Anal. Chem. 2008, 80, 2599-2605
Direct Assay of Enzymes in Heme Biosynthesis for
the Detection of Porphyrias by Tandem Mass
Spectrometry. Uroporphyrinogen Decarboxylase
and Coproporphyrinogen III Oxidase
†
†
†
‡
,†
Yuesong Wang, Paula Gatti, Martin Sad ı´ lek, C. Ronald Scott, Franti sˇ ek Ture cˇ ek,* and
Michael H. Gelb*,
†,§
Departments of Chemistry, Pediatrics, and Biochemistry, University of Washington, Seattle, Washington 98195-1700
We report new assays of enzymes uroporphyrinogen
decarboxylase (UROD) and coproporphyrinogen III oxi-
dase (CPO) in the heme biosynthetic pathway. The assays
were developed for use in clinical diagnostics of inherited
disorders porphyria cutanea tarda and hereditary copro-
porphyria, respectively. Electrospray ionization tandem
mass spectrometry is used to monitor the decarboxylation
of pentaporphyrinogen I or uroporphyrinogen III catalyzed
by UROD and to determine the enzyme activity in human
erythrocytes by measuring the production of copropor-
in infancy and is characterized by a very low level of UROD in
red blood cells. The sporadic form is more common, and UROD
activity is reduced in the liver only. Sporadic PCT is often
associated with liver disease caused by alcoholism, hepatitis C,
or estrogen intake by women. The PCT manifests itself by blisters,
which become ulcerated in areas of the skin exposed to sunlight,
especially on the face, ears, and hands. The affected areas of skin
tend to be fragile and show hyperpigmentation and hypertrichosis.
Coproporphyrinogen oxidase (CPO) is a mitochondrial enzyme
that catalyzes decarboxylative oxidation of two carboxyethyl
groups in coproporphyrinogen III to form vinyl groups in another
heme intermediate, protoporphyrinogen IX (Scheme 1). Mutations
in the gene encoding CPO (located on chromosome 3q11.2) result
in hereditary coproporphyria (HCP), an autosomal dominant acute
hepatic porphyria, which is characterized by acute attacks of
neurological dysfunction often provoked by drugs, fasting, men-
strual cycle, or infectious diseases.2
m
phyrinogen I or III. The K value for pentaporphyrinogen
I was measured as 0.17 ( 0.03 µM. A mass spectromet-
ric assay was also developed for the two-step decarboxy-
lative oxidation of coproporphyrinogen III to protopor-
phyrinogen IX catalyzed by CPO in mitochondria from
m
human lymphocytes (K ) 0.066 ( 0.009 µM). The
,3
assays show good reproducibility, use simple workup by
liquid-liquid extraction of enzymatic products, and em-
ploy commercially available substrates and internal stan-
dards.
Current methods for analyzing UROD and CPO deficiencies
are indirect as they rely on the analysis of porphyrin metabolites
in urine, feces, or erythrocytes. These approaches are often
frustrating because of variability of excretion or loss during
purification by chromatography. For many of the heme biosyn-
thetic enzymes, direct enzyme assays are either not readily
available for diagnosis or rely on a complex array of bioanalytical
Porphyrias are a group of rare diseases caused by enzyme
1
deficiencies in the heme biosynthetic pathway. Heme biosynthesis
starts from succinyl-CoA and glycine and involves eight enzy-
matic steps, of which seven are connected with known enzyme
4
-12
techniques
that often require separation of products by liquid
1
deficiencies in humans. The enzyme uroporphyrinogen decar-
chromatography.4 Fluorimetric assays have been developed but
because of poor sensitivity have given way to radiometric assays,
,6
7
boxylase (UROD) catalyzes stepwise decarboxylation of heme
precursor uroporphyrinogen III, which is converted to copropor-
phyrinogen III (Scheme 1). Lack of UROD is the basic cause of
porphyria cutanea tarda (PCT), the late skin form of porphyria,
with onset in adult life. PCT is the most common form of
porphyria, which occurs in two clinical familial and a sporadic
form. The familial forms of PCT are inherited as an autosomal
dominant trait, in which UROD is reduced in all tissues. A severe
form of PCT, hepatoerythropoietic porphyria (HEP), has its onset
8
which suffer from the lack of a commercially available source of
substrate.
(2) Brodie, M. J.; Thompson, G. G.; Moore, M. R.; Beattie, A. D; Goldberg, A.
Quant. J. Med. 1977, 46, 229-241.
(
(
3) Kuhnel, A.; Gross, U.; Doss, M. O. Clin. Biochem. 2000, 33, 465-473.
4) Jones, M. A.; Thientanavanich, P.; Anderson, M. D.; Lash, T. D. J. Biochem.
Biophys. Methods 2003, 55, 241-249.
(5) Francis, J. E.; Smith, A. G. Anal. Biochem. 1984, 138, 404-410.
6) McManus, J.; Blake, D.; Ratnaike, S. Clin. Chem. 1988, 34, 2355-2357.
(
*
To whom correspondence should be addressed. E-mail: turecek@
(7) Labbe, P.; Camadro, J. M.; Chambon, H. Anal. Biochem. 1985, 149, 248-
260.
chem.washington.edu (F.T.), gelb@chem.washington.edu (M.H.G.).
†
Department of Chemistry.
Department of Pediatrics.
Department of Biochemistry.
(8) Elder, G. H.; Evans, J. O. Biochem. J. 1978, 169, 205-214.
(9) Kardish, R. M.; Woods, J. S. J. Appl. Biochem. 1980, 2, 159-67.
(10) Li, F.; Lim, C. K.; Peters, T. J. Biochem. J. 1986, 239, 481-484.
(11) Grandchamp, B.; Nordmann, Y.; Elder, G. H.; Smith, S. G. Enzyme 1982,
28, 196-205.
(12) Gross, U.; Gerlach, R.; Kuehnel, A.; Seifert, V.; Doss, M. O. J. Inherited Metab.
Dis. 2003, 26, 565-570.
‡
§
(
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.
1
0.1021/ac702130n CCC: $40.75 © 2008 American Chemical Society
Analytical Chemistry, Vol. 80, No. 7, April 1, 2008 2599
Published on Web 02/23/2008