it through Whatman. No. 1 filter paper. (Whatman, Inc., Clifton, NJ), and recorded the MCD spec- trum. When necessary, we diluted the final extracts with 1.5.
CLIN. CHEM. 30/3, 391-394(1984)
Rapid Semiquantitative Measurement of Total Porphyrins in Urine and Feces by Magnetic Circular Dichroism Kathryn M. lvanetlch,1coon Movsowltz,’ and Michael R. Moore2 Magneticcirculardichroismis a suitable technique for semiquantitative determination of total porphyrins in urine and feces, being rapid, reliable, and involving little sample preparation. The sensitivity of the assay suffices to distinguish normal from above-normal concentrations of total porphyrins in urineand feces. Resultsfor urinecomparewell with those obtained by thin-layer chromatography of porphyrin esters (n = 42, r = 0.92) or ion-exchangecolumnchromatographyof porphyrinacids (n = 19, r = 0.80). AddItionalKeyphrases:coproporphyrin
uroporphyrin prochromatography, thin-layer chromatography,
toporphyrin ion-exchange
.
.
.
.
screening
.
porphyria
Traditional methods for determining the porphyrin content of urine, feces, and blood are time-consuming and laborious, including such steps as extraction, esterification, and separation by thin-layer chromatography (PLC) or liquid chromatography before quantification (1, 2). Clinically, the information yielded by such tedious assays may be far more detailed than is actually required for screening potential disorders such as the porphyrias and lead poisoning. We therefore sought to develop a rapid, semiquantitative assay for total porphyrins in physiological samples that would be sufficiently sensitive to distinguish between normal and above-normal concentrations of perphyrins in urinary and fecal samples. Magnetic circular dichroism (MCD) appeared suitable for this, in view of reports that urinary uroporphynin and coproporphyrin could be determined rapidly and quantitatively by this technique (3). We have extended the use of MCD to an assessment of the total porphyrin content in urinary and fecal samples from normal and porphyric patients. The reported results are compared with those from two standard techniques for porphyrin measurement involving TLC (4,5) and ion-exchange chromatography (6).
Materials and Methods Materials (and their sources) were: porphobilinogen, urocoproporphyrin, and protoporphyrin (Sigma Chemical Co., St. Louis, MO); hydrochloric acid, solvents, and silica gel TLC plates (Merck Chemicals, Darmstadt, F.R.G.); diethyl ether (certified anesthetic grade; Natal Cane By-Products, Ltd., Natal, R.S.A.); and ion-exchange columns specifically for porphyrins (Bio-Rad Laboratories, Richmond, CA). Urine and feces were obtained from porphyric patients and normal subjects. Materials
porphyrin,
‘Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, C.P., RS.A. 2Porphyria Research Unit, Department of Medicine, University of Cape Town Medical School. Current address: University of Glasgow, Dept. of Medicine, Western Infirmary, Glasgow Gil 6NT, Scotland, U.K. Received July 6, 1983; accepted January 3, 1984.
Methyl esters of the tn-, penta-, hexa-, and heptacarboxyl porphyrins, extracted from the feces of rats treated with hexachlorobenzene, were separated by TLC (4, 5). The respective PLC bands corresponding to the required porphyrin esters were scraped off the plates, extracted with an equivolume mixture of CHC13 and CH3OH, and the solvent was evaporated at 30-35 #{176}C in a rotary evaporator. The porphyrin methyl esters were redissolved in 5 mol/L HC1 and hydrolyzed for 24 h in the dark at room temperature. The resulting free porphyrin acids were diluted and used within 6 h. For absorbance spectroscopy we used a scanning spectrophotometer (Model 5230; Beckman Instrument8, Sunnyvale, CA). In the spectral studies we used cuvettes with 0.5or 1-cm pathlengths. The MCD studies involved use of a Jasco J4OA circular dichroism instrument with a 4-T (4000 G) permanent magnet.
Procedures We determined the concentrations of stock solutions of uroporphyrin, coproporphyrun, and protoporphyrin in 0.41.5 mol/L HC1 spectrophotometnically, using the millimolar absorptivities (L/mmol per cm) of the Soret maxima as follows: uroporphyrin, 541; coproporphyrin, 425;and protoporphyrin, 262 (7). The values for millimolar absorptivities that we used for determination of the remaining porphynns were as follows: heptacarboxyporphyrun, 512; hexacarboxyporphyrin, 483; pentacarboxyporphyrin, 454; and tricarboxyporphyrin, 350 L/mmol per cm.3 To prepare the urine samples for MCD, we acidified them to pH 0.4 with 5 or 12 mol/L HC1 and, where necessary, centrifuged (1500 x g, 5 miii) to remove any precipitate. For comparative purposes, we prepared porphyrin standards by adding known amounts of porphynins in 0.4 mol/L HC1 to acidified urine samples. To determine fecal porphyruns, we weighed between 0.1 and 0.2 g (wet wt.) of feces into a culture tube, noting the exact weight. After adding 5 mL of an equivolume mixture of glacial acetic acid, n-amyl alcohol, and diethyl ether (Dean’s solution), we vortex-mixed the suspension for 30 s, added 5 mL of 1.5 mol/L HC1, and vortex-mixed again for 30 s. After the layers separated, we removed the HC1 (lower) layer, filtered it through Whatman No. 1 filter paper (Whatman, Inc., Clifton, NJ), and recorded the MCD spectrum. When necessary, we diluted the final extracts with 1.5 mol/L HC1. We also prepared porphyrin standards by adding known amounts of porphyruns in 50-100 L of 1.5 molJL HC1 to feces before the initial addition of HC1. For comparative purposes, the porphyrin content of the physiological samples was also determined by TLC of porphyrin methyl esters (4,5), in which case the value for total porphyrin was obtained by simple addition of the values for the individual porphynins. For some urine samples, a total porphyrin value was also obtained from ion-exchange chro3The millimolan absorptivities were estimated by interpolation from those reported (7) for uroporphyrin, coproporphyrin, and protoporphyrin.
CLINICALCHEMISTRY,Vol.30, No. 3,
1984
391
matography
of the free acid porphyrins,
tion by absorbance
spectroscopy
followed by estima-
(6).
and Discussion
Results
Figure 1 illustrates a typical MCD spectrum of a porphyrin in acidic medium. We used the peak-to-trough intensity of the spectrum, in units of miulidegrees (m#{176}) per centimeter, to provide a measure of the porphyrin content of solutions. Figure 2 shows standard curves for determination by MCD of uroporphyrin and coproporphyrin in HCI. The same curves were obtained when the medium was urine adjusted to pH 0.4. These standard curves were linear to the highest porphyrin concentrations measured. We used a standard curve intermediate between those for uroporphyrin and coproporphyrin (Figure 2) to determine the porphyrin content of urine samples. Figure 3 shows the effect on the MCD spectrum of mixtures containing different relative amounts of uroporphyrin and coproporphyrin. When either porphyrin constituted more than 80% of the total, the results were within 5% of the expected value. In contrast, with mixtures
containing about equimolar amounts of the two porphyrms, the porphyrin content was underestimated by 15 to 20%. Because about 80% of the total porphyrin in normal urine consists of coproporphyrin, and because the urine of patients with disorders characterized by an increased porphyrin concentration (e.g., symptomatic porphyria) can contain more than 80% of its porphyrin as uroporphyrin plus heptacarboxylporphyrin, the MCD assay would appear to be operating at maximal efficiency under most circumstances. Figure 4 shows the effect of the concentration of tn-, penta-, hexa-, and heptacarboxyl porphyrins on the peak-totrough intensity of the MCD spectrum. Uroporphyrin and coproporphyrun are included in Figure 4 for comparison. Inasmuch as penta-, hexa-, and heptacarboxyl porphyrins may be present in urine (8), and in view of the similarity of their MCD spectra to those of uroporphyrin and coproporphyrin, it is evident that the method of Barth et al. (3) for the quantitative determination of these last two porphynins
MCD as % calCulated value 110
2.5
100
E
C., 0
E
0
420
wavelength (nm)
-2.5
100% Uroporphyrin
50% Uro: 50% Copro
RELATIVE CONCENTRATIONS Fig. 1. MCD spectrum ofuroporphyrin (860nrriol/L in 0.4 mol/L 0.4)
HCI,pH
100%
Coproporphyrin
OF PORPI-WRINS
Fig. 3. Percentage of calculated valueforthepeak-to-trough intensityof the MCD spectrum formixturesofuroporphynn and coproporphyrin in 0.4 moVL HCI, pH 0.4 X,expeflment 1;0, expenment 2
Lko
Iko
10
7-GOOH
Copro
1000
Concentration
Copro
2000
1000
(nmol/L)
Concentration
2000 (nmolIL)
FIg.2. Peak-to-troughintensityof the MCD spectrum as a function of the concentration (nmol/L) of uroporphynnand coproporphynn in 0.4
the concentration(nmol/L)of several porphynns in 0.4 mol/L HCI, pH
mol/LHCI,pH 0.4
0.4
392
CLINICAL
CHEMISTRY,
Vol. 30, No.3, 1984
Fig. 4. Peak-to-trough intensity of the MCD spectrum
as a functionof
in urine is inaccurate when other urinary porphyrins are present. Figure 5 shows that about 65% of the porphyrins are extracted from Dean’s solution (see Materials and Methods) into HC1, and that neither the efficiency of extraction of porphyrins into the HC1 layer nor their determination by MCD is affected by the presence of fecal material. Figure 5 also shows the two standard curves that we used for determining the porphyrin content of fecal samples. One standard curve (rmean in Figure 5) was exactly intermediate between the curves for the extraction of coproporphyrin and protoporphynin from Dean’s solution. The second standard curve was constructed with a bias towards protoporphyrin, i.e., for a molar ratio of protoporphyrin to coproporphyrin of 8 to 1, reflecting the results of an examination of fecal samples from 40 porphyrics and 50 subjects with normal porphyrin concentrations.4 Figures 6 and 7 compare results obtained from the determination of total porphyrins in urine by MCD with those from TLC and ion-exchange chromatography, respectively. The concentrations of total urinary porphyrin as determined by MCD in general correlate well with those determined by TLC and by ion-exchange chromatography. For urine samples, a plot of the results of the MCD vs the TLC assay has a slope of 1.08, y-intercept of 8.4, and a correlation coefficient (r) of 0.93 (n = 41) (Figure 6). For a plot of MCD results vs results for total porphyrins by column chromatography (Figure 7), the slope was 1.07 and they-intercept 77.3 (r = 0.80, n = 19). In view of the slopes of the above plots, MCD appears to overestimate the porphyrin content by about 7% relative to the other two methods. We do not know the basis of this difference, but it should be unimportant clinically. Table 1 lists the results we calculated for fecal samples, using each of the two standard curves shown in Figure 5 (molar ratios of 1:1 and 8:1 for protoporphyrin to coproporphyrin). Although, on the basis of the average molar ratio of 8:1 of protoporphyrin to coproporphyrin found for the feces of normal subjects and the porphyrics, we anticipated that the latter curve would provide greater accuracy, we found that using the 1:1 ratio standard curve gave far better agreement with the TLC results. For the latter standard curve, a plot of the MCD results vs TLC results gave a slope of 0.84 and ay-
1200
. 341
1000
#{149}2580.4607
200
400
molar ratios of 8 to 1 for fecal protoporphyrin to coproporphyrin were found for the 90 subjects investigated. However, extremely great interindividual differences were noted, with the ratios ranging from 45. Cogro 16
-
itfeces)
y
6N
(1:1) Cogro
Os)
P10*0
c-jo
a #{149} Plato (Deens i feces) 5
Porphyrin
(mnoles)
Fig. 5. Peak-to-trough intensity of the MCD spectrum forcoproporphyrin and protoporphynn (nanomoles) in 1.5 mol/L HCI and after extraction into1.5 mol/L HCI from an equal volume of Dean’s solution in the presenceand absenceof 0.1-0.2 g (drywt.) of feces Dean’ssolution is descnbed in the text
600
800
1200
1000
Thin layer chromatography
Fig. 6. Concentration(in nmol/L)of urinaryporphynns as determined MCD and by TLC
by
1930.1707
1000
800 . a
6o0 U
.
400
200
200
400 Ion exchange
4Average
