High-pressure liquid chromatography of plasma free acid porphyrins

ANALYTICAL

BIOCHEMISTRY

104, 268-276

(1980)

High-Pressure Liquid Chromatography Plasma Free Acid Porphyrins] MARIA

0. LONCAS

AND MAUREEN

Received

September

of

B. POH-FITZPATRICK

6. 1979

The separation and quantitation of plasma free acid porphyrins by high-pressure liquid chromatography and fluorescence is described. Porphyrins were extracted from plasma in a simple manner with a recovery >90%. They were separated by high-pressure liquid chromatography on a silica gel (10 pm) column. using a gradient of acetone:dilute acetic acid. Resolution of seven free acid porphyrin standards including coproporphyrins I and 111, bur not uroporphyrins I and III. was achieved in 12 min at picomolar concentrations. Plasma of patients with erythropoietic protoporphyria displayed protoporphyrin. Uroporphyrin was the only porphyrin found in plasma of eight patients with porphyria cutanea tarda. Normal plasma contained small amounts of uroporphyrin and/or traces of protoporphyrin.

Plasma free acid porphyrins have been identified and quantified mainly by the absorbance of partially purified porphyrins (I -3) corrected by a factor derived from the Rimington-Sveinsson formulas (4,5) to account for absorption due to contaminants. This technique has been criticized because the use of such factors in the quantitation of very low concentrations of porphyrins (as in plasma) increases the possibility of error (6). In addition, this method does not differentiate porphyrin isomers. Plasma porphyrins have also been determined as their methyl ester derivatives by thin-layer chromatography (tic)’ and fluorodensitometry (7.8). These methods, although they provide useful information, present the risk of incomplete esterification and porphyrin alteration or derivatization (7,9). ’ This work was supported by Research AM 18549 from the National Institutes of Health. Y Abbreviations used: tic, thin-layer chromatography; hplc. high-pressure liquid chromatography: EPP. erythropoietic protoporphyria: PCT. porphyria cutanea tarda: VP, variegate porphyria. 0003.26971801080268.09$02,00/O Copyright i(: 1980 hy Academic Pre\\. Inc. All righta of reproductmn m any form resel-ved.

High-pressure liquid chromatography (hplc) has been recently employed for the separation and quantitation of porphyrin methyl esters derived from urine and feces (IO- 16) and free acid porphyrins derived from urine (9,16). This technique has not been used to analyze plasma porphyrins. In this communication we describe a procedure for the purification of porphyrins from plasma of patients with porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP). and normal persons, in their free acid form, under conditions that preserve their absorbant and fluorescent characteristic and yield more than 90% recovery. The method is simple. It involves extractions, hplc. and fluorescence techniques for porphyrin resolution and quantitation. In a second paper, we will describe the more difficult purification of porphyrins from plasma of patients with variegate porphyria (VP). MATERIALS

AND METHODS

Heparinized or titrated blood from porphyric patients or normal people was cen268

PLASMA

FREE

ACID

PORPHYRIN

HIGH-PRESSURE

FIG. 1. Typical appearance of p&ma mixed with an equal volume of ethyl acetate:acetic acid (4: I.viv) followed by incubation of 0°C for 30 min and centrifugation at 4°C and 27,OOOq for IO min.

trifuged at 4°C and 27,OOO
LIQUID

CHROMATOGRAPHY

269

Purijcrrtiou (If’ plusr~tr porphyrirrs. Frozen plasma was thawed at 37”C, mixed with an equal volume of ethyl acetate:acetic acid (4: l.v/v) (18). placed in ice for 30 min, and centrifuged at 4°C and 27.000!: for IO min. Upon centrifugation. two liquid phases and two precipitates separated (Fig. I). The upper. ethyl acetate phase was placed aside, and the aqueous phase was extracted twice with 1 vol of ethyl acetate:acetic acid. The precipitates were combined and washed twice with ethyl acetate:acetic acid. using one-half of the original plasma volume. All the ethyl acetate samples were pooled. and porphyrins were then extracted from this pool with 1.5 N HCI, using one-fourth of its volume (19). As expected the aqueous acid phase then contained most of the porphyrins as evidenced by its red fluorescence under ultraviolet light. The organic phase was extracted further until no fluorescence was detected in the HCI layer. Two extractions proved to be sufficient to remove >95r+ of the porphyrins from the organic phase. The pH of the porphyrin-HCI solution was adjusted to 3.0 k.O.2 with saturated sodium acetate. and the porphyrins were recovered by extraction with ,V-butanol (70). using one-third of the volume of the aqueous phase. Under these conditions. one extraction removed >95’Y of the porphyrins. The butanol was then evaporated under vacuum over a 37’C bath. The dry porphyrins were reconstituted in a minimal volume of acetone:O. I 1\; HCI ( IO: I. v/vi ( 161,passedthrough a 0.45-lrn FH Millipore filter to remove the salt that formed upon evaporation of the solvent, and analyzed by hplc. Unless otherwise indicated, purification was performed at room temperature under red incandescent illumination. An outline of this procedure appears in Scheme I. b#icic~u~v

of’ pf),;l,ll~ritl

c.vtr-trc,tiom.

In

order to determine the yield of porphyrins extracted by the method outlined in Scheme I. known concentrations of free acid

270

LONGAS

AND

porphyrin standards were added to normal plasma which was negative for porphyrins by spectrofluorometric analysis of the diluted plasma (21) or by a rapid quantitative microfluorometric assay (22). The plasmaporphyrin mixture was incubated at 37°C for 30 min to allow porphyrin association with other plasma components (2325). The amount of porphyrins used ranged from 0.1 to 0.9 Fg/ml, and the type was determined by the porphyric plasma to be imitated. Thus, the preparation imitating the plasma of patients with EPP was normal plasma made 0.4 to 0.9 pg/ml in protoporphyrin, since the concentration of this porphyrin in the plasma of patients with EPP laid within these limits (Table 1).

POH-FITZPATRICK

The plasma of patients with PCT was imitated by adjusting normal plasma to 0.1-0.25 pg/ml in uroporphyrin. .Porphyrins were extracted as described above and analyzed by hplc. The picomolar quantities recovered were determined by a standard curve prepared using known porphyrins (Fig. 3). In 10 preparations, the yield was always >90%. Preparation oj:fLer-wid porphyrin stared urds. A mixture of porphyrin standards consisting of 8- to 4-carboxylic porphyrins of the isomer I series, coproporphyrin III, and mesoporphyrin (a dicarboxylic porphyrin migrating as protoporphyrin), 0.04 nM in each porphyrin, was dissolved in acet0ne:O.l N HCl for analysis by high-

PLASMA

FREE

ACID

PORPHYRIN

HIGH-PRESSURE

liquid

~hrotttcrto~rccplt~.

Dry porphyrins were reconstituted in acetone:O. 1 N HCl(l0: 1.v/v) ( 161, and analyzed in a Perkin-Elmer series 3 liquid chromatograph equipped with an LC-55 UV-VIS digital spectrophotometer, a Model 56 dualpen recorder and a Rheodyne 7105 loop injector valve (175-11.1capacity) from Rheodyne, California. Porphyrins were detected by absorbance at 403 nm and by fluorescence in a Perkin-Elmer fluorescence spectrophotometer Model 240A connected to the high-pressure liquid chromatograph and equipped with an R212F photomultiplier detector, and a xenon lamp power supply Model 150. A Perkin-Elmer Silica A 10 pm (0.26 x 25 cm) column was the stationary phase. Elution was performed

RESULTS Porphyritt stmtlwis. Figure 2 shows a typical resolution of the porphyrin standard mixture obtained with this technique in 12 min. Although coproporphyrins I and III are separable, uroporphyrins I and III could not be similarly distinguished whether chromatographed separately or together in solution. The peaks for 8- and 7-carboxyl porphyrins were usually close together. I~Pttti~c.Lltiotis

Subject

no.”

I. Pool of 10 2. 3. 4. 5. 6. 7. 8. 9. IO. Il. Pool of 4 12. 13. 14. 15. 16.

Type of plasma Normal PCT PCT PCT PCT PCT PCT PCT PCT EPP EPP EPP EPP EPP EPP EPP

Volume used (ml) 200 20.0 20.0 20.0 20.0 20.0 20.0 20.0 80.0 3.0 5.0 2.4 I.5 2.0 0.7 5.4

FREL

trtiti qtuitttitutioti

of‘plrrsttitr

The method for the isolation of plasma porphyrins outlined in Scheme 1 can be completed in 3 h. The concentrations of

porphyritls.

TABLE PLGhlA

271

CHROMATOGRAPHY

for 25 min with a linear gradient of acetone:dilute acetic acid (2-90% A) as follows: acetone:0.23 N acetic acid (70:30. v/v) delivered from pump B, and 10% acetic acid delivered from pump A in the reverse pump exchange mode.

pressure liquid chromatography. In separate experiments uroporphyrin 1 and uroporphyrin III were similarly analyzed. both separately and together. High-Pressure

LIQUID

ACID

1 PORPHYRIYS

Final volume (ml) 2.7 0.2 0.7 0.2 0.2 0.2 0.2 0.2 2.0 0.3 0.5 0.3 0.2 0.2 0.1 0.5

Porphyrin identified” Uroporphyrin Protoporphyrin Uroporphyrin Uroporphyrin Uroporphyrin Uroporphyrin Uroporphyrin Uroporphyrin Uroporphyrin Uroporphyrin Protoporphyrin Protoporphyrin Protoporphyrin Protoporphyrin Protoporphyrin Protoporphyrin Protoporphyrin

” Patients whose plasma was employed for porphyrin purification. Ir Determined by their retention time (t H ) on the hplc chromatograms. as compared acid porphyrin standards (Figs. 2 and 4); and by their maximal excitation and emission j Estimated on a standard curve of known free-acid porphyrins (Fig. 3).

Porphyrin concentration’ (&ml) 0.0016 0.0005 0.1’0 0. I50 0. I35 0.095 0.094 0.024 0.056 0.106 0.100 0.403 0.37’ 0.562 I.062 I.110 0.646

to the f H of known wavelengths (Table

free 2).

272

LONGAS

AND

POH-FITZPATRICK

plasma porphyrins determined by this technique (Table 1). except those of normal plasmas. are very close to the values obtained by the rapid microextraction and fluorometric method of Poh-Fitzpatrick and DeLeo (22). As illustrated in Fig. 4 and Tables 1 and 2, protoporphyrin was the only porphyrin detected in the plasma of 7 patients with EPP; uroporphyrin was the only porphyrin found in 8 plasma specimens from patients with PCT. and the porphyrins extracted from a pool of normal plasma from 10 different persons were uroporphyrin and traces of protoporphyrin. These results, except those obtained from the plasma of patients with PCT. are in agreement with the data reported by With (7) and by Day et al. (8). DISCUSSION The method presented in this paper utilized known techniques for the purification of porphyrins from biologic materials (18-201, which were modified to optima1 conditions for the isolation of pure freeacid porphyrins from plasma. For example, in the course of this work, we found that all porphyrins (2COOH to 8-COOHI in aqueous solutions at pH 3.0 2 0.20 pass into the organic phase when these solutions are shaken with N-butanol. Using the method outlined in Scheme 1, efficient extraction into such an aqueous solution was observed in all plasmas from normal or porphyric individuals tested, except in the case of plasma from patients with VP. These findings insured recovery of all the porphyrins from these plasmas, except from the plasma of patients with VP, free of contaminants that absorb light near 400 nm, or alter porphyrin fluorescence (Fig. 4, Table 2). The resolution of free acid porphyrins required 10% acetic acid as one of the eluting solvents (Fig. 2). Therefore, the instrument was always flushed with meth-

0

2

4

6

8

IO

12

14 16

MINUTES

FIG.

?.

phyrins Each

by peak

Resolution high-pressure represents

of 0.04

standard liquid nmol

free acid chromatography. of porphyrin

at a flow rate of 0.5 mlimin at room Full-scale deflection was 0.05 absorbance nm and I mV. The point of injection by the arrow. tions are described

All

other chromatographic in the text.

poreluted

temperature. units at 403 is indicated condi-

anol at the end of a day’s work. This was done to avoid corrosion of its metallic parts by chlorine ions leached from solvents such as chloroform or from acetone: 0.1 N HCI (lO:l, v/v) used to dissolve the porphyrins. Analysis of the N-butanol-porphyrin extracts by hplc generally resulted in poor porphyrin resolution. However, porphyrin from the plasma of patients with EPP

PLASMA

FREE

ACID

PORPHYRIN

HIGH-PRESSURE

LIQUID

273

CHROMATOGRAPHY

10.0 9.0 80

t

0

5

I, I3

lo

20

1 25

I, 30

35

I 40

I 45

PICOMOLES

I 50

I 55

11 60

65

I 70

I 75

81 80

05

11 90

95

I 100

OF PORPHYRIN

FIG. 3. Calibration curves of protoporphyrin (0) and uroporphyrin I KJ) obtained after high-pressure liquid chromatography of these standard free acid porphyrins. The ordinate represents the areas of the porphyrin peaks tin cm”) determined by fluorescence. The abscissa indicates the picomoles used. ChromatosraDhic conditions are described in the text and legend to Fig. 2. and Ruorometric conditions are outlined in the legend to Fig. 4. I

1

could be identified and quantified in Nbutanol because this plasma contained only protoporphyrin, whose retention time, absorbance, and fluorescence were not affected in N-butanol. It is interesting that each of the plasma specimens from eight patients with PCT displayed only uroporphyrin: even though some samples were analyzed several times, and porphyrins were concentrated by IOOfold (Table 1). The same results were obtained using direct esterification of porphyrins in plasma followed by tic of the esterihed porphyrins (7). This finding indicates either that the plasmas from patients with PCT used in our study contained only uroporphyrin. or that the amount of any other porphyrin present was below the threshold concentration detectable by the procedures employed. It is also possible that small amounts of 7-carboxyl porphyrin may have been hidden by the overwhelming peak of the 8-carboxyl porphyrin present. The discrepancy between the plasma porphyrins from patients with PCT determined by the method outlined in this communica-

tion and those reported by other investigators might also result from errors due to partial esterification, porphyrin structural alteration during purification, or real natural variability in the amounts and types of plasma porphyrins of these patients (7-9, 16.27). Analysis of individual normal plasmas by other investigators (7,8) as well as in this laboratory revealed that some specimens contained small quantities of uroporphyrin and others small amounts of protoporphyrin. uroporphyrin being more frequently found .I( The pool of normal plasma used in our investigations showed no porphyrins by other methods (2 1.22). Nonetheless, 0.00 I6 pg of uroporphyrin and 0.0005 pg of protoporphyrin/ml of plasma could be detected after the porphyrins were concentrated by IOO-fold (Table I). These results from normal plasma are in accord with those previously reported (7.8), and tend to con” Longas, M. 0.. and Poh-Fitzpatrick. published observation>.

M. B.. Un-

274

LONGAS

a , , , , , , , , , 0 2 4 6 8 IO 12 14 16

AND

cl,,

POH-FITZPATRICK

, ~,

0

2

4

/ ( ( ,

6

8

IO I2

=,

14 16

I

0

2

4

6

8

IO 12 14 16

MINUTES FIG. 4. Elution profile of free 0. I N HCI (IO: 1,v/v) t 17) and

acid plasma porphyrins. Dry injected in the high-pressure

at 403 592-612

nm.

recorded and

ditions

are

(rj om

= fluorescence for the excitatton

described

in the

text

and

with EPP. (b) plasma. Notice

Porphyrins extracted that normal plasma

concentration

(in

achieved ograph

by using (Table

&ml)

is

a larger

plasma

very

at 120 emission legend from shows

nmimin and wavelengths.

to Fig.

porphyrins liquid

10 mV with respectrvely.

2. tar Porphyrins

low

compared for

to the

other

purification

PCT. peaks

plasmas and

larger

in acetone: = absorption

bandpasses of 390-410 Chromatographic

isolated

plasma of patients with significant fluorescence

volume

were reconstituted chromatograph. (y)

from

plasma

tcj Porphyrins (c). although

analyzed injection

(Table in the

and con-

of patients from normal its porphyrin II. hplc

This

was

chromat-

I j.

firm our findings in the plasmas of patients with PCT. Since the porphyrins present in the plasmas of the patients with PCT were also concentrated by lOO-fold, traces of any porphyrin ~0.0005 pgiml should have been observed with the possible exception of 7COOH porphyrin. An attempt was made to resolve standard uroporphyrin isomers I and III. Unfortunately, under the conditions of the present work, these isomers eiuted as a single peak and their resolution represents a major project. Therefore. it was not possible to determine the isomer(s) of uroporphyrin present in the plasmas analyzed. The resolution and quantitation of plasma free acid porphyrins by high-pressure liquid

chromatography and fluorescence proved to be accurate. efficient, and reproducible. The high sensitivity of fluorescence detection and the simplicity of the method present the attractive possibility of utilizing this technique for clinical purposes not only with plasma specimens but also with other biologic materials as well.3 It is noteworthy that under the conditions described in this paper, hplc resolved mixtures of several different free acid porphyrins including isomers I and III of coproporphyrin in 12 min (Fig. 2). This is a rather quick and reliable technique applicable to identification and quantitation of types of porphyrins in biologic specimens which characterize the several porphyrias.

PLASMA

FREE

ACID

PORPHYRIN

HIGH-PRESSURE ‘TABLE

LIQUID

27s

CHROMATOGRAPHY

2

Excitation” Sample” Protoporphyrin

IX

Uroporphyrin

I (free

(free

acid

acid

standard)

from the extracts

plasma of patients with EPP: or eluted from tic plates’

Porphyrins

from

plasma

of patient5

.Y-butanol extracts or eluted Porphyrins from normal plasma: eluted from tic plates”

tnml

407-409

604 597759X

40.5 - 406

standard)

Porphyrins ,Y-butanol

the

with

JOY

ho4

3OS-406

5Y7-59x

PCT:

from tic plates’ ‘L’-butanol extract\.

407.5

1~ I.5

At K, of uroporphyrin” At K, of protoporphyrin

Emission”

Illrnl

5Y6

405 40’)

IX

- 2

SYX 604 -

” Porphyrins employed all cases.

in :V-butanol

ranged from the maximal

or acetone:

0.0016 to 0.05 &ml. and excitation and emission

” Determined on a Perkin-Elmer supply Model 10. a recorder

power were: length

slitwidth. of 0.5

0. I 3 HCI

X nm:

Model

scanning.

120

(10: I. v/v)

several (three wavelengths

and

IO mV

M. 0.. on tic

wish

to thank

scientific

and P&Fitzpatrick. plates.

M.

B..

unpublished

Hunter, 3.

Mrs.

Julia

Amrr.

Einbinder

5. 6.

./. C’lirr. With. T. eases:

for

her

M.

C. I~.., and

help-

R.. Thompson. J. A. A.. and

Kitchell.

B. S.

G. G.. Allen. Parker. S. (1973) E. I 1976),!.

k/i,t.

Proceedings.

R.,

I.

A..

2. ‘09-Z Porphyrin

16. in

International

Charalambides,

and A.

12.

Evans.

13.

‘Towill. 355. Gray. C.

Dts-

A..

a xenon lamp conditions cell

with

a path

and

H..

(1975)

N..

Jackson.

H..

Lim.

M.

Diseases:

S. (1976)

Proceedings.

Meeting. (Doss, M.,

H..

pp. York.

R. (1976)

Stall.

B/~,c~/tr~~r!.

1st. Freiburg, and Nouraeki,

Karger. Basel. H.. Matlin. S.

A.,

i/r High Pressure in Clinical Chemistry

C.

eds.1.

J. (1978)

Lim.

C.

71-77. A.

K..

C.

H.,

K.,

i/r High-Pressure

and

Stoll.


and

Press.

S. A..

./. ,J/ (‘/r~~j~rrtrro,<,r.

125,

Nicholson.

Liquid

and

Liquid t Dixon,

and 34SD. c‘.

Chromatography

in Clinical Chemistry (Dixon, P. F., Gray. Lim, C. K.. and Stall. M. S., eds.), pp.

C. H.. 79-85.

1975 279-

1-I.

Academic Press, London/New Petryka. A. J.. and Watson. BirK/lcrr/. 84, 173- 17X.

C.

York. J. t 1978)

A/rlll.

463-

15.

Carlson,

D.

(1976)

High-

1.. t 197x1 Jones.

C.

Porphyrin 1975

Gray.

t 1976) Porphyrin

Eales.

F..

R.

pp. 179-180. Jackson, A.

M. S.. London/New

(‘ht~rr~.

Human

May

P.. eds.). Evans. N..

P.

Ridge.

in Human

Towill. R. Chromatography

R.. C/i/t

S. L.. Rimington. C.,and Barnes. H. D. Sc~clrrd. /. C’lirr. Ltrh. frll~c~sr. 1, 2-I I. C., and Sveinsson. S. L. t l9_50) .S(.~III,/.

467. 8. Day. R. S., Pimstone, N. R., int. ./. 5i
B.

14. 213-215.

Lrth. frrl~rst. K. (1976) irr

I I.

36, 31-36.

Meeting. 1st. Freiburg. Germany, May (Doss, M.. and Nouraeki. P., eds.). pp. 280, Karger. Base]. 7. With. ‘I‘. K. ( 1977) C/i,!. SC,;. ,‘llr,/. Zlr
9.

In

observations.

International Germany.

J. C‘/;,I. Ptrthcd.

.sc,i. 45, 711-713. lwanov, E.. and Drenska, Sveinsson, (1949) Rimington.

in a cylindrical

i/f Porphyrin

F. S.. Davis,

RirKfrcvrr. 4.

The concentrations

for each determination. were identical,

equipped with Spectrotluorometric

temperature

Magnus.

discussions.

I. Schlenker.

7. Moore.

were used porphyrin

.I. 173. 693-696. IO. Lim. C. K.. Gray.

REFERENCES (1961)

I .5 x HCI.

spectrophotometer detector R777.

at room

ACKNOWLEDGMENT We

wjith

cm.

’ Longas. ” Mobility

ful

diluted

to five) sample\ of a particular

MPF-44A fluorescence .S6 and a photomultiplier

nm/min

were

K..

Pressure Chemistry C. K.. Academic

R.

E..

and

Dolphin,

in

Liquid Chromatography in Clinical (Dixon. P. F.. Gray, C. H., Lim, and Stall. M. S.. ed\.i, pp. 87-9s. Pres\. London/New York.

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J. C/ill.

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H. L. ( 1950) J. C‘lirl.

M., and Watson. 147, 615-684.

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Int~cst. .I. Ei~jl.

20. Fernandez, A. A.. Henry. R. J.. and Goldenberg. H. (1966) (./;!I. c’h~~/,~. 12. 463-474. 21. Poh-Fitzpatrick, J. Ltrh. Clirr.

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A. A. (1976)

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