mixture of porphyrin standards (Porphyrin Products, Logan, Utah) was analyzed with the same system. Mesoporphyrin was used as the 2-COOH porphyrin standard in this mixture because it was more stable than protoporphyrin. The retention time of mesoporphyrin was slightly less than that of protoporphyrin. Stool samples were also analyzed by high-performance liquid chromatography for their porphyrin content after a weighed portion of the sample was dissolved in the citric acid reagent.
A plasma sample was also taken from the patients with variegate porphyria for examination for the plasma porphyrin marker that has been described as specific for variegate porphyria.18 The chemical composition of this marker is uncertain. One milliliter of plasma was diluted with 9 ml of phosphate-buffered saline (0.9 N sodium chloride and 0.01 M sodium phosphate, pH 7.4) and transferred to optical-glass cuvettes with a 1-cm path length for spectrofluorometry. The fluorescence emission spectrum was scanned from 550 to 700 nm, with excitation set at 400 nm. The excitation spectrum was then scanned from 350 to 450 nm, with the emission set for the maximum determined by the scan.
Statistical Analysis
The results were analyzed by nonparametric testing with the two-sample Wilcoxon test from the NPAR1WAY procedure in the Statistical Analysis System (SAS Institute, Cary, N.C.).19 Corrections for multiple comparisons were performed by the Bonferroni method. Differences were considered significant if the P value was less than 0.05.
Results
The total porphyrin levels in bile in the three groups are shown in Figure 2Figure 2
Total Porphyrin Levels in Bile Samples from Control Subjects, Patients with Protoporphyria (PP); and Patients with Variegate Porphyria (VP). and Table 1Table 1
Bile Porphyrin Analysis in 10 Patients with Variegate Porphyria, 6 Patients with Protoporphyria, and 17 Controls.*. The mean level in the patients with variegate porphyria was significantly higher than that in the control subjects (P<0.00002). There was more than a ninefold difference between the highest value in a control subject and the lowest level in any patient with variegate porphyria. Consistent with previous findings, the mean bile concentration of total porphyrin in the patients with protoporphyria was significantly higher than that in the controls (P<0.002).The differences in the groups were similar when the results were expressed as the total bile porphyrin concentration relative to the bilirubin concentration of the same sample (Fig. 2). In the patients with variegate porphyria the mean ratio of total bile porphyrin concentration to bilirubin concentration was 210, as compared with 1.5 in the control subjects (P<0.00005). The range of values in the patients with variegate porphyria did not overlap with the values in the controls. The percentage of bile porphyrin present as porphyrinogen was similar in the patients with variegate porphyria and the controls (Table 1). In contrast, the percentage of porphyrin present as porphyrinogen was significantly lower in the patients with protoporphyria than in the control subjects (P<0.004).
The mean (±SE) fecal level of total porphyrin in the control subjects was 0.14±0.04 μmol per gram of dry weight (Fig. 3Figure 3
Fecal Levels of Total Porphyrin and Protoporphyrin in the Controls and Patients with Variegate Porphyria (VP).). The comparable value in the patients with variegate porphyria was 0.79±0.22 μmol per gram of dry weight (P<0.007), but four of these patients had levels that were within the range of the control group. In terms of fecal protoporphyrin alone, the mean level in the controls was 0.11 ±0.04 μmol per gram of dry weight and that in the patients with variegate porphyria was 0.47±0.13 μmol per gram of dry weight (P<0.03).The porphyrin profiles of the bile samples, as determined by high-performance liquid chromatography, were different in the controls and the patients with variegate porphyria. Most (79 percent on average) of the porphyrin in the bile samples from the control subjects was coproporphyrin (Table 1 and Fig. 4Figure 4
High-Performance Liquid Chromatography of Bile and Fecal Samples from a Control Subject and a Patient with Variegate Porphyria.), and only an average of 12 percent was protoporphyrin. In the bile samples from patients with variegate porphyria, protoporphyrin and other 2-COOH and 3-COOH porphyrins predominated.The profiles of porphyrins in feces were more similar than the profiles in bile between the control subjects and the patients with variegate porphyria (Fig. 4). The porphyrin profiles in the controls showed a complex mixture of 2-COOH and 3-COOH porphyrins in stool that were not present in bile (Fig. 4).
The plasma porphyrin marker that is specific for variegate porphyria was detected in 8 of the 10 patients with variegate porphyria.
Discussion
We found that porphyrin levels in bile were high in all 10 patients with variegate porphyria we studied. The mean level in these patients was 95 times that in the control subjects. Coproporphyrin was the predominant porphyrin in the bile samples from the controls, confirming earlier studies.20 In contrast, the bile samples from the patients with variegate porphyria contained predominantly protoporphyrin and other 2-COOH and 3-COOH porphyrins. Earlier studies21 , 22 showed that a fraction of the porphyrin in the bile from patients with variegate porphyria is conjugated to protein. We did not test for a porphyrin—protein conjugate in bile, but one of the 2-COOH or 3-COOH porphyrin peaks may be due to such a conjugate.
The biochemical diagnosis of variegate porphyria has heretofore relied on the detection of increased urinary excretion of porphobilinogen and δ-amino-levulinic acid during acute attacks in patients with a compatible history and physical findings.1 2 3 4 , 23 Fecal porphyrin levels may remain elevated when symptoms have subsided.24 However, measurement of fecal porphyrin would have led to a misdiagnosis in 4 of the 10 patients we studied, since their levels were within the range of those in the control subjects.
This discrepancy between the sensitivity of the measurement of porphyrins in bile and its sensitivity in feces is due to the fact that a fraction of the porphyrin in feces is not of biliary origin. This is demonstrated by the difference between porphyrin profiles in bile and those in feces in the control subjects (Fig. 4). Bacterial metabolism alters fecal porphyrin content, and studies of the suppression of bacterial flora12 have demonstrated wide variations in fecal porphyrin levels both among normal persons and within individuals. Ingested food adds to the porphyrin content of feces13; 8 to 83 percent of ingested heme is converted to porphyrins.25
Two other means of diagnosing variegate porphyria merit discussion. The plasma of patients may contain a porphyrin marker that may be diagnostic.18 However, this marker was not detected in two of our patients. The demonstration of decreased protoporphyrinogen oxidase activity in cultured skin fibroblasts, liver tissue, or peripheral-blood lymphocytes is another way of diagnosing variegate porphyria,8 9 10 but the assay is difficult because of nonenzymatic oxidation of protoporphyrinogen. In addition, the levels of enzyme activity in patients with variegate porphyria are approximately 50 percent of normal, and these values may overlap with normal values. For these reasons, the assay is in clinical use in only a few laboratories.
In conclusion, analysis of bile porphyrin permits clear differentiation between control subjects and patients with variegate porphyria. We speculate that such analysis may be useful not only in asymptomatic patients suspected of having variegate porphyria but also in family members of patients with the disease.
Supported in part by a research grant (DK26466) and a research core center grant (DK34931) from the National Institutes of Health.
Presented in part at the annual meeting of the American Association for the Study of Liver Disease in Chicago, November 1990.
Source Information
From the Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, University of Minnesota, Minneapolis (M.K.W., J.R.B.); the Section of Gastroenterology, St. Paul Ramsey Medical Center, St. Paul, Minn. (G.M.L.); the Department of Medicine, Minneapolis Medical Research Foundation, Minneapolis (M.E.); and the Department of Medicine, Abbott—Northwestern Hospital, Minneapolis (C.A.P.). Address reprint requests to Dr. Bloomer at Box 36 UMHC, Harvard St. at River Rd., Minneapolis, MN 55455.
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- Citing Articles (15)
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R.J. Hift, G. Todd, P.N. Meissner, R.E. Kirsch. (2003) Administration of oral activated charcoal in variegate porphyria results in a paradoxical clinical and biochemical deterioration. British Journal of Dermatology 149:6, 1266-1269
CrossRef2
J.Thomas Hindmarsh, Linda Oliveras, Donald C. Greenway. (1999) Biochemical differentiation of the porphyrias. Clinical Biochemistry 32:8, 609-619
CrossRef3
Frank, McGrath, Poh-Fitzpatrick, Hawk, Christiano. (1999) Mutations in the translation initiation codon of the protoporphyrinogen oxidase gene underlie variegate porphyria. Clinical and Experimental Dermatology 24:4, 296-301
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Yolanda V. Scarlett, David A. Brenner. (1998) Porphyrias. Journal of Clinical Gastroenterology 27:3, 192-198
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JOSEPH R BLOOMER. (1998) Liver metabolism of porphyrins and haem. Journal of Gastroenterology and Hepatology 13:3, 324-329
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Michael R Moore. (1998) The Biochemistry of Heme Synthesis in Porphyria and in the Porphyrinurias. Clinics in Dermatology 16:2, 203-223
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(1995) Technical Report. Clinical Chemistry and Laboratory Medicine 33:7, 453-464
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Karl Jacob, Manfred O. Doss. (1995) Excretion Pattern of Faecal Coproporphyrin Isomers I—IV in Human Porphyrias. Clinical Chemistry and Laboratory Medicine 33:12, 893-902
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Jun-Bi Tu. (1994) Theory and practice of psychopharmacogenetics. American Journal of Medical Genetics 54:4, 391-397
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G. J. J. Beukeveld, L. Meerman, J. R. Huizenga, E. E. A. Venekamp-Hoolsema, C. H. Gips, B. G. Wolthers. (1994) Determination of Porphyrins in Bile Using High Performance Liquid Chromatography and Some Clinical Applications. Clinical Chemistry and Laboratory Medicine 32:3, 153-160
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C. LONG, S.J. SMYTH, J. WOOLF, G.M. MURPHY, A.Y. FINLAY, R.G. NEWCOMBE, G.H. ELDER. (1993) Detection of latent variegate porphyria by fluorescence emission spectroscopy of plasma. British Journal of Dermatology 129:1, 9-13
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