ANALYTICAL
BIOCHEMISTRY
84,
173-178 (1978)
A New Rapid Method for Isolation of Naturally Occurring Porphyrins and Their Quantitation after High Performance Liquid Chromatography Z. University
J. PETRYKA
of Minnesota
AND
C. J.
WATSON
Unit for Teaching and Research at Northwestern Minneapolis. Minnesota 55407
Received
Hospital,
April 4, 1977: accepted August 29, 1977
High performance liquid chromatography (HPLC) has been used to separate porphyrin methyl esters isolated from porphyric patients. Fecal porphyrins were esterified directly with boron trifluoride; urinary porphyrins were absorbed on talcum and then treated with boron trifluoride. HPLC permits rapid, efficient, and quantitative detection and identification of porphyrins in complex natural mixtures.
The isolation and quantitative determination of porphyrins of urine and feces have always been complex and time consuming. Technical developments have led progressively to simpler, faster, and more specific methods. In the main, evolution has been via the solvent fractionation method (SFM) (l), countercurrent distribution (2), column chromatography of free or esterified porphyrins after preliminary SFM (3,4), or thin-layer chromatography (tic) (5), followed by spectrodensitometry (SD) (6,7) and, more recently, high performance liquid chromatography (HPLC) (8- 12). Successive improvements have shortened the time of analysis from 1 or 2 days to 1 or 2 hr. The change in the analytical method has also involved change in preliminary treatment of the samples; originally slow extractions (1) and esterification involving varying loss have been supplanted by a more direct method of preliminary treatment, followed by a single analytical technique. The recent methods (89) of direct esterification with methanol/ HzS04, separation on HPLC, and final quantitation require 25-36 hr (8). In the present study a new method is described for isolation and esterification of urinary and fecal porphyrins, followed by HPLC and spectrophotometric quantitation (13); the new method is very rapid and requires only 2-3 hr. MATERIALS
AND METHODS
The reagents were all of analytical reagent grade with the exception of HPLC solvents purchased from Burdick and Jackson Laboratories, Inc. Boron trifluoride in methanol was purchased from Aldrich Chemical Co. 173
0003-2697/78/0841-0173$02.00/O Copyright “1 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
174
PETRYKA
AND
WATSON
Porphyrin esters (5) were used as standards. A PPorasil HPLC column, L/sin. in diameter, was used. The solvent system for porphyrin separation was: benzene:ethyl acetatechloroform (70:10:20, v/v/v). The solvent flow initially at 2 ml/min was changed to 3 ml/min after 5-COOH porphyrins were eluted. HPLC instrument Model 6000A and the HPLC columns have been purchased from Waters Associates, Inc., Milford, Mass. A Beckmann Model 25 spectrophotometer equipped with a micro-flow cell was used as a detector and was set at 400 nm. Also, this instrument was equipped with a stop-flow switch which permitted scanning of the absorption spectrum of each one of the eluted porphyrins. Fecal porphyrin quantitation. The method is shown schematically in Fig. 1. Feces (1 g) are mixed with BF, in methanol (15 ml) and refluxed for 20 min on a hot water bath; after cooling to room temperature, chloroform (200 ml) is added, and the mixture is shaken well. The precipitate is filtered off and’washed three times with chloroform (or until no red fluorescence
Chloroform 1 4
Ir.ction rilt.~ with
thxe,,gh. chloroform
s"mpr.t.
to &yn...
Di..olv. inr..u?d
I
"“6
tiltor
the
for
dry vvlly
..tur.r.d in
residue Of
"ecu0
sdvmht
BPLC
&mly.. qu.utativ.1y qu.ntit.tiv.1y 0
.a6 mP3.c
I
FIG. 1. Schematically fecal porphyrins.
shown,
the method
for esterification
and isolation
of urinary
and
HPLC QUANTITATION
OF NATURAL
PORPHYRINS
175
is detectable in the chloroform). The chloroform washes and the main filtrate are combined; the precipitate is discarded. The chloroform filtrate is washed once with 1% ammonia and once with water, filtered through chloroform-saturated filter paper, and evaporated to dryness at 50°C. The dry residue is dissolved in a measured volume of the solvent used for HPLC. Five or ten microliters of the solution is injected into the HPLC instrument for the separation. Eluted porphyrins are recorded as absorbance peaks on a spectrophotometer; peak heights are measured, concentrations are read from the standard curves, and the total porphyrin amount in the sample is calculated. Urinary porphyrin quantitation. Urine (100 ml) acidified with glacial acetic acid to pH 5 is mixed with talcum powder (15 g), well shaken, and left standing at room temperature for 15 min. The collected talcum powder is filtered off (there should be no red fluorescence in the residual urine), mixed with BF, in methanol (30 ml), and refluxed on a hot water bath for 20 min. After cooling to room temperature, chloroform (200 ml) is added and the mixture is shaken well. The precipitate is filtered off and washed three times with chloroform (until no red fluorescence is detectable in chloroform washes). The washes and the main filtrate are combined; the
FIG. 2. Recorded tracing of a mixture of 2-, 4-, 5-, 6-, 7-, and Scarboxylic porphyrin methyl esters. The spectrophotometric detector was set at 400 nm. Change in flow rate after S-COOH is necessary for faster elution and more pronounced peaks.
176
PETRYKA
AND WATSON
precipitate is discarded. The chloroform filtrate is washed once with 1% ammonia and once with water until neutral, filtered through a filter saturated with chloroform, and finally evaporated to dryness. The dry residue is dissolved in a measured volume of the HPLC solvent and quantitated as described above for fecal porphyrins. RESULTS AND DISCUSSION
The calibration curves were prepared for protoporphyrin, coproporphyrin, uroporphyrin, and tri-, penta-, hexa-, and heptacarboxylic porphyrin methyl esters. An example of the elution scan is shown in Fig. 2. The calibration curves were prepared for areas under each peak and for the height of each peak. A very good correlation has been found between peak heights and the amount of porphyrin used, as shown in Fig. 3; therefore, the calibration curves plotted against the heights of peaks have been used throughout. The variation for three runs was less than 2%, except for very small quantities (less than 50 ng); particularly for porphyrins with a larger number of carboxylic groups, it increased to 5% (see Fig. 3). The efficiency of the esterification of porphyrins added to feces and urine varied from 90 to 97%. The total time required to obtain the essential data,
FIG. 3. Calibration curves for standard porphyrin esters. Number of carboxylic groups is indicated by each curve; heights (millimeters) of the peaks are plotted against micrograms of porphyrins.
HPLC
QUANTITATION
OF NATURAL
177
PORPHYRINS
as above, varied from 2.25 to 3 hr which is quite advantageous in comparison with the other previously mentioned methods (l-7), including the reported HPLC method (8). The detector flow through the column can be stopped, and the absorption spectrum of any eluted porphyrin or any other absorbing compound can be scanned; therefore, any experimental error is avoided. The flow rate after S-COOH porphyrins has been changed from 2 to 3 mYmin in order to retain the shape of the peaks for porphyrins with six to eight carboxyl groups and to shorten the elution time. The same effect can be achieved with a solvent gradient (8); however, this requires a special attachment, and in our experience, the change of flow rate was a reasonable and effective solution. HPLC for analytical and preparative separation of porphyrins has already been used by a number of students of porphyrin chemistry. A Symposium on Application of HPLC in Clinical Chemistry (10) contained a few new methods for porphyrin separation. Within the last year methods for porphyrin separation have been described in several publications (8,9). The present method has been designed especially as an analytical method for quantitative determination of the various porphyrins in urine and feces in respect to diagnosis and basic aspects of porphyria. Special significance of the method lies in quantitation offecal porphyrins, as the available meth-
FIG.
4. Calibration
millimeters)
is plotted
curves for standard against nanomoles
porphyrin esters; of porphyrin.
area under
each peak
(square
178
PETRYKA
AND WATSON
ods, at least within practical limits, are laborious and, as in the SFM, provide information as to the usually dominant porphyrin(s), i.e., uro-, copro-, and proto-groups. Newer methods for diagnosis of porphyria refer to profiles of excreted porphyrins (6,8- 10); the profiles of urinary and fecal porphyrins characterize more precisely the different types of porphyria. The analytical results of the above HPLC method provide the profiles and also the exact quantitative values for each porphyrin. Slight modification of the method will make it applicable to quantitation of porphyrins in blood and tissues. The profiles of the porphyrins excreted suffice for diagnosis of porphyria and type represented. Porphyrins characteristic of porphyria cutanea tarda and porphyria variegata (isocoproporphyrins and porphyrin x, respectively) have been isolated. They can be separated and quantitated (8) in the appropriate fraction obtained by HPLC. Molar extinction coefficients for porphyrins with four to eight COOH groups are very similar (14); therefore, the calibration curves from Fig. 3 expressed as area under the peaks against nanomolar amounts of porphyrins are in close proximity (Fig. 4). ACKNOWLEDGMENTS Supported by a grant from the Alice Tweed Tuohy Foundation, Santa Barbara, and by individual grants from Mrs. Vera Bowman and Mrs. Eugene Leonard of Minneapolis.
REFERENCES 1. Schwartz, S., Berg, M. H., Bossenmaier, I., and Dinsmore, H. (1960) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 8, pp. 221-293, Interscience, New York and London. 2. Falk, J. E., and Dresel, E. L. B. (1960) Biochim. Biophys. Acta 39, 458. 3. Martinez, C. A., and Mills, G. C. (1971) Clin. Chem. 17, 199. 4. Watson, C. J., Schwartz, S., and Hawkinson, V. (1945) J. Biol. Chem. 157, 345. 5. Cardinal, R. A., Bossenmaier, I., Petryka, Z. J., Johnson, L., and Watson, C. J. (1968) J. Chromatogr. 38, 100-105. 6. Grosser, Y., and Eales, L. (1973) S. Afr. Med. J. 47, 2162. 7. Novacek, V. M. (1973) Amer. Lab. 5 (3), 85. 8. Evans, N., Jackson, A. H., Matlin, S. A., and Towill, R. (1976) J. Chromatogr. 125, 345-355. 9. Horchner, P., and Rietveld, T. (1976) J. Chromatogr. 123, 414-418. 10. Evans, N., Jackson, A. H., Matlin, S. A., and Towill, R. (1976) in High Pressure Liquid Chromatography in Clinical Chemistry (Dixon, P. F., Gray, C. H., Lim, C. K., and Stoll, M. S., eds.), pp. 71-78, Academic Press, London. 11. 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. 79-86, Academic Press, London. 12. Carlson, 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. 87-96, Academic Press, London. 13. Petryka, Z. J., and Watson, C. J. (1976) Fed. hoc. 35, 1459. 14. Smith, K. M. (1975) Porphyrins and Metalloporphyrins, Elsevier, Amsterdam.
BIOCHEMISTRY
84,
173-178 (1978)
A New Rapid Method for Isolation of Naturally Occurring Porphyrins and Their Quantitation after High Performance Liquid Chromatography Z. University
J. PETRYKA
of Minnesota
AND
C. J.
WATSON
Unit for Teaching and Research at Northwestern Minneapolis. Minnesota 55407
Received
Hospital,
April 4, 1977: accepted August 29, 1977
High performance liquid chromatography (HPLC) has been used to separate porphyrin methyl esters isolated from porphyric patients. Fecal porphyrins were esterified directly with boron trifluoride; urinary porphyrins were absorbed on talcum and then treated with boron trifluoride. HPLC permits rapid, efficient, and quantitative detection and identification of porphyrins in complex natural mixtures.
The isolation and quantitative determination of porphyrins of urine and feces have always been complex and time consuming. Technical developments have led progressively to simpler, faster, and more specific methods. In the main, evolution has been via the solvent fractionation method (SFM) (l), countercurrent distribution (2), column chromatography of free or esterified porphyrins after preliminary SFM (3,4), or thin-layer chromatography (tic) (5), followed by spectrodensitometry (SD) (6,7) and, more recently, high performance liquid chromatography (HPLC) (8- 12). Successive improvements have shortened the time of analysis from 1 or 2 days to 1 or 2 hr. The change in the analytical method has also involved change in preliminary treatment of the samples; originally slow extractions (1) and esterification involving varying loss have been supplanted by a more direct method of preliminary treatment, followed by a single analytical technique. The recent methods (89) of direct esterification with methanol/ HzS04, separation on HPLC, and final quantitation require 25-36 hr (8). In the present study a new method is described for isolation and esterification of urinary and fecal porphyrins, followed by HPLC and spectrophotometric quantitation (13); the new method is very rapid and requires only 2-3 hr. MATERIALS
AND METHODS
The reagents were all of analytical reagent grade with the exception of HPLC solvents purchased from Burdick and Jackson Laboratories, Inc. Boron trifluoride in methanol was purchased from Aldrich Chemical Co. 173
0003-2697/78/0841-0173$02.00/O Copyright “1 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
174
PETRYKA
AND
WATSON
Porphyrin esters (5) were used as standards. A PPorasil HPLC column, L/sin. in diameter, was used. The solvent system for porphyrin separation was: benzene:ethyl acetatechloroform (70:10:20, v/v/v). The solvent flow initially at 2 ml/min was changed to 3 ml/min after 5-COOH porphyrins were eluted. HPLC instrument Model 6000A and the HPLC columns have been purchased from Waters Associates, Inc., Milford, Mass. A Beckmann Model 25 spectrophotometer equipped with a micro-flow cell was used as a detector and was set at 400 nm. Also, this instrument was equipped with a stop-flow switch which permitted scanning of the absorption spectrum of each one of the eluted porphyrins. Fecal porphyrin quantitation. The method is shown schematically in Fig. 1. Feces (1 g) are mixed with BF, in methanol (15 ml) and refluxed for 20 min on a hot water bath; after cooling to room temperature, chloroform (200 ml) is added, and the mixture is shaken well. The precipitate is filtered off and’washed three times with chloroform (or until no red fluorescence
Chloroform 1 4
Ir.ction rilt.~ with
thxe,,gh. chloroform
s"mpr.t.
to &yn...
Di..olv. inr..u?d
I
"“6
tiltor
the
for
dry vvlly
..tur.r.d in
residue Of
"ecu0
sdvmht
BPLC
&mly.. qu.utativ.1y qu.ntit.tiv.1y 0
.a6 mP3.c
I
FIG. 1. Schematically fecal porphyrins.
shown,
the method
for esterification
and isolation
of urinary
and
HPLC QUANTITATION
OF NATURAL
PORPHYRINS
175
is detectable in the chloroform). The chloroform washes and the main filtrate are combined; the precipitate is discarded. The chloroform filtrate is washed once with 1% ammonia and once with water, filtered through chloroform-saturated filter paper, and evaporated to dryness at 50°C. The dry residue is dissolved in a measured volume of the solvent used for HPLC. Five or ten microliters of the solution is injected into the HPLC instrument for the separation. Eluted porphyrins are recorded as absorbance peaks on a spectrophotometer; peak heights are measured, concentrations are read from the standard curves, and the total porphyrin amount in the sample is calculated. Urinary porphyrin quantitation. Urine (100 ml) acidified with glacial acetic acid to pH 5 is mixed with talcum powder (15 g), well shaken, and left standing at room temperature for 15 min. The collected talcum powder is filtered off (there should be no red fluorescence in the residual urine), mixed with BF, in methanol (30 ml), and refluxed on a hot water bath for 20 min. After cooling to room temperature, chloroform (200 ml) is added and the mixture is shaken well. The precipitate is filtered off and washed three times with chloroform (until no red fluorescence is detectable in chloroform washes). The washes and the main filtrate are combined; the
FIG. 2. Recorded tracing of a mixture of 2-, 4-, 5-, 6-, 7-, and Scarboxylic porphyrin methyl esters. The spectrophotometric detector was set at 400 nm. Change in flow rate after S-COOH is necessary for faster elution and more pronounced peaks.
176
PETRYKA
AND WATSON
precipitate is discarded. The chloroform filtrate is washed once with 1% ammonia and once with water until neutral, filtered through a filter saturated with chloroform, and finally evaporated to dryness. The dry residue is dissolved in a measured volume of the HPLC solvent and quantitated as described above for fecal porphyrins. RESULTS AND DISCUSSION
The calibration curves were prepared for protoporphyrin, coproporphyrin, uroporphyrin, and tri-, penta-, hexa-, and heptacarboxylic porphyrin methyl esters. An example of the elution scan is shown in Fig. 2. The calibration curves were prepared for areas under each peak and for the height of each peak. A very good correlation has been found between peak heights and the amount of porphyrin used, as shown in Fig. 3; therefore, the calibration curves plotted against the heights of peaks have been used throughout. The variation for three runs was less than 2%, except for very small quantities (less than 50 ng); particularly for porphyrins with a larger number of carboxylic groups, it increased to 5% (see Fig. 3). The efficiency of the esterification of porphyrins added to feces and urine varied from 90 to 97%. The total time required to obtain the essential data,
FIG. 3. Calibration curves for standard porphyrin esters. Number of carboxylic groups is indicated by each curve; heights (millimeters) of the peaks are plotted against micrograms of porphyrins.
HPLC
QUANTITATION
OF NATURAL
177
PORPHYRINS
as above, varied from 2.25 to 3 hr which is quite advantageous in comparison with the other previously mentioned methods (l-7), including the reported HPLC method (8). The detector flow through the column can be stopped, and the absorption spectrum of any eluted porphyrin or any other absorbing compound can be scanned; therefore, any experimental error is avoided. The flow rate after S-COOH porphyrins has been changed from 2 to 3 mYmin in order to retain the shape of the peaks for porphyrins with six to eight carboxyl groups and to shorten the elution time. The same effect can be achieved with a solvent gradient (8); however, this requires a special attachment, and in our experience, the change of flow rate was a reasonable and effective solution. HPLC for analytical and preparative separation of porphyrins has already been used by a number of students of porphyrin chemistry. A Symposium on Application of HPLC in Clinical Chemistry (10) contained a few new methods for porphyrin separation. Within the last year methods for porphyrin separation have been described in several publications (8,9). The present method has been designed especially as an analytical method for quantitative determination of the various porphyrins in urine and feces in respect to diagnosis and basic aspects of porphyria. Special significance of the method lies in quantitation offecal porphyrins, as the available meth-
FIG.
4. Calibration
millimeters)
is plotted
curves for standard against nanomoles
porphyrin esters; of porphyrin.
area under
each peak
(square
178
PETRYKA
AND WATSON
ods, at least within practical limits, are laborious and, as in the SFM, provide information as to the usually dominant porphyrin(s), i.e., uro-, copro-, and proto-groups. Newer methods for diagnosis of porphyria refer to profiles of excreted porphyrins (6,8- 10); the profiles of urinary and fecal porphyrins characterize more precisely the different types of porphyria. The analytical results of the above HPLC method provide the profiles and also the exact quantitative values for each porphyrin. Slight modification of the method will make it applicable to quantitation of porphyrins in blood and tissues. The profiles of the porphyrins excreted suffice for diagnosis of porphyria and type represented. Porphyrins characteristic of porphyria cutanea tarda and porphyria variegata (isocoproporphyrins and porphyrin x, respectively) have been isolated. They can be separated and quantitated (8) in the appropriate fraction obtained by HPLC. Molar extinction coefficients for porphyrins with four to eight COOH groups are very similar (14); therefore, the calibration curves from Fig. 3 expressed as area under the peaks against nanomolar amounts of porphyrins are in close proximity (Fig. 4). ACKNOWLEDGMENTS Supported by a grant from the Alice Tweed Tuohy Foundation, Santa Barbara, and by individual grants from Mrs. Vera Bowman and Mrs. Eugene Leonard of Minneapolis.
REFERENCES 1. Schwartz, S., Berg, M. H., Bossenmaier, I., and Dinsmore, H. (1960) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 8, pp. 221-293, Interscience, New York and London. 2. Falk, J. E., and Dresel, E. L. B. (1960) Biochim. Biophys. Acta 39, 458. 3. Martinez, C. A., and Mills, G. C. (1971) Clin. Chem. 17, 199. 4. Watson, C. J., Schwartz, S., and Hawkinson, V. (1945) J. Biol. Chem. 157, 345. 5. Cardinal, R. A., Bossenmaier, I., Petryka, Z. J., Johnson, L., and Watson, C. J. (1968) J. Chromatogr. 38, 100-105. 6. Grosser, Y., and Eales, L. (1973) S. Afr. Med. J. 47, 2162. 7. Novacek, V. M. (1973) Amer. Lab. 5 (3), 85. 8. Evans, N., Jackson, A. H., Matlin, S. A., and Towill, R. (1976) J. Chromatogr. 125, 345-355. 9. Horchner, P., and Rietveld, T. (1976) J. Chromatogr. 123, 414-418. 10. Evans, N., Jackson, A. H., Matlin, S. A., and Towill, R. (1976) in High Pressure Liquid Chromatography in Clinical Chemistry (Dixon, P. F., Gray, C. H., Lim, C. K., and Stoll, M. S., eds.), pp. 71-78, Academic Press, London. 11. 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. 79-86, Academic Press, London. 12. Carlson, 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. 87-96, Academic Press, London. 13. Petryka, Z. J., and Watson, C. J. (1976) Fed. hoc. 35, 1459. 14. Smith, K. M. (1975) Porphyrins and Metalloporphyrins, Elsevier, Amsterdam.
