T H E AMEHICAN JOURNAL OF CLINICAL PATIIOLOGV
Vol. 44, No. 2
Copyright © 1005 by The Williams & Wilkins Co.
Printed in U.S.A.
TOTAL URINARY PORPHYRINS
FRANK S. SCHLENKER, P H . D . , N. ANN TAYrLOR, B.S., AND
CYNTHIA L. KITCHELL, B.S.
Veterans Administration Medical Teaching Group Hospital, Memphis, Tennessee
2.5 N HC1, and 2.5 ml. of acetone-etheracetic acid. The eluate is collected in a
graduated 15-ml. centrifuge tube.
To enhance extraction into the aqueous
acid phase, 2 to 3 ml. of ether are added to
the eluate and the combination is shaken
vigorously. The tubes are kept cool to
minimize pressure developed by the evaporation of ether. Phase separation, an essential
precaution for maximal extraction, is complete in approximately 5 min. The upper
ether layer is removed by suction and the
acid aqueous porphyrin solution is diluted
to 5 ml. with water and mixed.
The absorbance, A, for the diluted eluate
(0.5 N HC1) is found at 380 mM, 430 m/x,
and maximal mju (400 to 406 mju), using
1-cm. Pyrex cuvets, reading against 0.5 N
HC1 in both spectrophotometers.
The capacity of Florisil, activated at 500
F., to adsorb and release porphyrins under
specified conditions, forms the basis of a
procedure for the determination of this class
of metabolites in urine and plasma.1-4
The average recovery of known amounts
of copro- and uroporphyrin isomers I and
III, added to urine and ranging from 1.00
to 10.00 fig. per column, was 86 per cent
calculated as total porphyrin.3 An attempt
was made to improve the completeness of
elution. A Beckman DU was used for
measuring absorbance, A, as was a Bausch
and Lomb Spectronic 505 recording spectrophotometer in this study.
MATERIALS
1. Hydrochloric acid, 2.5 N (analytical
reagent).
2. Cdacial acetic acid (USP).
3. Diethyl ether (USP).
4. Acetone (analytical reagent).
5. Eluting solution acetone-ether-acetic
acid, 5:5:1 (volumes).
6. Florisil, 60 to 100 mesh, activated at
500 F.
CALCULATIONS
1. Corrected A. = 2 A mnXj ,„M — (/1 3S0 ,m
"T" ^1430
PROCEDURE
Air-dried Florisil, 1 ml., is poured into a
7 to 8 mm. by 340 to 350 mm. chromatographic tube with glass wool holding the
adsorbent in place. The column is preconditioned with 1 ml. of 2.5 N HC1. When
it is completely drained of acid, an aliquot
of urine (pH 1 to 2) containing from 1 to 10
Mg- of total porphyrin is added. Entrapped
urine is removed by flushing the Florisil
with 2 to 5 ml. of water. After this washing,
a gentle suction is applied at the lower
orifice which withdraws excess water.
The adsorbed porphyrins are eluted by
adding the following reagents in succession
without mixing above the adsorbent: 2.5
ml. of acetone-ether-acetic acid, 1 ml. of
Received, December 26, 1964.
ni(i)
2. Total porphyrin
a. /ig./ml. diluted eluate = corr. A
X 0.80
b. /ig./ml. urine = corr. A X 0.80 X
eluate dilution, ml.
sample, ml.
c. Mg-/24 hr. = jug./ml. urine X total
24-hr. output, ml.
Porphyrin elution. A number of organic
solvents were used singly and in various
combinations, in an attempt to increase the
yield of porphyrin from Florisil. Also,
stronger organic acids were used to replace
acetic acid in the acetone-ether-acetic acid
reagent. None of these combinations improved recovery of porphyrin.
Eventually a few trials indicated that a
nearly complete elution could be achieved
with the solvents already in use, provided
the acetoue-ether-acetic acid reagent was
added to the column in equally divided
portions of 2.5 ml. each. Before the second
189
190
SCHLENKER
portion was used, however, the column was
reconditioned with 1 ml. of 2.5 N HCl.
Recovery of porphyrin from aqueous solution. In order to test further the reliability
of this technic, methyl esters of coproand uroporphyrin isomers I and III were
hydrolvzed in*7 N HCl for 24 to 36 hr. and
ET
Vol. 44
AL.
diluted to 0.5 N. A mixture containing
equal volumes of the 4 solutions was prepared. This composite solution contained
approximately equal quantities of the copro
and uro isomers. Aliquots containing from
0.3 to 1.56 tig. of total porphyrin were added
to a number of duplicate columns. One
TABLE 1
RECOVERY OF COPHO- AND UROPORPHYRIN
ISOMERS I AND I I I
PROM AQUEOUS SOLUTIONS
Per Cent of Recovery
Single acetone ether-acetic acid*
Divided acetone-ether-acetic acid*
DU
Porphyrin
Range
Average
DU
Ct
Ut
77-97
80
85-97
92
C
81-90
83
U
89-94
91
C
91-114
99
C
93-109
100
U
93-100
98
U
95-100
98
* T h e amount of total porphyrin varied from 0.3 to 1.50 ng. per column.
f Each sample contained approximately equal amounts of isomers I and I I I .
TABLE 2
R A T I O OP SPECTRONIC 505 AND DU
ABSOHBANCE V A L U E S *
Porphyrin Mixture
Sample
< 0.7 Mg./column
Standard, water
Control, urinef
P a t i e n t , urine
> 0.7 pg./column
> 1.0 pg./column
per cent
per cent
per cent
99 ± S.D. 11
102 ± S.D. 10
96 ± S.D. 12
99 ± S.D. 3.5
100 ± S.D. 2.4
101 ± S.D. 3.2
100 ± S.D. 2.8
100 =fc S.D. 2.4
99 ± S.D. 4.4
A, 505
f Laboratory personnel.
TABLE 3
RECOVERY OF T O T A L P O R P H Y R I N FROM U R I N E OF P A T I E N T S *
Per Cent Recovery
ng. per Column
Average Recovery. Summary
tig. per Dayt
Average
Range
0.10-0.27
0.32-0.53
19-32
38-64
121
100
75-176
77-125
0.16-0.53 Mg113 ± S.D. 2 4 . 5 %
0.59-0.87
1.17-1.57
1.54-4.08
74-104
141-189
185-562
100
97
94
83-118
92-107
80-102
0.59-4.68 Mg.
97.0 ± S.D. 7 . 8 %
* Combined results from DU and Spectronic 505.
t Calculated for a 10-ml. aliquot, and daily o u t p u t of 1.2 liters.
Aug. 1965
191
TOTAL URINARY PORPHYRINS
RECOVBKY OP T O T A L PORPHVHIN
FROM U U I N B S OP N O N - P A T I E N T S *
Per Cent Recovery
*ig. per Column
Average Recovery, Summary
Mg. per Dayt
Average
Range
0.12-0.30
0.28-0.54
14-30
34-03
102
110
70-150
89-150
0.70-0.84
1.18-2.58
3.49-9.88
84-100
142-310
40G-118G
99
98
97
84-109
90-105
91-104
0.12-0.54 jug.
100 ± S.D. 24%
0.70-9.SS|ug.
9S ± S.D. 3 . 5 %
* Combined results from D U and Spectronic 505.
f Calculated for a 10-ml. aliquot, and a daily o u t p u t of 1.2 liters.
column was eluted with 5 ml. of acetoneether-acetic acid, and the other was handled
as described under Procedure.
The efficiency of elution can be improved
by adding acetone-ether-acetic acid in 2
portions. The average recovery from pure
solution was increased from 88 to 99 per cent
through the use of the split elution technic
(Table 1).
Recovery of porphyrin added to urine. A
number of specimens of urine were obtained
from patients and non-patients, and amounts
of the combined porphyrin solution, from
0.16 ng to 9.88 Mg-, were added to 10 ml.
of this urine. These mixtures and their
appropriate urine blanks were processed as
outlined above. The eluates were read in
both spectrophotometers. The absorbance
values of the 2 instruments were in good
agreement (Table 2).
The recovery of porphyrin added to a wide
variety of urine specimens was essentially
the same in 2 series using patient and nonpatient urines (Tables 3 and 4). When the
amount of total urinary porphyrin per
column was 0.54 ng. or less, the procedure
gave erratic results as a consequence of the
variable degrees of eluate contamination
by the urinary pigments. In the absence of
pigment, amounts of porphyrin in the 0.3ng. range were obtained from the column
with an efficiency of at least 90 per cent.
Recording spectrophotometer. A continuous
curve furnishes a more comprehensive and
detailed record of the absorption characteristics of a solution. Thus, curves in Figure
440 420 400 380 360 340
I
I
I
F I G . 1. Acid concentration of
all solutions, 0.5 N HC1. Curve 1,
urine diluted 5:0 ml.; curve 2, pure
copro Til diluted 1:0 ml.; curve 3,
5 ml. of urine plus 1 ml. of pure
copro I I I solution; curve 4, same
solution as used for curve 3, but
chroma tographed.
192
SCHLENKER ET AL.
1 illustrate individual aspects of the wave
length-absorbance relation of a randomly
chosen urine in the presence and absence of
2.34 ixg. of copro III, calculated from DU
absorption values. The urine specimen was
adjusted to 0.5 N with concentrated HCl.
Below 356 mix this acid urine (diluted 5 ml.
to 6 ml. with 0.5 N HCl) had an absorbance
greater than 1.0; however, above 356 mix
absorbance decreased rapidly, being 0.65
at 370 mix, and 0.27 at 380 mM (Fig. 1,
Curve 1).
An acid copro III solution (0.5 N) diluted
1 to 6 with 0.5 N HCl gave Curve 2, which
calculated to 2.4 ^g- per ml. One milliliter of
this copro standard was added to 5 ml. of
the acidified urine, and the wave lengthabsorption curve was determined directly
on the mixture. The somewhat lower porphyrin content of this sample, 2.16 jug.,
was the result of the high A at 380 m/x
caused by the presence of urinary pigments
(Curve 3). When a similar combination
was chromatographed, the removal of
pigment during the process resulted in a
lower absorbance at 380 mxi, resulting in a
recovery of 2.4 jug., i.e., all of the added
copro III (Curve 4).
SUMMARY
In previously recommended procedures,
the removal of porphyrin from Florisil aver-
Vol.
44
aged approximately 86 per cent when eluted
with a single 5-ml. aliquot of acetone-etheracetic acid. By elution with two 2.5-ml.
portions of acetone-ether-acetic acid, interspersed with 1 ml. of 2.5 N HCl porphyrin,
recovery from urine was raised to an average
ranging from 97 to 98 per cent when the
quantity of total porphyrin per column was
in excess of approximately 0.6 ng.
Acknoivledgments. Some of the porphyrin esters
used in this s t u d y were synthesized by Dr. S. F .
McDonald; others were isolated from biological
materials by Dr. T. K. With and the present
authors. The esters had melting points and extinction coefficients in agreement with presently
accepted values. Figure 1 was reproduced by H.
Schonert from the original tracings.
REFERENCES
1. Schlenker, F . S., Davis, C. L., and Kitchell, C.
L.: Total content of porphyrin in urine.
Am. J. Clin. P a t h . , 32: 103-106, 1959.
2. Schlenker, F . S., Davis, C. L., and Kitchell, C.
L.: Plasma porphyrin. Am. J. Clin. Path.,
36: 31-36, 1961.
3. Schlenker, F . S., Davis, C. L., and Kitchell, C.
L.: Urinary total, aqueous and ether-soluble
porphyrins. Am. J. Clin. P a t h . , 39: 531-540,
1963.
4. Waldron, H . A.: Plasma porphyrins in lead
workers. Brit. J. Indust. Med., 81: 315-317,
1964.