Direct assay of δ-Aminolevulinic acid dehydratase in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry

Article abstract of DOI:10.1021/ac101111m

We report a new assay of human δ-aminolevulinic acid dehydratase (ALAD), an enzyme converting δ-aminolevulinic acid (ALA) into porphobilinogen. The assay is developed for use in the clinical diagnosis of δ-aminolevulinic acid dehydratase-deficient porphyria, a rare enzymatic deficiency of the heme biosynthetic pathway. The assay involves the incubation of erythrocyte lysate with the natural substrate, ALA, followed by quantitative in situ conversion of porphobilinogen to its butyramide, and liquid-liquid extraction into a mass spectrometer-friendly solvent. Quantitation of the butyrylated porphobilinogen is done by electrospray ionization tandem mass spectrometry, using a deuterium labeled internal standard. The assay stays well within the range wherein ALAD activity is linear with time. The K_m of ALAD for ALA was measured as 333 μM, and the V_max was 19.3 μM/h. Average enzyme activity among a random sample of 36 anonymous individuals was 277 μmol/L erythrocyte lysate/hour with a standard deviation of 90 μmol/L erythrocyte lysate/hour. The tandem mass spectrometric assay should easily detect the enzyme deficiency, which causes a reduction of activity by 95-99%. The assay shows good reproducibility and low background, requires a simple workup, and uses a commercially available substrate.

Full text of DOI:10.1021/ac101111m

Anal. Chem. 2010, 82, 6730–6736
Direct Assay of δ-Aminolevulinic Acid Dehydratase
in Heme Biosynthesis for the Detection of
Porphyrias by Tandem Mass Spectrometry
†
‡
,†,§
,†
John R. Choiniere, C. Ronald Scott, Michael H. Gelb,*
and Franti sˇ ek Ture cˇ ek*
Departments of Chemistry, Pediatrics, and Biochemistry, University of Washington, Seattle, Washington 98195-1700
We report a new assay of human δ-aminolevulinic acid
dehydratase (ALAD), an enzyme converting δ-aminole-
vulinic acid (ALA) into porphobilinogen. The assay is
developed for use in the clinical diagnosis of δ-aminole-
vulinic acid dehydratase-deficient porphyria, a rare en-
zymatic deficiency of the heme biosynthetic pathway. The
assay involves the incubation of erythrocyte lysate with
the natural substrate, ALA, followed by quantitative in situ
conversion of porphobilinogen to its butyramide, and
liquid-liquid extraction into a mass spectrometer-friendly
solvent. Quantitation of the butyrylated porphobilinogen
is done by electrospray ionization tandem mass spectrom-
etry, using a deuterium labeled internal standard. The
assay stays well within the range wherein ALAD activity
is linear with time. The Km of ALAD for ALA was
measured as 333 µM, and the Vmax was 19.3 µM/h.
Average enzyme activity among a random sample of 36
anonymous individuals was 277 µmol/L erythrocyte
lysate/hour with a standard deviation of 90 µmol/L
erythrocyte lysate/hour. The tandem mass spectromet-
ric assay should easily detect the enzyme deficiency,
which causes a reduction of activity by 95-99%. The
assay shows good reproducibility and low background,
requires a simple workup, and uses a commercially
available substrate.
Scheme 1
ecules of δ-aminolevulinic acid (ALA) (Scheme 1). ALAD is the
first enzyme in the pathway to be found in the cytosol of the cell,
rather than the mitochondrion.
ADP is passed on in an autosomal recessive pattern. Homozy-
gous patients experience a drop in enzyme activity of 95-99%. A
heterozygous deficiency results in an enzyme activity level roughly
5
0% of nonaffected people but does not cause ADP. However, the
heterozygotes, thought to be as prevalent as 1 to 2% of certain
populations, are potentially at risk for greater negative effects of
lead poisoning. Lead poisoning results in a lack of ALAD activity
as lead reversibly replaces zinc at the enzyme’s active sites.
1
3
Heme-based proteins, such as the cytochrome enzymes and
hemoglobin, play a vital role in human life. The central molecule
of these proteins, heme, is made by the human body in an eight-
step, enzyme-assisted pathway. Genetic deficiency in any one of
the last seven of these enzymes defines any one of seven rare
Clinically, ADP typically presents with acute abdominal pain and
peripheral neuropathy, similar to other acute porphyrias.
Given its similarities to other porphyrias, ADP is difficult to
diagnose. It is differentiated from acute intermittent porphyria
4
(AIP), deficiency in the subsequent enzyme of the pathway, only
1
genetic disorders known collectively as the porphyrias. Deficiency
in the second enzyme, δ-aminolevulinic acid dehydratase (ALAD),
by increased urinary excretion of PBG. Current assays for ALAD
deficiency are, for the most part, based on a method developed
5
is the cause of ALAD-deficient porphyria (ADP), sometimes
by Mauzerall and Granick in 1956. This technique involves
2
referred to as Doss porphyria. ALAD catalyzes the formation of
reacting the enzymatic product with p-dimethylaminobenzalde-
porphobilinogen (PBG), a pyrrole intermediate, from two mol-
hyde, or Erlich’s reagent, to create a compound detectable by
6-10
colorimetry or fluorimetry.
A common alternative is a coupled-
*
To whom correspondence should be addressed.
Department of Chemistry.
Department of Pediatrics.
†
‡
§
(3) Bird, T. D.; Hamernyik, P.; Nutter, J. Y.; Labbe, R. F. Am. J. Hum. Genet.
1979, 31, 662–668
Department of Biochemistry.
.
(
1) Anderson, K. E.; Sassa, S.; Bishop, D. F.; Desnick, R. J. Disorders of Heme
Biosynthesis: X-Linked Sideroblastic Anemia and the Porphyrias. In The
Metabolic and Molecular Basis of Inherited Disease, 8th ed.; Scriver, C. R.,
Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 2000;
Chapter 124, pp 2993-2999 and 3005-3008.
(4) Doss, M.; Sassa, S. The Porphyrias. In Laboratory Medicine. The Selection
and Interpretation of Clinical Laboratory Studies; Noe, D. A., Rock, R. C.,
Eds.; Williams and Wilkins: Baltimore, MD, 1994; Chapter 26, pp 535-
553.
(5) Mauzerall, D.; Granick, S. J. Biol. Chem. 1955, 219, 435–446
(6) L u¨ o¨ nd, R. M.; Walker, J.; Neier, R. W. J. Org. Chem. 1992, 57, 5005–5013
(7) Sassa, S.; Fujita, H.; Kappas, A. Pediatrics 1990, 86, 84–86
.
(2) Doss, M.; von Tiepermann, R.; Schneider, J.; Schmid, H. Klin. Wochenschr.
.
1979, 57, 1123–1127
.
.
6730 Analytical Chemistry, Vol. 82, No. 15, August 1, 2010
10.1021/ac101111m  2010 American Chemical Society
Published on Web 06/28/2010

1
1,12
enzyme assay,
wherein porphobilinogen is carried further
Whole blood (3 mL) was then transferred to a 15 mL polypropy-
lene centrifuge tube; 9 mL of 0.9% w/v saline solution was added.
The sample was gently shaken, and the tube was centrifuged at
600g for 10 min. The nonerythrocyte-containing supernatant was
discarded, followed by another addition of 9 mL of saline. The
tube was inverted a few times to gently remix the sample, followed
by a second centrifugation at 600g for 10 min. Again, the
supernatant was discarded, and the cells were washed and
centrifuged a third time. Following this third washing, 2.5 mL of
red blood cells were drawn from the bottom of the tube and placed
in a new 15 mL centrifuge tube. The tube was frozen in a dry
ice/acetone bath for 10 min and then brought back to room
temperature in a water bath. This was repeated two additional
times in order to ensure complete cell lysis. The resulting lysate
was centrifuged at 12 000g for 10 min, prior to being split into 50
µL aliquots, stored in 600 µL polypropylene microfuge tubes at
down the biosynthetic pathway to produce hydroxymethylbilane,
which spontaneously cyclizes to uroporphyrinogen I, which is then
oxidized and detected as uroporphyrin I by fluorimetry. None of
the current methods detect porphobilinogen directly, putting them
at a clear disadvantage compared to a method that does.
Furthermore, it is exceedingly difficult by these methods to
distinguish this specific enzyme deficiency from those upstream
or downstream.
Previous work in our laboratory has focused on developing
procedures for testing enzyme activity levels in human samples
using tandem mass spectrometry as a common analytical platform
1
3-15
for both clinical diagnostics and newborn screening.
The
methods involve selecting an appropriate biological source of
enzyme, typically either isolated and lysed blood cells for
diagnostics or dried blood spots for screening, and incubating this
sample with the enzyme’s substrate, which is often synthetically
designed in lab. The enzymatic product is then quantified by
selected-reaction monitoring tandem mass spectrometry, using a
mass-differentiated internal standard such as a deuterium-labeled
isotopologue or a homologue. This method takes advantage of
the speed and sensitivity offered by mass spectrometry over
spectroscopic methods, as well as the specificity and selectivity
offered by the use of a tandem instrument. The unique masses
used for each assay’s product and standard allow for easy
multiplexing, as the products of many different assays can be
combined into one injection without any need for chromatographic
separation. In particular, we have developed tandem mass spec-
-80 °C. Prior to use in assays, the sample tubes were brought
back to room temperature and diluted with 450 µL of 18MΩ
deionized water. A 50 µL aliquot of this solution was used for
assays corresponding to 5 µL of red blood cell lysate per assay.
Internal Standard Synthesis. Butyrylated porphobilinogen-
d
7
(d
7
-But-PBG) was synthesized using butyric anhydride-d14
,
which was made from butyryl chloride-d
7
and sodium butyrate-
d
d
7
7
(both C/D/N Isotopes, 98% D) as follows. Sodium butyrate-
(1 g, 8.54 mmol) was added under stirring to anhydrous
tetrahydrofuran (50 mL) under an argon atmosphere. Butyryl
chloride-d (1 g, 8.80 mmol) was added dropwise, and the
7
system was allowed to react for 72 h at room temperature.
Sodium chloride was filtered off, the solvent was evaporated
in vacuo, and the residue was purified by short-path vacuum
distillation. GC/MS analysis showed that the synthesized
butyric-d14 anhydride was >98% pure and its D content was 98%.
14
trometry assays for the detection of acute intermittent porphyria,
porphyria cutanea tarda, hepatoerythropoietic porphyria, and
1
5
hereditary coproporphyria, which are caused by deficient
enzymes in the later stages of the heme biosynthetic pathway. In
an effort to provide clinical laboratories with a complete cassette
of porphyria assays based on a single analytical platform, we
developed a new procedure for the direct assay of ALAD which
is reported here.
The butyrylated-d
7
porphobilinogen internal standard was
synthesized from 25.5 mg (8.4 × 10- mol) of porphobilinogen
using three molar equivalents of butyric-d14 anhydride in 5 mL
of 0.25 M sodium phosphate buffer (pH 6.8) for 30 min at room
temperature. HCl (1 M) was added to reduce the pH to ∼2.0,
followed by ammonium sulfate (50% w/w of aqueous phase).
The product was extracted with 10 2 mL portions of ethyl
acetate. The solvent was removed by evaporation under a
5
EXPERIMENTAL SECTION
Materials. All water used was purified by a Millipore Milli-Q
1
8MΩ filtering system. Porphobilinogen was purchased from
Frontier Scientific (Provo, UT). Butyryl-d chloride and sodium
butyrate-d were supplied by C/D/N Isotopes (Pointe-Claire,
Quebec). δ-Aminolevulinic acid, dithiothreitol, and all other
chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Isolation of Erythrocytes. Red blood cells were isolated and
lysed according to a procedure previously used in this laboratory.
All procedures regarding human blood followed Institutional
Review Board (IRB) protocols and received an approval (IRB
7
stream of N
for purification by HPLC. A 10 mL/min gradient of 100:0 to
:100 H O/ACN over 30 min using a 100 × 20 mm reverse-
phase C18 column achieved adequate separation to isolate d
2
, and the resulting solid was reconstituted in water
7
0
2
7
-
14
But-PBG, which eluted at 10.4 min. No acid was necessary in
the HPLC solvents to separate the compound of interest from
the impurities. The chemical stability of the internal standard
1
was checked by HPLC-MS and H NMR after 16 months of
2
5200). Blood was drawn into a vacuum sealed tube with heparin.
storage at -80 °C, and the sample was found to be unchanged.
Assay Protocol. An amalgamation of previously reported
assays was adapted to be compatible with electrospray ionization
(
(
8) Anderson, P. M.; Desnick, R. J. J. Biol. Chem. 1979, 254, 6924–6930
9) Wigfield, D. C.; Farant, J.-P. Clin. Chem. 1981, 27, 100–103.
.
(
10) Wigfield, D. C.; Farant, J.-P.; Goldberg, C.; MacKeen, J. E. J. Anal. Toxicol.
981, 5, 57–61.
11) Giampetro, P. F.; Desnick, R. J. Anal. Biochem. 1983, 131, 83–92
12) Bishop, D. F.; Desnick, R. J. Method Enzymol. 1986, 123, 339–345
13) Gelb, M. H.; Ture cˇ ek, F.; Scott, C. R.; Chamoles, N. A. J. Inherited Metab.
Dis. 2006, 29, 397–404
14) Wang, Y.; Gatti, P.; Sadilek, M.; Scott, C. R.; Ture cˇ ek, F.; Gelb, M. H. Anal.
Chem. 2008, 80, 2599–2605
15) Wang, Y.; Scott, C. R.; Gelb, M. H.; Ture cˇ ek, F. Anal. Chem. 2008, 80,
606–2611
5
,11
1
and tandem mass spectrometry.
Three hundred microliters of
(
(
(
.
0
.25 M sodium phosphate buffer (pH 6.80), 50 µL of 20 mM
.
dithiothreitol in buffer, and 50 µL of 10-fold diluted red blood cell
lysate were combined in a 2.0 mL polypropylene microfuge tube,
and the mixture was preincubated at 37 °C for 15 min. Then, 100
µL of 5 mM ALA in buffer was added, and the mixture was
incubated for 60 min. After incubation, 5 µL of n-butyric anhydride
.
(
(
.
2
.
Analytical Chemistry, Vol. 82, No. 15, August 1, 2010 6731

Figure 1. Fraction of porphobilinogen butyramide extracted in consecutive extractions by anhydrous n-butanol (open circles) and water-
saturated n-butanol (full circles).
was added and allowed to react for 30 min. Following this in situ
derivatization, 5 µL of a 275 µM solution of d -But-PBG in buffer
was added, as well as 100 µL of 1 M HCl to reduce the pH to
2. Four hundred and fifty microliters of water-saturated
n-butanol and ca. 300 mg of ammonium sulfate were then
added; the mixture was vortexed for 30 s until the ammonium
sulfate dissolved, followed by centrifugation for 3 min at 13 200g
to separate the layers. Two hundred microliters of the n-butanol
supernatant was removed with a syringe to a new 600 µL
polypropylene tube and stored at -80 °C. The solution was
mixed with 200 µL of methanol containing 2% (v/v) formic acid
immediately before analysis.
Mass Spectrometry. Analysis was conducted by flow injection
with 10 µL injections using a Waters Quattro Micro triple
quadrupole mass spectrometer in positive ion mode. Solvent flow
to the spectrometer’s ESI ion source came from a Waters 1525u
HPLC pump, flowing methanol containing 1% (v/v) formic acid
at 100 µL/min The instrument was operated using MassLynx
software with the following settings: electrospray ionization
capillary voltage, 4.00 kV; cone voltage, 38 V; extractor, 2 V; RF
lens voltage, 200 mV; source temperature, 80 °C; desolvation
temperature, 300 °C; desolvation gas flow, 500 L/h; dwell time,
to ethyl acetate. The cross-solubility of water and n-butanol
(roughly 10% soluble) was overcome using n-butanol that had
previously been saturated with water. Under these conditions, 89%
of porphobilinogen butyramide was extracted in two steps,
including 80% in the first extraction (Figure 1). One extraction
was, therefore, deemed to be sufficient for the assay. There was
no significant difference in extractability between the enzymatic
product and the deuterated internal standard. Using anhydrous
n-butanol decreased the extraction yield to 52% in the first step
(Figure 1).
7
∼
Previous fluorimetric assays for ALAD have shown that
16
dithiothreitol (DTT) increased enzymatic activity, by preventing
possible inhibition caused by the level of lead in the analyzed
blood. However, DTT may suppress the ESI-MS analyte signal
and, as such, could negatively affect the assay results. Experi-
mental runs with and without DTT showed that the inclusion of
2 mM DTT resulted in a 16% increase of enzyme activity; thus,
DTT was included in the final assay mixture. Zinc was also
considered as an assay additive, since zinc is used by the enzyme
as an active site cofactor. Due to the insolubility of zinc phosphate,
this additive required the use of a carbonate buffer. However,
using a carbonate buffer rather than phosphate caused nearly a
250 µs; collision energy, 20 eV; collision gas was argon at 1.97
9
0% drop in activity, and addition of zinc resulted in a further
mTorr.
decrease by up to 35% (Figure 2). Thus, the assays were run in
the phosphate buffer system, without any added zinc.
RESULTS AND DISCUSSION
Since a tandem quadrupole mass spectrometer may not be
available for immediate analysis in some clinical laboratories, a
study was carried out to determine the viability of transporting a
blood sample to a laboratory with the necessary instrumentation.
The relevant shipping conditions were duration and temperature;
as such, freshly drawn whole blood samples were stored at either
room temperature or at 4 °C for a set of time intervals ranging up
to 8 days (Figure 3). Temperatures below 0 °C were not
considered, as any such storage conditions would prematurely
Assay and Sample Workup Conditions. Analysis by elec-
trospray ionization mass spectrometry mandates that the sample
be in a compatible solvent free of involatile salts and buffers.
Therefore, extraction of the enzymatic product into a suitable
solvent was necessary. However, porphobilinogen is ionic or
zwitterionic at all relevant pH values, so derivatization was
necessary to allow extraction. We found that porphobilinogen
butyramide was effectively extracted into n-butanol. In situ
conversion of porphobilinogen to a butyramide was quantitative
by treating the assay mixture with 5 µL of butyric anhydride for
(
16) Sassa, S.; Granick, S.; Kappas, A. Ann. N.Y. Acad. Sci. 1975, 244, 419–
439
30 min. Of the extraction solvents used, n-butanol was superior
.
6732 Analytical Chemistry, Vol. 82, No. 15, August 1, 2010

Figure 2. Comparison of enzyme activity levels using different buffer systems with or without the inclusion of dithiothreitol.
Figure 3. Measured activity levels of heparinized blood stored at 4 °C for a number of days before erythrocyte isolation and enzyme assay
measurement.
lyse the desired erythrocytes, making it impossible to isolate them.
This experiment was conducted using two separate sets of blood
samples, one for each of the two most common blood preserva-
tives (heparin and EDTA). Results show that, while some activity
is lost within a week at room temperature (regardless of preserva-
tive), storing with heparin at 4 °C had no effect on the measured
enzyme activity level (Figure 3). Furthermore, both preservatives
allowed the retention of enzyme activity, although heparinized
samples gave slightly higher levels. Thus, samples should be
preserved in heparin and kept at 4 °C for any required shipping,
to ensure the most reliable determination of enzyme activity.
Mass Spectrometry. Porphobilinogen was found to be readily
protonated, sodiated, or potassiated by electrospray ionization.
Protonated porphobilinogen readily eliminated ammonia in the
electrospray source and on collisional activation. For example, at
5 eV laboratory collision energy, the (M + H)+ ion completely
dissociated. However, due to the above-described solubility
issues, underivatized porphobilinogen was not a suitable analyte
for this assay. Electrospray of porphobilinogen butyramide
+
+
mainly produced (M + Na) and (M + K) ions which were
+
less prone to dissociation than the (M + H) ion. The (M +
+
Na) ion showed the highest intensity for both the derivatized
product and internal standard and was, therefore, used for MS/
+
MS analysis. The (M + K) ions, albeit also abundant, showed
intensity variations in different assay runs. Collision-induced
+
dissociation of the (M + Na) ions gave fragments at m/z 275
Analytical Chemistry, Vol. 82, No. 15, August 1, 2010 6733

Figure 4. Collision induced dissociation mass spectra at 20 eV of (M + Na)+ ions from (top) porphobilinogen butyramide (m/z 319) and (bottom)
porphobilinogen butyramide-d (m/z 326).
7
Scheme 2
precursor ions fragmented to form the same product ion was
beneficial because it increased the duty cycle of the measure-
ments, thereby increasing the assay efficiency.
Blank measurements were carried out with samples that lacked
erythrocytes or the δ-aminolevulinic acid substrate. In each of
these blank measurements, no detectable signal for the m/z 319
f 188 transition was found by MS/MS. Blank correction in the
ALAD assays was, therefore, unnecessary.
ALAD Enzyme Kinetics. The assay conditions were opti-
mized by varying the assay time, amount of sample, and concen-
and 282 for the butyrylated enzymatic product and internal
standard, respectively, due to loss of 45 Da (COOH) (Figure
tration of substrate to determine the K
m
and Vmax. The time
course of the assay was found to be linear up to 4 h (Figure
S1, Supporting Information). A 15 min preincubation period was
necessary to warm up the blood sample before the addition of
the substrate. On the basis of the time dependence, we chose a
4
). The most abundant fragment ion at m/z 188 by loss of 131 Da
from the butyrylated product was due to a combined elimination
of butyramide and CO . The transitions monitored by MS/MS
2
were m/z 319 f 188 for the enzymatic product and 326 f 188
for the internal standard (Figure 4). Scheme 2 shows the
tentative scheme for the elimination involving a proton transfer
from the proximate carboxyl group onto the butyramide moiety.
The fact that both the deuterated and nondeuterated (M + Na)+
6
0 min incubation for standard assays which produced 800-1000
pmol of porphobilinogen to be readily quantified by tandem mass
spectrometry. Experiments were done to find the optimal amount
of red blood cell lysate to use. The assay showed a linear product
6734 Analytical Chemistry, Vol. 82, No. 15, August 1, 2010

Figure 5. (Top panel) Chart of enzyme activity of 36 random erythrocyte samples assayed by tandem mass spectrometry. The data error bars
are one standard deviation of triplicate measurements. The red dashed line indicates the presumed ALAD activity in a sample from a homozygotic
affected patient. (Bottom panel) Distribution of activities among the 36 samples.
formation with erythrocyte lysate volume (Figure S2, Supporting
Information). The most convenient amount was found to be 50
µL of diluted lysate corresponding to 5 µL of the erythrocyte
fraction.
of red blood cell lysate, was used for all samples. The 36 samples
showed ALAD activities ranging from 140 to 500 µmol/L eryth-
rocyte lysate/hour. The distribution of activities is shown in Figure
5. The mean enzyme activity was 277 µmol/L erythrocyte lysate/
hour, with a standard deviation of 90 µmol/L erythrocyte lysate/
hour. The relative standard deviation between injections of the
same assay was 3%, while assay-to-assay (using the same enzyme
source sample) RSD was 13%. Figure 5 further shows that a 5%
enzyme activity due to an ADP-affected patient would appear at
2.9 standard deviations of the sample mean and would be readily
distinguished from those of healthy individuals.
17,18
Taking into account K
m
values previous reported
for this
enzyme from slightly different biological sources, which ranged
from 160 µM in bovine liver to 600 µM in human fetal erythro-
cytes, the Michaelis-Menten enzyme kinetics parameters were
determined by varying the concentration of substrate in the range
of 100 µM to 3 mM, with triplicate assays at each concentration
level, and the enzyme activity at each concentration was fitted to
a nonlinear least-squares model (Figure S3, Supporting Informa-
tion). The K
to be 19 µM/h. Given these values, the concentration used for
the assay was set at 1 mM, roughly three times the K , to
ensure enzyme saturation and reduce any possibility of inter-
sample variance in enzyme velocity.
Clinical Sample Analysis. Following the determination of the
relevant parameters, the assay was applied to 35 clinical blood
samples, of unknown but varied ages and storage conditions, along
with the original blood sample used to elucidate the assay
parameters. An equal volume of red blood cells, and subsequently
m
was found to be 340 µM, and the Vmax was found
CONCLUSIONS
Tandem mass spectrometry has been shown to be an effective
and efficient means for the quantitation of ALAD in red blood
cells. The method requires only a one-step liquid-liquid extraction
out of aqueous reaction buffer into n-butanol and dilution with
acidified methanol. Blood samples need not be processed or
assayed immediately if stored with heparin at 4 °C and, thus, can
be shipped to a laboratory capable of tandem mass spectrometric
analysis without difficulty. Due to the high specificity of tandem
mass spectrometry, resulting from the analytes differing in mass
from other compounds in the heme biosynthetic pathway, the
assay extract could easily be combined with extracts from other
m
(
(
17) Sassa, S. Enzyme 1982, 28, 133–145
18) Chang, C. S.; Sassa, S. Blood 1985, 65, 939–944
.
.
Analytical Chemistry, Vol. 82, No. 15, August 1, 2010 6735

porphyria assays for multiplexed detections in a single injection
into the mass spectrometer, improving efficiency. This method
offers advantages over earlier more cumbersome, time-consuming,
and less specific methods. In spite of some efforts, we have been
unable to locate patients who had been previously diagnosed with
Doss porphyria. It is hoped that the specific assays developed
in this laboratory, together with the increasing availability of
tandem mass spectrometers in clinical laboratories, will contribute
to improved diagnostics of porphyrias in clinical practice.
ACKNOWLEDGMENT
Support of this work by the NIH-NIDDK (Grant R01 DK067859)
is gratefully acknowledged. We thank Dr. Martin Sadilek for
technical assistance with mass spectrometric measurements.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
1
9
Received for review April 27, 2010. Accepted June 18,
2010.
(19) Akagi, R.; Kato, N.; Inoue, R.; Anderson, K. E.; Jaffe, E. K.; Sassa, S. Mol.
Genet. Metab. 2006, 87, 329–336
.
AC101111M
6736 Analytical Chemistry, Vol. 82, No. 15, August 1, 2010