An internal standard for porphyrin analysis

ANALYTICAL

BIOCHEMISTRY

l&$360-365

(1984)

An Internal Standard

for Porphyrin

Analysis

ROBERT E. CARLSON, RAMAM SIVASOTHY,DAVID DOLPHIN,’ MELVYN BERNSTEIN,* AND ALLY SHIVJI* Department of Chemistry, University of British Columbia, Vancouver, British Columbia MT 1 Y6, and *Department of Pathology, University of British Columbia, Vancouver, British Columbia V6T 1 W5. Canada Received December 20, 1983 The routine clinical analysis of the porphyrin precursors of heme requires an internal standard to determine the efficiency of the analytical procedure used. 2-Vinyl-4-hydroxymethyldeuteroporphyrin IX has been prepared as an internal standard. Its application to porphyrin analysis has been demonstrated using high-performance liquid chromatographic resolution of the uroto protoporphyrins in normal and potphyric urine.

The biosynthesis of heme, iron protoporphyrin IX, involves the octacarboxylate uroporphyrinogen III, which undergoes sequential decarboxylations through the hepta-, hexa-, and pentacarboxylate porphyrinogens to the tetracarboxylate porphyrinogen coproporphyrinogen III. This, in turn, undergoes two successive oxidative decarboxylations to give protoporphyrinogen IX which is then enzymatically oxidized to protoporphyrin IX (1) (1). The porphyrias are a group of metabolic diseases involving the heme biosynthetic pathway, and some of these diseases are characterized by overproduction of these macrocyclic intermediates in a specific pattern (2). The porphyrinogens are readily oxidized to the corresponding porphyrins and the analysis of these porphyrins from body fluids, tissue and excreta provides a method for the clinical diagnosis of the prophyrias based on porphyrin accumulation and excretion pattern (3). The routine clinical analysis of these compounds requires that the quantity, in addition to the pattern, of excretion be measured. This, in turn, requires the use of an internal standard. Since all of the clinically important porphyrins contain at least two carboxyl groups,

it is essential that an internal standard contain this functional group. This ensures that the efficiency of manipulations which involve the carboxyl groups (e.g., esterification) can be monitored. Furthermore, the vinyl groups of protoporphyrin are especially labile, being sensitive to both acid-catalyzed hydration, giving I’-hydroxyethyl side chains, and to photochemical autoxidation via singlet oxygen (4). Porphyrins are excellent photosensitizers for ‘02 - ‘02; thus protoporphyrin catalyzes its own destruction. In order to ensure that photochemical degradation is not occurring, an internal standard should contain a vinyl side chain. In addition, the internal standard should be a porphyrin with X,,, and t,,, similar to the uro- through protoporphyrins. Finally, the internal standard cannot be a naturally occurring porphyrin. Thus mesoporphyrin (Z), which has been suggested as an internal standard (3), is unacceptable since it can be formed by the microbial degradation of protoporphyrin in feces (2). This paper describes the preparation and use of 2-vinyl-4-hydroxymethyldeuteroporphyrin (3), which is readily prepared from protoporphyrin IX (1) as an internal standard for porphyrin analyses.

’ To whom correspondence should be addressed.

0003-2697184 $3.00 Copyright Q 1984 by Academic Press, Inc. All ri#tts of npmduclion in any form remved.

360

AN INTERNAL

STANDARD

FOR PORPHYRIN

ANALYSIS

361

1) NoBH,

and

MATERIALS

6, the ring isomer

A

AND METHODS

Preparation of 2-vinyl-4-hydroxymethyldeuteroporphyrin IX dimethyl ester (3J2 A solution of sodium borohydride (50 mg) in methanol (2 ml) was added to a stirred solution of the mixed 2,4-positional isomers of formylvinyldeuteroporphyrin dimethyl ester (7 and 8) (50 mg; prepared from protoporphyrin IX via photoprotoporphyrin using a modification (5) of the method of Inhoffen et al. (4)) in dichloromethane (25 ml). The solution was stirred for 1 h. The reaction was monitored by thin-layer chromatography on silica gel (dichloromethane/ether, 20/l, v/v; &of start2 The internal standard (3) is available from Porphyrin Products, Logan, Utah.

ing material, 0.68; product, 0.05). On completion of the reaction, excess borohydride was destroyed by the dropwise addition of acetic acid. The organic phase was washed with water

362

CARLSON

and dried over sodium sulfate, and the solvent was removed in vacua. The mixed hydroxymethyl isomers (3 and 6) were separated by chromatography on silica gel using dichloromethane/ether (10/l, v/v) as eluant. The isomer which eluted first was the required 2vinyl-4-hydroxymethyldeuteroporphyrin IX dimethyl ester (3) (60% isomer yield). Anal. Calcd for C37H40N406: C, 70,69; H, 6.44; N, 9.10. Found: C, 71.04; H, 6.27; N, 9.66. X,, E(log) 403 (5.19) 502 (4.12), 536 (4.00), 572 (3.83), 628 (3.65). ‘H NMR 6 (100 MHz), 3.24 (t, 4H, propionate CH& 3.50 (s, 9H, ring methyls), 3.66 (s, CH, ester-CH,), 4.30 (t, 4H, propionate CH& 5.80 (s, 2H, hydroxymethyl CH& 6.24 (m, 2H, vinyl CH& 8.19 (dd, lH, vinyl CH), 9.92 (4S, meso H). Preparation of the internal standard as the dicarboxylic acid. A concentrated solution was prepared by dissolving 0.1 mg of the diester (3) in 1% KOH in methanol (1.5 ml) and shaking overnight, in the dark, in a water bath (37°C). The mixture was then diluted with an equal volume of water, and any unhydrolyzed material was extracted with chloroform (30 ml). The aqueous phase, containing the porphyrin diacid, was used as a stock solution for porphyrin analysis. The stock solution was stored at 4°C in the dark. No change was observed in the internal standard over a period of 5 months when prepared and stored under the above conditions. Urine sample preparation for normal phase chromatography. The preparation of the urine sample for porphyrin analysis was begun by adding 10 ~1 of the internal standard solution to 5 ml of urine. HCl (5 N) was then added to the specimen to a final pH of 2.9 f 0.1. The urine sample was then added to a l-g acid-washed florisil column. The sample was washed onto the column with 2 ml of 1 N HCl and washed with 5 ml of 1 N HCl. It was further washed with 5 ml of ethanol/l N HCl (19/l, v/v), followed by 2 ml of methanol. The porphyrins were then eluted with ammonium hydroxide/methanol ( l/ 19, v/v). The eluate was esterified with HzSOdmethanol, 1/

ET

AL.

20, v/v (overnight reaction) (6), followed by dilution with 5 ml of water and extraction with dichloromethane (2 X 5 ml). The organic phase was washed with ammonium bicarbdnate solution, evaporated to dryness under a gentle stream of air, and finally dissolved in ethyl acetate for analysis by HPLC. Extraction of porphyrins from biological materials for reverse-phase chromatography. Porphyrins from urine, stool, and liver homogenates were extracted and purified, alter the addition of a known aliquot of the internal standard, using Sephadex G-l 5 (7). Samples were acidified with HCl to pH 2 and applied to a column of Sephadex G- 15 preequilibrated with water. Porphyrins containing carboxyl groups bind strongly to the support thus allowing for their separation from proteins and other pigments, which we removed by eluting the column with water. The porphyrin fraction was then eluted with a small volume of acetone/O. 15 N HC1(3/4, v/v), followed by water. Because of the differences in volume gains of Sephadex in acetone and water, the column volume decreases when acetone is used. This effect appears to trap some porphyrins in the hollow matrix of the support. In order to obtain a quantitative recovery it is necessary to follow these steps: (i) elute porphyrins with eluting agent (approx 1 ml for 10-l 5 ml of G- 15); (ii) wash with a small volume of water (0.5-0.75 ml); and (iii) elute again with 1 ml of the acetone/HCl eluant, followed by sufficient water to remove all porphyrins. The porphyrin fraction is then ready for injection into the HPLC system. Chromatography. The isocratic HPLC system has been described previously (7). The isocratic solvent system was composed of liquid chromatography-grade methyl acetate/ heptane (4/6, v/v) at a flow of 1.5 ml/min. The gradient HPLC system was a HewlettPackard 1084b instrument with a variablewavelength detector which was used at 403 nm. A Waters Associates ~Porasil column was used. The solvent flow was 1.5 ml/min. The solvents used were liquid chromatography-

AN INTERNAL

STANDARD

FOR PORPHYRIN

363

ANALYSIS

FIG. 1. HPLC resolution of the uro- to protoporphyrins and the internal standard isomers on normal phase. The uro- to protoporphyrins are identified by their number of carboxylate groups (8-2). The internal standard (3) is IS. The internal standard isomer (6) is ISI.

grade heptane and ethyl acetate. The gradient was composed of Time (min)

Ethyl acetate content

O-8 8-12 12-20

40% Linear gradient 40-60% 60%

Reverse phase analysis was performed using the method described by Ford et al. (8). However, the concentration of the phosphate buffer was reduced to 0.05 M instead of 0.1 M as reported. The flow rate was 1.3 ml/min using a Waters Associates PBondapak C 18 column. Under these conditions the internal standard was well resolved, appearing between coproand mesoporphyrins with a retention time of 16.5 min (Fig. 2). RESULTS

AND

experiments indicated that none of the most readily available or easily synthesized porphyrins (e.g., photoprotoporphyrin (4) or 2,4dihydroxymethyldeuteroporphrin IX (5)) could be used as internal standards because they would interfere with uro- to protoporphyrin quantification. However, our analysis of these compounds allowed us to specifically synthesize a compound which met all the requirements for an internal standard.

DISCUSSION

High-performance liquid chromatography is the method of choice for porphyrin analysis. Consequently, our choice of an internal standard has been based on its use in an HPLC system. The controlling factor in the choice of an internal standard is that its chromatographic mobility must be such that it does not interfere with any of the other porphyrins likely to be found in the sample. Preliminary

rneso

;i0

10

15

zomin

2. HPLC resolution of the uro- to protoporphyrins on reverse-phase. The porphyrins are identified by their number of carboxylate groups (8-2). Mesoporphyrin IX(meso) and the internal standard (IS) are also included. FIG.

364

CARLSON

The internal standard, 2-vinyl-4-hydroxymethyldeuteroporphyrin IX (3) was synthesized from protoporphyrin as outlined in Scheme 1 (5). Figure 1 illustrates the chromatographic relationships of the internal standard and its 2,4-positional isomer (2hydroxymethyl-4-vinyldeuteroporphyrin 1X(6)) on normal phases. The 4-hydroxymethyl compound (3) was chosen as the internal standard because it could be consistently separated from the penta- and hexacarboxylate porphyrins (Fig. 2), while the 2-hydroxymethyl isomer (6) overlapped the hexacarboxylate porphyrin with some of the solvent systems tested. The internal standard also serves an additional useful function because its vinyl sub-

ET AL.

stituent is subject to photooxidation. Consequently, the appearance of a new peak which elutes ca. l-2 min past uroporphyrin octamethyl ester, in combination with a decrease in the internal standard content of the sample, is indicative of conditions which would also be expected to effect the quantitation of protoporphyrin IX. Figure 3 illustrates the application of the internal standard to the analysis of urinary porphyrins from control and porphyric patients. Note that the internal standard does not interfere with the analysis of any of the desired porphyrins and that the quantity of internal standard used is sufficient to give adequate results for both normal and elevated NORMAL

A

,.I! IS

4

0

5

15

TIME ;iIN)

TIME (MIN)

TIME ItdIN)

4 COPROPOW-IYRIA

PORFtiYRIA

CUTANEA (WENT

TARM PHASE]

TIME(MIN)

FIG. 3. HPLC analysis of normal and porphyric urine using the internal standard. The uro- to protoporphyrins are identified by their number of carboxylate groups (8-2). The internal standard is IS. The chromatography was performed on normal phase.

AN INTERNAL

STANDARD

porphyrin samples. In these studies we have chosen to purify urine samples on florisil or Sephadex before HPLC analysis. For those who prefer to inject urine directly into the HPLC system the internal standard can, of course, simply be added before the injection. The internal standard can be used to monitor the recovery of compounds during their isolation and after their separation by chromatography. Using the techniques described under Materials and Methods, it was found that, after florisil chromatography, esterification with methanol and sulfuric acid, and extraction with dichloromethane, there was a quantitative recovery of both the internal standard and all of the porphyrins used in this study (see Fig. 1). With the Sephadex column extraction method it was found that, if these samples were exposed to light, there was a change in the proportion of the internal standard to the uro-copro porphyrins by between lo-15%. Protoporphyrin was also found to decrease by a comparable amount. This reflects the fact that the internal standard, like protoporphyrin, is photosensitive. The internal standard was used during the extraction of porphyrins from a variety of biological samples. The overall recovery of the internal stan-

FOR PORPHYRIN

ANALYSIS

365

dard parallelled that of the porphyrins being extracted, and was over 90% in all cases. ACKNOWLEDGMENTS This work was supported by the United States National Institutes of Health (AM 17989) and the Canadian Medical Research Council.

REFERENCES 1. Bogorad, L. (1979) in The Porphyrins (Dolphin, D., ed.), Vol. VI, pp. 125-l 78, Academic Press, New York. 2. Eales, L. (1979) in The Porphyrins (Dolphin, D., ed.), Vol. VI, pp. 665-804, Academic Press, New York. 3. Gray, C. H., Lim, C. K., and Nicholson, D. C. (1976) in High Pressure Liquid Chromatography in Clinical Chemistry (Dixon, P. F., Gray, C. H., Lim, C. K., and Stoll, M. S., eds.), pp. 79-85, Academic Press, London/New York. 4. Inhoffen, H. H., Brockman, H., and Bleisnev, K. M. (1969) Ann. Chem. 730, 173-185. 5. DiNello, R. K., and Dolphin, D. (1981) J. Biol. Chem. 256, 6903-6912. 6. Carbon, R. E., and Dolphin, D. (1976) in High Pressure Liquid Chromatography in Clinical Chemistry (Dixon, P. F., Gray, C. H., Lim, C. K., and Stoll, M. S., eds.), pp. 37-95, Academic Press, London/ New York. 7. Shivji, A., and Bernstein, M. (1981) Clin. Biochem. 14, P3. 8. Ford, R. E., Ou, C., and Ellefson, R. D. (1981) Clin. Chem. 27,397-401.