Direct assay of enzymes in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry. Porphobilinogen deaminase

Article abstract of DOI:10.1021/ac702244x

We report a new assay of human porphobilinogen deaminase (PBGD). Deficiency in this enzyme activity causes acute intermittent porphyria, the most common disorder of heme biosynthesis. The assay involves incubation of blood erythrocyte lysate with porphobilinogen, the natural PBGD substrate. Two subsequent enzymes in the heme biosynthetic pathway, uroporphyrinogen III synthase and uroporphyrinogen decarboxylase, are deactivated by heating so that their activity does not interfere with the PBGD assay. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is used to monitor the production of uroporphyrinogen I and thus measure the PGBD activity. A simple and efficient workup using liquid-liquid extraction with >90% product recovery was employed to avoid separation by liquid chromatography. The assays show good reproducibility (±3.3%) and linear dependence of the uroporphyrinogen I formation on incubation time and protein amount. The K_m of PGBD for porphobilinogen was measured as 11.2 ± 0.5 μM with V_max of 0.0041 ± 0.0002 μM/min·mg of hemoglobin). The coefficient of variation of PBGD activity among several unaffected individuals (12%) is significantly lower than the decrease due to acute intermittent porphyria (50%).

Full text of DOI:10.1021/ac702244x

Anal. Chem. 2008, 80, 2606-2611
Technical Notes
Direct Assay of Enzymes in Heme Biosynthesis for
the Detection of Porphyrias by Tandem Mass
Spectrometry. Porphobilinogen Deaminase
Yuesong Wang,† C. Ronald Scott,‡ Michael H. Gelb,*,†,§ and Frantisˇek Turecˇ ek*,†
Departments of Chemistry, Pediatrics, and Biochemistry, University of Washington, Seattle, Washington 98195
uroporphyrinogen I, which becomes significant when UROS is
deficient. Uroporphyrinogen III and uroporphyrinogen I can be
further converted to other intermediates by uroporphyrinogen
decarboxylase (UROD) in the cycle of heme biosynthesis.
AIP has an incidence of 5 in 100 000 in the United States and
most other countries but is more common in some European
countries, such as Sweden, Britain, and Ireland. An even much
higher incidence of 210 in 100 000 was reported in a chronic
psychiatric population in the United States.2 Although half-normal
PBGD activity due to AIP is sufficient to maintain functional heme
synthesis in most situations, it can become the rate-limiting step
for the heme formation when the affected individual is exposed
to endocrine factors, steroid hormones, and certain drugs, such
as antipyrine, ketoconazole, mephenytoin, etc., to show clinical
manifestation.3
Several assays have been developed to determine the activity
of PBGD that were based on UV-vis spectrophotometry,4 fluo-
rimetry,5 or the combination of HPLC and fluorimetry.6-8 However,
because of the formation of multiple products in the pathway and
high background in the spectrophotometric measurements, most
of the previous assays could not provide a sensitive and selective
method for the diagnosis of AIP. Another recent study used
HPLC-mass spectrometry to monitor the level of PBG in patients
treated with recombinant PBGD.9 However, studies which are
based on measuring increased substrate levels are not well-suited
to distinguish defects of PBGA from those of enzymes downstream
in the biosynthetic pathway. Product-based direct assays of PBGD
suffer the essential difficulty of having to detect HMB, which is
labile and difficult to prepare in a pure state. These properties
severely hamper measurements of response and method calibra-
tion. The difficulties with HMB can be overcome by inactivating
We report a new assay of human porphobilinogen deami-
nase (PBGD). Deficiency in this enzyme activity causes
acute intermittent porphyria, the most common disorder
of heme biosynthesis. The assay involves incubation of
blood erythrocyte lysate with porphobilinogen, the natural
PBGD substrate. Two subsequent enzymes in the heme
biosynthetic pathway, uroporphyrinogen III synthase and
uroporphyrinogen decarboxylase, are deactivated by heat-
ing so that their activity does not interfere with the PBGD
assay. Electrospray ionization tandem mass spectrometry
(ESI-MS/MS) is used to monitor the production of uropor-
phyrinogen I and thus measure the PGBD activity. A
simple and efficient workup using liquid-liquid extraction
with >90% product recovery was employed to avoid
separation by liquid chromatography. The assays show
good reproducibility ((3.3%) and linear dependence of
the uroporphyrinogen I formation on incubation time and
protein amount. The Km of PGBD for porphobilinogen was
measured as 11.2 ( 0.5 µM with Vmax of 0.0041 (
0.0002 µM/(min‚mg of hemoglobin). The coefficient of
variation of PBGD activity among several unaffected
individuals (12%) is significantly lower than the decrease
due to acute intermittent porphyria (50%).
Acute intermittent porphyria (AIP), the most common acute
porphyria, results from the reduced activity of the enzyme
porphobilinogen deaminase (PBGD).1 PBGD is the third enzyme
in heme biosynthesis and catalyzes the stepwise condensation of
four molecules of porphobilinogen (PBG) to form hydroxymeth-
ylbilane (HMB, Scheme 1). HMB can be converted to uropor-
phyrinogen III by intramolecular rearrangement and ring closure
by the fourth enzyme, uroporphyrinogen III synthase (UROS).
An alternative pathway is spontaneous cyclization of HMB to
(2) Tishler, P. V.; Woodward, B.; O’Connor, J.; Holbrook, D. A.; Seidman, L.
J.; Hallett, M.; Knighton, D. J. Am. J. Psychiatry 1985, 142, 1430-1436.
(3) Ohkubo, H.; Musha, H.; Okuda, K. Dig. Dis. Sci. 1979, 24, 700-7044.
(4) Grandchamp, B.; Nguyen, P.; Grelier, M.; Nordmann, Y. Clin. Chim. Acta
1976, 70, 113-118.
* To whom correspondence should be addressed. E-mail: turecek@
chem.washington.edu (F.T.), gelb@chem.washington.edu (M.H.G.). Fax: 206-
685-8665 (F.T.), 206-685-8665 (M.H.G.).
(5) Magnussen, C. R.; Levine, J. B.; Doherty, J. M.; Cheesman, J. O.; Tschudy,
† Department of Chemistry.
D. P. Blood 1974, 44, 857-868.
‡ Department of Pediatrics.
(6) Lim, C. K.; Li, F.; Peters, T. J. J. Chromatogr. 1988, 429, 123-153.
(7) Schoenfeld, N.; Mamet, R. J. Chromatogr. 1991, 570, 51-64.
(8) Erlandsen, E. J.; Jorgensen, P. E.; Markussen, S.; Brock, A. Scand. J. Clin.
Lab. Invest. 2000, 60, 627-634.
§ Department of Biochemistry.
(1) Kappas, A.; Sassa, S.; Galbraith, R. A.; Nordmann, Y. The Porphyrias. In
The Metabolic and Molecular Basis of Inherited Disease, 7th ed.; Scriver, C.
R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York,
1995; Vol. II, Chapter 66, pp 2103-2159.
(9) Sardh, E.; Rejkjaer, L.; Andersson, D. E. H.; Harper, P. Clin. Pharmacokinet.
2007, 46, 335-349.
2606 Analytical Chemistry, Vol. 80, No. 7, April 1, 2008
10.1021/ac702244x CCC: $40.75 © 2008 American Chemical Society
Published on Web 02/23/2008

Scheme 1
UROS, the next enzyme in the pathway, and monitoring the
production of uroporphyrinogen I, which arises by spontaneous,
nonenzymatic, cyclization of HMB (Scheme 1). In the absence of
UROS, assaying with PBG and monitoring the formation of
uroporphyrinogen I provides an indirect but unequivocal measure
of PBGD activity. UROS is deactivated by short heating to 56 °C
while PBGD is not affected.10,11 In general, assaying enzyme
activities provides a specific diagnostic tool for inborn errors of
metabolism, as reported for several diseases.12-14
In spite of several different approaches to assaying PBGD, it
is advantageous to use methods that are based on a single
instrumental platform. To this end, we have been developing
tandem mass spectrometric assays of enzymes in biological
samples such as dried blood spots and cell lysates for the
biochemical analysis of inborn errors of metabolism.13,14 Tandem
mass spectrometry offers the advantages of analytical sensitivity,
selectivity, low background, and speed and is also ideally set up
for multiplex analysis,12 whereby the products of several different
enzymes can be quantified during a single infusion into the
instrument without time-consuming chromatographic separation.
As tandem quadrupole mass spectrometers are becoming standard
equipments in clinical laboratories, new assays using electrospray
ionization tandem mass spectrometry (ESI-MS/MS) represent a
useful and attractive alternative to the existing methods. Our
previous communication reported on ESI-MS/MS quantification
of enzymes UROD and coproporphyrinogen oxidase in human
blood.15 We envision using ESI-MS/MS to directly quantify all of
the enzymes in the heme biosynthetic pathway for the molecular
analysis of porphyrias. The method utilizes a designed, often
synthetic, substrate for the selected enzyme that is added to a
biological sample. After incubation, the amount of enzyme-
generated product is quantified, along with a mass-differentiated
internal standard, by selective detection with ESI-MS/MS. In this
study, we report a direct enzyme assay for PBGD using ESI-MS/
MS.
EXPERIMENTAL SECTION
Materials and Methods. Uroporphyrin I dihydrochloride (cat.
no. U830-1) and heptacarboxylporphyrin I dihydrochloride (cat.
no. H885-ID) were purchased from Frontier Scientific Inc. (Logan,
UT). Porphobilinogen (cat. no. P226) was purchased from Sigma-
Aldrich (St. Louis, MO). Anticoagulant tubes with EDTA dipo-
tassium salt (cat. no. 367899), lithium heparin (cat. no. 367886),
ACD solution B (trisodium citrate, 13.2 g/L, citric acid, 4.8 g/L,
and dextrose 14.7 g/L, 1.0 mL, cat. no. 364816) were obtained
from BD (Franklin Lakes, NJ). Hydrochloric acid, 1-butanol, and
formic acid were from EMD Chemicals Inc. (San Diego, CA). All
other chemicals were from Sigma-Aldrich (St. Louis, MO).
Isolation of Erythrocytes. Human blood was drawn in a
vacuum sealed tube with EDTA (BD Vacutainer, cat. no. 367899).
(10) Tsai, S. F.; Bishop, D. F.; Desnick, R. J. J. Biol. Chem. 1987, 262, 1268-
1273.
(11) Dresel, E. I. B.; Tooth, B. E. Nature 1954, 174, 271.
(12) Gerber, S. A.; Scott, C. R.; Turecˇek, F.; Gelb, M. H. J. Am. Chem. Soc. 1999,
121, 1102-1104.
(13) Li, Y.; Scott, C. R.; Chamoles, N. A.; Ghavami, A.; Pinto, B. M.; Turecˇek,
F.; Gelb, M. H. Clin. Chem. 2004, 50, 1785-1796.
(14) Gelb, M. H.; Turecek, F.; Scott, C. R.; Chamoles, N. A. J. Inherited Metab.
Dis. 2006, 29, 397-404.
(15) Wang, Y.; Gatti, P.; Sadilek, M.; Scott, C. R.; Turecˇek, F.; Gelb, M. H. Anal.
Chem. 2008, 80, http://dx.doi.org/10.1021/ac702130n.
Analytical Chemistry, Vol. 80, No. 7, April 1, 2008 2607

Whole blood (3 mL) was transferred to a 15 mL plastic centrifuge
tube. Nine milliliters of NaCl solution (0.9%, w/v) was added, and
the sample was centrifuged at 600g for 10 min at room temper-
ature. The supernatant above the erythrocyte pellet was discarded.
To the pellet was added 9 mL of NaCl solution, and the cells were
resuspended by gently inverting the tube several times followed
by centrifugation. This washing step was repeated once more. To
the washed erythrocyte pellet was added NaCl solution to bring
the volume to 3 mL. After resuspending the cells, the suspension
was frozen in a dry ice/acetone bath. The tube was placed in a
water bath at room temperature until the solution was fully thawed.
This freeze/thaw process was repeated twice. The suspension of
lysed cells was centrifuged at 12 000g for 10 min at room
temperature. The supernatant was divided into 100 µL aliquots in
polypropylene microfuge tubes, which were stored at -80 °C (no
loss in PBGD activity was observed when cell lysates were stored
frozen up to 4 months). Before use, an aliquot was thawed and
diluted 5-fold with distilled water. Sixty microliters (∼2 mg) of
this diluted stock is used for the PBGD assay (see text).
instrument operating in positive multiple reaction monitoring
(MRM). Ion scanning was carried out with the MassLynx software
with the following settings: capillary voltage, 3.5 kV; cone voltage,
120; extractor, 3; rf, 0; source temperature, 80 °C; desolvation
temperature, 350 °C; dwell time, 100 ms; desolvation gas, 500 L/h;
collision gas, 0.21 L/h; collision energy, 57 eV. The product ion
monitored was due to the combined loss of CH2COOH and COOH
from the respective precursor MH+ ions (Figure 1). MRM data
for product (coproporphyrin) m/z 831 f m/z 727 and internal
standard (heptaporphyrin I) m/z 787 f m/z 683 were collected.
The sample (10 µL of the 150 µL sample in 1-butanol) was infused
into the mass spectrometer with a Waters 1525 binary HPLC pump
equipped with a sample injection loop. After injection, the solution
of 60% methanol (methanol/acetonitrile/formic acid, 90:10:0.1,
v/v/v) and 40% water (water/formic acid, 100:0.2 v/v) was infused
at 200 µL/min as the flow solvent. The amount of product was
calculated from the ratio of integrated ion peak intensities of
product to internal standard according to the calibration curve.
The ESI-MS/MS responses were calibrated using mixtures of
uroporphyrin I and heptaporphyrin I at different molar ratios and
a fixed total amount of 300 pmol that were added to the incubation
matrix with lysed erythrocytes, followed by standard extraction
workup. The calibration curve is given in Figure S4 (Supporting
Information).
Storage of Assay Buffer and Substrate. Tris-HCl buffer
(100 mM, pH 8.2) and PBG (1 mM) were stored into 1.5 mL
microfuge tubes at -20 °C.
PBGD Enzyme Assay. PBGD assay buffer (50 µL; 100 mM
Tris-HCl, pH 8.2) was added to a 1.5 mL polypropylene microfuge
tube followed by 60 µL (∼2 mg of hemoglobin) of diluted red
blood cell lysates and 100 µL of distilled water. The tube was
capped and incubated in a water bath at 56 °C for 30 min to
inactivate enzymes UROS and UROD, and the solution was then
cooled in an ice bath. The capped tubes were centrifuged at 1500g
for 30 s to spin down the water on the cap back to the aqueous
phase. Porphobilinogen (40 µL of a 1 mM solution) was added to
the tubes, and they were incubated at 37 °C for 60 min. The water
bath chamber was covered with foil to exclude light. After
incubation, the reaction was quenched by placing the tubes in an
ice bath. Ammonium formate buffer solution (150 µL; 1.0 M, pH
3.17) was added to adjust pH to the appropriate range for
extraction, followed by 1-butanol (400 µL) and heptacarboxypor-
phyrin I (15 µL of a 20 µM solution in 1 M HCl) as the internal
standard. The mixture was placed on a vortex mixer for 30 s before
being centrifuged at 15 000g for 3 min. The top 300 µL of
supernatant was transferred using a pipettor with a polypropylene
tip to a new 1.5 mL polypropylene microfuge tube containing 150
µL of ammonium formate (20 mM, pH 3.17). After the first
centrifugation, proteins were clearly located at the interface of
the 1-butanol and water layers. The organic phase should be
carefully transferred to a new tube without touching the interface.
In order to keep the assay consistent and avoid experimental
errors, the volume of the transferred organic layer was kept
constant and smaller than the total volume of the 1-butanol phase.
The mixture was placed on a vortex mixer for 30 s and centrifuged
again for 3 min. The top 150 µL of the butanol layer was
transferred to another microfuge tube for ESI-MS/MS analysis.
Hemoglobin Measurement. The amount of hemoglobin in
red blood cell lysates was measured by using colorimetric
determination at 400 nm with a QuantiChrom hemoglobin assay
kit (BioAssay Systems, cat. no. DIHB-250, Hayward, CA).
Mass Spectrometry. Most ESI-MS and MS/MS experiments
were carried out on a Waters Acquity TQD tandem quadrupole
RESULTS AND DISCUSSION
The ESI-MS/MS assay of PBGD relies on the production of
uroporphyrinogen I which occurs by spontaneous cyclization of
HMB (Scheme 1) after the enzymatic pathway to uroporphyrino-
gen III has been blocked by deactivating UROS. Spontaneous
cyclization of HMB is fast with a half-time of <4 min at 37 °C,16
which guarantees virtually complete conversion during our
incubation time of 60 min. Human UROS has been reported to
be unstable to heat, with the half-life of 4 and 1 min at 45 and
60 °C, respectively.10,11 Thus, after heating at 56 °C for 30 min
UROS is completely deactivated, while the activity of PBGD is
not affected. Heat treatment also inactivates the next enzyme in
the heme biosynthetic pathway (UROD) and thus prevents
depletion of uroporphyrinogen I by subsequent enzymatic decar-
boxylation.
Uroporphyrinogen I (1) is not detected directly but is con-
verted to the more stable uroporphyrin I (2, Scheme 2) by
spontaneous oxidation on exposure to air and light. Product 2
shows favorable properties for electrospray ionization and collision-
induced dissociation (CID) which are both necessary for tandem
mass spectrometric analysis. Both UROS deactivation and uropor-
phyrinogen oxidation were studied in detail to ensure robustness
and reproducibility of the assay procedure, as described below.
UROD deactivation prevents enzymatic formation of heptacar-
boxylporphyrinogen I. Therefore, heptacarboxylporphyrin was
chosen as an internal standard because of its similarity to
uroporphyrin I.
The ESI-MS/MS assay was based on monitoring the products
of ion dissociation of protonated uroporphyrin I, (M + H)+ at m/z
831. On CID, the latter sequentially eliminates COOH and CH2-
COOH neutral fragments to produce an abundant ion fragment
(16) Battersby, A. R.; Fookes, C. J. R.; Gustafson-Potter, K. E.; McDonald, E.;
Matcham, G. W. J. J. Chem. Soc., Perkin Trans. 1 1982, 2427-2444.
2608 Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

Figure 1. ESI-MS/MS spectra of (M + H)+ ions from (a) uroporphyrin I at m/z 831 and (b) heptacarboxylporphyrin at m/z 787.
Scheme 2
at m/z 727 (Figure 1a) which was used for monitoring. Heptac-
arboxylporphyrin (3) that was used as an internal standard
undergoes an analogous dissociation of its (M + H)+ ion (m/z
787) to form an abundant product ion at m/z 683 (Figure 1b).
Although we did not study the mechanism of these ion dissocia-
tions, it is of interest to note that the intermediate fragments due
to loss of either COOH or CH2COOH (e.g., m/z 786 and 772,
respectively, Figure 1) are very weak and indicate facile dissocia-
tion following the loss of the first neutral radical. The fact that
the same neutrals are lost from 2 and 3 is advantageous, because
it allows one to use MRM based on precursor ion or neutral loss
scans. The response in ESI-MS/MS of 3 relative to that of 2 was
determined to be 1.47:1, with a 3.1% coefficient of variation.
We note that monitoring the formation of the enzymatic
product is superior to monitoring the decrease of the substrate
concentration for two reasons. First, product formation is entirely
Analytical Chemistry, Vol. 80, No. 7, April 1, 2008 2609

Figure 2. Porphyrin formation in assays with (empty bars) and
without (filled bars) heat deactivation of UROS and UROD.
due to enzyme activity and thus product detection can be expected
to have a very low blank level, as confirmed by the present data
(see below). Second, it is advantageous to maintain a high and
steady substrate concentration to ensure that the measurements
are performed in the initial velocity stage of the enzyme reaction
(see below).
The effect of blocking UROS prior to the PBGD assay was
studied in detail. Four parallel triplicate experiments were carried
out in which two assays included heating at 56 °C and two assays
were performed without heat deactivation. ESI-MS/MS analysis
(Figure 2) indicated that about 40% ((2.2%) of uroporphyrin was
lost in the assays performed without heat deactivation. In addition,
the assays carried out without heat inactivation showed an
increased production of heptacarboxylporphyrin, which is an
intermediate product of UROD-catalyzed decarboxylation of
uroporphyrinogen I.15 These results suggested that heat treatment
is an important step to deactivate both UROS and UROD to
achieve a robust and reproducible assay of PBGD.
Considerable attention was also paid to sample workup fol-
lowing incubation with the goal of optimizing product and internal
standard recovery. A standard workup procedure for the isolation
of porphyrins is liquid-liquid extraction into ethyl acetate.11,17,18
However, partitioning of 2 between the organic and aqueous phase
is largely dependent on pH for different organic solvents. For
example, extraction of 2 into ethyl acetate is efficient only in a
narrow pH range of 3.0-3.2,18 whereas 2 is very little soluble in
a range of other organic solvents. We found 1-butanol as the
optimum choice for the described assays because of its high
extraction efficiency (over 90%) over a relatively large pH range
of 2.2-3.8.13 1-Butanol is sufficiently volatile to be evaporated in
the ESI source at a somewhat increased droplet desolvation
temperature for mass spectrometric analysis of 2. Ammonium
formate buffer, which is volatile and thus compatible with ESI,
was used to adjust the pH of the aqueous layer into an optimum
range for good recovery yields. These were studied in detail in
blank experiments with or without blood and incubation. Figure
S1 (Supporting Information) shows that the percent conversion
by oxidation to uroporphyrin I in blank assays that contained blood
Figure 3. (a) Activity of PBGD measured in erythrocytes (∼2 mg
of hemoglobin) as a function of the incubation time. Reactions were
carried out at 37 °C using 16 µM PBG. Error bars are shown for
triplicate analyses. (b) Activity of PBGD measured in erythrocytes as
a function of the amount of erythrocytes. Reaction conditions as in
(a). Error bars are shown for triplicate analyses.
was >90% and was nearly constant in the period of 5-70 min.
This showed that the oxidation of uroporphyrinogen I to uropor-
phyrin I was complete and that the latter was stable in solution
under the workup conditions. In contrast, blank assays that were
performed without blood showed a much slower oxidation rate
(Figure S1) and reached 90% recovery yield only after a period of
60 min. It is possible that the fast oxidation of uroporphyrinogen
I to uroporphyrin I is catalyzed by Fe ions from the blood sample.
The amount of product in the PBGD assays increased linearly
with reaction time from 15 to 150 min (Figure 3a). This indicated
that the enzyme reaction was in its initial velocity stage. The PBG
substrate conversion was around 11% after 60 min, and this time
was chosen for standard assays. We note that PBGD showed a
substantially higher stability by comparison with UROD and
coproporhyrinogen oxidase, which were studied previously using
ESI-MS/MS assays.15 In addition, the PBG substrate appears to
be more stable than porphyrinogen substrates which are gener-
ated in situ by chemical reduction.19 All this resulted in good
reproducibility of data points ((3.3% mean SD) and a tight
correlation (r2 ) 0.9996, Figure 3a). The amount of product also
showed a linear dependence on the amount of red cell lysate,
measured as hemoglobin by colorimetry at 400 nm, which was
used for the assay. In the range of 0.08-3.5 mg of hemoglobin
from whole blood we obtained a tight linear dependence (r2 )
(17) Scott, C. R.; Labbe, R. F.; Nutter, J. Clin. Chem. 1967, 13, 493-500.
(18) Fernandez, A. A.; Henry, R. J.; Goldenberg, H. Clin. Chem. 1966, 12, 463-
(19) Jones, M. A.; Thientanavanich, P.; Anderson, M. D.; Lash, T. D. J. Biochem.
Biophys. Methods 2003, 55, 241-249.
474.
2610 Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

As a final practical note, we investigated the stability upon
storage of blood samples. Blood was collected in standard
commercial tubes in the presence of various anticoagulants, e.g.,
(1) lithium heparin, (2) ACD (trisodium citrate, citric acid, and
dextrose), and (3) dipotassium salt of EDTA. All samples were
found to give similar PBGD activities without apparent difference
after having been stored with the anticoagulant at 4 °C for 2 days
(Figure S3, Supporting Information). No loss of activity was
observed after storage at -80 °C for 4 months. Thus, PBGD has
proven to be a stable enzyme that can be assayed in normal blood
samples after weeks of storage under the above-described ap-
propriate conditions.
Figure 4. Lineweaver-Burk plot of reciprocal PBGD velocity in
erythrocytes (∼2 mg of hemoglobin) over reciprocal concentration of
porphobilinogen (PBG).
CONCLUSIONS
Electrospray ionization tandem mass spectrometry has been
shown to provide a platform for robust, sensitive, and selective
assays of PBGD in human blood samples. Samples can be stored
for prolonged time and assayed using a simple procedure that
does not require chromatographic separation. Following clinical
tests including samples from affected patients, the ESI-MS/MS
assay should be suitable for diagnostics of AIP and, in combination
with the procedures developed for UROD and coproporphyrinogen
III oxidase,15 could be employed for the multiplex detection of
several porphyrias using a single instrumental platform. We
envisage that direct determination of PBGA activity should be
preferred over measuring the amount of excreted porphyrins.
1.000, Figure 3b). From the latter, 2 mg of hemoglobin was chosen
for the standard assay to provide an amount of PBGD that could
be reliably assayed by ESI-MS/MS with a tandem quadrupole
mass spectrometer. Comparison of standard and blank assays (no
substrate or no hemoglobin) gave the product peak intensity over
150 times of that of the blank. Thus, no blank subtraction was
necessary in data evaluation.
Plots of the reaction rate versus the substrate concentration
showed the classical hyperbolic behavior for the PBGD assay
yielding values of KM of 11.2 ( 0.5 µM and Vmax of 0.0041 ( 0.0002
µM/(min‚mg of hemoglobin), respectively (r2 ) 0.9968, Figure
4). This value is close to that from an early fluorimetric determi-
nation (13.8 µM)5 but higher than the value from an HPLC-based
assay (6.2 mM).8 The average value for the standard PBGD assay
was 1.01 ( 0.12 pmol/(min‚mg of hemoglobin) of uroporphyrin
I, as determined in blood samples from four healthy individuals
(Figure S2, Supporting Information). The coefficient of variation
among the healthy individuals from these measurements, CV )
12%, is significantly lower than the expected loss of activity in
affected patients (ca. 50%). Thus, by virtue of its robustness and
reproducibility, the ESI-MS/MS assay appears to be suitable for
clinical diagnostics of patients manifesting AIP.
ACKNOWLEDGMENT
This work was supported by a Grant from the NDDKsNational
Institutes of Health (DK67869).
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review October 30, 2007. Accepted January
10, 2008.
AC702244X
Analytical Chemistry, Vol. 80, No. 7, April 1, 2008 2611