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Determination of Urinary Porphyrin by DEAE-Cellulose Chromatography and Visual Spectrophotometry

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Determination of Urinary Porphyrin by DEAE-Cellulose Chromatography and Visual Spectrophotometry A n n a l s o f C l i n i c a l a n d L a b o r a t o r y S c i e n c e , Vol. 4, No. 1 Copyright © 1974, Institute for Clinical Science Determination of Urinary Porphyrin by DEAE-Cellulose Chromatography and Visual Spectrophotometry MARTHA I. WALTERS, P h .D.* Ohio Valley Hospital, Steubenville, OH 43952 ABSTRACT A simple, reliable and rapid method for the isolation and determination of urinary porphyrins is described. Both uroporphyrin and coproporphyrin are quantitatively adsorbed by DEAE cellulose. Urobilinogen, urobilin, and bili­ rubin are removed with sodium acetate. The isolated porphyrins, eluted with 1 N HC1, are measured spectrophotometrically, and the excretion is related to that of creatinine. Porphyrin excretion was studied in normal adults and children, and in patients with disorders of porphyrin metabolism ( e.g., in lead poisoning, hemolytic anemia, hepatic disease and porphyria) in order to eval­ uate the method. The coefficient of variation of replicate analyses of a sample containing 200 ¡xg porphyrin per gm creatinine was 3.5 percent. The correlation coefficient between 83 duplicate analyses was 0.993. Introduction or adsorption onto an adsorbant followed Patients with porphyria show great in­ by elution. Methods combining extraction creases in the excretion of uroporphyrin, and adsorption have been the most com­ coproporphyrin and their precursors indi­ monly used,3’12 but procedures employing cating a primary abnormality of porphyrin ion-exchange chromatography,11’13 electro­ synthesis (figure 1). In addition to these phoresis,4’10 and thin-layer chromatogra­ disorders, there are a number of diseases phy15’16 also have been described. Most of showing moderate increases of excreted these methods do not yield a clean separa­ coproporphyrin. These diseases, known as tion of porphyrin from other pigments or the porphyrinurias, include hemolytic ane­ are complicated and time-consuming to mia, hepatic disease and lead poisoning. perform. Because of the wide variety of conditions After separation, spectrophotometric or where porphyrin excretion is increased, it fluorometric methods have been used to would be of value to devise a simple, rapid measure the concentration of the porphyrin. and accurate laboratory procedure for the Filter fluorometric methods tend to be non­ detection of urinary porphyrins. specific and have the drawback that non­ There are several approaches for isolat­ porphyrin fluorescence is difficult to detect. ing porphyrins, such as solvent-extraction Spectrophotometric and spectrofluoromet- * Present address: Consolidated Biomedical ric methods, on the other hand, are based Laboratories, Columbus, OH 43216. on measurements at several wavelengths so 2 9 3 0 WALTERS Glycine the remaining bilirubin. The porphyrins are ----------- ► AL A -v—AI A Succinate a l a s |AL.or then eluted with HC1 and the concentra­ tion of porphyrin in the eluate is calculated PBGy-PBG I ppeDa from spectrophotometric absorbances using Heme * Rimington’s equations.14 A Series-Ill ---------(tetrapyrro le) | porphyrin: I PROTO •< --------COPRO --------URO I R eagen ts Serie s- I porphyrin : DEAE cellulose (0.2 gm). Packets of URO cellulose* are preweighed to be available for use. I COPRO Sodium acetate (25 percent-saturated). excreted Distilled water is added to a bottle of F i g u r e 1 . The general pathway of porphyrin and heme synthesis (ALA—S-aminolevulinic acid, sodium acetate, and the mixture is wanned PBG—porphobilinogen, S—synthetase, D—dehy- to 60° until all of the crystalline material is drase, Da—deaminase) . 6 ’ 7 in solution. The mixture is cooled slowly to room temperature, then one volume of that corrections can be made for non­ the saturated supernatant solution above porphyrin absorbance or fluorescence. Rim- the crystals is mixed with three volumes of ington’s procedure, involving visible spec­ distilled water. trophotometry, eliminates the need for HCl (2 N ). Approximately 167 ml of con­ standards since absorptivities and correc­ centrated HC1 are diluted to 1000 ml with tion factors have been well established.14 distilled water. Spectrofluorometric techniques are con­ HCl (I N). The 2 N HCl is diluted 1:1 sidered to be highly sensitive and thus well- with distilled water. adapted for detecting low levels of por­ HCl ( 0.001 N ). The 1 N HCl is diluted phyrin; however, the availability of the 1:1000 with distilled water. spectrofluorometer instrumentation in most clinical laboratories limits the use of this Special Apparatus technique. A narrow band-pass (<2 nm) spectro­ The method presented in this article photometer with a one cm cuvette is re­ utilizes cellulose column chromatography quired. It is imperative that the instrument to separate the porphyrins from hemoglobin be checked regularly for wavelength ac­ and the bile pigments. Porphyrin in the curacy with didymium or holmium oxide eluate is measured spectrophotometrically. filters. The combined chromatographic, spectro- The chromatographic column is con­ photometric procedure is a quantitative structed by pulling a taper on one end of a method that can be completed in less than 13 mm ID X 300 mm glass tubing. Just 15 minutes. before use, a small loose plug of glass wool is inserted into the end of the tube and dry Principle cellulose is added without packing. A piece An aliquot (10 ml) of a 24-hour urine of rubber tubing and a screw clamp are sample is passed through a DEAE-cellulose fitted onto the tapered end of the tube. column where both uroporphyrin and Distilled water is added to wet the cellu­ coproporphyrin are quantitatively ad­ lose, and the screw clamp is closed with sorbed. A water wash of the column re­ the liquid level at the top of the cellulose moves hemoglobin and some of the yellow column. chromogens. A sodium acetate wash then removes urobilin, urobilinogen and most of * Eastman. DETERMINATION OF URINARY PORPHYRIN 31 Procedure A 24-hour urine sample is collected with­ F i g u r e 2. Typical spectral absorption out a preservative, the total volume (TV) curves of DEAE-cel- is measured and the creatinine concentra­ lulose column eluates tion is determined. If the urine contains a containing high con­ centrations of copro­ precipitate, 2 N HC1 is added to adjust the porphyrin (402 nm pH below 5, and the mixture is warmed to maximum) or uropor­ 37°. The pH of the clarified urine sample phyrin (405 nm max­ imum ). Solid line, is readjusted to approximately 7. Ten ml from a highly pig­ (UV) of urine is then carefully added mented urine sample down the side of the prepared column. The of a patient with liver disease. Broken line, screw clamp is opened to allow for a steady from a patient with flow of droplets. Keeping fluid in the col­ acute intermittent umn, the urine is followed by one 1 0 ml porphyria. water wash and two 1 0 ml washes with w a v e l e n g t h , sodium acetate. Additional sodium acetate washes may be required if the effluent The amount of porphyrin excreted per stream of highly pigmented samples is not day is then related to creatinine excretion colorless. After a final wash with 10 ml of (as determined by the Jaffe reaction) and 0.001 N HC1, the screw clamp is closed. is expressed as ¡xg prophyrin per gm cre­ Elution of the porphyrins is begun by add­ atinine. ing 2.5 ml of 2 N HC1 and collecting about one ml of eluate into a 15 ml graduate. The S u b jects screw clamp is closed for three to five min­ In order to evaluate the DEAE cellulose utes to allow elution to occur. While ob­ method, urine samples were collected from serving with a long-range UV lamp, the 15 apparently healthy adults who were 18 eluted porphyrins are completely washed to 56 years of age, and from 15 apparently into the graduate with no fewer than two healthy children who were one to 14 years 2.5 ml aliquots of 1 N HC1. The volume of of age. Samples were also collected from the eluate (EV) is recorded, and absorb­ 53 clinical patients and grouped into 47 ances at 380, 400 to 406 and 430 nm are porphyrinurias and 6 porphyrias. The por­ read against a water reference. phyrinuria group had 27 patients who were children (ages one to 8 years) with lead Calculations poisoning, 10 patients (ages five to 58 The wavelength in the Soret region (fig­ years) with acute hemolytic anemia and 10 patients (ages 15 to 78 years) with liver ure 2 ) where maximum absorbance of the disease. In the group with liver disease eluate occurs determines which of Riming- were two with hepatitis, three with chole­ ton’s equations applies: 14 lithiasis and five with alcoholic cirrhosis. 402, ng coproporphyrin/day Urine samples from all 10 of the patients with liver disease were positive for bili­ 2 A max (A38O "I- A 430) ^ EV TV rubin. In the group of six porphyrias, four = 1.832 X 0.673 ÏÏV had acute intermittent porphyria and two had porphyria cutanea tarda. Results of 405, ug uroporphyrin/day porphyrin excretion for the normal adults 2 Amaj (A 380 + A 430) v EV v Vp-tr and children and the patient groups are 1.944 X 0.6 A UV A presented in tables I, II, III, and IV. 3 2 WALTERS T A B LE I P o r p h y r in E x c r e t io n in N o r m a l A d u l t s a n d C h il d r e n Adults Children ng/day ng/gm age I sex ng/day pg/gm age/sex 26* 27f 18/F 41 31 5 /F 59 31 30/M 18 34 10/F 24 35 35/F 47 35 14/F 60 41 26/F 15 52 5 /F 49 46 27/F 41 81 5 /F 56 51 27/M 1 1 84 8 /M 26 52 21/F 2 85 2 /F 82 53 20/M 28 89 3/M 56 57 19/F 2 2 96 6 /M 8 8 72 24/M 6 107 1/M 75 78 .
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