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C H A P T E R
94
Disorders of Propionate and
Methylmalonate Metabolism
Wayne A. Fenton
_
Roy A. Gravel
1. Propionyl coenzyme A (CoA) Ð formed in the catabolism
of several essential amino acids (isoleucine, valine,
methionine, threonine), odd-chain fatty acids, and cholesterol Ð is metabolized primarily by enzymatic conversion
to methylmalonyl CoA, which is subsequently isomerized
to succinyl CoA. This sequence depends on the activity of
several enzymes (see Fig. 94-2): propionyl CoA carboxylase, methylmalonyl CoA racemase, and methylmalonyl
CoA mutase. Propionyl CoA carboxylase requires biotin
as a cofactor, whereas methylmalonyl CoA mutase
requires adenosylcobalamin (AdoCbl), a cobalamin (Cbl;
vitamin B12) coenzyme.
2. Propionyl CoA carboxylase and methylmalonyl CoA
mutase are oligomeric enzymes. Propionyl CoA carboxylase is composed of nonidentical subunits (a and b); biotin
binds to the a subunit. The holocarboxylase contains six a
and six b subunits (a6b6). The a subunit is encoded by a
gene on chromosome 13 (NM_000282) in humans, the b
subunit by a gene on chromosome 3 (NM_000532). Methylmalonyl CoA mutase is a dimer of identical subunits (a2),
encoded by a gene on chromosome 6 (NM_000255).
3. Inherited de®ciency of propionyl CoA carboxylase activity
in humans results from genetically distinct defects at four
loci. Isolated de®ciency is caused by mutations at the a and
b loci coding for the carboxylase subunits. De®ciency of
multiple biotin-dependent carboxylases occurs in two
forms: one resulting from de®ciency of holocarboxylase
synthase (the enzyme that attaches biotin to apocarboxylase subunits), the other from de®ciency of biotinidase (the
enzyme that cleaves biotin from the lysine residue in the
carboxylase to which the biotin is attached). Multiple
carboxylase de®ciency is discussed in detail in Chapter
156.
4. Isolated de®ciency of propionyl CoA carboxylase, a major
cause of the ketotic hyperglycinemia syndrome, results in
the accumulation of propionate in blood and of 3hydroxypropionate, methylcitrate, tiglylglycine, and unusual ketone bodies in urine. Two complementation groups,
pccA (OMIM 232000) and pccBC (OMIM 232050) have
been de®ned among propionyl CoA carboxylase±de®cient
patients. These groups correspond to mutations affecting
A list of standard abbreviations is located immediately preceding the index in each
volume. Nonstandard abbreviations used in this chapter include:
AdoCbl ˆ adenosylcobalamin; cbl ˆ cobalamin metabolism locus (cblA, cblB,
etc.); Cbl ˆ cobalamin; CN-Cbl ˆ cyanocobalamin; CoA ˆ coenzyme A; CPS
I ˆ carbamylphosphate synthetase I; DMB ˆ dimethyl benzimidazoyl; H4folate ˆ tetrahydrofolate; IF ˆ intrinsic factor; MeCbl ˆ methylcobalamin; MeH4folate ˆ N5-methyltetrahydrofolate; mut ˆ methylmalonyl CoA mutase locus;
OH-Cbl ˆ hydroxocobalamin; OLCFA ˆ odd-numbered long-chain fatty acids;
pcc ˆ propionyl CoA carboxylase locus; TC (I, II, or III) ˆ transcobalamin (I, II,
or III).
5.
6.
7.
8.
_
David S. Rosenblatt
genes coding for the a subunit and the b subunit,
respectively, of the carboxylase apoprotein. Clinically,
the disorder is characterized by severe metabolic ketoacidosis, which often appears in the neonatal period and
requires vigorous alkali therapy and protein restriction.
Oral antibiotic therapy to reduce gut propionate production also may prove useful.
Multiple carboxylase de®ciency (OMIM 253270) leads to
impaired activity of four biotin-dependent enzymes: acetyl
CoA carboxylase, propionyl CoA carboxylase, 3-methylcrotonyl CoA carboxylase, and pyruvate carboxylase. The
clinical hallmarks of this disorder include ketoacidosis, a
diffuse erythematous rash, alopecia, seizures, hypotonia,
and developmental retardation (see Chapter 156).
Inherited de®ciency of methylmalonyl CoA mutase activity
in humans is caused by mutations at many different loci.
Isolated de®ciency results from mutations at the apomutase locus (OMIM 251000) and at two loci coding for gene
products required, speci®cally for the biosynthesis of
AdoCbl. Combined de®ciency of mutase and of the other
major Cbl-dependent enzyme in mammalian cells, methionine synthase (formally, N5-methyltetrahydrofolate:homocysteine methyltransferase), results from inherited defects
in Cbl transport and from three distinct defects in the
intracellular pathway of Cbl coenzyme synthesis affecting
the synthesis of both AdoCbl and methylcobalamin
(MeCbl), the coenzyme required by methionine synthase.
These several defects in intracellular Cbl metabolism are
discussed in detail in Chapter 155.
Neonatal or infantile metabolic ketoacidosis is the clinical
hallmark of isolated methylmalonyl CoA mutase de®ciency. Cells from some apomutase-de®cient children have
no functional mutase (designated mut 0); cells from others
contain a structurally altered mutase with reduced af®nity
for AdoCbl and with reduced stability (mut 2). Such
children exhibit methylmalonic acidemia and methylmalonic aciduria that do not respond to Cbl supplementation
but can sometimes be treated with dietary protein
restriction. Carnitine, as well as oral antibiotic therapy
to reduce gut ¯ora, may be effective as well.
Two abnormalities in AdoCbl synthesis only, designated
cblA (OMIM 251100) and cblB (OMIM 251110), lead to
impaired methylmalonyl CoA mutase activity and are
characterized by a clinical and chemical picture virtually
identical to that seen in apomutase-de®cient children. In
most cblA patients and some cblB patients, pharmacologic
supplements of CN-Cbl or hydroxocobalamin (OH-Cbl)
produce distinct reductions in methylmalonate accumulation and offer a valuable therapeutic adjunct to dietary
protein limitation.
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PART 9 / ORGANIC ACIDS
9. Three other distinct defects Ð cblC (OMIM 277400), cblD
(OMIM 277410), and cblF (OMIM 277380) Ð lead to
impaired synthesis of both AdoCbl and MeCbl and,
accordingly, to de®cient activity of both methylmalonyl
CoA mutase and methionine synthase. Such children have
methylmalonic aciduria and homocystinuria. Most children with a cblC mutation appear to be more severely
affected clinically than the two known sibs in the cblD
group, although a number of cblC patients have had an
onset of disease in adult life. Major clinical problems in
cblC patients include failure to thrive, developmental
retardation, and such hematologic abnormalities as
megaloblastic anemia and macrocytosis. The precise defect
in the cblC and cblD patients is not yet known, but it
involves an early step in the intracellular metabolism of
cobalamins. The defect in cblF cells involves impaired
ef¯ux of free Cbl from lysosomes. Therapy includes
protein restriction, pharmacologic doses of OH-Cbl, and
betaine supplementation.
10. The discriminating biochemical features of the known
forms of inherited methylmalonic acidemia are shown in
Table 94-4.
11. All of the disorders of propionate and methylmalonate
metabolism for which there are adequate data are inherited as autosomal-recessive traits. Heterozygotes for
the following defects can be detected biochemically: pccA,
mut 0, mut2, and cblB. Genetic complementation analyses
with somatic cell heterokaryons have been particularly
useful in demonstrating genetic heterogeneity and in
con®rming the existence of autosomal-recessive inheritance among the propionic acidemias and the methylmalonic acidemias. Precise molecular defects have been
described for pccA, pccBC, mut 0, and mut2 patients.
12. Prenatal detection of fetuses with propionyl CoA carboxylase de®ciency, methylmalonyl CoA apomutase de®ciency,
and defective synthesis of AdoCbl or of both coenzymes is
best done using assays in cultured amniotic cells and gas
chromatographic/mass spectroscopic determinations on
amniotic ¯uid or maternal urine.
Methylmalonic acid and its immediate precursor, propionic acid,
are detectable in only trace amounts in normal human blood, urine,
and cerebrospinal ¯uid. The minuscule quantities of these
compounds in extracellular ¯uids have obscured the key role
that these acids play in human metabolism. Biochemists
investigating animal nutrition have been interested in propionate
metabolism for many years because ruminants derive most of their
energy requirements from the oxidation of propionate and acetate
produced by bacterial fermentation in their rumens.1 Although
propionate and methylmalonate are of little quantitative importance in humans as direct sources of energy, these acids, found
intracellularly largely as their coenzyme A (CoA) esters, are vital
intermediates in the catabolism of fat and protein.
Several independent, and seemingly unrelated, lines of
evidence drew the attention of the physician and the clinical
investigator to the study of propionate and methylmalonate metabolism. In 1959 and 1960, several groups reported that adenosylcobalamin (AdoCbl), one of the coenzyme forms of cobalamin (Cbl)
(vitamin B12), is an essential cofactor in the enzymatic conversion
of L-methylmalonyl CoA to succinyl CoA.2± 4 Shortly thereafter,
patients with acquired Cbl de®ciency were shown to excrete large
amounts of methylmalonic acid in their urine.5,6 The methylmalonic aciduria was rapidly reversed by administration of
physiologic doses of Cbl and was attributed to an acquired block
in methylmalonate catabolism caused by inadequate amounts of
the needed Cbl coenzyme.
In 1961, Childs and associates7 described a young boy with
recurrent attacks of severe ketoacidosis who had elevated
concentrations of glycine and several other amino acids in his
blood and urine. A series of detailed metabolic studies demonstrated that the attacks were precipitated by protein feeding and
more speci®cally by ingestion of the branched chain amino acids,
methionine and threonine. Because elevation in plasma glycine
level was the most striking biochemical abnormality, the disorder
was called ketotic hyperglycinemia. Later evidence has established that this disorder is caused by an inherited defect in the
catabolism of propionate, not by a primary abnormality in glycine
utilization or biosynthesis.8,9
Since 1967, a number of critically ill children have been
described who draw these seemingly disparate observations
together and focus attention on the enzymes and coenzymes that
participate in the pathway responsible for the formation of
propionate and its conversion to succinate. Oberholzer,10 Stokke,11
and their colleagues described infants with profound metabolic
acidosis and hyperglycinemia (or hyperglycinuria) who excreted
huge amounts of methylmalonic acid in their urine, but who were
not Cbl de®cient. Subsequently, Rosenberg and his colleagues8
reported that urine from the index patient with ketotic hyperglycinemia and from his affected sister contained no methylmalonic
acid. This observation indicated that primary methylmalonic
acidemia and ketotic hyperglycinemia were different disorders
with identical clinical manifestations.3
The latter group and Lindblad et al.12,13 also described children
with ketoacidosis and methylmalonic acidemia who were not Cbl
de®cient, but who responded to administration of pharmacologic
doses of cyanocobalamin (CN-Cbl) or its coenzyme with a marked
decrease in concentration of urinary methylmalonic acid. The
index patient8 was subsequently shown to suffer from a primary
defect in AdoCbl synthesis,14,15 not from a defect of the apoenzyme that catalyzes the conversion of methylmalonyl CoA to
succinyl CoA.
These observations and others, which will be discussed in
detail subsequently, emphasize that numerous inherited abnormalities in the metabolic pathway for propionate and methylmalonate occur and that these defects lead to profound illness and, in
many cases, death due to a disturbed acid-base balance or
developmental failure. The study of these disorders has led to
important insights in our understanding of the role of this pathway
in humans and has illustrated, once again, that a group of clinically
identical disorders can be produced by several different mutations
affecting the synthesis of related apoenzymes and coenzymes.
Several reviews of this subject matter have been published.16±21
BIOCHEMICAL PATHWAYS
Propionate Metabolism
Formation of Propionate and Methylmalonate. Most of the
propionic acid used by ruminant animals is formed by bacterial
fermentation in the rumen.1 By contrast, nonruminant mammals
derive most of their propionate from the catabolism of lipid and
protein. As noted in Fig. 94-1, catabolism of the branched chain
amino acid isoleucine leads to the formation of propionyl CoA, as
does the degradation of methionine and threonine.22 Studies with
[13C]valine in a patient with methylmalonic acidemia23 and with
the valine catabolite [13C]isobutyrate in rats24 indicate that valine
is also a propionate precursor and is not catabolized directly to
methylmalonyl CoA, as had been suggested. Catabolism of these
amino acids accounts for much of the propionate formed in
humans; data presented by Thompson et al.25 indicate that their
contribution is on the order of 50 percent in patients with inborn
errors of propionate or methylmalonate metabolism. Other sources
of propionate include b-oxidation of fatty acids with an odd
number of carbon atoms, which ultimately leads to the formation
of 1 mole of propionyl CoA per mole of fatty acid.25± 27
Degradation of the side chain of cholesterol also leads to the
synthesis of propionyl CoA, but this pathway appears to be of little
quantitative signi®cance.28 Finally, there have been suggestions
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
cycle and is ultimately glycogenic because of its conversion to
pyruvate by way of oxaloacetate. The sum of these reactions may
be written as follows:
Propionate ‡ ATP ! pyruvate ‡ 4H‡ ‡ ADP ‡ Pi
In bacteria, propionate is formed from pyruvate by reversal of
the reaction sequence just described,26 but in mammalian systems
the equilibrium of the system is far in the direction of propionate
catabolism, rather than biosynthesis.
Apoenzymes
that gut bacteria may contribute a substantial amount of
propionate, at least under some circumstances. These ®ndings
are based on the ability of oral antibiotics to decrease urine29 and
plasma25 propionate concentrations in some propionic and
methylmalonic acidemia patients. From stable isotope studies in
patients with methylmalonic aciduria and propionic aciduria,
Leonard estimated that about 50 percent of propionate production
is due to amino acid catabolism, 25 percent is due to the odd chain
fatty acid catabolism, and 25 percent is due to bacterial activity in
the gut.30
Methylmalonyl CoA is synthesized essentially only from
propionyl CoA (Fig. 94-1). Propionate has long been known to
be glycogenic in animals,31 but the pathway by which propionate
is converted to carbohydrate became clear only when Lardy and
Adler demonstrated that liver mitochondria contain enzymes that
synthesize succinate from propionate.31 The discovery in 1955 that
methylmalonate is an intermediate in the formation of succinate
from propionate (Fig. 94-1) provided an important further step in
the characterization of this pathway.32,33
Kaziro and Ochoa ®rst de®ned the individual steps of
propionate catabolism in animal tissues and characterized the
enzymes involved.26 Propionyl CoA, formed either by the degradative reactions discussed above or by the enzymatic esteri®cation
of propionate itself,34 may be considered the precursor of this
reaction sequence (Fig. 94-2). Three enzymatic reactions are
responsible for the conversion of propionyl CoA to succinyl CoA.
The ®rst involves the carboxylation of propionyl CoA to
methylmalonyl CoA,34,35 a reaction catalyzed by propionyl CoA
carboxylase (EC 6.4.1.3). Although two stereoisomers of methylmalonyl CoA exist, only the D form is produced in the
carboxylation reaction.36,37 This isomer is not a substrate for the
subsequent mutase reaction and must be racemized to the L
con®guration by another enzyme, methylmalonyl CoA racemase
(EC 5.1.99.1).38 The third reaction, catalyzed by methylmalonyl
CoA mutase (EC 5.4.99.2), isomerizes L-methylmalonyl CoA to
succinyl CoA.39 The latter compound enters the tricarboxylic acid
Propionyl CoA Carboxylase. This enzyme, ®rst crystallized
from pig heart,40 has been puri®ed to homogeneity from bovine
kidney41 and human liver.42,43 The enzyme is composed of
nonidentical subunits (a and b): the required cofactor, biotin, is
bound exclusively to the larger (a) subunit. The molecular masses
of the human enzyme are 750 to 800 kDa for the native form,
72 kDa for the a subunit, and 56 kDa for the b subunit. In the
enzyme from Mycobacterium smegmatis, each mole contains 6
moles of biotin.44 This suggests that the native enzyme is a
hexamer of protomers, each protomer containing a single a and a
single b subunit. The size of the human enzyme implies a similar
structure, namely an (ab)6 quaternary structure.
The carboxylation of propionyl CoA occurs in a two-step
reaction.26 In the ®rst step, which requires adenosine triphosphate
(ATP) and Mg2‡ and is stimulated by K‡, bicarbonate is attached
to the ureido (N 1) nitrogen of biotin in the apoenzyme±biotin
complex (Fig. 94-3), forming a carboxybiotin±apoenzyme intermediate. This complex, in turn, reacts with propionyl CoA to
transfer the carboxyl group from biotin to the second carbon of
propionyl CoA, forming D-methylmalonyl CoA. As with other
biotin-catalyzed carbon dioxide ®xation reactions, the biotin
molecule is directly responsible for the transfer of the carboxyl
group.45
The complementary DNAs (cDNAs) for the a and b subunits
have been cloned from human46±48 and rat liver 49±51 libraries. A
2.9-kb messenger RNA (mRNA) codes for the a chain and a major
2.0-kb mRNA codes for the b chain.46,50 Based on the sequence of
the full-length cDNAs, the human a chain contains 703 amino
acids52 and the b chain 539 amino acids.53,54 As is the case for
most nuclear-encoded mitochondrial proteins, the a and b subunits
are synthesized on free cytoplasmic polyribosomes as larger
precursors, bearing cleavable N-terminal leader peptides.55 The Nterminus of the mature human a subunit has been determined and
implicates a leader peptide of 26 amino acids.48 In studies on the
rat enzyme, the positively charged leader peptides of both subunits
were shown to direct mitochondrial uptake and processing in
vitro.51,56 Assembly of the mature a and b subunits into the
oligomeric holoenzyme is expected to occur after mitochondrial
import and removal of the leader peptides have occurred.
The polypeptide sequences of the a and b subunits have
revealed a number of highly conserved regions by comparison
with related enzymes from bacteria to mammals. Most striking is
the near universal tetrapeptide, Ala-Met-Lys-Met, that de®nes the
biotin-binding site (Lys residue) of the a subunit.47,51 In addition,
the a subunit contains a recognizable biotin carboxylase domain
Fig. 94-2 Enzymatic details of the major catabolic pathway for
propionyl CoA and methylmalonyl CoA. Succinyl CoA has several
metabolic fates, including oxidation through the tricarboxylic
acid cycle and condensation with glycine to form d-aminolevulinic
acid. Two coenzymes act in the reaction sequence: biotin, in
the carboxylation of propionyl CoA, and adenosylcobalamin
(AdoCbl), in the isomerization of methylmalonyl CoA to succinyl
CoA.
Fig. 94-1 Precursors of and the major catabolic pathway for
propionate and methylmalonate. The free acids are derived from
their CoA esters by hydrolysis. A number of clinical disorders arise
from errors at various steps in these pathways. Broken arrows
indicate the presence of several reactions.
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2168
PART 9 / ORGANIC ACIDS
Fig. 94-3 A model for the mammalian propionyl CoA carboxylase
protomer containing two nonidentical subunits (a and b), a biotin
carrier site on the a subunit, and multiple substrate and effector
sites.
and, within it, an ATP binding site57; the b subunit contains
sequences related to carboxybiotin and propionyl CoA binding
domains (Fig. 94-3) . Expression of the C-terminal 67 amino acids
of the human a subunit in Escherichia coli results in its biotinylation by the bacterial biotin ligase, BirA, or by coexpressed
human holocarboxylase synthase.58
The human PCCA gene, encoding the a subunit, has been
mapped to chromosome 13q32.46,59 The PCCA gene is at least 100
kbp long, but its intron±exon organization remains to be
elucidated. The PCCB gene, encoding the b subunit, is on
chromosome 3q13.3-q2246,49 and has been positioned within a
YAC contig containing the blepharophimosis-ptosis-epicanthus
inversus syndrome locus.60 The PCCB gene contains 15 exons,
ranging in size from 57 to 183 bp.61
Methylmalonyl CoA Racemase. This enzyme owes its discovery
to the observation that methylmalonyl CoA synthesized chemically is a substrate for the mutase reaction (Fig. 94-2), whereas
methylmalonyl CoA formed enzymatically from the carboxylation
of propionyl CoA will not react with the mutase unless it is ®rst
heated. Ultimately, the demonstration that heating converts
D-methylmalonyl CoA to DL-methylmalonyl CoA led to the
conclusion that only the L form of the ester will react with the
mutase enzyme. This interpretation was con®rmed by separating
mutase activity from racemase activity using Sephadex chromatography.26,38,62 The racemase has been puri®ed extensively from
sheep liver.38 It has no known cofactor requirements and catalyzes
the conversion of D- to L-methylmalonyl CoA by inducing a shift
in the a-hydrogen atom.38,62
Methylmalonyl CoA Mutase. In 1955, Flavin et al.32 and Katz
and Chaikoff33 observed independently that the isomerization of
methylmalonyl CoA to succinyl CoA was catalyzed by an enzyme
found in sheep kidney and rat liver. The chemical analogy between
this isomerization reaction and the isomerization of glutamate to
b-methylaspartate in bacteria,63 along with the demonstration by
Barker and his colleagues64,65 that a coenzyme form of Cbl was
needed for the latter reaction, led to the ®nding in several
laboratories that a Cbl coenzyme is also required for the isomerization of methylmalonyl CoA.2 ±4 The enzyme, originally called
methylmalonyl CoA isomerase but now designated methylmalonyl
CoA mutase, was ®rst crystallized from sheep kidney and bacteria.
More recently, it has been puri®ed to homogeneity from human
placenta66 and human liver.67 From both human sources, the
enzyme appears to be a dimer (145 to 150 kDa) of identical
subunits (72 to 77 kDa). The holoenzyme contains 1 mole AdoCbl
per mole of subunit,67 the Cbl cofactor being very tightly bound to
the apoenzyme (Km of the puri®ed, expressed human enzyme for
AdoCbl is 5 10 8 M).68 Under certain conditions, the human
enzyme displays complex kinetics with regard to the binding of
methylmalonyl CoA and AdoCbl, leading to the thesis that the
active sites of the dimeric enzyme are not equivalent.69 In this
regard, it is signi®cant that hydroxocobalamin (OH-Cbl) appears
to act as both a competitive and an irreversible inhibitor of human
mutase.69
As indicated in Figure 94-2, the isomerization reaction could
occur by exchange of a hydrogen for either the free carboxyl group
or the CoA-carboxyl moiety of methylmalonyl CoA. Studies using
isotopically labeled substrate demonstrated convincingly that it is
the CoA-carboxyl group that is transferred 70 through an
intramolecular isomerization.71,72 The role of the Cbl coenzyme
in this reaction has been established by electron paramagnetic
resonance and ultraviolet (UV)-visible spectroscopy. As suggested
by Halpern,73 AdoCbl serves as the source of a pair of free
radicals, generated initially by homolysis of the cobalt±carbon
bond. Although no evidence for such radicals has been obtained
with holoenzyme alone, in the presence of substrate, a tightly
exchange-coupled free radical pair can be observed.74,75 One
electron is clearly on cob(II)alamin, as further identi®ed by its UVvisible spectrum.76 The nature of the other radical species is less
clear, but isotope effect data suggest that it is substrate derived,
rather than a radical form of the 50 -deoxyadenosyl group.76 For
mutase from Propionibacterium shermanii, about 20 to 25 percent
of the cofactor is present in a radical form under steady-state
conditions, and stopped-¯ow kinetic observations suggest that
homolysis is much faster than the overall reaction.76 This implies
that a later, slow step (such as product release) must be rate
controlling, a conclusion supported by tritium isotope effect
studies77 and rationalized by structural investigations of the
enzyme.
The mutase subunit is synthesized as a larger cytoplasmic
precursor, bearing a 3- to 4-kDa cleavable leader peptide.78 In a
cell-free system, the precursor is imported by mitochondria via an
energy-dependent mechanism and cleaved to its mature form by a
divalent cation-dependent protease (the mitochondrial processing peptidase). In intact cells, the precursor is rapidly (halflife ˆ 6±9 min) converted to its mature form.78
Both cDNA and genomic DNA encoding human mutase have
been cloned, as have the corresponding murine sequences.79±82
The cDNA sequence predicts a leader peptide 32 amino acids long
that is strongly positively charged (4 Arg, 2 Lys, 1 Glu).80,82
Analysis of Southern blots of DNA from human/hamster somatic
cell hybrids and in situ hybridization, using the mutase cDNAs as
probes, mapped the human gene for mutase and the MUT locus to
region 6p12-p21.283 and uncovered one highly informative
restriction fragment length polymorphism.79
Mutase from P. shermanii has been puri®ed, cloned, and
overexpressed in E. coli. It is an ab heterodimer with a single
active subunit (a) that binds one molecule of AdoCbl.84,85 The a
subunit is about 60 percent identical (75 percent similar) to the
human enzyme, with many of the nonconservative substitutions
occurring in the N-terminal region.86 Several forms of P.
shermanii mutase have been crystallized and their radiographic
structures solved (PDB codes 1REQ±5REQ).87±89 Based on these,
mutase consists of two major domains, with an N-terminal region
involved in subunit±subunit interaction and an interdomain linker
(see Fig. 94-10). The ®rst domain is a (ba)8 TIM barrel, with the
substrate binding site threaded through its center. The second
domain is the so-called Cbl-binding domain, by virtue of its close
sequence and structural homology with the Cbl-binding domain of
methionine synthase.90 It is a (ba)5 barrel that has a groove for
binding the 5,6-dimethylbenzimidazolyl (DMB) side chain of Cbl,
the stabilized histidine side chain that displaces the DMB from the
lower axial position of the Cbl, and interaction sites for the lower
face of the corrin ring. The interface between these domains
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
Fig. 94-4 Minor pathways of propionate catabolism. Note that both pathways can ultimately generate acetyl CoA. The signi®cance of these
minor pathways is discussed in the text.
accommodates the upper face of the corrin and the 50 deoxyadenosyl group.
Comparison of substrate-free and substrate-bound structures
indicates both how substrate and product come and go from the
active site and how the enzyme protects the radicals at its active
site.88 In the substrate-free (open) conformation, four of the ba
pairs in the ®rst domain have moved as a rigid body, relative to the
other four and the second domain, to split open the TIM barrel and
provide access to the active site and the binding sites for the
pantotheine chain of the substrate. In the bound (closed)
con®guration, however, the domain has come back together to
close the active site around the methylmalonyl group, the rest of
the substrate blocks access through the middle of the barrel, the
active site cavity is reduced in volume, and the binding site for the
50 -deoxyadenosyl group is destroyed. Thus, the substrate-triggered
conformational change appears to simultaneously drive homolysis
of the cobalt±carbon bond while sequestering the radicals formed
at the active site, permitting a free-radical rearrangement protected
from the surrounding aqueous environment. Because a corresponding opening of the TIM barrel must occur before product can
be released, it has been suggested that this is likely the slow step in
the overall forward reaction.88
Alternative Pathways of Propionate Metabolism
Although the catabolism of propionate to succinate through
methylmalonate is the major pathway for propionate utilization in
mammalian systems, alternative pathways exist. Propionyl CoA
can replace acetyl CoA as a ``primer'' for long-chain fatty acid
synthesis91 and lead to the formation of odd-chain fatty acids,
notably heptanoate, nonanoate, and undecanoate. There are also
alternative catabolic mechanisms, one of which is described in Fig.
94-4.26 The ®rst step in this sequence involves the formation of an
a,b-unsaturated fatty acid, acrylyl CoA, which is subsequently
hydrated, leading to the formation of either lactyl CoA or b-OHpropionyl CoA. The former compound is hydrolyzed to lactate,
thus providing a second means by which propionate may be
converted to pyruvate. Catabolism of b-OH-propionyl CoA leads
ultimately to the synthesis of acetyl CoA or b-alanine, compounds
discussed in Chapter 91. In addition, propionyl CoA may condense
with oxaloacetate to form methylcitrate in a reaction analogous to
the biosynthesis of citric acid from acetyl CoA and oxaloacetate.92
These alternative pathways are of little quantitative importance in
normal subjects but become much more prominent in patients with
blocks in the major pathway of propionate metabolism.93±95
Coenzymes
Biotin. Biotin is widely distributed in plants and animal tissues
and is readily synthesized by a variety of microorganisms. It was
®rst isolated from egg yolk in 1936 by the Dutch biochemist
Kogl,96 and its structure was de®ned soon thereafter by du
Vigneaud and colleagues.97 Our understanding of this watersoluble cofactor is inextricably linked with the evolution of our
knowledge concerning avidin, an egg white protein that binds
biotin very tightly. Comprehensive reviews on biotin98,99 and on
biotin-dependent enzymes100,101 exist. The reader is referred to
Chapter 156 for further discussion of biotin.
Structure and Function. Biotin is the essential cofactor of the
four bicarbonate-utilizing carboxylases in mammals. Structurally,
it is composed of fused imidazole and thiophene rings to which is
attached an n-valeric acid side chain; it has a molecular mass of
244 daltons. It is covalently attached to carboxylases through a
linkage between the carboxyl group and the e-amino group of a
lysine residue in a well-de®ned biotin-binding domain, which is
highly conserved among carboxylases from bacteria to mammals
(Fig. 94-3).102 Its role is to act as the carboxyl carrier in the
carboxylation of substrate molecules (Fig. 94-5).
The four biotin-dependent carboxylases are enzymes of
intermediary metabolism.103 One of these, acetyl CoA carboxylase, occurs in two forms104,105 and is a cytosolic enzyme. It
catalyzes the key step in long-chain fatty acid biosynthesis, the
formation of malonyl CoA from acetyl CoA. The three other
biotin-dependent carboxylases are found in the mitochondrial
matrix, where they catalyze critical steps in amino acid and
organic acid metabolism. Pyruvate carboxylase is a key enzyme of
gluconeogenesis; b-methylcrotonyl CoA carboxylase and propionyl CoA carboxylase are involved in the catabolism of amino
acids, and the latter also performs the ®nal step in the oxidation of
fatty acids of odd-numbered chain lengths and cholesterol.
All four carboxylases are biotinylated by the same enzymatic
reaction (reviewed in Chapter 156). This is con®rmed genetically
by the inherited metabolic disorder biotin-responsive multiple
carboxylase de®ciency, in which the activity of all four biotindependent carboxylases is impaired.106 The responsible enzyme,
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PART 9 / ORGANIC ACIDS
Fig. 94-5 Schematic representation of biotin uptake
and metabolism by tissue cells. Neither the mechanism by which biotin is transported across the
plasma membrane nor that by which it enters the
mitochondrion is well understood. Abbreviations:
bio 5 biotin; ACC 5 acetyl CoA carboxylase;
PC 5 pyruvate carboxylase; MCC 5 b-methylcrotonyl CoA carboxylase; PCC 5 propionyl CoA carboxylase.
holocarboxylase synthase, has been puri®ed from bovine liver and
is found in both the mitochondria and cytosol.107,108 Although
mitochondrial apocarboxylases are expected to be biotinylated
subsequent to their import, it has been shown experimentally that
biotinylation of the a subunit of propionyl CoA carboxylase can be
accomplished in either compartment.109 Holocarboxylase synthase
shows a striking ability to cross species barriers and is fully
interchangeable with the orthologous E. coli biotin ligase BirA,
with which it shares sequence similarity across its biotin binding
region.110,111 Either enzyme will biotinylate the E. coli biotin
carboxyl carrier protein, a component of the bacterial acetyl CoA
carboxylase, or the 67±amino acid, biotin-carrier component of
the human propionyl CoA carboxylase a subunit.58,111,112
Biotin remains bound to the carboxylases until they are
degraded. On proteolysis of the enzymes, biotin is released from
small biotinylated peptides or from biocytin (biotinyl lysine) by
the enzyme biotinidase. The latter enzyme, widely distributed in
tissues and most abundant in serum, has been puri®ed to
homogeneity.113,114 In addition to its biocytin hydrolase activity,
the enzyme has been shown to have a biotinyl-transferase activity
in which biotin is transferred from biocytin to lysine-containing
acceptors, including poly-lysine and histones, the latter suggesting
a novel physiologic role for biotin.115,116 (discussed in detail in
Chapter 156).
Absorption and Distribution. It seems likely that free biotin is
formed in the intestinal lumen, either by enzymatic hydrolysis of
ingested, protein-bound biotin or by release from intestinal
microorganisms. Biotinidase may play a role in releasing biotin
from foods.117 Saturable, carrier-mediated, sodium-dependent
systems for biotin transport and absorption have been demonstrated in human118 and rat119 intestine and in human intestinal
brush-border membranes.120 In addition, a second, sodiumindependent system has been described in basolateral membrane
vesicles from human intestine,121 thus accounting for both
intestinal absorption and release into the portal circulation.
Although nonmediated transport can occur, particularly at low
pH, it seems likely that the mediated system predominates at
physiologic biotin concentrations and pH. Biotin in blood appears
to be largely free.122 Although experiments in cultured cell
systems have been rather uninformative concerning the mechanism of cellular transport, basolateral membrane vesicles prepared
from rat123 or human124 liver show a sodium-dependent, carriermediated transport, as do brush-border membrane vesicles from
human placenta,125 suggesting that biotin transport is generally a
mediated process.
Biotin De®ciency. Spontaneous biotin de®ciency has almost
never been reported in humans, probably because the daily
requirement is very small (estimated at l20 mg/day) and because
intestinal microorganisms synthesize suf®cient amounts of the
cofactor even in the absence of nutritional sources. Biotin
de®ciency has been reported, however, in patients with the short
bowel syndrome being fed exclusively by parenteral alimentation.126 Experimental biotin de®ciency has been produced in
animals and humans by ingestion of large amounts of egg white,
which contains the potent biotin binder avidin.127 Under these
conditions, cutaneous pallor, dermatitis, depression, lassitude,
muscle pains, hyperesthesia, and ®nally anemia and electrocardiographic changes developed in four experimental human subjects.
All these symptoms and signs were reversed rapidly by
administration of 150 to 300 mg biotin daily for several days. In
animals, experimental biotin de®ciency has been shown to produce
decreased activity of biotin-dependent carboxylases in tissues.98± 100
Cobalamin (Vitamin B12). The structure and function of this
compound have intrigued students of human biology since 1926,
when Minot and Murphy demonstrated that oral administration of
crude liver extract was effective in the treatment of pernicious
anemia.128 In 1948, this anti-pernicious anemia factor was isolated
from liver and kidney129,130 and was named vitamin B12.
Administration of as little as 1 mg of the vitamin daily was shown
to prevent relapse of pernicious anemia. Although the vitamin is
widely distributed in animal tissues, there is strong evidence that it
is synthesized only in microorganisms found in soil, water, or the
rumen and intestine of animals. For further information on Cbl, the
reader is referred to Chapter 155. For comprehensive reviews of
Cbl chemistry and metabolism, the reader is referred elsewhere.131
Structural Features. The isolation of vitamin B12 culminated in
the elucidation of its three-dimensional structure by Hodgkin and
co-workers using x-ray crystallographic techniques.132 Vitamin
B12, or, as it is now of®cially designated, cobalamin, is composed
of a central cobalt atom (Co) surrounded by a planar corrin ring
and a complex side chain extending down from the corrin plane
consisting of a 5,6-dimethylbenzimidazolyl phosphoribosyl moiety (Fig. 94-6). The benzimidazole is linked to the Co atom
through one of its nitrogens, whereas the phosphate is bonded to
the D ring of the corrin. The molecule is completed by coordinate
linkage of one of several different radicals to the Co nucleus from
above the corrin plane. Thus, CN-Cbl or, more strictly, a-(5,6dimethylbenzimidazolyl)-cobamide cyanide, is formed by the
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
Fig. 94-7 Reactions catalyzed by cobalamin coenzymes in mammalian tissues. Note the speci®city of adenosylcobalamin for the
isomerization of methylmalonyl CoA and of methylcobalamin for the
methylation of homocysteine. Me-H4folate 5 N 5-methyltetrahydrofolate; H4folate 5 tetrahydrofolate.
Fig. 94-6 Structure of adenosylcobalamin (AdoCbl). R 5
CH2CONH2; R0 5 CH2CH2CONH2. Other radicals that may be
coordinately linked to the cobalt atom include CH3 (methylcobalamin), OH2 (hydroxocobalamin), and CN2 (cyanocobalamin). (Reprinted with permission from Babior BM: Cobamides as cofactors:
Adenosylcobamide dependent reactions, in: Cobalamin Biochemistry and Pathophysiology. New York, Wiley, 1975, p 141.)
attachment of a cyanide radical to the Co atom. Although this
compound is the most common commercial form of the vitamin, it
is an artifact of isolation and does not occur naturally in
microorganisms, plants, or animal tissues. Many other cobalamins
can be formed by replacement of the cyanide radical, but only four
have been isolated from mammalian tissue: OH-Cbl, glutathionylcobalamin (GSCbl), methylcobalamin (MeCbl), and AdoCbl.
The latter two compounds are unique for two reasons: They are the
only two compounds in nature known to have a direct carbon±
cobalt bond, and they are the only two forms of Cbl known to act
as speci®c coenzymes in mammalian systems.
The structure and nomenclature of the cobalamins are further
complicated by oxidation and reduction of the Co atom. In OHCbl, the Co atom is trivalent [cob(III)alamin], and this compound
has been called vitamin B12a. When the Co is reduced to a divalent
state [cob(II)alamin], the molecule is called vitamin B12r and, in
the monovalent state [cob(I)alamin], it is called vitamin B12s.
These oxidation-reduction states are important because there
appear to be speci®c reductase enzymes that sequentially convert
cob(III)alamin to cob(I)alamin, with cob(II)alamin acting as an
intermediate.133 The Co atom must be reduced to its monovalent
state prior to formation of MeCbl or AdoCbl.
Cobalamin Coenzymes. In 1958, Barker and his colleagues
demonstrated that the glutamate mutase reaction in Clostridium
tetanomorphum required vitamin B1263 and, more speci®cally, that
the active coenzyme form of the vitamin was AdoCbl.64,65 One
year later, Smith and Monty reported that the analogous isomerization of methylmalonyl CoA to succinyl CoA was defective in the
liver of Cbl-de®cient rats.2 They suggested that Cbl is a cofactor
for the latter isomerization system, a thesis borne out by Gurnani
et al.3 and Stern and Friedmann,4 who showed in vitro that the
activity of methylmalonyl CoA mutase in liver from Cbl-de®cient
animals could be restored to normal by the addition of AdoCbl, but
not by CN-Cbl or other vitamin B12 analogues (Fig. 94-7). For
several years, because AdoCbl was the only known coenzyme
form of vitamin B12, it was designated coenzyme B12.
In 1966, Weissbach and his colleagues134 demonstrated that
MeCbl is a cofactor in the complex reaction by which homocysteine is methylated to methionine (Fig. 94-7). This reaction
requires S-adenosylmethionine and N5-methyltetrahydrofolate
(Me-H4folate), as well as the methionine synthase apoenzyme
and MeCbl. The mechanism of homocysteine methylation probably involves the following sequence: Me-H4folate is converted to
tetrahydrofolate (H4 folate) by transferring its methyl group to a
reduced Cbl prosthetic group on the methionine synthase
holoenzyme; in turn, the methyl group is transferred from MeCbl
to homocysteine, leading to the formation of methionine135,136
(see Chapter 155).
The conversion of methylmalonyl CoA to succinyl CoA and
the methylation of homocysteine to methionine are the only Cbldependent reactions that have been demonstrated conclusively in
mammalian systems. Poston reported that AdoCbl acts as a
cofactor in the enzymatic reaction by which a-leucine is
isomerized to b-leucine,137 but this has not been con®rmed in
other laboratories. In microorganisms, several other enzymes
require AdoCbl138,139: glutamate mutase, diol dehydrase, glycerol
dehydrase, ethanolamine ammonia-lyase, and ribonucleotide
reductase. In addition, MeCbl is involved in the formation of
methane and acetic acid and the fermentation of lysine in bacteria.
Cbl Absorption and Distribution. The Cbl vitamins have a unique
and highly specialized mechanism of intestinal absorption that has
been reviewed in detail.140±142 The ability to transport physiologic
quantities of the vitamin depends on the combined action of
gastric, ileal, and pancreatic components. The gastric substance,
called intrinsic factor (IF) by Castle,138 who ®rst demonstrated its
existence, is a glycoprotein, synthesized by gastric parietal cells,
that binds cobalamins in the intestinal lumen after they have been
released from dietary protein by acid and peptic hydrolysis.
Subsequently, the IF±Cbl complex interacts through its protein
moiety with a speci®c ileal receptor protein, called cubilin.143 The
vitamin is transcytosed across the ileal epithelial cells and appears
in the portal blood bound to transcobalamin II (TC II), the
transport protein for newly absorbed vitamin.140,144,145 When
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PART 9 / ORGANIC ACIDS
labeled Cbl is administered intravenously or orally, most of it is
immediately bound to TC II and disappears from the plasma in a
few hours.146,147 Two other Cbl-binding proteins, transcobalamin I
(TC I) and transcobalamin III (TC III), are also found in serum. TC
I and TC III are glycoproteins of the R-binder, or haptocorrin,
family, which carry the majority of Cbl found in plasma, but their
physiologic role is unclear. For example, only a small fraction of
newly absorbed Cbl binds to TC I, and this component turns over
very slowly. MeCbl is the major circulating Cbl species,
accounting for 60 to 80 percent of total plasma Cbl; OH-Cbl
and AdoCbl make up the remainder.148 Because over 90 percent of
total plasma Cbl is bound to TC I, it is clear that most of the
circulating MeCbl travels with this R-binder. This Cbl distribution
pattern is puzzling, particularly in the face of evidence indicating
that AdoCbl accounts for approximately 70 percent of total hepatic
Cbl, whereas MeCbl constitutes a mere 1 to 3 percent.148 A
preponderance of AdoCbl is also present in such other tissues as
erythrocytes, kidney, and brain. The physiologic signi®cance of
these widely different fractional amounts of Cbl compounds in
extracellular and intracellular compartments remains obscure.
Transcobalamin II facilitates Cbl uptake by mammalian
tissues. Finkler and Hall149 showed that CN-Cbl bound to TC II
was accumulated by HeLa cells much more rapidly than free CNCbl or CN-Cbl bound to TC I, IF, or other binding proteins. Such
TC II±mediated uptake has been subsequently con®rmed in a
variety of cell types, both in vivo and in culture.141 These ®ndings,
coupled with the observations in vivo that TC II disappeared from
plasma as TC II±Cbl was absorbed150 and appeared in lysosomal
fractions of hepatic151 and kidney152 cells, led to the proposal that
the circulating TC II±Cbl complex is recognized by a speci®c,
widely distributed plasma membrane receptor, a hypothesis
supported by considerable experimental evidence. YoungdahlTurner and associates153 showed that the complex binds to a
speci®c, high-af®nity (Ka 10 10 M) cell surface receptor on
cultured skin ®broblasts, that the TC II±Cbl complex is
internalized intact via adsorptive endocytosis,154 and that the
degradation of TC II and release of Cbl from the complex occur as
a result of lysosomal protease activity.153,154 Cbl then exits from
the lysosome and is either converted to MeCbl and bound to the
methionine synthase in the cytosol or enters the mitochondrion,
where, after reduction and adenosylation, it is bound to
methylmalonyl CoA mutase (Fig. 94-8).155,156
The intricate process just described is surely the most widely
distributed physiologic means by which mammalian cells obtain
Cbl, but it is not the only one. Hepatocytes, for instance, contain a
surface receptor for asialoglycoproteins, and this receptor interacts
with TC I±Cbl (and perhaps TC III±Cbl) complexes, thereby
providing a second potential means by which this particular tissue
obtains Cbl.157 There is also evidence that at least some tissues are
capable of taking up free (unbound) Cbl if the concentration of
unbound vitamin is increased to suf®ciently high concentrations.
In cultured ®broblasts, this uptake process for free Cbl is saturable,
Ca2 independent, and sensitive to inhibitors of protein synthesis
and sulfhydryl reagents.158 Its functional role under most
circumstances is probably negligible, however.
Coenzyme Biosynthesis and Compartmentation. Because
methylmalonyl CoA mutase, the mammalian enzyme dependent
on AdoCbl, is a mitochondrial protein,159 whereas the MeCbldependent methionine synthase is cytoplasmic,160 it becomes
important to relate the cellular biology of the vitamin to its cellular
and molecular chemistry. The chemical pathway of AdoCbl
synthesis was de®ned initially in bacteria.133,161 Three enzymes
are required for coenzyme synthesis: two reductases and an
adenosyltransferase. The reductases are ¯avoproteins that require
NAD as a cofactor. The ®rst (EC 1.6.99.8) is responsible for
converting cob(III)alamin (i.e., OH-Cbl) to cob(II)alamin, and the
second (EC 1.6.99.9) for catalyzing the further reduction to
cob(I)alamin. The latter compound and ATP are substrates for an
adenosyltransferase (EC 2.5.1.17), which completes the synthesis
of AdoCbl. Neither of the reductases has been puri®ed extensively,
but the adenosyltransferase has. It has an optimal pH of 8, requires
Mn2‡, and has a Km of 1 10 5 M for cob(I)alamin and
1.6 10 5 M for ATP.161 The biosynthetic steps leading to
MeCbl formation are somewhat analogous, likely involving
reduction followed by methylation directly by the methionine
synthase apoenzyme.162,163
Evidence has accumulated that indicates that mammalian cell
metabolism of Cbl may proceed by a very similar set of reactions
(Fig. 94-8). In 1964, Pawalkiewicz et al.164 showed that human
liver and kidney homogenates could convert CN-Cbl to AdoCbl.
Several years later, AdoCbl synthesis from OH-Cbl was observed
in HeLa cell extracts incubated with ATP and a reducing system
that presumably bypassed the enzymatic reduction of OH-Cbl
[cob(III)alamin] to cob(I)alamin.165 Subsequently, Mahoney and
Rosenberg166 demonstrated the synthesis of both AdoCbl and
MeCbl by intact human ®broblasts growing in a tissue culture
medium containing OH-[57Co]Cbl. This system was subsequently
characterized in cell extracts.167,168 As with the HeLa cell system,
chemical reductants were employed to bypass both Cbl
Fig. 94-8 General pathway of the cellular
uptake and subcellular compartmentation of
Cbl, and of the intracellular distribution and
enzymatic synthesis of Cbl coenzymes. TC
II 5 transcobalamin II; OH-Cbl 5 hydroxocobalamin;
GSCbl 5 glutathionylcobalamin;
MeCbl 5 methylcobalamin; MeFH4 5 methyltetrahydrofolate; FH4 = tetrahydrofolate;
AdoCbl 5 adenosylcobalamin; CblIII, CblII,
CblI ˆ cobalamins with cobalt valence of 31,
21, and 11, respectively.
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
reductases.168 Such extracts synthesized AdoCbl, thereby demonstrating that the adenosyltransferase found in bacteria also exists in
normal human cells. These experiments also revealed that the
adenosyltransferase was mitochondrial in location, implying
that both the synthesis and cofactor activity of AdoCbl take
place in this organelle. In contrast, MeCbl synthesis takes place in
the cytosol in conjunction with the methionine synthase reaction
(Fig. 94-8).
Metabolic Abnormalities in Cbl De®ciency. The biochemical
abnormalities in plasma and urine of patients with Cbl de®ciency
re¯ect the dysfunction of the enzymes dependent on Cbl
coenzymes. The ®rst relevant observation in this context was the
demonstration by Cox and White5 and by Barness and his
colleagues6 that methylmalonic acid excretion in the urine was
distinctly increased in Cbl-de®cient patients with classic pernicious anemia. The methylmalonic aciduria in these patients was
reversed rapidly by administration of physiologic doses of Cbl,
indicating that repletion of Cbl restored the methylmalonyl CoA
mutase reaction to normal. Later, Cox et al. reported that patients
with Cbl de®ciency also have distinctly increased amounts of
propionic acid in the urine, this abnormality again being reversed
by treatment.169 Interestingly, they also found excessive amounts
of acetic acid in the urine of Cbl-de®cient subjects. The
mechanism of this abnormality is not clear, because acetate does
not participate in the major pathway of propionate catabolism. The
®nding could re¯ect increased utilization of the alternative
pathways of propionate metabolism in the face of a block in the
major pathway, because each of the alternative routes leads
eventually to the formation of acetyl CoA (Fig. 94-4). Excessive
excretion of homocystine also has been documented in Cblde®cient patients,170,171 as has combined methylmalonic aciduria
and homocystinuria.172 The latter report is particularly interesting
because it documents congenital but not hereditary Cbl de®ciency,
in this instance due to acquired Cbl de®ciency in the offspring of a
strictly vegetarian mother who also was de®cient in the vitamin. A
number of such cases have now been reported.21
DISEASE STATES
The Propionic Acidemias
In 1961, Childs et al.7 described a male infant with episodic
metabolic ketoacidosis, protein intolerance, and remarkably
elevated plasma glycine concentration. Several hundred children
with similar clinical and biochemical ®ndings have since been
described. Many of these children were subsequently found to
have methylmalonic acidemia173; a few had b-ketothiolase
de®ciency (see Chapter 93). However, the patient described by
Childs et al., and many reported subsequently, had propionic
acidemia due to a primary and speci®c de®ciency of propionyl
CoA carboxylase activity (Fig. 94-2). This conclusion was derived
independently from the description of a patient with massive
propionate accumulation in blood,174 from another with impaired
propionate oxidation in leukocytes9 and defective carboxylase
activity in ®broblast extracts,175 and from a third with both
propionic acidemia and defective carboxylase activity.176 We now
recognize that propionyl CoA carboxylase de®ciency also occurs
in children with inherited abnormalities in biotin metabolism,
leading to the de®ciency of multiple biotin-dependent carboxylases (see Chapter 156). Hence, we must now use the term
propionic acidemias to refer to this heterogeneous group of related
inborn errors. As will be discussed subsequently, a similar
heterogeneity exists among the methylmalonic acidemias.
Propionyl CoA Carboxylase De®ciency. Clinical Manifestations. As mentioned above, this disorder was originally referred to
as ketotic hyperglycinemia. E.G., the patient described by Childs
and Nyhan and their colleagues,7,177,178 presented with dehydration, lethargy, and coma on the ®rst day of life. He was found to be
severely ketoacidotic and responded slowly to massive alkali
replacement. The clinical course was characterized by recurrent
attacks of ketoacidosis, precipitated by infections or protein
ingestion, and by developmental retardation, electroencephalographic (EEG) abnormalities, and osteoporosis. The patient had
episodic neutropenia and thrombocytopenia prior to death at age 7.
A sister (A.G.) also became ketotic and acidotic during the ®rst 4
days of life, but the course of her condition has been modi®ed
dramatically because of the extensive experience gained in
studying her brother. Although she has had mild attacks of
ketoacidosis during intercurrent infections, maintenance on a lowprotein diet has resulted in little need for hospital care and normal
somatic and mental development up to 15 years of age.179
In 1968, Hommes and his colleagues174 described a male infant
with hyperventilation, are¯exia, and grunting at 60 h of age. There
was a profound metabolic acidosis (arterial pH 6.98), and despite
administration of massive amounts of sodium bicarbonate and
tris(hydroxymethyl)-aminomethane, the infant died on the ®fth
day of life. Leukocytes and platelets were normal. Postmortem
examination showed only a fatty liver and degeneration of
Purkinje cells and the granular layer of the cerebellum.
Subsequent descriptions of patients with propionic acidemia
have con®rmed that most patients present in the newborn period
with severe metabolic acidosis manifested by refusal to feed,
vomiting, lethargy, and hypotonia; dehydration, seizures, and
hepatomegaly occur less often.180,181 Other patients have presented later, either with acute encephalopathy or episodic
ketoacidosis or with developmental retardation apparently uncomplicated by attacks of ketosis or acidosis.182,183 A 5-year-old boy
presented with a fatal necrosis of the basal ganglia without either
metabolic acidosis or hyperammonemia.184 Propionic acidemia
has been identi®ed in a 29-year-old man who presented initially
with adult-onset chorea and dementia.185 Still other children, with
almost complete de®ciency of propionyl CoA carboxylase activity
as measured in extracts of cultured ®broblasts, have had no clinical
abnormalities whatever and have been identi®ed only during
family studies.186,187 No satisfactory explanation for this striking
lack of clinical enzymatic correlation exists at present.
Based on a survey of 65 patients with propionic acidemia, Wolf
et al.181 reported that the clinical course of symptomatic patients is
characterized by repeated relapses, usually precipitated by
excessive protein intake, constipation, or intercurrent infection.
Treatment of these children has been quite dif®cult, and neurologic
sequelae have been common. Among the neurologic complications often observed, developmental delay, focal and general
seizures, cerebral atrophy, and EEG abnormalities have been the
most prominent. Surtees et al.183 also have reported a high
prevalence of neurologic sequelae, including dystonia, severe
chorea, and pyramidal signs, particularly in patients who survive
longer. The cranial computer tomographic and magnetic resonance
imaging ®ndings in propionic acidemia were reviewed by
Bergman et al., who showed spectroscopic abnormalities, speci®cally an increase in glutamine/glutamate, even when the patients
appeared to be stable.188 Walter et al. described 11 newborn
patients with elevated blood ammonia levels and neurologic
symptoms; only 4 had clinically important acidosis.189 Leukopenia
and thrombocytopenia, perhaps due to marrow suppression by one
or more of the toxic metabolites produced, is also not uncommon.
Parathyroid hormone resistance and B-cell lymphopenia was
described in a 7-week-old patient.190
Biochemical Abnormalities. Childs and Nyhan7,177,178,191 studied
their index patient extensively. Because of the hyperglycinemia,
they focused their attention on the pathways of glycine formation
and utilization but found no consistent abnormalities. Normal
hemoglobin concentration in the peripheral blood indicated that
the pathway from glycine to d-aminolevulinic acid was not
blocked. Slices of the patient's liver incorporated [14C]glycine into
protein and carbon dioxide as well as slices of rat liver did.
Salicylate and benzoate were normally conjugated with glycine,
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2174
PART 9 / ORGANIC ACIDS
and the glutathione concentration of whole blood was normal.
Although the rate of conversion of tritiated glycine to serine in vivo
was slower than in controls, this difference may have re¯ected the
enlarged glycine pool rather than a speci®c block in the conversion
of glycine to serine.191
Moreover, several observations suggested an abnormality in the
catabolism of the branched chain amino acids, methionine, and
threonine. Plasma concentrations of valine, isoleucine, and leucine
were elevated intermittently; administration of leucine, valine,
isoleucine, threonine, and methionine each precipitated attacks of
ketoacidosis, but no other amino acids were toxic. Menkes192
reported that the urine contained large amounts of butanone (a
four-carbon ketone that is a by-product of isoleucine catabolism)
and the longer chain ketones, pentanone and hexanone. These
long-chain ketones were not detected in the urine of patients with
ketosis due to diabetes, starvation, or ketogenic diets. Because
isoleucine, valine, threonine, and methionine are all precursors of
propionate, a defect in propionate metabolism seemed likely, but
patient E.G. died before any other studies of propionate catabolism
could be performed. Subsequently, Hsia et al.9 demonstrated a
striking defect in propionate catabolism in A.G., the affected sister
of E.G. When leukocytes isolated from her peripheral blood were
incubated with [3-14C]propionate, negligible quantities of 14CO2
were evolved as compared with values in controls, but her cells
oxidized methylmalonate and succinate normally. Identical
®ndings were obtained using ®broblasts grown in tissue culture.
These data showed that the primary metabolic defect in E.G. and
A.G. was in the conversion of propionyl CoA to D-methylmalonyl
CoA, a reaction catalyzed by propionyl CoA carboxylase. This
conclusion was con®rmed subsequently by direct assay of
carboxylase activity in ®broblast extracts.175
In their child with lethal neonatal acidosis, Hommes et al.174
found that the serum propionic acid concentration was 400 mg/dl
(5.4 mM), a value more than 100 times that reported in normal
infants. The liver contained fatty acids with 15 and 17 carbon
atoms in addition to the even-chain fatty acids found in control
livers. From these data, Hommes et al. also postulated a defect in
propionyl CoA carboxylation in their patient.
Subsequent investigations have con®rmed and extended these
early ®ndings. Analysis of body ¯uids in several additional
patients92±94,176 showed that propionate accumulation in blood
and urine occurs regularly, its magnitude being related to the
severity of the clinical course and the time at which sampling is
performed. Ando and colleagues92 have stressed that other
propionate derivatives also accumulate in urine. These include
methylcitrate, which is probably formed from the intramitochondrial condensation of propionyl CoA with oxaloacetate92;
propionylglycine, which results from the conjugation of propionate with glycine93; b-hydroxypropionate, an intermediate in one
of the alternative pathways of propionate catabolism94 (Fig. 94-4);
and tiglic acid,193 an isoleucine catabolite several steps proximal
to the block. Although the exact amounts of these compounds in
urine have not always been determined, they appear to account for
a small fraction of the propionate pool that accumulates in vivo in
this disease. Their presence may be important in mitigating the
toxic effects of propionate excess. Wendel et al. showed elevated
levels of odd-numbered fatty acids (OLCFA) in the erythrocyte
lipids of ®ve patients with propionic acidemia and suggested that
OLCFA levels re¯ect the continuous burden of propionyl CoA
toxicity within cells and could serve as a means of evaluating the
quality of long-term metabolic control.194
Other compounds, not directly concerned with the propionate
pathway, also have been found in signi®cantly increased amounts.
In addition to hyperglycinemia and hyperglycinuria, which were
discussed earlier, marked hyperammonemia has been documented
in several patients,181,195 and a distinct correlation between plasma
propionate and blood ammonia has been noted in two patients.196
The Enzymatic Defect. The molecular pathology of propionic
acidemia is both complex and interesting. Cell extracts from a
number of affected patients share a common ®nding, namely,
reduction in propionyl CoA carboxylase activity to 1 to 5 percent
of that in controls.197±199 Because the enzyme is composed of two
independently encoded enzyme subunits, the causative mutations
will necessarily occur in one of two genes. This was ®rst illustrated
in complementation experiments in which ®broblast heterokaryons
formed between pairs of affected cell lines were assayed for
recovery of functional propionyl CoA carboxylase by ®xation of
14
C-propionate.200±202 Two major complementation groups, pccA
and pccBC, were identi®ed, the latter group showing intragroup
complementation (subgroups pccB and pccC) compatible with the
occurrence of interallelic complementation (see Fig. 94-11). It was
shown subsequently that patients in the pccA group have a primary
defect in the PCCA gene encoding the a subunit of propionyl CoA
carboxylase, whereas patients in the pccBC group and subgroups
have defects of the PCCB gene encoding the b subunit.203,204
There are several unusual features of mutant ®broblast lines
from patients belonging to the different complementation groups.
First, many individuals in the pccA group lack detectable a subunit
protein; when this is the case, they invariably lack detectable
b subunits as well.203,205 This has been explained by the inherent
instability and consequent degradation of the b subunit in the
absence of a subunit with which to assemble to form the native
enzyme. Among a-minus/b-minus cell lines, some lack a subunit
mRNA but contain b subunit mRNA, con®rming the assignment of
the pccA complementation group to mutations of the PCCA
gene.204 Second, a number of pccBC or subgroup ®broblasts lack
b subunits but have a subunits. These have unstable b subunit
protein due to mutation, although b subunit protein is present in at
least some cases.205,206 Importantly, the small amount of residual
propionyl CoA carboxylase activity observed in extracts of most
mutant ®broblasts appears to be present even in those with absent
a or b subunits. This suggests that the ``background'' activity is
due to the minimal activity of other carboxylases acting on
propionyl CoA as substrate, not to propionyl CoA carboxylase
itself.207 A third unusual feature of propionic acidemia is that
many heterozygotes of the pccBC group or subgroups have
propionyl CoA carboxylase activity indistinguishable from that in
controls, whereas obligate heterozygotes of the pccA group have
the expected 50 percent of control activity.199 This has proved to
be due to relative differences in the synthesis of the enzyme
subunits. It has been shown that b subunits are synthesized in fourto ®vefold excess over a subunits, so that heterozygotes for nulltype b subunit mutations still have more than enough wild-type
b subunits to interact with the limiting amount of a subunits to
form normal amounts of carboxylase enzyme.205 Conversely, any
reduction in the amount of a subunit, as in pccA heterozygotes, is
directly re¯ected in a proportionate reduction in carboxylase
activity.199 All three of these features of mutant cell lines can be
used diagnostically to identify the affected gene, but caution is
warranted because patients with point mutations in either gene
may have both subunits present, despite having a defective
holoenzyme.
Pathologic Physiology. A defect in the carboxylation of propionate provides a satisfactory explanation for many of the ®ndings
reported in this disorder. This defect would be expected to lead to
an elevated concentration of propionate in the blood and an
inability of leukocytes to catabolize propionate to carbon dioxide.
Because isoleucine, valine, threonine, and methionine are
precursors of propionate, such a block also should lead to the
observed protein and speci®c amino acid intolerance. The
appearance of long, odd-chain fatty acids in the liver suggests
that when propionyl CoA carboxylation is blocked, odd-chain
fatty acid biosynthesis may be augmented because propionyl CoA
is the ``primer'' for such compounds. Finally, the presence of such
compounds as butanone, methylcitrate, b-hydroxypropionate,
propionylglycine, and tiglic acid very likely results from reversal
of reactions proximal to the primary carboxylase block or from
increased utilization of alternative pathways.
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
It is not at all clear from the foregoing, however, why some
patients have a severe and often life-threatening course, and others
are only mildly affected clinically. Major differences in dietary
protein uptake and quality, in the contributions of gut bacteria to
total propionate load, or in the activity of alternative mechanisms
for propionate disposal are possible explanations for the wide
clinical spectrum, but the prominent intrafamilial differences in
severity are not easily explained this way.186 Furthermore, several
other features of the disease are not adequately explained by the
block in propionate catabolism. The ketosis produced in E.G. by
leucine is not understood, because this amino acid is not
catabolized to propionate. However, it is ketogenic in normal
subjects, suggesting that its effect in E.G. was nonspeci®c. The
cause for the hyperglycinemia seen in many, but not all, of these
patients also has not been adequately de®ned. Because the infant
described by Hommes et al.174 with massive propionic acidemia
never demonstrated signs of hyperglycinemia, the latter cannot be
ascribed simply to the acidosis or ketosis. Numerous theses have
been put forth in explanation. For example, one or more products
of isoleucine catabolism may interfere with glycine cleavage or
glycine±serine interconversion.191,208,209 Ando et al.92 speculated
that methylcitrate cleavage in the cytosol may yield propionate and
glyoxylate, the latter being used as a substrate for glycine
overproduction. Impaired glycine conjugation systems have been
suggested, but no data in support of this notion have been
forthcoming. Because plasma glycine concentration may increase
in sick children with negative nitrogen balance of many causes,210
the hyperglycinemia may be nonspeci®c. The hyperammonemia
often observed in this disorder has been the subject of considerable
investigation. It appears likely that this secondary but clinically
important ®nding results from inhibition of the ®rst enzyme of
the urea cycle, mitochondrial carbamyl phosphate synthetase
(CPS I), by the organic acids and CoA esters that accumulate
intramitochondrially behind the block in propionyl CoA carboxylation. This conclusion rests on data from studies with
experimental animals and animal tissues. For example, propionate
inhibits ureagenesis in rat liver slices when ammonia, but not
citrulline or aspartate, is the nitrogen-donating substrate.211
Administration to rats of suf®ciently large amounts of propionate
or methylmalonate to produce hyperammonemia is associated
with a marked decrease in hepatic concentration of N-acetyl
glutamate,212 the required allosteric effector of CPS I, probably by
competitively inhibiting N-acetyl glutamate synthetase.213 That
such CPS I inhibition occurs in vivo as well as in vitro is supported
by case reports that describe selective impairment of CPS I activity
in the livers of patients with propionic acidemia214 or methylmalonic acidemia.215
Genetics. As a prelude to mutation identi®cation in propionic
acidemia, it is ®rst necessary to identify the affected gene. This is
most readily done by conducting complementation tests and
determining whether the affected patient belongs in the pccA or
pccBC group or a subgroup. Determination of the subgroup (e.g.,
pccB or pccC vs. pccBC) is not speci®cally required, but does
provide insight into the functional impact of mutations affecting
the b subunit (see Fig. 94-11). Alternatively, as described above,
demonstration of some of the peculiarities of propionyl CoA
carboxylase activity or of mRNA or enzyme subunit expression
can also reveal the affected gene. Thus, the demonstration of
normal enzyme activity in parents of an affected child would be
compatible with mutations in the PCCB gene, although obtaining
the converse, 50 percent activity, does not necessarily implicate
the PCCA gene. Absence of both a and b subunits by Western
blotting does identify the PCCA gene as responsible for the
disease. Other more straightforward ®ndings, such as absence or
abnormality of one of the two mRNA species or polypeptide, also
will identify the affected gene. Performing the required experiments is worthwhile because there is a great diversity of mutations
in the PCCA gene, and although the PCCB gene shows bias toward
a small number of mutations that account for about 30 to 60
percent of alleles in different populations, there remains a large
diversity of mutations that account for the rest.
Mutations in the PCCA Gene. Nineteen disease-causing mutations have been identi®ed in the PCCA gene, eight of which fail to
produce a complete a subunit. There are four splicing mutations,
two nonsense mutations and two small deletions causing frameshifts. All four splicing mutations cause exon skipping. Three
of them Ð 1771IVS-2del9, 1824IVS+3del4, and 1824IVS‡
3insCTÐ affect the same exon.216 The nonsense mutations,
R288X and S537X, and the small deletions, 700del5 and
1115del4, are expected to produce truncated proteins. However,
®broblasts from a patient homozygous for 1115del4 failed to show
mRNA by Northern blot, although it could be detected by reverse
transcriptase polymerase chain reaction (RT-PCR). A similar
®nding was made for a patient heteroallelic for R288X and
700del5, indicating that both mutant mRNA species are
unstable.217 These results suggest a propensity for mutations
disrupting normal mRNA translation to produce mRNA-minus
outcomes, as has been noted for other genes.
An unusual ®nding in patient cells with mRNA destabilizing
mutations at both alleles is the detection of an RT-PCR product
showing an 84 bp insertion at nucleotide 1209, containing two inframe stop codons.217 The insertion is an anomalous exon derived
by aberrant splicing within the adjacent intron. The cryptic
transcript is part of the background ``noise'' of abnormal mRNAs
occurring at very low level in normal cells. Cell lines in which the
84 bp insertion is detected as a predominant (but low-level) species
share at both alleles severely deleterious mutations, which are
consistent with rendering the normally structured mRNA species
unstable and rapidly degraded. In a study of 12 mutant cell lines, 4
showed the characteristic transcript. The mutations included a
splicing mutation (1671IVS ‡ 5G ! C), a nonsense mutation
(R288X, three occurrences), and two small deletions (700del5,
1115del4) causing frameshifts.217 Screening for the 84 bp
insertion by RT-PCR may provide a diagnostic bene®t, given the
high proportion of such cell lines.
The remaining mutations cause amino acid substitutions, which
are expected to produce inactive or unstable a subunits, although
most have yet to be evaluated in expression experiments. Two
subunits with point mutations, G643R and Cdel687, have been
expressed in E. coli and shown to abolish biotin binding.57
Another mutation, M348K, has been shown to reduce the
intramitochondrial stability of the mutant a chain without
appreciably affecting its import.218 This a chain failed to be
biotinylated, as also was found for two other nearby mutations,
D343G and G354V, and one near the N-terminus, A50P,57
indicating that all four mutations likely result in unstable a chains
in vivo, accounting for the lack of biotinylation, because the biotin
binding domain is far removed toward the C-terminus of the
protein.
Mutations in the PCCB Gene. Twenty-eight disease-causing
mutations have been identi®ed in the PCCB gene. There are 16
missense mutations, three nonsense, three insertions, and one
complex insertion/deletion (ins/del). Five other mutations cause
disease through disruption of normal splicing. The most frequent
mutation in white individuals is ins/del,219±221 present in 32
percent of alleles of mixed white heritage. A similar value also was
obtained in a study involving 29 patients of Spanish or Latin
American heritage.61 Two other mutations have been found in
signi®cant frequency in the same populations. These are 1170insT
and E168K, with combined frequencies of 14 percent and 17
percent, respectively, in Spanish and Latin American
patients.61,222 The remaining mutations occur singly or have
been found in only two or three patients among whites.
Six mutation have been identi®ed among Japanese patients.
Two of them, R410W and T428I, appear to be prevalent, occurring
in 25 and 31 percent of alleles, respectively.221,223 There are also
two splicing mutations (IVS4‡3del4 and IVS12‡3del8), both of
2175
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2176
PART 9 / ORGANIC ACIDS
which result in exon skipping; one nonsense mutation (R499X),
and one other point mutation (R165W).54,223,224 Of these, R410W
and R165W also have been found in whites.61,225
The ins/del mutation is a complex mutation resulting from a
deletion of 14 nucleotides and their replacement by 12 nucleotides
unrelated to those that were lost. The outcome is the loss of an Msp
I site, which has been used diagnostically, and generation of a
frameshift that results in the production of an unstable, truncated
protein.220 The origin of the ins/del is not obvious, but it is
attractive to speculate that it is derived from duplication of nine
nucleotides just upstream of the site, with random ®lling of three
more nucleotides.219 The R410W mutation occurs in the same
location and also results in loss of the Msp I site. This leaves
ambiguous the results of diagnosis by Msp I digestion of PCR
products. Dot blotting can be used to distinguish these possibilities,219 or PCR products that show at least one allele not cleaved
by Msp I can be sequenced.
Two amino acid substitutions, L519P and R512C, are unusual
in that they are associated with apparently absolute de®ciency of b
subunits.61 Both are present in patients with null-type second
alleles (1170insT and ins/del, respectively) and both patients' cells
are negative for the b subunit by Western blot. Other point
mutations, including R44P, G131R, R165W, E168K, R410W, and
A497V are associated with unstable b subunits, which appear
reduced in quantity and may show smaller-sized fragments by
Western blot.61,223 The abundance of b subunit mutations that are
CRM negative (in the presence of detectable a subunit) highlights
the affected gene (PCCB) and focuses the analysis for mutations.
The identi®cation of mutations in the PCCB gene has made it
possible to assess the basis of interallelic complemention that
de®nes the pccB and pccC subgroups. Each complementing cell
line would have one, if homozygous, or two candidate mutations
that could be responsible for the interallelic complementation
observed between mutant subgroups. The identity of the
complementing allele in several cell lines was determined by
microinjecting b subunit cDNA plasmids containing the candidate
mutations into ®broblasts of each subgroup and assaying for
recovery of 14C-propionate metabolism by autoradiography.225,226
The results paralleled the original complementation results, and
the distribution of complementing alleles shows a pattern
suggestive of functional domains within the b subunit. For
example, two mutations from the pccB subgroup in the N-terminal
half of the protein, dupKICK140 and P228L, are both complementing alleles. These mutations reside in the putative carboxybiotin binding domain by homology with related sequences in the
12S subunit of P. shermanii transcarboxylase. Similarly, two
complementing alleles from the pccC subgroup, Idel408 and
R410W, are close together in the C-terminal half of the protein.
They are located in a region of high homology with the propionyl
CoA binding site of the P. shermanii 12S subunit. These ®ndings
suggest the importance of b±b interactions in the enzyme and
indicate the bene®t of determining subgroup complementation
within the PCCB gene as a way of assessing functional
characteristics of the enzyme.
A particular dif®culty for investigating the impact of mutations
is the requirement for a two-subunit expression system and
successful biotinylation to obtain functional propionyl CoA
carboxylase. So far, this has been achieved in only one instance.
Kelson et al.227 coexpressed cDNAs encoding the a and b subunits
in E. coli, along with the chaperonin proteins GroES and GroEL to
facilitate folding and assembly, and obtained fully assembled,
biotinylated propionyl CoA carboxylase. Using this system, they
evaluated the Japanese T428I mutation in the b subunit and
showed that it resulted in complete obliteration of enzyme activity.
Diagnosis and Mutation Analysis. A defect in propionate
carboxylation must be considered in any child in whom ketosis
or acidosis develops in the neonatal period. Other inborn errors of
metabolism must be ruled out, as must the more common causes of
acidosis in the newborn period. Determinations of propionic acid
and its metabolites in blood or urine and studies of propionyl CoA
carboxylase activity in leukocyte or ®broblast extracts are required
for de®nitive diagnosis. The enzymatic test is, in fact, the only
absolutely speci®c one, because propionate accumulation can
occur in patients with defects of methylmalonate metabolism as
well as in those with propionyl CoA carboxylase de®ciency. Such
assays on cord blood leukocytes should allow immediate diagnosis
in a high-risk newborn. Prenatal diagnosis has been accomplished
reliably by measuring carboxylase activity in cultured amniotic
¯uid cells228 or chorionic villous biopsies,229 by measuring
[14C]propionate ®xation in amniotic ¯uid cells,230 or by measuring
methylcitrate in amniotic ¯uid.231
The identi®cation of the speci®c gene defect, in PCCA or
PCCB, responsible for the enzyme de®ciency is important for
monitoring future pregnancies and to contribute to genotype±
phenotype correlations. The gene assignment can be achieved
most unequivocally through complementation analysis. However,
this is an esoteric procedure that is not readily available in most
laboratories.200 ±202 Other methods can be used that take advantage
of some of the peculiarities of each complementation group,
although the techniques do not apply universally. For example,
estimation of PCC activity in parents' ®broblasts will indicate
PCCB gene mutations if the activity is within the normal range,
although this does not apply in all cases of b subunit defects.199
Western blot, or 3H-biotin labeling in the case of the a subunit, will
indicate defects of the PCCA gene, if both the a and b subunits are
absent, or implicate the PCCB gene if only the b subunit is absent,
reduced in quantity, or partially degraded.205 Finally, Northern blot
or semiquantitative RT-PCR will identify the defective gene if the
level or mobility of one or the other mRNA species is adversely
affected.204
After identi®cation of the defective gene, cell lines can be
analyzed for mutation by conventional techniques. In the PCCB
gene, it is worthwhile to ®rst screen for common mutations. These
include ins/del, which appears widespread, and 1170insT and
E168K, which are frequent in Spanish and Latin American
populations.61,219± 221 In Japanese patients, the common mutations
include R410W and T428I.221,223 When screening for the ins/del
by loss of the Msp I site, it should be recalled that R410W is also
associated with loss of the same site. Although ins/del has not
been observed in Japanese patients, R410W and R165W do occur
in both Japanese and white individuals. It would be important to
con®rm the mutation by sequencing if it is identi®ed through loss
of the Msp I site. The PCCA gene does not have predominant
mutations. Because a number of patients have had null mutations
on both alleles, however, it is worthwhile to test for the
characteristic 84-bp insertion detectable by RT-PCR in patients
with absent or unstable mRNA.217
For determining the identity of unknown mutations, genomic
DNA can be analyzed in the case of the PCCB gene. With the
structure of the PCCB gene completed, it is possible to amplify
each of the 15 exons and ¯anking intronic sequences for analysis
of PCR products.61 Alternatively, RT-PCR and sequencing have
been used to identify mutations at the mRNA level222,223 and to
examine the effect of splicing mutations.54,224 In the case of the
PCCA gene, where complete genomic structure is still lacking,
analysis of overlapping cDNA segments generated by RT-PCR has
been the method of choice.57,217,218
Treatment. A low-protein diet (0.5±1.5 g/kg per day) or one
selectively reduced in the content of propionate precursors appears
to be the best treatment for the disorder at this time. Such diets will
minimize the number of attacks of ketoacidosis but will not
necessarily prevent them or allow normal development in all
patients. Because fasting has been shown to increase the excretion
of propionate metabolites in patients, frequent feeding has been
recommended.27 Attacks of ketoacidosis should be treated
vigorously by withdrawing all dietary protein and administering
sodium bicarbonate parenterally; glucose is also required to avoid
catabolism. Acute attacks, particularly those accompanied by
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
hyperammonemia, have been treated with peritoneal dialysis.232
Total parenteral nutrition also has been used to treat critically ill
patients.233 Because propionyl CoA carboxylase requires biotin as
a coenzyme and because some patients' cells show a biotindependent increase in enzyme activity,234 it is possible that certain
patients could improve when given supplementary biotin, but no
clear example of a biotin-responsive patient with isolated
propionyl CoA carboxylase de®ciency has yet been documented.
On the other hand, dramatic biotin responsiveness has been
described in several children in whom propionyl CoA carboxylase
de®ciency is part of the constellation now called multiple
carboxylase de®ciency (see Chapter 156).
Two additional therapeutic adjuncts deserve mention. Roe and
Bohan reported marked, transient clinical improvement in a child
with propionic acidemia given a single oral dose (100 mg/kg) of
L-carnitine.173 Because this child's urinary hippurate concentration increased markedly after carnitine, because free plasma
carnitine was reduced in three other patients with propionic
acidemia,235 and because urinary propionylcarnitine was present
in large amounts in such patients,235 these workers proposed that
patients with propionic acidemia have a relative carnitine
de®ciency. Subsequently, Wolff et al.236 observed that L-carnitine
supplements signi®cantly reduced the ketogenic response to
fasting in patients with propionic acidemia. Thus, it appears that
long-term L-carnitine supplementation in these patients warrants
serious consideration. To date, no results of long-term treatment
with carnitine have been published.
The recognition that gut bacteria may contribute signi®cantly
to propionate production in at least some individuals25 has led to
the suggestion that speci®c antimicrobial therapy may be of
clinical bene®t to some children with propionic acidemia by
reducing the total amount of propionate in their serum and
tissues.237 Metronidazole (10 mg/kg) has been reported to reduce
fecal propionate substantially, concomitant with a reduction in the
anaerobic bacterial count.237 Plasma propionate also decreased by
50 to 60 percent in two patients, whereas urinary excretion of
propionate metabolites was reduced by an average of 34 percent in
four.237 No comment was made on the effect of this treatment on
the clinical status of these patients. Others have reported using a
similar treatment, but also without any ®rm evidence of clinical
ef®cacy183 (see the later subsection on Diagnosis and Treatment
under the section on The Methylmalonic Acidemias). Further
study is clearly required to establish whether such therapy
improves the management of acute episodes of metabolic
decompensation or provides any long-term bene®t with respect
to growth, mental development, or neurologic outcome for this
condition.
In a retrospective study of 17 patients (12 early-onset, 5 lateonset) from a single hospital over 20 years, 7 patients died (5
early-onset, 2 late-onset).238 The neurologic outcome of the
surviving patients was felt to be satisfactory, and was even better
for early-onset patients. The investigators felt that other treatments
such as liver transplantation or somatic gene therapy could
improve the quality of life of propionic acidemia patients in the
future.
Not unexpectedly, successful application of therapy, particularly in mildly affected patients, is leading to the survival of
women with propionic acidemia into their child-bearing years.
One report documents a relatively uneventful pregnancy and
delivery in a woman mildly affected with propionic acidemia
treated with protein restriction and carnitine supplementation.239
Other such pregnancies are likely to occur; each will need to be
handled individually according to the mother's speci®c clinical
and biochemical status.
Multiple Carboxylase De®ciency. In 1971, Gompertz et al.240
reported a male infant (J.R.) thought to have speci®c de®ciency of
the mitochondrial, biotin-dependent enzyme, b-methylcrotonyl
CoA carboxylase (Fig. 94-5). This infant developed a diffuse,
erythematous skin rash at 5 weeks of age and was admitted to the
hospital at 5 months of age because of a worsening rash, recurrent
vomiting, irritability, and a mild metabolic acidosis. His urine,
which smelled like ``tomcats' urine,'' was analyzed for organic
acids and was found to contain large excesses of b-methylcrotonylglycine, tiglylglycine, and b-hydroxyisovaleric acid. When he
was given 10 mg biotin (about 100 times the estimated human
requirement) by mouth daily for several days, the rash, vomiting,
irritability, and abnormal urine metabolites all disappeared
dramatically. Several years later, it became clear that J.R. had
multiple Ð not speci®c Ð carboxylase de®ciency. His reanalyzed
urine contained metabolites characteristic of propionyl CoA
carboxylase de®ciency, as well as b-methylcrotonyl CoA carboxylase de®ciency241; his cultured ®broblast extracts were de®cient
in pyruvate carboxylase,202 as well as in propionyl CoA and
b-methylcrotonyl CoA carboxylase242,243; and supplementation of
the ®broblast growth medium with biotin led to complete
correction of the de®ciency of all three biotin-dependent
enzymes.202,242,243 Subsequently, more than 50 children with
multiple carboxylase de®ciency have been described.106 These
children are now known to suffer from defects in one of two steps
in biotin metabolism: the transfer of biotin to apocarboxylases,
catalyzed by holocarboxylase synthase244± 247; or the hydrolysis of
biocytin, the biotin-containing product of degraded holocarboxylases, to release biotin, catalyzed by biotinidase.248± 250
As a group, children with holocarboxylase synthase de®ciency
tend to present in the ®rst days or weeks of life with feeding
dif®culties, hypotonia, lethargy, and seizures; some have a diffuse
skin rash or alopecia.106 The ®rst described patient with multiple
carboxylase de®ciency, J.R., proved to have a defect in holocarboxylase synthase. Early studies showed that his enzyme had a
reduced af®nity for biotin when assayed in vitro.251,252 This was
con®rmed by the identi®cation of a point mutation in the biotinbinding region of the enzyme and the production of a biotinresponsive mutant enzyme when it was expressed in E. coli.253,254
Several additional mutations have been identi®ed in patients, many
of them clustering within the biotin-binding region of the
enzyme.253±257
A second, larger group of children is de®cient in biotinidase
activity.248±250 These children usually present later in life (mean
age of onset 3 months) with a variety of neurologic problems
(seizures, hypotonia, developmental delay, hearing loss, optic
atrophy). Although a large number of mutations have been
identi®ed in the biotinidase gene, several are highly prevalent and
account for the majority of alleles in patients.258±260
Both groups respond dramatically to biotin supplements
(10 mg daily) with prompt and sustained clinical improvement.
Thus, multiple carboxylase de®ciency differs markedly from
isolated propionyl CoA carboxylase de®ciency in response to
biotin and, hence, in long-term prognosis. It should be emphasized, however, that the clinical presentations may be very similar.
For this reason, urinary metabolite identi®cation is of important
therapeutic signi®cance.
For more information on multiple carboxylase de®ciency, the
reader is referred to Chapter 156.
The Methylmalonic Acidemias
In 1967, Oberholzer,10 Stokke,11 and their colleagues described
critically ill infants with profound metabolic ketoacidosis and
developmental retardation who accumulated huge amounts of
methylmalonate in their blood and urine. These children had none
of the hematologic or neurologic stigmata of Cbl de®ciency, failed
to respond to Cbl supplements, and excreted much larger amounts
of methylmalonate than those observed in patients with pernicious
anemia.6,7 They were presumed to have a congenital defect of
methylmalonyl CoA racemase or of the methylmalonyl CoA
mutase apoenzyme (Fig. 94-2). Shortly thereafter, Rosenberg,261
Lindblad,12,13 and their co-workers reported children with similar
clinical presentations whose methylmalonic aciduria responded
dramatically to pharmacologic but not physiologic amounts of
CN-Cbl or AdoCbl. Such children were found subsequently to
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2178
PART 9 / ORGANIC ACIDS
have a primary defect of AdoCbl synthesis that resulted in
impaired mutase activity.14,15 The array of different biochemical
and clinical disturbances of methylmalonate metabolism was
broadened still further in 1969 and 1970, when Mudd,262
Goodman,263 and their associates described children with
methylmalonic aciduria whose clinical and chemical ®ndings
differed from those described above. Ketoacidosis was not present,
and the increased methylmalonate excretion was accompanied by
homocystinuria, cystathioninuria, and hypomethioninemia. This
biochemical constellation was interpreted as evidence for
defective synthesis of both Cbl coenzymes, with secondary
impairment of AdoCbl-dependent methylmalonyl CoA mutase
and MeCbl-dependent methionine synthase (Fig. 94-7). These
early descriptions, coupled with a body of data to be discussed
below, have demonstrated that there are many different biochemical bases for inherited forms of methylmalonic acidemia: two
distinct defects of the mutase apoenzyme, one producing complete
mutase de®ciency (mut 0), the other partial de®ciency (mut ); two
distinct defects of AdoCbl synthesis, one probably due to
de®ciency of a mitochondrial Cbl reductase (cblA), the other to
de®ciency of mitochondrial cob(I)alamin adenosyltransferase
(cblB); and three distinct defects of both AdoCbl and MeCbl
synthesis due to abnormal cytosolic or lysosomal metabolism of
cobalamins (cblC, cblD, and cblF). Patients with lesions
producing methylmalonic acidemia only (mut 0, mut , cblA,
cblB) share many clinical features and will be discussed as a
group; discussion of the other group of patients whose lesions
produce methylmalonic acidemia and homocystinuria (cblC, cblD,
and cblF) will follow.
Methylmalonyl CoA Mutase De®ciency. Clinical and Laboratory Presentation. More than 100 children with isolated mutase
de®ciency have been documented. Although, as mentioned above,
there are four known etiologies for such de®ciency, the clinical
®ndings in affected patients from the four groups are remarkable
more for their similarities than for their differences. Matsui et al.
surveyed 264 the natural history in 45 such patients: 15 were mut 0;
5 were mut , 14 were cblA, and 11 were cblB. There were
approximately equal numbers of males and females in each group.
Information was obtained from questionnaires completed by the
patients' physicians, published reports, unpublished communications, and personal experience. The most common signs and
symptoms at the onset of clinical dif®culty were lethargy, failure to
thrive, recurrent vomiting, dehydration, respiratory distress, and
muscular hypotonia (Table 94-1). Little interclass difference was
observed for these major clinical manifestations or for such less
common ones as developmental retardation, hepatomegaly, or
coma. Patients in the mut 0 class, however, presented earlier than
those in the other groups (Fig. 94-9). Whereas 80 percent of
children in the mut0 class became ill during the ®rst week of life,
Table 94-1 Clinical Presentation in 45 Patients wth
Methylmalonic Acidemia
Fig. 94-9 Age at clinical onset in 45 patients with methylmalonic
acidemia. Inset numbers denote percentages of patients in each
group. (Reprinted with permission from Matsui SM, Mahoney MJ,
Rosenberg LE: The natural history of the inherited methylmalonic
acidemias. New Engl J Med 308:857, 1983.)
less than half the children in the three other groups presented
during this interval. Furthermore, clinical onset occurred in 90
percent of mut0 patients before the end of the ®rst month, whereas
onset beyond the ®rst month was observed in an appreciable
fraction of patients in each of the other groups. A survey of 20 mut
patients has reached similar conclusions.265
The laboratory ®ndings in affected patients at the time that
methylmalonic acidemia (with or without aciduria) was ®rst
documented are shown in Table 94-2. As expected, serum Cbl
concentrations were routinely normal. Metabolic acidosis, with
blood pH values as low as 6.9 and serum bicarbonate concentrations as low as 5 mEq/L, was observed in the majority of patients
in all four groups. Ketonemia or ketonuria was found in 80 percent
of patients, with hyperammonemia being only slightly less
common, occurring in 70 percent of affected patients. Leukopenia,
thrombocytopenia, and anemia were the only other manifestations
that were noted in 50 percent or more of this group of patients.
Earlier case reports266 reported that hypoglycemia occurs in about
40 percent of affected patients. Inadvertently, this parameter was
not assessed in this survey.
It should be mentioned that mutase de®ciency is not always
associated with serious clinical consequences. Ledley et al.267
reported eight children, between the ages of 18 months and 13
years, who had methylmalonate accumulation in blood and urine,
Table 94-2 Laboratory Findings in 45 Patients with
Methylmalonic Acidemia
Mutant Class
Signs and Symptoms
at Onset
cblA
cblB
mut 2
mut 0
Total
Lethargy
Failure to thrive
Recurrent vomiting
Dehydration
Respiratory distress
Muscular hypotonia
Developmental retardation
Hepatomegaly
Coma
78
75
58
64
89
44
36
11
50
83
86
86
86
67
57
33
67
29
100
40
80
100
50
33
25
0
40
85
77
77
62
55
91
65
57
38
84
73
73
71
67
63
47
41
40
Numerical values represent percentages of patients in each group.
Reprinted with permission from reference 264.
Mutant Class
Findings at Clinical
Onset
cblA
cblB
mut 2
mut 0
Total
Normal serum cobalamin
Metabolic acidosis
Ketonemia/ketonuria
Hyperammonemia
Hyperglycinemia/glycinuria
Leukopenia
Anemia
Thrombocytopenia
100
100
78
50
70
70
10
75
100
88
67
83
83
45
45
45
100
100
100
80
40
60
0
40
100
85
85
75
70
62
58
40
100
92
81
71
68
60
55
50
Numerical values represent percentages of patients in each group.
Reprinted with permission from reference 264.
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
but had no symptoms. Presumably, these apomutase-de®cient
patients have an enzyme defect so ``leaky'' that homeostasis is not
compromised. At least some of these individuals continued to be
symptom free 7 years after the initial report.268
Another report determined that patients, initially ascertained by
a newborn screening program with methylmalonic aciduria urine
levels of less than 1400 mmoles/mmole creatinine, had normal
somatic and cognitive outcomes.269
Conversely, another group of patients appear to have
methylmalonic acidemia without a demonstrable defect in mutase
activity, at least as measured in cultured cells. Although the
elevations of methylmalonate are relatively mild as compared with
mutase-de®cient patients, they are chronic and usually are
discovered on laboratory workup for failure to thrive or
developmental retardation.270 Because Cbl metabolism also
appears to be normal in this group, the cause of the disease
remains an enigma. Roe et al. have described a patient with
psychomotor delay, methylmalonic aciduria without episodes of
metabolic acidosis, and methylmalonic semialdehyde dehydrogenase de®ciency.271 It is possible that others in this group have
the same defect.
Chemical Abnormalities In Vivo. Large amounts of methylmalonic acid have been found in the urine or blood of all reported
patients. Whereas normal children and adults excrete less than
0.04 mmole (5 mg) methylmalonate daily, children with isolated
methylmalonic acidemia have excreted from 2.1 to 49 mmoles
(240 to 5700 mg) in a 24-h period. Their plasma concentrations of
methylmalonate, almost undetectable in normal subjects, have
ranged from 0.22 to 2.9 mM (2.6±34 mg/dl). In the few patients
in whom it was measured, the cerebrospinal ¯uid concentration of
methylmalonate equaled that of plasma (for references to early
case reports, the reader is referred elsewhere266). It is important to
note that patients with mild, late-onset, or ``benign''267 disease
may have much lower levels, particularly when clinically
asymptomatic.267,268 No relationship between the quantities of
methylmalonate accumulated in body ¯uids and the etiology of
mutase de®ciency (i.e., apoenzyme vs. coenzyme de®ciency) has
been reported. Methylmalonate is surely the major, but not the
only, abnormal metabolite found in body ¯uids of these patients.
Because propionyl CoA carboxylation is reversible, propionate
and some of its precursors (butanone) or metabolites
(b-hydroxypropionate and methylcitrate) also accumulate in blood
and urine,8,92,93,272,273 although their amounts are small compared
with that of methylmalonate.
Several groups have studied the relationship between protein or
amino acid loading and methylmalonate accumulation in these
patients. Without exception, administration of protein or amino
acids known to be precursors of propionate and methylmalonate,
such as methionine, threonine, valine, or isoleucine, has resulted in
augmented methylmalonate accumulation and, in some instances,
ketosis or acidosis.8,10±12 When Cbl-responsive patients are given
supplements of this vitamin, such augmentation by methylmalonate precursors is lessened considerably.274 All these ®ndings
suggest that patients with discrete defects at the mutase step have a
major block in the utilization of methylmalonyl CoA that is
expressed as methylmalonate accumulation.
Localization of Enzymatic Defects. Because the conversion of
propionate to succinate is blocked in each of the methylmalonic
acidemias, an early screening test for these disorders measured the
ability of intact peripheral blood leukocytes or cultured ®broblasts
to oxidize [14C]propionate to 14CO2 and compared this with the
oxidation of [14C]succinate to 14CO2.261 By including estimation
of [14C]methylmalonate oxidation as well, this test can distinguish
between de®ciency of propionyl CoA carboxylase and of
methylmalonyl CoA mutase. Incorporation of [14C]propionate
into trichloroacetic acid±precipitable material by intact cultured
cells has replaced the more cumbersome 14CO2 evolution
technique.230,275 Further discrimination among the methylmalonic
Table 94-3 Methylmalonyl CoA Mutase Activity in Liver
Homogenates from Patients with Methylmalonic Acidemia
Enzymatic Activity*
Subjects
Controls (3)
Patients
1
2
3
4
Without Added
AdoCbl
535±866
1
8
3
80
With Added AdoCbl
(4 3 1025 M)
799±1058
3
33
7
1368
*Assayed by measuring conversion of D L -[3H]methylmalonyl CoA to [3H]succinyl CoA. Values expressed as picomoles of succinate formed per milligram
protein per 30 min.
Reprinted with permission from reference 276.
acidemias has depended on studies of Cbl uptake and AdoCbl
formation by intact cultured ®broblasts, on assays of mutase
activity in cell extracts, and on genetic complementation studies
with cultured cell heterokaryons.
Mutase Apoenzyme De®ciency. Morrow and colleagues276 provided the ®rst evidence in vitro for apoenzyme abnormalities and
for biochemical heterogeneity among the methylmalonic acidemias. In four patients who had died, they studied mutase activity
in liver homogenates by measuring the conversion of DL[3H]methylmalonyl CoA to [3H]succinyl CoA (Table 94-3).
Activity was barely detectable in three and showed no response
when AdoCbl was added at concentrations suf®cient to saturate
the normal enzyme. In the fourth, mutase activity was restored to
control values by AdoCbl. These ®ndings were interpreted as
evidence for a mutase apoenzyme defect in the ®rst three patients
and for defective AdoCbl synthesis in the fourth. These ®ndings
were con®rmed subsequently in studies with cultured ®broblasts.277 Cells from the ®rst three patients synthesized AdoCbl
normally but had much reduced mutase activity in extracts
regardless of the amount of AdoCbl added; cells from the fourth
had a distinct defect in AdoCbl synthesis.
Subsequently, it has become clear that two general types of
apomutase defects exist. In one type, designated mut 0 and
constituting about two thirds of the mut complementation group,
mutase activity in extracts of cultured ®broblasts is undetectable
( < 0.1 percent of control), even when assayed in the presence of
AdoCbl concentrations greatly in excess of that normally required
to saturate the enzyme.277±279 When CRM was sought by
radioimmunoassay under steady-state conditions in cell lines
from 21 such patients, 12 had no immunologically identi®able
mutase protein (CRM ), whereas 9 had reduced amounts of CRM
ranging from 1 to 40 percent of that found in control extracts.280 In
a follow-up study,281 cells from this group of patients were pulse
labeled to determine how amounts of newly synthesized mutase
protein, detected by speci®c immunoprecipitation, compared with
the CRM values obtained under steady-state conditions. As
expected, all CRM‡ mutants had easily detectable newly
synthesized mutase. Of 11 CRM lines, however, 5 had amounts
of newly synthesized mutase ranging from barely detectable to
nearly half that seen in controls. Thus, some apomutase mutations
lead to the synthesis of unstable mutase proteins, which are rapidly
degraded intracellularly. One other result of this study bears
mention. Using a pulse-chase experimental protocol, mitochondrial import and cleavage of the apomutase precursor were studied
in control lines and in 38 lines from mut mutants that synthesized
mutase protein. In one of the 38 mutant lines, an N-terminal
deletion resulted in failure of the mutant mutase to be taken up by
mitochondria.281,282 All the others underwent normal mitochondrial uptake and processing.
2179
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2180
PART 9 / ORGANIC ACIDS
Fig. 94-10 Schematic representation of the secondary structure of
human methylmalonyl CoA mutase, predicted by homology modeling based on the radiographic structure of the P. shermanii mutase a
subunit. Helices are denoted by rectangles, b strands by bold
arrows, and coils or turns by a thin line. For each of the two main
domains, the ba pairs are labeled. The AdoCbl is represented by the
horizontal thick line labeled Cbl (the position of the corrin ring)
attached to an almost vertical thick line indicating the DMB side
chain as it extends into the Cbl-binding domain. The approximate
positions of a number of characterized missense mutations
determined in mut patients are indicated on the structure by small
ellipses. Although many mutations affecting AdoCbl binding are
located in the Cbl-binding domain, several, including R93H, Y231N,
and R369H, are in the N-terminal (ba)8 barrel domain. (Redrawn
with permission from Thoma NH, Leadley PF: Homology modelling
of human methylmalonyl-CoA mutase: A structural basis for point
mutations causing methylmalonic aciduria. Protein Sci 5:1922,
1996.)
The second type, designated mut , involves a structurally
abnormal mutase apoenzyme. The mutant apoenzymes in extracts
of these cells retain maximally 2 to 75 percent of control activity,
have a Km for AdoCbl approximately 200 to 5000 times normal,
show a normal Km for methylmalonyl CoA, and exhibit increased
thermolability relative to control enzyme.278,279,283 By radioimmunoassay, the amount of immunologically reactive mutase
protein in these extracts ranges from 20 to 100 percent of
control.280 Because pairwise crosses between mut 0 and mut
generally yield noncomplementing heterokaryons and because
there are affected individuals who appear to be mut 0 /mut
compound heterozygotes, both mutant types re¯ect abnormalities
of the locus coding for the apomutase structural gene.278,279 The
identi®cation of nonsense and missense mutations within the
mutase gene that lead to absent enzyme or abnormal enzyme
kinetics have ®rmly established this conclusion.
Ledley and his colleagues ®rst described the molecular changes
in mut patients. They surveyed a number of patient ®broblasts and
found no evidence for gross rearrangements at the genomic level,
but reductions in mRNA in some lines.284 A number of point
mutations were described, including nonsense changes that lead to
a mut 0 phenotype,282 missense mutations that also generate a mut 0
phenotype,285,286 and a missense mutation that generates a mut
phenotype.287 Each of these changes was con®rmed as a mutation
causing de®ciency of mutase activity by expressing the variant
protein, either in a mut 0 ®broblast line285,286 or in Saccharomyces
cerevisiae cells.287 Interestingly, one of the mut 0 lines was able to
complement a number of the other mut 0 and mut lines, although
not all,286 an example of interallelic complementation. To date, 28
mutations and 2 benign sequence changes have been identi®ed,
some of which have been characterized by expression in cultured
cells or in E. coli.68,288,289 A common mutation (G717V) was
found in ®ve black patients of African and African-American
ancestry,290,291 and in a series of patients from Japan, six patients
carried the same mutation (E117X).292
Figure 94-10 is a linear representation of the structure of
human mutase,86 based on the crystal structure of the P. shermanii
homologue.87 On it are indicated the locations of a number of the
missense mutations in mutase identi®ed so far. The effects of
some of these on mutase activity have been rationalized in terms
of the predicted three-dimensional structure of the human
enzyme.68,86,289,293 The easiest to explain are the mut0 mutations
G630E and G703R, affecting residues that line the binding pocket
for the DMB side-chain of AdoCbl. Although both change ¯exible
glycine residues to charged ones, the main effect is to introduce
bulky side chains into the narrow binding pocket, effectively
blocking access to it and preventing AdoCbl binding.293 Some
changes in the Cbl-binding domain (G623R, G626C) appear to
affect the positioning of His627, whose side-chain provides the
essential bottom ligand to the bound AdoCbl,86,293 whereas others
likely affect the position or interaction of the ba strands that form
the Cbl-binding domain. One of these, G717V, results in a highly
unstable protein, in addition to modifying AdoCbl binding.68
Mutations in the N-terminal TIM barrel, particularly those
producing a mut phenotype, are less easily explained. W105R
appears to affect the substrate channel in the TIM barrel, whereas
A377E, V368D, and R369H are in the likely dimer interface. The
interaction between dimerization or dimer stability and AdoCbl
binding, revealed by the mut phenotype of R369H,68 remains
unexplained.
Even less apparent is an explanation of the interallelic
complementation supported by two mutations in the N-terminus
of mutase, R93H and G94V. R93H was identi®ed in homozygous
form in a cell line that showed interallelic complementation with a
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
AdoCbl normally when incubated with OH-[57Co]Cbl, ATP, and
a reducing system designed to bypass the Cbl reductases (Fig.
94-8). Subsequent biochemical167 and genetic complementation295±297 studies have differentiated two mutant classes among
patients defective in AdoCbl synthesis. One class, which contains
the index Cbl-responsive patient, is designated cblA, and may be
due to a de®ciency of one of the mitochondrial Cbl reductases,
perhaps an NADPH-linked aquacobalamin reductase.298 Complementation has been demonstrated between several cblA lines,
raising the possibility of interallelic complementation in this
disorder as well.299 The second, designated cblB, has been shown
to result from a speci®c de®ciency of cob(I)alamin adenosyltransferase.168
Fig. 94-11 Model for interallelic complementation between pccB
and pccC mutations causing propionic acidemia. The schematic
shows a pair of b subunits aligned head to toe so that two functional
domains are produced, with the carboxybiotin site coming from one
subunit and the propionyl CoA binding site coming from the other.
In the case in which each b subunit contains complementing
mutations Ð near the N-terminus of one subunit for dupKICK140
and near the C-terminus of the other subunit for R410W Ð the
outcome is that only one of the two functional sites is inactivated.
The second functional site retains activity despite the presence of a
mutation on each subunit.
number of other mut lines,286 and G94V was found in
heterozygous form in a cell line that appeared to show interallelic
complementation with the G717V mutation in vivo.68 Subsequent
in vitro experiments with mutant proteins expressed in E. coli have
con®rmed its ability to complement a range of other mut cell lines
(J. Janata and W. Fenton, unpublished observations). This region
of mutase is the least homologous between the human and
P. shermanii enzymes. In the bacterial enzyme, this region forms a
long element that wraps around the TIM domain of the other
subunit, and thus appears to be involved with subunit±subunit
interactions. How this relates to AdoCbl binding (G94V is mut )68
and how these mutations modify the effects of mutations in the
distant Cbl-binding domain to produce interallelic complementation are questions that may only be answered by crystal structures
of the human enzyme and these mutant versions.
It seems clear from the available structures, however, that
complementation is not simply a matter of bringing together
unaffected regions from each subunit of a mutase dimer to form
one ``normal'' active site,293 as may be the case for the b subunits
of PCC (Fig. 94-11). The active site of mutase is clearly contained
in a single subunit, with no possibility for sharing elements of it
between subunits.87 Moreover, the combined active site model
predicts that the kinetic parameters of the restored site should be
the same as wild type. This is not what is observed in vivo for the
complementing G94V/G717V pair, where the ``complemented,''
presumably heterodimeric, enzyme has a Km at least 10-fold lower
than either of its homodimeric parents, but 100-fold higher than
wild type.68 Similar results have been obtained in vitro by
combining the individually expressed mutant enzymes (J. Janata
and W. Fenton, unpublished observations), suggesting a more
complex mechanism for restoration of activity upon interallelic
complementation between mutase mutants.
Finally, it should be mentioned that the only patient thus far
reported to have methylmalonyl CoA racemase de®ciency294 has
been restudied and shown conclusively to be a mut mutant
biochemically and genetically.17
Defective Synthesis of AdoCbl. A series of observations by
Rosenberg,14 Mahoney,15 and their colleagues on the ®broblasts of
the index patient with Cbl-responsive methylmalonic acidemia
led to the demonstration of a primary defect in AdoCbl synthesis
in intact cells, resulting in a de®ciency of mutase activity.
Although intact cells were unable to convert OH-[57Co]Cbl to
Ado[57Co]Cbl, cell-free extracts from this line synthesized
Pathophysiology. All studies in vivo and in vitro in patients with
methylmalonic acidemia due to speci®c methylmalonyl CoA
mutase de®ciency indicate that the primary block in the conversion
of methylmalonyl CoA to succinyl CoA explains the accumulation
of methylmalonate in blood and urine; the augmentation of
methylmalonate excretion and the precipitation of ketosis by
protein, amino acids, or propionate; and the excretion of longchain ketones formed in the catabolism of branched chain amino
acids. However, the primary block does not explain several
important physiologic disturbances: the acidosis, hypoglycemia,
hyperglycinemia, and hyperammonemia. Oberholzer et al.10
pointed out that the concentration of methylmalonate in the blood
(no more than 3 mM) could not alone explain the acidosis and
suggested other possibilities. They proposed that an accumulation
of CoA, ``trapped'' intracellularly as methylmalonyl CoA, could
lead to an insuf®ciency of this widely utilized coenzyme and,
secondarily, to impaired carbohydrate metabolism and subsequent
acidosis. Alternatively, they suggested that an excess of methylmalonyl CoA, a known inhibitor of pyruvate carboxylase,300 could
interfere with gluconeogenesis and lead directly to hypoglycemia
and indirectly to excessive catabolism of lipid, with ketosis and
acidosis. Halperin et al.301 showed that methylmalonate inhibited
the transmitochondrial shuttle of malate and argued that impairment of this key step in gluconeogenesis could lead to hypoglycemia. As discussed earlier for de®ciencies of b-ketothiolase
and propionyl CoA carboxylase, the mechanism of the hyperglycinemia and hyperammonemia so often observed in children with
any one of these disorders probably re¯ects inhibition of the
intramitochondrial glycine cleavage enzyme and of CPS I,
respectively, by the accumulated organic acids or their CoA
esters.208,209,211± 215 Thus, as shown in Fig. 94-12, each of the
major secondary biochemical abnormalities in the propionic and
methylmalonic acidemias can be explained satisfactorily by
inhibition of speci®c intramitochondrial processes by the accumulated organic acids and esters.
As a further consideration, about half the reported patients with
isolated methylmalonic acidemia also show pancytopenia.264 One
report suggests that methylmalonate inhibits growth of marrow
stem cells in a concentration-dependent fashion.302
By comparing and contrasting the ®ndings in patients with
isolated mutase de®ciency with those in patients with Cbl
de®ciency (as in classic pernicious anemia), it has been possible
to shed some light on the mechanism responsible for the
hematologic and neurologic abnormalities in the latter disorder.
Thus, the absence of megaloblastic anemia in any patient with
isolated mutase de®ciency militates against any involvement of
this enzyme in the typical megaloblastosis seen in Cbl de®ciency.
Similarly, the cerebellar and posterior column abnormalities so
often encountered in Cbl-de®cient patients have never been
observed in patients with methylmalonic acidemia due to speci®c
mutase dysfunction. Therefore, the notion that neurologic
dysfunction in pernicious anemia re¯ects aberrant incorporation
of odd-chain or branched-chain fatty acids into myelin because of
a block in the propionate pathway has little to recommend it. It
appears likely, then, that abnormalities in the Cbl-dependent
methionine synthase account for the hematologic and neurologic
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2182
PART 9 / ORGANIC ACIDS
Fig. 94-12 Proposed mechanisms of hypoglycemia, hyperglycinemia, and hyperammonemia in
patients with inherited de®ciencies of b-ketothiolase (see Chapter 93), propionyl CoA carboxylase,
or methylmalonyl CoA mutase. Inhibitory effects of
the enlarged intramitochondrial pools of acyl CoA
esters (such as propionyl CoA) or their respective
free acids on selected mitochondrial functions are
shown by the numbered dashed lines corresponding to the following enzymatic or shuttle-mediated
reactions: (1) pyruvate carboxylase; (2) the transmitochondrial malate shuttle; (3) the glycine cleavage enzyme; (4) carbamyl phosphate synthetase I;
and (5) N -acetylglutamate synthetase.
abnormalities in Cbl-de®cient patients. This matter will be
discussed further when we consider that group of patients with
methylmalonic acidemia and homocystinuria.
Genetic Considerations. Each of the four etiologic bases for
speci®c methylmalonyl CoA mutase de®ciency (mut 0, mut , cblA,
and cblB) is almost certainly inherited as an autosomal-recessive
trait. This conclusion is based on the following ®ndings. First,
approximately equal numbers of affected males and females are
encountered in each group.264 Second, no instance of vertical
transmission from affected parent to affected child has been
reported. Third, interclass heterokaryons formed between cell
lines from different etiologic groups (i.e., mut 0 cblA) complement each other, whereas intraclass heterokaryons (i.e.,
mut 0 mut 0) generally do not (with the exception of interallelic
complementation); thus, each mutant class behaves as a recessive
in culture.295±297 Fourth, cell lines from heterozygotes for the
mut0, mut , and cblB mutations show partial mutase apoenzyme
de®ciency279 and partial adenosyltransferase de®ciency,168 respectively. And ®fth, among a large group of mut mutants studied,
some have inherited a genetically different mutant allele from each
parent, thereby being compound heterozygotes rather than true
homozygotes.279,284,285
It is not possible to de®ne with any precision the prevalence of
these disorders in the general population. A survey of newborns in
Massachusetts has suggested that methylmalonic acidemia may
occur in 1:48,000 infants.303 A similar survey in Quebec yielded
1:61,000 infants.304 Because this study screened urines from
infants 3 to 4 weeks of age and because it is known that many
children with methylmalonic acidemia die in the ®rst week of life
from ketoacidosis or hyperammonemia or both, the true
prevalence must be greater.
Diagnosis, Treatment, and Prognosis. Because simple colorimetric assays for urinary methylmalonate and more complex gas
chromatography±mass spectrometry assays for serum and urinary
methylmalonate are now available, it should no longer be dif®cult
to make a diagnosis of methylmalonic acidemia once this
condition is considered. Other sources of neonatal or infantile
ketoacidosis must be ruled out. If excessive amounts of
methylmalonate are found in the urine, Cbl de®ciency can be
excluded by direct measurement of serum Cbl concentration.
Con®rmation and etiologic designation (i.e., mut or cbl) depend on
studies with cultured cells and extracts therefrom.17,305 Prenatal
detection of methylmalonic acidemia has been accomplished in
two ways: by measurement of methylmalonate in amniotic ¯uid
and maternal urine at mid-trimester306,307 and by studies of mutase
activity and Cbl metabolism in cultured amniotic ¯uid
cells.295,307,308 Assays of [14C]propionate utilization230 in uncultured chorionic villous biopsy specimens have proven unsatisfactory, however.309 Mutase apoenzyme307,308 and AdoCbl
synthesis230,306 de®ciencies have been identi®ed prenatally.
Two treatment regimens for children with methylmalonic
acidemia exist and should be used in tandem. A diet restricted in
protein (or a special formula restricted in amino acid precursors of
methylmalonate) should be instituted as soon as life-threatening
problems such as ketoacidosis, hypoglycemia, or hyperammonemia have been addressed; and supplementary Cbl (1±2 mg CNCbl or, preferably, OH-Cbl intramuscularly daily for several days)
should be given as soon as the diagnosis of methylmalonic
acidemia is made (or even seriously considered). Such measures
should decrease the circulating concentrations of methylmalonate
and propionate. Even Cbl-unresponsive children with delayed
development have been shown to improve markedly when treated
with careful dietary protein restriction.310,311 As discussed above
for patients with propionyl CoA carboxylase de®ciency, Roe and
associates235,312,313 have pointed out that L-carnitine supplements
may be a useful therapeutic adjunct in patients with methylmalonic acidemia, presumably by repleting intracellular and extracellular stores of free carnitine that are depleted in affected
patients because of exchange with excess methylmalonyl CoA and
propionyl CoA. Likewise, oral antibiotic therapy may prove useful
here as well. Thompson and his colleagues report that three
patients showed subjective improvement in alertness and appetite
following brief metronidazole therapy237; longer treatment periods
have resulted in signi®cant improvements in other patients,
including decreased number and severity of acidotic episodes,
increased appetite, decreased vomiting, growth acceleration, and
improved behavior.29,314 Total parenteral nutrition also has been
used in at least one reported case.233
The previously mentioned survey264 suggests that both the
response to Cbl supplements and the long-term outcome in
affected patients depend considerably on the nature of the
biochemical lesion causing the methylmalonic acidemia. As
shown in Figure 94-13, essentially none of the children designated
mut 0 or mut responded to Cbl supplements with a distinct
decrease in blood or urinary methylmalonate, whereas over 90
percent of the cblA and about 40 percent of the cblB patients
showed such a response. Given the complete absence of mutase
activity in cells from the mut 0 group, it is not surprising that they
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
Fig. 94-13 Biochemical response to cobalamin supplementation in
45 patients with methylmalonic acidemia. MMA refers to the
concentration of methylmalonate. The supplementation protocol
generally used 1 mg CN-Cbl parenterally daily for 7 to 14 days. Inset
numbers denote the percentage of patients in each group.
(Reprinted from Matsui SM, Mahoney MJ, Rosenberg LE: The
natural history of the inherited methylmalonic acidemias. New Engl
J Med 308:857, 1983.)
Fig. 94-14 Long-term outcome in 45 patients with methylmalonic
acidemia. The ages of the patients surveyed ranged from a few
weeks to 14 years. (Reprinted with permission from Matsui SM,
Mahoney MJ, Rosenberg LE: The natural history of the inherited
methylmalonic acidemias. New Engl J Med 308:857, 1983.)
were regularly Cbl-unresponsive in vivo. The disappointing
absence of response in four mut patients presumably means
that even parenteral Cbl supplements could not drive tissue
concentrations of AdoCbl suf®ciently high to increase signi®cantly their mutase holoenzyme activity. The fraction (60 percent)
of cblB patients unresponsive to Cbl supplements presumably has
such complete adenosyltransferase de®ciency that AdoCbl synthesis cannot be augmented by Cbl supplements, as it apparently can
in the cblB patients with leaky mutations that permit responsiveness in vivo. Patients in the cblA group were uniformly responsive,
suggesting either that the responsible mutations are generally
leaky, thereby allowing mass action to result in more AdoCbl
synthesis, or that alternative pathways of Cbl reduction that require
high substrate concentrations exist in cells. It should be
emphasized that clinical responsiveness in vivo does not mean
complete correction of mutase de®ciency. Even in a patient whose
clinical improvement is dramatic, Cbl administration only reduces,
rather than eliminates, methylmalonate excretion. Studies with
cultured cells from a variety of patients with methylmalonic
acidemia17,297 suggest that raising holomutase activity to only 10
percent of normal values by supplementing the growth medium
with OH-Cbl results in distinct augmentation of propionate
pathway activity (or, conversely, in a distinct decrease in the
magnitude of the metabolic block). Some patients in the cblB
group, unresponsive to CN-Cbl or OH-Cbl in vivo, might be
expected to respond to AdoCbl itself, but published reports on two
patients suggest that this logical alternative is ineffective.315,316
The long-term outlook for affected patients is revealing. As
noted in Fig. 94-14, the mut0 group has the poorest prognosis, with
60 percent deceased and 40 percent distinctly impaired developmentally at the time of the survey. Shevell et al. reported similar
®ndings.265 In sharp contrast, the cblA patients (i.e., the group
biochemically most responsive to Cbl supplements) had the best
outcome: 70 percent were alive and well at ages up to 14 years.
The cblB and mut groups were intermediate, with about equal
fractions in each group being found in the alive and well, the alive
and impaired, or the deceased category.
It is interesting, albeit anecdotal, that the index patient in the
cblA group (now over 30 years old) discontinued Cbl supplements
at age 9 years despite advice to the contrary. In the ensuing
years, his development and general health have remained
excellent despite the high concentration of methylmalonate in
his blood and his continued excretion of very large amounts of
methylmalonate.
Perhaps, as in some other inherited metabolic disorders,
treatment of methylmalonic acidemia is most critical during the
early years of life, making expert clinical management in the early
weeks or months of life most important. There have been several
reports of ``metabolic stroke'' in patients following episodes of
metabolic decompensation.317 ±319 Three of the patients317,318
belonged to the Cbl-responsive cblA group, but were not being
treated at the time. Extrapyramidal signs, particularly dystonia,
were accompanied by bilateral lucencies of the globus pallidus and
persisted after the acute crisis had passed. In one case, the dystonia
has been gradually progressive over a period of 7 years without
visible progression of the neurologic lesions.319
Another complication of long-term survival of some methylmalonic acidemia patients is chronic renal failure.320 One report
has indicated that 8 of 12 non±Cbl-responsive patients (1±9 years
of age) had a reduced glomerular ®ltration rate, with ®ve severely
affected.321 In one of these, ``greatly improved metabolic control''
over a period of 18 months led to increased, but still impaired,
renal function.321 Signi®cantly, the index cblA patient referred to
above returned for treatment of moderate renal dysfunction due to
biopsy-proven interstitial nephritis. We do not yet know what
impact better metabolic control and Cbl supplementation may
have in this and similar cases.
A 7-year-old boy with mut methylmalonic aciduria developed
persistent lactic acidosis and multiorgan failure. He was shown to
have glutathionine de®ciency and responded to treatment with
high-dose ascorbate.322
The feasibility of prenatal therapy with Cbl supplements also
has been demonstrated. Ampola et al.307 showed that administration of Cbl supplements to a woman carrying a Cbl-responsive,
affected fetus resulted in signi®cant reduction in maternal
excretion of methylmalonate. Other cases also have been
reported.323,324 However, the value of this regimen over one in
which therapy is instituted immediately postnatally remains to be
established.
Because of the poor prognosis of early-onset severe methylmalonic aciduria, liver transplantation has been attempted in a
limited number of patients.325± 328 Although liver transplantation
appears to protect against acute metabolic decompensation,
biochemical correction is incomplete and it is not certain that
2183
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2184
PART 9 / ORGANIC ACIDS
there will be complete protection against the renal and neurologic
complications. Finally, it must be noted that preliminary steps have
been taken toward somatic gene therapy for mutase de®ciency.329,330 Besides all the usual questions of safety and longterm stability of response that surround somatic gene therapy, two
important issues remain unanswered for mutase: How much
activity must be restored in vivo to normalize the biochemical
hallmarks of the disease? And, does correction of the defect in the
liver, for example, lead to reversal or amelioration of the
pathologic changes in other organ systems, such as the kidneys,
and overall clinical improvement? Much work remains to be done
before we can address these problems.
these children suffered from a defect in cellular metabolism of Cbl
such that both Cbl-dependent enzyme activities (mutase and
methionine synthase; see Fig. 94-7) were de®cient. The methylmalonic aciduria in these children is distinctly less severe than that
encountered in children with isolated mutase de®ciency. It is
important to note that elevations of homocystine may be
unremarkable in some cblC patients, even when acutely ill. One
of the cblF patients had no detectable homocystinuria despite a
cellular de®cit in methionine synthase activity,344 although all the
others have shown homocystinuria.345±347 Moreover, neither
hyperglycinemia nor hyperammonemia has been reported in any
of the cblC, cblD, or cblF patients.
Combined De®ciency of Methylmalonyl CoA Mutase and
Methionine Synthase. Clinical and Laboratory Presentations.
Many patients with inherited combined methylmalonic acidemia
and homocystinuria have been the subject of individual case
reports.263,331±347 Cells from these children comprise three
biochemically and genetically distinct complementation groups,
designated cblC, cblD, and cblF.167,295,296,348
Clinical ®ndings have varied widely among the more than 100
known patients in the cblC group, including some who have been
diagnosed only in adult life. In a review of 50 patients, 44 had
onset in the ®rst year of life and 6 had onset after 6 years of age.349
The median age of onset was 1 month, and the range was from
birth to 14 years. Thirteen of the early-onset patients died, with a
mean age of death at 9.6 months and a range of 1 to 47 months.
The early-onset patients had feeding dif®culties, hypotonia, failure
to thrive, seizures, microcephaly, developmental delay, cortical
atrophy, hydrocephalus, nystagmus, pigmentary retinopathy, and
decreased visual acuity. Blood ®ndings included megaloblastic
anemia, thrombocytopenia, leukopenia, and neutropenenia. In
some patients there was renal failure, sometimes with a hemolyticuremic syndrome. The later-onset patients presented in childhood
or adolescence with acute neurologic ®ndings, which included
decreased cognitive performance, confusion, dementia, delirium,
myelopathy, and tremor. Only one late-onset patient in this series
had pigmentary retinopathy. Hematologic abnormalities were seen
in half the late-onset patients. Signi®cantly, serum Cbl and folate
concentrations are generally normal in cblC patients.
Neither of the two brothers in the cblD group263 had any
clinically signi®cant problems until later in life. The older brother
came to medical attention because of severe behavioral pathology
and moderate mental retardation at 14 years of age. He also had a
poorly de®ned neuromuscular problem involving his lower
extremities. His then 2-year-old brother was asymptomatic,
although biochemically affected. No hematologic abnormalities
were noted in either sib.
Five patients have been reported in the cblF group. The ®rst
two (both female) were small for gestational age and had
methylmalonic aciduria, poor feeding, growth retardation, and
persistent stomatitis.344,345 One had minor facial anomalies,
dextrocardia, and abnormal Cbl absorption from the gut. The
other had a persistent rash, macrocytosis, and elevated homocysteine and died suddenly despite a good biochemical response to
Cbl treatment. A male cblF patient had recurrent stomatitis in
infancy, arthritis at age 4, and confusion, disorientation, and a
pigmentary dermatitis at age 10.347 Another boy had aspiration
pneumonia at birth, hypotonia, lethargy, hypoglycemia, thromobocytopenia, and neutropenia. A native Canadian girl was
diagnosed at 6 months of age because of anemia, failure to thrive,
developmental delay, recurrent infections, low serum Cbl, and Cbl
malabsorption.346 As a group, the cblF patients have responded
well to treatment with Cbl.
Localization of Defective Cellular Metabolism of Cbl. It has long
been clear that patients in the cblC and cblD groups have a defect
in cellular metabolism of Cbl. This conclusion is based on the
following data: total Cbl content of liver, kidney, and cultured
®broblasts is markedly reduced262,332,350,351; the ability of cultured
cells to retain 57Co-labeled CN-Cbl352 or to convert 57Co-labeled
CN-Cbl or OH-Cbl to AdoCbl and MeCbl is markedly
impaired15,296; activity of methylmalonyl CoA mutase and
methionine synthase in cultured cells is de®cient, such de®ciency
being improved by supplementation of the growth medium with
OH-Cbl296,297,353; and the mutase and methionine synthase
apoenzymes in cells from affected patients appear to be
normal.262,263,296,297,354 The precise nature of the metabolic defect
in the cblC and cblD classes remains elusive, but some progress
has been made. Because these mutant cells demonstrate normal
receptor-mediated adsorptive endocytosis of the TC II±Cbl
complex and normal intralysosomal hydrolysis of TC
II,17,153,154,296 perusal of Fig. 94-8 makes it clear that the defects
in the cblC and cblD cells affect some step or steps subsequent to
cellular uptake, common to the synthesis of both coenzymes, and
prior to the binding of the Cbl coenzymes to their respective
apoproteins. Signi®cantly, cblC (and, to a lesser extent, cblD) cells
use CN-Cbl less well than OH-Cbl353,355 and are unable to convert
CN-Cbl to OH-Cbl, a step shown in normal cells to be a metabolic
prerequisite for the synthesis of both AdoCbl and MeCbl.355 The
latter results have been interpreted as evidence for a defect in a
cytosolic cob(III)alamin reductase, which is required for reducing
the trivalent cobalt of Cbl prior to alkylation.355 In both cblC and
cblD ®broblast extracts, partial de®ciencies of CN-Cbl b-ligand
transferase and microsomal cob(III)alamin reductase have been
described by Pezacka.356,357 Watanabe and colleagues have
described a partial de®ciency of a mitochondrial NADH-linked
aquacobalamin reductase in cblC ®broblast extracts.298 The
suggestion that glutathionyl Cbl may be an intermediate in
the reductive pathway358 provides another potential site for the
mutation in one of these groups. Finally, it should be pointed out
that the distinction between the cblC and cblD classes is based ®rst
and foremost on complementation studies that de®ne the two
classes as unique,296 although essentially only one example of
cblD exists. The biochemical differences between them appear to
be quantitative rather than qualitative, with the cblC group having
more severe metabolic derangements (and, at the same time, more
severe clinical involvement) than the sibs designated cblD. Thus,
the possibility must be considered that cblD is an allele of cblC
that shows interallelic complementation.
Studies using cultured ®broblasts from two patients in the cblF
group are of particular interest. As with cells from cblC and cblD
patients, both mutase and methionine synthase activities were
impaired, and AdoCbl and MeCbl contents were reduced. In
contrast to the cblC and cblD mutants, however, the cblF cells
accumulated unmetabolized, non±protein-bound CN-Cbl in lysosomes.359 These ®ndings indicate that cblF cells are de®cient in
the mediated process by which Cbl vitamers exit from lysosomes
after being taken up by receptor-mediated endocytosis.
The biochemical features of patient ®broblasts that distinguish
the various methylmalonic acidemias are summarized in
Table 94-4.
Chemical Abnormalities In Vivo. In addition to the methylmalonic aciduria and homocystinuria that characterize the cblC, cblD,
and cblF groups of patients, some have shown hypomethioninemia
and cystathioninuria. This constellation of chemical abnormalities,
plus the normal serum Cbl values, led to the proposal262,331 that
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
Table 94-4 Salient Biochemial Features of Cultured Fibroblasts from Patients with
the Various Methylmalonic Acidemias
Mutant Class
Biochemical Parameter
mut 0
mut2
cblA
cblB
Studies with intact cells
[14C]propionate oxidation
[14C]MeH4F ®xation
MeCbl synthesis
AdoCbl synthesis
Conversion of CN-Cbl to OH-Cbl
Lysosomal ef¯ux of free Cbl
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
‡
Enzyme activities in cell extracts*
Mutase holoenzyme
Mutase total enzyme
Met synthase holoenzyme
Met synthase total enzyme
Cob(I)alamin adenosyltransferase
cblC
cblD
cblF
‡
‡
‡
‡
nt
nt
‡
‡
‡
*Holoenzyme is defined as that enzyme activity measured in the absence of added cofactor; total enzyme
is that activity measured in the presence of saturating concentrations of cofactor. Abbreviations:
‡ ˆ normal;
ˆ markedly deficient or undetectable; ˆ partially deficient; nt ˆ not tested;
MeCbl ˆ methylcobalamin;
AdoCbl ˆ adenosylcobalamin;
MeH4F ˆ N 5-methyltetrahydrofolate;
CN-Cbl ˆ cyanocobalamin; OH-Cbl ˆ hydroxocobalamin.
Pathophysiology. The megaloblastic anemia so commonly
observed in the cblC patients almost surely re¯ects the enzymatic
disturbance of methionine synthase. This can be stated with some
assurance because patients with isolated methylmalonyl CoA
mutase de®ciency (mut 0, mut , cblA, cblB) more severe than that
encountered in the cblC patients exhibit no such hematologic
dysfunction. The early and severe central nervous system
abnormalities encountered in the cblC group probably re¯ect the
methionine synthase abnormality as well, in that such patients do
not have the severe metabolic ketoacidosis that probably accounts
for the neurologic problems in patients with mutase de®ciency
only. Thus, patients with severe, inherited dysfunction in the
Fig. 94-15 Summary scheme of inherited defects of propionate and
methylmalonate metabolism. The circled numbers and their key
signify the nine general sites at which abnormalities have been
identi®ed. Abbreviations: PCC 5 propionyl CoA carboxylase;
MCC 5 b-methylcrotonyl CoA carboxylase; PC 5 pyruvate carbox-
ylase; ACC 5 acetyl CoA carboxylase; MUT 5 methylmalonyl CoA
mutase; CblIII 5 cob(III)alamin (e.g., OH-Cbl); CblI 5 cob(I)alamin;
AdoCbl 5 adenosylcobalamin; MeCbl 5 methylcobalamin; MS 5
methionine synthase.
2185
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2186
PART 9 / ORGANIC ACIDS
synthesis of both Cbl coenzymes resemble closely patients with
exogenous Cbl de®ciency, both groups having prominent hematologic and neurologic manifestations resulting from the blocked
methionine synthase system.
Genetic Considerations. Because equal numbers of affected
males and affected females exist in the cblC group, because
females have been as seriously affected as males, and because cells
from affected patients behave as recessives in complementation
studies,295 it seems safe to predict that this disorder is inherited as
an autosomal-recessive trait. The mode of inheritance of the cblD
and the cblF mutations cannot yet be de®ned with certainty,
because of the paucity of known patients. Identi®cation of
heterozygotes for cblC, cblD, or cblF has not yet been
accomplished. One additional contribution of the somatic cell
genetic studies used to characterize these disorders deserves
mention. The locus coding for the human methionine synthase
structural gene was originally mapped to chromosome 1 using
human±hamster hybrids.354 This assignment has been con®rmed
with the cloning of the gene.360±363
Diagnosis, Treatment, and Prognosis. The combination of
methylmalonic aciduria, homocystinuria, and normal serum Cbl
concentrations is the triad needed to distinguish patients in the
cblC, cblD, or cblF groups from those with isolated mutase
de®ciency, with other causes of homocystinuria such as cystathionine synthase de®ciency or N5,10-methylenetetrahydrofolate
reductase de®ciency, with Cbl de®ciency, or with the cblE and
cblG mutations affecting only methionine synthase (see Chapters
88 and 155). Such distinctions, easily con®rmed by cell studies,
are critical because appropriate therapy depends on them. Whereas
exogenous Cbl de®ciency will respond dramatically to physiologic
amounts of Cbl and certain forms of homocystinuria will respond
to supplements of pyridoxine or folate, successful treatment of
cblC, cblD, or cblF patients may demand administration of very
large amounts (up to 1 mg daily) of OH-Cbl.263,333,335 ±337,344
Such treatment has resulted in dramatic decreases in urinary
methylmalonate (and less dramatic decreases in urinary homocystine) in patients who have received it.364 Supplementation with
betaine can reduce homocysteine levels and restore methionine,
although the clinical effects of this treatment are unclear.365
Early diagnosis and prompt institution of therapy with Cbl
supplements (and betaine) may be the only way to change the
outcome of these patients, which, at least in the case of the cblC
group, has been dismal thus far.366 Documentation of experience
with such treatments will be particularly important in assessing
the clinician's ability to modify the natural history of these
disorders.
OVERVIEW
The panorama of variants leading to methylmalonic aciduria is
shown in Fig. 94-15, juxtaposed to those seen in inherited forms of
propionic acidemia.
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CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM
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