Clinical Biochemistry, Vol. 32, No. 8, 609–619, 1999 Copyright © 1999 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/99/$–see front matter PII S0009-9120(99)00067-3 Biochemical Differentiation of the Porphyrias J. THOMAS HINDMARSH,1,2 LINDA OLIVERAS,1 and DONALD C. GREENWAY1,2 1Division of Biochemistry, The Ottawa Hospital, and the 2Department of Pathology and Laboratory Medicine, University of Ottawa, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada Objectives: To differentiate the porphyrias by clinical and biochem- vals for urine, fecal, and blood porphyrins and their ical methods. precursors in the various porphyrias and in normal Design and methods: We describe levels of blood, urine, and fecal porphyrins and their precursors in the porphyrias and present an subjects and have devised an algorithm for investi- algorithm for their biochemical differentiation. Diagnoses were es- gation of these diseases. Except for Porphyria Cuta- tablished using clinical and biochemical data. Porphyrin analyses nea Tarda (PCT), our numbers of patients in each were performed by high performance liquid chromatography. category of porphyria are small and therefore our Results and conclusions: Plasma and urine porphyrin patterns reference ranges for these should be considered were useful for diagnosis of porphyria cutanea tarda, but not the acute porphyrias. Erythropoietic protoporphyria was confirmed by approximate. erythrocyte protoporphyrin assay and erythrocyte fluorescence. Acute intermittent porphyria was diagnosed by increases in urine Materials and methods delta-aminolevulinic acid and porphobilinogen and confirmed by reduced erythrocyte porphobilinogen deaminase activity and nor- REAGENTS AND CHEMICALS mal or near-normal stool porphyrins. Variegate porphyria and he- reditary coproporphyria were diagnosed by their characteristic stool porphyrin patterns. This appears to be the most convenient diag- Porphyrin standards were obtained from Porphy- nostic approach until molecular abnormalities become more exten- rin Products Inc. (Logan, UT). All solvents were of sively defined and more widely available. Copyright © 1999 The high performance liquid chromatography (HPLC) Canadian Society of Clinical Chemists grade. KEY WORDS: Porphyria; porphyrins; feces; urine; EQUIPMENT blood; plasma; erythrocytes; renal failure; abdominal pain; neuropathy; erythema; urticaria; photodermatitis. HPLC was performed using a Varian 5500 (Var- ian Canada Inc., Mississauga, ON) equipped with a Introduction Shimadzu RF-1501 spectrofluorometer (Shimadzu Scientific Instruments, Inc., Columbia, MD). The he biochemical differentiation of the porphyrias column was a Perkin-Elmer (Norwalk, CT) reversed Tresides, at present, with the measurement of phase Percospher 3 C18 Brownlee, 33 ϫ 4.6 mm. porphyrins and their metabolites in urine, feces and Data analysis was performed using Dionex A1-450 blood (1). Enzyme tests are often technically difficult chromatography software (Dionex Canada Ltd., and require tissues such as cultured fibroblasts, Oakville, ON). lymphocytes, or liver biopsy material and therefore, except for the use of erythrocyte porphobilinogen PLASMA PORPHYRINS deaminase in acute intermittent porphyria, they are rarely used (2). Genetic analysis produces a precise Plasma porphyrin analysis was performed accord- diagnosis in some of the porphyrias provided the ing to the method of Hindmarsh et al. (4). Heparin- patient has one of the previously described point ized plasma was used, samples being centrifuged mutations, but this technique is currently confined immediately after collection and frozen at Ϫ20° C. to a few research laboratories (3). As a consequence Stock standards were prepared using porphyrin of our role as a major reference laboratory for markers in porphyrin-free plasma obtained from our porphyrin analysis performing approximately 2500 hospital blood transfusion service; 2-vinyl 4-hy- tests per year, we have assembled reference inter- droxymethyl-deuteroporphyrin IX was used as an internal standard. The recovery of individual por- Correspondence: Dr. J.T. Hindmarsh, The Ottawa Hos- phyrins added to porphyrin-free plasma varied from pital, General Campus, 501 Smyth Road, Ottawa, Ontario 89% to 114% except for coproporphyrin-III, whose K1H 8L6, Canada. recovery varied from 71% to 99%. Recoveries of Manuscript received July 13, 1999; revised August 13, protoporphyrin were low, therefore, no attempt was 1999; accepted August 17, 1999. made to quantify this fraction. Between-run preci- CLINICAL BIOCHEMISTRY, VOLUME 32, NOVEMBER 1999 609 HINDMARSH, OLIVERAS, AND GREENWAY sion varied from 5.4% to 13.2%, coproporphyrin-III Urine ALA and porphobilinogen (PBG) were mea- being the least precise. sured by the method of Mauzerall and Granick (9) using a BioRad kit (BioRad Laboratories, Missis- URINE PORPHYRINS sauga, Ontario, Canada). Urine porphyrins were measured by a reversed SCREENING TESTS phase HPLC method based upon that of Johnson et al. (5). To achieve separation of the I and III isomers Urine was screened for increased porphyrin con- of uroporphyrin and coproporphyrin, we substituted tent by spectrophotometric scanning of an acidified their mobile phase with a gradient ofa1mol/L sample between 350 and 415 nm and measuring the ammonium acetate solution (pH 5.16) and metha- peak absorption at 405 nm. The molar absorptivity nol, varying from 27% (starting) to 90% (ending) of was then used to obtain the total porphyrin content methanol V/V. Direct standardization was used with (10) and a “cut off” of 150 nmol/d was used to ensure uroporphyrin-I, uroporphyrin-III, heptacarboxyl-I, a maximal detection of normal subjects. Currently hexacarboxyl-I, pentacarboxyl-I, coproporphyrin-I, in our service laboratory we use a “cut off” of 110 and coproporphyrin-III. Recoveries of standard por- nmol/d to ensure maximal detection of abnormal phyrins added to urine samples varied from 92% to subjects (11). Fecal screening was performed by 115%. Between-run precision for the various frac- extracting interfering substances from an acidified tions varied between 7% and 10%. Samples were sample with diethylether and scanning the remain- preserved at 4° C with 5-g sodium carbonate per ing aqueous fraction in a spectrophotometer. Peak daily collection in brown glass bottles. absorption was measured and a “cut off” of 35 nmol/g wet feces was used (12). We prefer spectrophotomet- FECAL PORPHYRINS ric absorption screening techniques rather than flu- orometric screening methods because we have en- Our fecal porphyrin method that was based upon countered too many false positive results with the the reversed phase HPLC method of Pudek et al. (6), latter. but using direct standardization with uroporphyrin-I, uroporphyrin-III, heptacarboxyl-I, hexacarboxyl-I, Results pentacarboxyl-I, coproporphyrin-I and copropor- phyrin-III, deuteroporphyrin, mesoporphyrin, and Tables 1, 2, and 3 report plasma, urine, and fecal protoporphyrin. We used a 3 ϫ 0.43 cm (ID) cartridge porphyrin and precursor results in a variety of analytical column containing 3-␮m octadecylsilyl par- porphyrias and include our normal reference inter- ticles. The solvent system was a gradient ofa1mol/L vals. The data on Table 1 have been published solution of ammonium acetate and methanol varying previously (4). All data were derived from samples from 27% (starting) to 90% (ending) of methanol V/V. we received in our role as a major porphyrin refer- Recovery of standards added to samples varied from ence laboratory and were checked for proper preser- 75% to 102%. Random samples of feces were preserved vation and transportation (acid pH for urine ALA at Ϫ20° C in opaque containers. Between-run CVs and PBG measurements, alkaline pH for urine por- varied from 10% to 15%. phyrin samples, samples frozen and transported in dry-ice for plasma porphyrins, samples frozen and ERYTHROCYTE PROTOPORPHYRIN wrapped in aluminum foil for fecal porphyrins). All abnormal data include the complete range of results Erythrocyte protoporphyrin was measured by the encountered by our laboratory for a particular dis- method of Piomelli (7). Blood samples were collected ease classification. Disease stratification was made in EDTA tubes (Becton Dickinson Canada, Inc., by an experienced clinician and porphyrin chemist Mississauga, ON) wrapped in aluminum foil and (J.T.H.) after discussion with the referring physician preserved at 4° C. Our between-run CV for this and perusal of porphyrin and precursor patterns in method was 10%. When defining our normal refer- urine and feces and erythrocyte porphobilinogen ence interval, all results from samples with hemat- deaminase activities. ocrit Յ 0.35 were excluded. NORMAL REFERENCE INTERVALS ERYTHROCYTE FLUORESCENCE “Normal” samples were chosen by visual inspec- Erythrocyte fluorescence was demonstrated by tion of the results of all specimens processed. In the briefly viewing an erythrocyte smear (diluted with case of urine and fecal samples, those designated 154 mmol/L saline, if necessary) using an Olympus “normal” had all tested negative by our screening fluorescence microscope with a BG3 (405 nm) exci- tests (10,12). The reference intervals were deter- tation filter. mined as a central 95% interval of ranked data Erythrocyte porphobilinogen deaminase activity using a nonparametric method (13). Modest eleva- was measured in duplicate using delta-aminolevu- tions above the apparently normal range were occa- linic acid (ALA) as substrate (8). Between