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Technical Briefs |
1 MRC Bioanalytical Science Group, School of Biological and Chemical Sciences, Birkbeck College, Malet St., London WC1E 7HX, United Kingdom; fax 44-20-7631-6384, e-mail a.gorchein{at}sbc.bbk.ac.uk
Increased porphobilinogen (PBG) in urine is pathognomonic of an attack or "crisis" of acute porphyria (acute intermittent porphyria, variegate porphyria, hereditary coproporphyria); the absence of increased urinary PBG in a suspected attack excludes the diagnosis. Some patients, especially with acute intermittent porphyria, excrete excess PBG even in remission, but in an attack, this increases above their generally characteristic "basal" concentrations (1). Thus, "screening" of urine for excess PBG and its measurement have an essential role in the initial diagnosis and management of such patients and in their continuing care.
Current screening tests for PBG in urine include the Watson–Schwartz test (2) and derivatives. It is performed by mixing 1 mL of urine with 1 mL of p-dimethylaminobenzaldehyde in acid solution (Ehrlich’s reagent). A reddish-mauve colored compound (
max, 553–555 nm) is formed. The test lacks specificity, however, because of interference from urobilinogen or other endogenous compounds or by drugs and their metabolites. The test also lacks sensitivity, so a concentration of PBG that is 10-fold above normal may be required for detection.
Specificity and sensitivity of the method are increased by adsorbing the PBG on an ion-exchange resin in a column, washing off interfering compounds, eluting the PBG, and reacting it with Ehrlich’s reagent (3). This allows quantification of PBG but this "gold standard" method is relatively time-consuming.
Resin can be directly added to the urine (4), but this method requires time-consuming steps, including centrifugation and transfer of the supernatant to another container. More recently a proprietary screening method (5) has become available that uses a syringe prepacked with ion-exchange resin. The urine sample is adsorbed, washed, and eluted through a series of procedures involving the addition and removal of filters and final color formation with Ehrlich’s reagent. A color chart and a dye color calibrator are provided for visual assessment.
The procedure described here is based on the original method of Mauzerall and Granick (3), but the resin is held in a permeable sack, like a small tea bag, instead of in a column. This provides a sensitive, specific, convenient, and rapid method that can be used for screening and for quantification.
A length of nylon bolting cloth (25-µm mesh size; approximately 25 cm x 25 cm; Sefar; Satake UK Ltd.) was folded over end to end and machine-stitched with cotton thread and a fine needle to provide several rows of sacks (Fig. 1
). A cut with fine scissors 2 mm below the horizontal stitch line of the top row, left open at the top, yielded the next row of open sacks. Each sack was filled with 0.5 mL of resin suspension, which was washed down the side with ion-free water until the upper part of the sack was free of resin. After gentle blotting, each row of sacks was closed with a stitch line; sacks were stored in ion-free water at 4 °C. Individual sacks (12 mm x 24 mm, with 1.5–2 mm margins) were cut as required. After use, the sacks were briefly rinsed with ion-free water and kept in a saturated solution of sodium acetate for batch regeneration of the resin, followed by washing with water; they were reusable for 
4 cycles.
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Urine (1 mL; adjusted, if necessary, to pH 6–8 with aqueous ammonia solution) was dispensed into a 7-mL screw-stoppered plastic bottle (e.g., 7-mL Sterilin), and 1 mL of ion-free water was added. A sack containing Dowex-2 (x8 crosslinked, 400 dry-mesh size; acetate form) was added, and the bottle was shaken for 3 min manually (for multiple samples, a mechanical shaker would be more convenient). The liquid was discarded, and the resin-containing sack was washed twice by shaking briefly with aliquots of 2–3 mL of ion-free water, which were poured off to waste. The PBG was then eluted by shaking the sack for 3 min with 2 mL of 0.2 mol/L acetic acid. The sack was then taken out (to be regenerated and reused as described above). Ehrlich’s reagent (2 mL; 1 g of p-dimethylaminobenzaldehyde in 50 mL of 6 mol/L HCl) was added, the bottle was stoppered, and the contents were mixed rapidly by inversion. Maximum color was reached within 5 min and could be determined spectrophotometrically for quantification or visually by comparison with a color chart according to photographs of quantitative yields.
Urine from 20 anonymous patients receiving drug therapy, including antihypertensive medication (bendrofluazide, amiloride, atenolol, enalapril, ramipril, amlodipine, doxazosin), psychotropic drugs (diazepam, chlorpromazine, prochlorperazine, sertraline), antibiotics (amoxicillin, clarithromycin, metronidazole), as well as omeprazole, alendronate, and gliclazide, were tested. A sample from a patient with obstructive jaundice was also tested. None of the eluates showed any color after reaction with Ehrlich’s reagent, in contrast to two samples from a porphyric patient diluted to give a concentration of 4 µmol/L PBG, run in the same batch. A urine sample from a patient with acute intermittent porphyria in long-term remission, but with continuing excretion of PBG, had a PBG concentration of 25 µmol/L. Serial dilutions were made with nonporphyric urine samples and electronic absorption spectra (450–600 nm) of the Ehrlich reaction products were obtained with a Pye-Unicam SP1800 double-beam recording spectrophotometer. PBG concentrations were calculated from A553. A linear response (y = 23.353x + 0.5017; R2 = 0.9965) down to 2 µmol/L was obtained by spectrophotometry. With visual inspection, concentrations of 3–5 µmol/L were unequivocally detectable.
In another assay, PBG was added to give concentrations of 5, 15, and 25 µmol/L to nonporphyric urine samples, which were then subjected to the entire procedure. Recovery of added PBG was 69–72%, uncorrected for loss in the "dead volume" of the sacks, as described below; duplicate samples differed by 3–4%. The response flattened at PBG concentrations >30 µmol/L, presumably because of a capacity limitation of the resin in the sacks, which was 25% of that used in this author’s application of the column method of Mauzerall and Granick (3), but this could be readily overcome by testing such urine samples at higher dilution.
The procedure removed 
70% of the PBG compared with the column method. A further elution under the same conditions yielded another 15–20%, consistent with retention in the dead volume (0.50–0.55 mL) of the resin-containing sack. Although the total color yield was less than that of column methods after a single elution step with 2 mL of 0.2 mol/L acetic acid, this was offset by the simplicity and increased speed of the procedure.
Columns are generally eluted with at least 3 column volumes, but only 2 mL of eluate is used in the Ehrlich reaction. The reaction concentration with the present method is therefore likely to be greater, with a corresponding increase in sensitivity.
In summary, a simple modification of the method of Mauzerall and Granick (3) extends its application as a sensitive and specific screening test for urinary PBG.
References
-aminolaevulinic acid and porphobilinogen. J Biol Chem 1956;219:435-446.The following articles in journals at HighWire Press have cited this article:
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  M. Roshal, J. Turgeon, and P. M. Rainey Rapid Quantitative Method Using Spin Columns to Measure Porphobilinogen in Urine Clin. Chem., February 1, 2008; 54(2): 429 - 431. [Abstract] [Full Text] [PDF]
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