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.o r 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 . 56/M 7 140 ll/M 92 80 35/M 26 145 3/F 177 89 33/M 30 193 4/M 79 1 0 1 18/F 23 232 3 /F 139 108 26/F 105 362 2 |/M

( 73 61 28 118 40 24 24 8 6

* Porphyrin excretion calculated as /xg/day. t Porphyrin excretion expressed as #ig/gm creatinine.

Procedure and Discussion between fluorescence and porphyrin con The correlation coefficient (r) compar centration. ing duplicate results found in analyzing 83 Fractions of the effluent stream repre normal and patient samples was 0.993. The senting each stage of the procedure were good correlation between duplicates indi collected from several urine samples which cates that the adsorption, washing and were found to contain very high concentra elution procedures were reproducible. The tions of porphyrin. The solutions, made coefficient of variation for a urine sample 1 N with respect to HC1, were examined containing 200 ¡i.g porphyrin per gm cre spectrophotometrically. None of the frac atinine, analyzed six times, was 3.5 percent. tions from any of these urine samples had This indicates that the method has a satis the spectral characteristics of porphyrin. factory degree of precision. Thin layer chromatography16 was done on During the course of the study, and par solvent extracts prepared from the original ticularly with respect to the urine samples effluent fractions, and none of the chromat from normal children and from children ograms had fluorescent spots. These results with lead poisoning, brilliant fluorescence indicate that porphyrin had not been eluted was often observed in the eluate of a sam from the column prematurely. ple, but the result expressed as ¡¡.g por The wavelength absorption maximum phyrin excreted per day could be very low. was 405 nm for eluates obtained in four of The discrepancy between the observed and the cases of porphyria, suggesting that uro measured data was noted even in samples porphyrin was the predominant pigment that were known to represent adequate 24- present (table IV). Thin layer chromato hour collections. When porphyrin excretion graphic results of the urines and their elu was related to creatinine excretion, how ates indicated that the amount of uropor ever, excellent correlation was observed phyrin present was much higher than that DETERMINATION OF URINARY PORPHYRIN 33

TA BL E II TA BL E III

P o r p h y r in E x c r e t io n in C h i l d r e n P o r p h y r in E x c r e t io n in D i s e a s e s w it h w it h L e a d P o is o n in g D is o r d e r s o f P o r p h y r in M e t a b o l is m

X-ray Hemolytic Anemia Liver Disease Blood Pb History skeletal Porphyrin Age (ttg/dl) of pica survey (ng/gm) 166* 239 178 246 2 50 __— 80 225 270 2 1 40 —— 98 273 314 2 40 + — 104 273 395 3 i 40 + + 115 336 486 4 40 —— 1 2 1 386 513 3 .4 0 + — 130 500 587 2 40 + — 145 3726 696 3 50 + — 147 4580 1176 8 60 + — 161 3 50 + + 163 * ^g porphyrin/gm creatinine. 1 1 60 + + / - 179 50 224 l i + + Acid (2 N HC1) was added to dissolve the 1 60 + — 240 1 90 + — 240 precipitate, and it was found that por 2 40 + — 251 phyrin was not adsorbed onto the cellulose 3 60 + — 289 unless the pH had been readjusted to near 2 80 + + 300 3 70 + + 301 neutrality. Porphyrin was completely ad U 90 + + 307 sorbed from untreated clear urine samples,, 3 50 - — 319 l i 50 + + 339 however, which varied in pH from 5 to 8. 3 50 + + 341 6 80 + + 373 Normal Range l i 60 + + 1135 2 170 + + 2471 Porphyrin excretion found with the 1 60 + + 3111 DEAE-cellulose method for normal adults 2 280 + + 5123 (table I) ranged from 27 to 108 fig per gm creatinine (mean, ±S.D., 61 ± 24 p.g per of coproporphyrin. Application of the uro gm creatinine). Using the conventional porphyrin equation in calculating the spec- means of expressing porphyrin excretion,. trophotometric data necessarily ignores the coproporphyrin present. An extraction T A B L E IV method3 was therefore used to separate the T o t a l P o r p h y r in C oncentrations in t h e P o r p h y r ia s a s D e t e r m i n e d b y B o th D EA E-Cel- porphyrins in these samples, and the sum LULOSE AND ETH E R E x TRACTION-Al UMINA of the individual uroporphyrin and copro A d s o r p t io n T e c h n iq u e s porphyrin concentrations were compared to DEAE- the “total” porphyrin values found in the cellulose Ether extrac Predominant eluates. The percentage difference between method tion method? Isomer16 total concentrations found with the extrac tion and DEAE cellulose methods averaged 500* 375f COPRO 611 573 URO 114 percent (range, 79 to 141 percent). 1618 1357 URO 2530 1789 COPRO Sou rces o f Error 3230 2986 URO 7750 9697 URO Phosphate precipitates in urine and ad sorbs the porphyrins; therefore, it is im * ng porphyrin/gm creatinine calculated using the appropriate Rimington equation. perative that phosphates be in solution t Mg porphyrin/gm creatinine as the sum of th& prior to DEAE-cellulose chromatography. individual uro- and coproporphyrin values. 3 4 WALTERS

the range was from 26 to 177 /xg per day The high excretion of coproporphyrin (mean, ±S.D., 73 ± 40 /xg per day). found in uncomplicated hemolytic anemia Porphyrin excretion in normal children can be explained solely from increased (table I ) ranged from 31 to 362 /xg per gm erythropoietic heme synthesis. In regenera creatinine (mean, ±S.D., 118 ± 86 /xg per tive hemolytic anemia, erythroid hyper gm creatinine). Expressed as /xg per day, plasia may be marked, and erythropoietic the range was from 2 to 105 (mean, ±S.D., activity may be increased to more than six 28 ± 24 fig per day). times that of normal. The two highest values for porphyrin excretion found in the Resume of Clinical Interpretations group with hemolytic anemia (table III) The pathway of porphyrin and heme were obtained from siblings at the time of synthesis, illustrated in figure 1, shows that their sickle cell crises. These exceptionally the porphyrin found in normal urine arises high levels would be consistent with the from the series-I porphyrin. The series-I extremely high erythropoietic activity that porphyrin, which is thought to be a by is characteristic of severe hemolytic anemia. product of series-III porphyrin, is not util The well-known increase of copropor ized by the body but is excreted in bile.6 phyrin excretion in cases of lead poisoning A small amount of the series-I porphyrin is has been shown to be due to disturbances reabsorbed and excreted in urine, where it in heme synthesis.5 Lead inhibits the activ is present primarily as coproporphyrin. ity of a number of enzymes resulting in a Since heme is the prosthetic group of high excretion of both ALA and copropor hemoglobin, myoglobin, the cytochromes, phyrin. Some investigators have attempted catalase and other chromoproteins, high to use elevated ALA and porphyrin levels production of the series-I porphyrin would for the diagnosis of lead poisoning, but they accompany any high activity of heme syn have been unable to find a good correlation thesis. Due to an expanding blood volume, between them and blood-lead levels. This an increase in muscle mass or liver tissue, lack of correlation was found in this study increased porphyrin excretion would there with several children (table II) whose por fore normally be expected in children dur phyrin excretion levels were not markedly ing periods of growth. Hsia and Page8 elevated, e.g., were lower than 200 /xg per showed daily coproporphyrin excretions gm creatinine. Although some of these chil closely correlating with body weight in dren had a history of pica, the X-ray films children and suggested that body weight generally did not show increased density reflected blood volume. They found that of the long bones, yet all of these children children excreted less than 100 /xg of por had blood-lead levels greater than the 40 phyrin per day and that levels were sub /xg per dl which is considered to be the stantially lower than in adults. Similar low threshold limit for toxicity.1 Vincent and values for daily porphyrin excretion were Ullman17 point out the fallacy of trying to found for normal children in this study (2 correlate blood-lead with hematological or to 105 /xg per day). In terms of porphyrin urinary findings because it is tissue-lead per gm creatinine, however, the excretion rather than circulating-lead that exerts an levels shown in table I tended to be higher inhibitory effect on heme synthesis. (range to 363) and more variable (S.D., In a study of alcoholic cirrhosis, Watson ±86) than in adults. It is possible that the et al18 found some correlation between porphyrin:creatinine ratio as a means for urine porphyrin and urobilinogen, but they expressing porphyrin excretion is a more found no correlation between porphyrin realistic reflection of overall heme synthesis and jaundice or ascites. It has long been in growing children. known that there is increased copropor DETERMINATION OF URINARY PORPHYRIN 35

phyrin excretion in liver disease. It is pos 5. G ib s o n , S. a n d G o l d b e r g , A.: Defects in sible that in bile obstruction, porphyrin is haem synthesis in mammalian tissues in ex perimental lead poisoning and experimental merely diverted from the bile and excreted porphyria. Clin. Sci. 38:63-72, 1970. in urine. It may be that urinary porphyrin 6 . G o l d b e r g , A. a n d R i m in g t o n , C.: Diseases of Porphyrin Metabolism, Charles C Thomas, arises in complex liver disease from several Springfield, IL, pp. 27-42, 1962. mechanisms operating simultaneously. In 7. H a r r is , J. W. a n d K e l l e r m e y e r , R. W .: The the present study the urine samples from Red Cell, Production, Metabolism, Destruc all of the cases of liver disease (table III) tion: Normal and Abnormal. The Common wealth Fund, Cambridge, p. 40, 1970. contained bilirubin suggesting that obstruc 8 . H s i a , D. Y. a n d P a g e , M.: Coproporphyrin tion, whether or not intrahepatic or extra- studies in children. I. Urinary coproporphyrin hepatic, was a common component. excretion in normal children. Proc. Soc. Exp. Biol. Med. 85:86-88, 1954. Excessive excretion of porphyrins in the 9. K a u f m a n , L. a n d M a r u e r , H. S.: Biochemi congenital porphyrias is thought to be due cal defects in two types of human hepatic porphyria. New Eng. J. Med. 283:954-958, to defective heme synthesis.2 Kaufman and 1970. Mauer9 found the specific defect in acute 10. L o c k w o o d , W. H. a n d D a v is , J. L .: Paper intermittent porphyria to be enhanced syn electrophoresis of urinary porphyrins. Clin. thesis of ALA in liver tissue and suggested Chim. Acta 7:301-308, 1962. 11. M a r t in e z , C. A. a n d M i l l s , G . C.: Spectro- that the cause of porphyria cutanea tarda fluorometric determination of porphyrins in might be a failure of the feedback mecha urine. Clin. Chem. 17:199—205, 1971. 12. M a u z e r a l l , D. a n d G r a n ic k , S .: The occur nism regulating heme synthesis. Excretion rence and determination of S-aminolevulinic of the porphyrin precursors differ in the acid and porphobilinogen in urine. J. Biol. different types of porphyria, therefore a Chem. 229:435—446, 1956. 13. M i l l s , G . C.: Routine analyses of porphyrins screening test for porphobilinogen alone is in urine. Clin. Chem. 7:165-174, 1961. not adequate for diagnostic purposes. A 14. R im in g t o n , C.: Spectral-absorption coeffi reliable test for porphyrin is essential for cients of some porphyrins in the Soret-band detecting the porphyrias. region. Biochem. J. 75:620-623, 1960. 15. S a m u e l s , S . a n d V e l i z , G . : Diagnosis of a case of acute intermittent porphyria with the R eferences aid of ithin-layer chromatography-thin-layer 1. C h i s o l m , J.: Screening techniques for undue electrophoresis of urine. Clin. Chem. 27:51-52, lead exposure in children: biological and prac 1971. tical considerations. J. Pediat. 79:719-725, 16. S c o t t , C. R., L a b b e , R. F., a n d N u t t e r , J.: 1971. A rapid assay for urinary porphyrins by thin- 2. E l d e r , G . H., G r a y , C. H., a n d N ic h o l s o n , layer chromatography. Clin. Chem. 23:493- D. C.: The porphyrias: a review. J. Clin. Path. 500, 1967. 25:1013-1033, 1972. 17. V i n c e n t , W. F. a n d U l l m a n n , W. W.: The 3. F e r n a n d e z , A. A., H e n r y , R . J., a n d G o l d e n - measurement of S-aminolevulinic acid in urine. b e r g , H .: Assay of urinary porphyrins. Clin. Ann. Clin. Lab. Sci. 2:31-36, 1972. Chem. 22:463-474, 1966. 18. W a t s o n , C. J., S u t h e r l a n d , D., a n d H a w k - 4. F i s c h l , J., E ic h h o r n , F ., R u t t e n b e r g , A., in s o n , V.: Studies of coproporphyrin. V. The a n d M a j o r , C.: Conventional and preparative isomer distribution and per diem excretion of electrophoretic separation of some urinary por the urinary coproporphyrin in cases of cirrho phyrins and porphyrin precursors. Clin. Chem. sis of the liver. J. Lab. Clin. Med. 37:8-28, 26:331-334. 1970. 1951.