Enzymes involved in branched-chain amino acid metabolism in humans

Abstract

Branched-chain amino acids (leucine, isoleucine and valine) are structurally related to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid. Similar to fatty acid oxidation, leucine and isoleucine produce acetyl-coA. Additionally, leucine generates acetoacetate and isoleucine yields propionyl-coA. Valine oxidation produces propionyl-coA, which is converted into methylmalonyl-coA and succinyl-coA. Branched-chain aminotransferase catalyzes the first reaction in the catabolic pathway of branched-chain amino acids, a reversible transamination that converts branched-chain amino acids into branched-chain ketoacids. Simultaneously, glutamate is converted in 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coA intermediates, which then follow separate catabolic pathways. Human tissue distribution and function of most of the enzymes involved in branched-chain amino acid catabolism is unknown. Congenital deficiencies of the enzymes involved in branched-chain amino acid metabolism are generally rare disorders. Some of them are associated with reduced pyruvate dehydrogenase complex activity and respiratory chain dysfunction that may contribute to their clinical phenotype. The biochemical phenotype is characterized by accumulation of the substrate to the deficient enzyme and its carnitine and/or glycine derivatives. It was established at the beginning of the twentieth century that the plasma level of the branched-chain amino acids is increased in conditions associated with insulin resistance such as obesity and diabetes mellitus. However, the potential clinical relevance of this elevation is uncertain.

Introduction

It has been long known that insulin resistance is associated with increased plasma level of branched-chain amino acids (leucine, isoleucine, and valine) but the clinical consequences of such elevation are uncertain. Congenital deficiencies of the enzymes involved in the catabolism of branched-chain amino acids usually cause neurological phenotypes suggesting an important contribution of these amino acids to the function of the central nervous system. Recurrent episodes of ketoacidosis with vomiting and lethargy often triggered by infections are another common manifestation of altered branched-chain amino acid metabolism. Data on metabolism of branched-chain amino acids in humans may help to understand their physiological relevance.

Branched-chain amino acids show some structural similarity to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid (Fig. 1). Branched-chain aminotransferase (BCAT) catalyzes a reversible transamination reaction that converts branched-chain amino acids into branched-chain ketoacids (Fig. 2). The branched-chain ketoacid dehydrogenase (BCKD) complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coenzyme A (coA) derivative esters, which undergo separate catabolic pathways depending on the initial substrate. Ultimately, leucine renders acetoacetate and acetyl-coA, isoleucine yields propionyl-coA and acetyl-coA and the end product of valine catabolism is propionyl-coA. Propionyl-coA from isoleucine and valine catabolism is converted into methylmalonyl-coA and succinyl-coA (Fig. 3).

Fig. 1

Branched-chain amino acids and branched-chain keto acids

Fig. 2

The branched-chain aminotransferase reaction

Fig. 3

The end-products of branched-chain amino acid metabolism

Branched-chain aminotransferase

BCAT catalyzes the reversible transamination reaction that converts a branched-chain amino acid into its respective branched-chain ketoacid. The pair glutamate/2-ketoglutarate (α-ketoglutarate or 2-oxoglutarate) is an obligate partner of the transamination reaction. In one direction, 2-ketoglutarate receives the amino group of the branched-chain amino acid, being converted into glutamate. At the same time, the branched-chain amino acid is converted into its cognate branched-chain ketoacid. Leucine yields 2-keto-isocaproate, isoleucine renders 2-keto-3-methylglutarate, and valine is converted into 2-keto-isovalerate. In the opposite direction of the transamination reaction catalyzed by BCAT, one branched-chain ketoacid (2-keto-isocaproate, 2-keto-3-methylglutarate or 2-keto-isovalerate) receives the amino group of glutamate. As a result, 2-keto-isocaproate generates leucine, 2-keto-3-methylglutarate produces isoleucine, and 2-keto-isovalerate gives rise to valine. Simultaneously, glutamate is converted into 2-ketoglutarate.

Aminotransferases in general catalyze the transfer of the α-amino group from an amino acid to 2-ketoglutarate to produce glutamate. These enzymes channel the formation of glutamate from many amino acids. Alanine aminotransferase (ALT) transfers the amino group of alanine to 2-ketoglutarate while aspartate aminotransferase (AST) transfers the amino group of aspartate to 2-ketoglutarate, both generating glutamate. At the same time, alanine aminotransferase forms pyruvate while aspartate aminotransferase produces oxaloacetate. The BCAT reaction produces branched-chain amino acids from glutamate, which yields its amino group to the branched-chain ketoacids to form branched-chain amino acids. Therefore, most amino acids may ultimately give rise to branched-chain amino acids via transamination reactions.

All aminotransferases including BCAT need pyridoxine (vitamin B6) as a cofactor. The transamination reaction is accompanied by interconversion between the pyridoxal 5′-phosphate (PLP) and the pyridoxamine 5′-phosphate (PMP) forms of the enzyme. The pyridoxal 5′-phosphate form reacts with the branched-chain amino acid and the reaction proceeds through several steps until the formation of the branched-chain ketoacid product and the pyridoxamine 5′-phosphate form of the enzyme. Pyridoxal 5′phosphate functions as a carrier of amino groups at the active site of the aminotransferases (Goto et al. 2005).

There are two isoenzymes of human BCAT, cytosolic and mitochondrial. Human tissue distribution of BCAT has been investigated in very few studies. In one study, the message for the cytosolic isoform is restricted to the brain while mitochondrial BCAT mRNA is widely present in human tissues, including skeletal muscle, kidney cortex, brain, heart, subcutaneous adipose tissue, stomach, colon, ileum, and liver. Human skeletal muscle has the most abundant BCAT message followed by kidney and brain whereas liver has low level of mitochondrial BCAT mRNA (Suryawan et al. 1998). However, other study finds BCAT protein and enzyme activity in human liver, being greater than the activity of the BCKD complex (Taniguchi et al. 1996). BCAT activity in human pancreas has not been fully investigated (Suryawan et al. 1998). In obese individuals, the amount of mitochondrial BCAT in adipose tissue increases after surgical weight loss compared with levels before surgery (She et al. 2007). In the human brain, mitochondrial BCAT is confined to the vasculature, being present in endothelial cells and tunica media while cytosolic BCAT is restricted to neurons, being identified in all regions of brain examined while astrocytes show no labeling (Hull et al. 2012). The brain of patients with Alzheimer disease, vascular dementia, and dementia with Lewy bodies shows increased expression of mitochondrial BCAT relative to controls (Ashby et al. 2016).

The X-ray crystal structure of human mitochondrial BCAT has been solved, showing that the enzyme is a homodimer. The three-dimensional structures of the cytosolic and mitochondrial isoforms of human BCAT complexed with gabapentin have also been reported (Goto et al. 2005). Human BCAT isoenzymes have a redox-sensitive CXXC motif located outside of the active site. Cytosolic BCAT has an overall redox potential lower than the mitochondrial isoenzyme. Site-directed mutagenesis to the active thiols of the CXXC motif demonstrates that they are crucial to protein folding. Inhibitors of human mitochondrial BCAT have been described, but their clinical relevance is unclear (Borthwick et al. 2016).

Two separate genes encode the two human BCAT isoenzymes, BCAT1, located to 12p, encodes cytosolic BCAT while the BCAT2 gene, sited on chromosome 19, encodes the mitochondrial isoform (Bledsoe et al. 1997).

Congenital deficiency of BCAT

A disease-causing mutation in the BCAT2 gene has been reported in a patient presenting with headache and memory impairment. Plasma level of valine and leucine is elevated. Brain magnetic resonance imaging (MRI) shows abnormal symmetric signals in the white matter. After treatment with vitamin B6, the plasma level of branched-chain amino acids especially valine decreased and brain MRI lesions improved. The patient carries two heterogeneous mutations in the BCAT2 gene, resulting in decreased BCAT activity. Each change is identified in his father and mother, suggesting an autosomal recessive disorder. Mutation analysis of the genes encoding the BCKD complex was normal (Wang et al. 2015).

Branched-chain ketoacid dehydrogenase complex

BCKD complex structure

The BCKD complex is a multiprotein enzyme composed of three subunits, E1, E2, and E3. A kinase and a phosphatase are attached to the E1 subunit and regulate the complex activity (Nobukuni et al. 1989).

The E2 subunit constitutes the center of the complex to which the E1 and E3 components are attached. The E2 subunit consists of 24 identical elements, being a homo-24-meric structure that has three domains: the core domain, the binding domain, and the lipoyl domain (lipoic acid-bearing domain). The binding domain attaches the subunits E1 and E3 to E2. The lipoyl domain is essential to substrate channeling within the complex. The core domain contains the active site (Chuang et al. 2006).

The E1 subunit of the BCKD complex is a tetramer composed of two α and two β components (α2β2). E1 binds thiamine pyrophosphate, possessing two thiamine-binding pockets located between α and β subunits. The crystal structure of the E1 subunit has been determined, showing a tetrahedral arrangement of the two α and two β subunits (Indo et al. 1987).

E3 is a homodimeric flavoprotein that contains a bound molecule of flavin adenine dinucleotide (Litwer, Herring, and Danner 1989). This subunit is common to three human α-ketoacid dehydrogenase complexes: pyruvate dehydrogenase (PDH), 2-ketoglutarate dehydrogenase, and BCKD, which catalyze the oxidative decarboxylation of their substrates, pyruvate, 2-ketoglutarate, and the branched-chain ketoacids, respectively (Indo et al. 1987). The PDH complex renders acetyl-coA from pyruvate. The 2-ketoglutarate dehydrogenase complex yields succinyl-coA from 2-ketoglutarate in the tricarboxylic acid cycle. The BCKD complex produces different branched-chain acyl-coA intermediates depending on the branched-chain ketoacid being decarboxylated. 2-Keto-isocaproate (from leucine) is converted into isovaleryl-coA. 2-Keto-3-methylglutarate (from isoleucine) is transformed into 2-methylbutyryl-coA. 2-Keto-isovalerate (from valine) yields isobutyryl-coA. The three enzymatic complexes have similar structural organization, consisting of three subunits. E1 and E2 components are specific for each complex while the E3 subunit is a flavoprotein shared by the three complexes. Therefore, a dysfunctional E3 protein leads to a combined deficiency of the three enzymes (multiple carboxylase deficiency) (Chuang et al. 2006). The isolation and sequencing of cDNA encoding the entire protein sequence of human E3 subunit has been reported (Otulakowski and Robinson 1987).

BCKD complex reaction

Human BCKD complex is likely located on the matrix side of the inner mitochondrial membrane and catalyzes the irreversible oxidative decarboxylation of the branched-chain ketoacids (2-keto-isocaproate derived from leucine, 2-keto-3-methylglutarate obtained from isoleucine, and 2-keto-isovalerate from valine). This reaction produces CO₂, NADH, and the respective branched-chain acyl-coA intermediates with a 1:1:1 stoichiometry. The action of BCKD complex generates isovaleryl-coA from 2-keto-isocaproate (leucine), 2-methylbutyryl-coA from 2-keto-3-methylglutarate (isoleucine), and isobutyryl-coA from 2-keto-isovalerate (valine) (Indo et al. 1987; Otulakowski and Robinson 1987; Chuang et al. 2006).

The global reaction of the BCKD complex is:

$$\begin{array}{*{20}l} {2{\text{-keto-isocaproate}}} \hfill & {} \hfill & {\text{isovaleryl-coA}} \hfill \\ {2{\text{-keto-}}3{\text{- methylglutarate}} + {\text{CoA}} + {\text{NAD}}^{ + } } \hfill & \xrightarrow{{\rm Thiamine, \; Mg}^{++}} \hfill & {2{\text{-methylbutyryl-coA}} + {\text{CO}}_{2} + {\text{NADH}} + {\text{H}}^{ + } } \hfill \\ {2{\text{-keto-isovalerate}}} \hfill & {} \hfill & {{\text{isovaleryl-coA}} .} \hfill \\ \end{array}$$

The reaction takes place in several steps. First, the E1 subunit of the BCKD catalyzes the decarboxylation of the branched-chain ketoacid, releasing carbon dioxide and a branched-chain acyl moiety. Second, the newly formed acyl group is transferred by the lipoic acid-bearing domain of E2 to the core of the complex where is attached to coenzyme A by the catalytic domain of E2, generating a branched-chain acyl-coA ester that is the product of the reaction. During this process, lipoate is reduced. Third, the E3 component re-oxidizes the reduced lipoate using NAD⁺ that is converted to NADH, thereby completing the functional cycle of the complex.

The decarboxylation of the branched-chain ketoacid is catalyzed by the E1 subunit, a thiamine-dependent branched-chain α-ketoacid dehydrogenase (branched-chain α-ketoacid decarboxylase). The decarboxylation activity of the E1 subunit needs Mg²⁺. The E1α element contains the catalytic site while the role of the E1β component of the decarboxylase remains to be clarified. The absence of the E1β subunit results in instability of the E1α subunit (Nobukuni et al. 1991). Human mitochondrial BCAT is associated with the E1 subunit of the BCKD complex, establishing a metabolon for branched-chain amino acid metabolism. This association facilitates the channeling of branched-chain ketoacids from BCAT to the E1 subunit of the BCKD complex. In the presence of BCAT, E1-catalyzed decarboxylation of branched-chain ketoacids is enhanced 12-fold (Islam et al. 2007).

The resulting branched-chain acyl moiety is transported from E1 to the core of the complex by the lipoyl-bearing domain of E2. In the core of the complex, the active site of E2 (dihydrolipoamide acyltransferase or dihydrolipoyl transacylase) catalyzes the transfer of the branched-chain acyl group from the lipoyl moiety to coenzyme A, generating a branched-chain acyl-coA ester and leaving lipoate in a reduced state (Indo et al. 1987; Otulakowski and Robinson 1987; Chuang et al. 2006).

Reoxidation of lipoate is accomplished by the E3 subunit (dihydrolipoamide dehydrogenase or dihydrolipoamide:NAD⁺ oxidoreductase), a flavoprotein that utilizes NAD⁺ as the electron acceptor (Indo et al. 1987; Otulakowski and Robinson 1987; Chuang et al. 2006).

BCKD complex human tissue distribution

Human tissue distribution of the BCKD complex has been scarcely investigated. In one study, the highest activity of the human BCKD complex is found in kidney, followed by liver, brain, and heart. Skeletal muscle, stomach, and colon have similar activity and they have activity of the enzyme compared to kidney, liver, brain, and heart. Subcutaneous adipose tissue and small intestine show the lowest BCKD complex activity. Human pancreas tissue was not available (Suryawan et al. 1998). The activity of the BCKD complex in human liver is lower than that of BCAT (Taniguchi et al. 1996).

BCKD complex kinase and phosphatase

The activity of the BCKD complex is regulated by phosphorylation and dephosphorylation catalyzed by a kinase and a phosphatase, respectively. The BCKD kinase inhibits the complex whereas the BCKD phosphatase activates it. The BCKD phosphatase has been expressed in Escherichia coli and purified in the soluble form, showing to be a monomeric protein that requires Mn²⁺ ions for activity. Mg²⁺ and Ca²⁺ ions do not support catalysis (Wynn et al. 2012).

The genes that encode components of the BCKD complex are listed in Table 1.

Table 1 Genes that encode components of the branched-chain keto acid dehydrogenase (BCKD) complex

Congenital deficiency of BCKD kinase

Inactivating homozygous mutations in the gene encoding BCKD kinase (BCKDK) have been identified in families with autism, epilepsy, and intellectual disability by whole exome sequencing. Affected patients show reduced plasma level of branched-chain amino acids (Novarino et al. 2012).

Congenital deficiency of the BCKD complex (maple syrup urine disease)

Maple syrup urine disease (MSUD) is an autosomal recessive disorder usually diagnosed by newborn screening (Axler and Holmquist 2014). Disease-causing mutations in the genes encoding the subunits E1α, E1β, E2, and E3 of the BCKD complex have been identified. Mutations in the subunits E1α, E1β, and E2 cause MSUD types 1A, 1B, and 2, respectively. Mutations in the E3 subunit cause multiple dehydrogenase deficiency, as E3 is the subunit shared by the three 2-ketoacid dehydrogenase complexes (PDH, 2-ketoglutarate dehydrogenase and BCKD) (Wynn et al. 1998). In the general population, the incidence of MSUD is approximately 1 in 150,000 live births. In the Old Order Mennonites of southeastern Pennsylvania, the incidence is much higher, approximately 1 in 358 births. MSUD in this ethnic group is caused by a founder mutation in the E1α subunit of the BCKD complex (Matsuda et al. 1990).

Severe MSUD usually appears during the neonatal period, but patients with milder clinical features may present later in life. The disease may manifest only intermittently with episodic symptoms (Axler and Holmquist 2014). During infections and other catabolic states, patients experience vomiting and neurological deterioration with lethargy and coma. Brain edema is a common complication of the acute metabolic crisis associated with MSUD. Patients with MSUD may have a maple syrup odor in urine (Wynn et al. 1998). Intellectual disability is a long-term consequence of severe MSUD. Mental retardation is common and not improved by liver transplantation. Depression, anxiety, inattention, impulsivity, and hyperactivity also occur. Patients may experience movement disorders, including dystonia, simple motor tics, choreoathetosis, and parkinsonism. Osteoporosis is frequent among adolescents with MSUD (Chuang et al. 2006). Affected children with the intermittent form of the disease show normal development and normal intelligence, but infections trigger ataxia or drowsiness. They are asymptomatic between episodes (Axler and Holmquist 2014).

Patients with MSUD show elevated baseline level of branched-chain amino acids and branched-chain ketoacids in plasma and urine that increase during metabolic crises (Wynn et al. 1998). Plasma concentration of leucine is generally higher than that of valine and isoleucine. l-alloisoleucine may be present in plasma (Frazier et al. 2014). Patients with the episodic form of the disease have normal laboratory tests between episodes (Axler and Holmquist 2014).

Patients with MSUD have a low leucine tolerance and current therapy relies on a lifelong restriction of this amino acid in the diet. Nutrition guidelines have been issued (Frazier et al. 2014). In addition, thiamine supplementation, sodium phenylbutyrate, and metformin have been proposed as drug therapy. Sodium phenylbutyrate activates the BCKD complex by inhibiting BCKD kinase. Therapy with sodium phenylbutyrate lowers plasma level of branched-chain amino acids and branched-chain ketoacids both in patients with low-severity MSUD and in control subjects, but has no significant effect on patients with severe MSUD (Brunetti-Pierri et al. 2011). In vitro investigations using fibroblasts derived from MSUD patients show that metformin reduces 2-keto-isocaproate (derived from leucine) concentration compared to untreated cells. Thiamine (vitamin B1) supplementation has been used commonly to treat MSUD since 1976, but its efficacy as only therapy is unproven and patients on thiamine supplementation have to adhere to leucine-restricted diet (Frazier et al. 2014).

Liver transplantation has been performed in selected patients. In 2000, an orthotopic liver transplantation was carried out in a patient with MSUD and liver failure triggered by hepatitis A infection. After liver transplantation, leucine tolerance improved and the patient tolerated an unrestricted diet, indicating that human liver is able to oxidize branched-chain ketoacids. However, plasma levels of branched-chain amino acids, branched-chain ketoacids, and l-alloisoleucine remained elevated after the transplant (Bodner-Leidecker et al. 2000). In addition, liver transplantation does not always prevent branched-chain amino acid elevation during acute catabolic states and does not reverse neurological damage or mental retardation. Mentally impaired patients show no improvement after liver transplantation (Mazariegos et al. 2012).

Congenital deficiency of the E3 subunit of the α-ketoacid dehydrogenase complexes (multiple carboxylase deficiency)

The E3 subunit (lipoamide dehydrogenase) is common to PDH, 2-ketoglutarate dehydrogenase, and BCKD complexes. Consequently, congenital deficiency of this subunit causes simultaneous deficiency of the three enzymatic complexes. Multiple carboxylase deficiency causes progressive neurological deterioration. The biochemical phenotype is characterized by congenital lactic acidosis, elevated plasma concentration of pyruvate and alanine, and increased urinary excretion of branched-chain ketoacids and 2-ketoglutarate. In one patient with multiple carboxylase deficiency, serum and urine acyl-carnitine profiles show increased levels of propionyl-carnitine (C3-carnitine), and two hydroxy-C5-carnitine (OH-C5-carnitine) species, 3-hydroxy-isovaleryl-carnitine and 3-hydroxy-2-methylbutyryl-carnitine, compared to a healthy patient (Maeda et al. 2008).

Enzymes involved in leucine catabolism

Leucine catabolic pathway proceeds through a series of enzymatic reactions to produce finally acetoacetate and acetyl-coA. BCAT catalyzes the reversible conversion of leucine into the branched-chain ketoacid 2-keto-isocaproate. The BCKD complex catalyzes the irreversible oxidative decarboxylation of 2-keto-isocaproate into isovaleryl-coA. Isovaleryl-coA dehydrogenase converts isovaleryl-coA into 3-methylcrotonyl-coA. 3-Methylcrotonyl carboxylase generates 3-methylglutaconyl-coA from 3-methylcrotonyl-coA. 3-Methylglutaconyl-coA hydratase yields 3-hydroxy-3-methylglutaryl-coA. Finally, 3-hydroxy-3-methylglutaryl-coA lyase catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl-coA to acetyl-coA and acetoacetate (Fig. 4).

Fig. 4

Leucine catabolic pathway

Leucine pathway: isovaleryl-coA dehydrogenase

Isovaleryl-coA dehydrogenase catalyzes the third step in leucine catabolism, the oxidation of isovaleryl-coA to 3-methylcrotonyl-coA. This enzyme has been purified from human liver, revealing to be a homotetrameric flavoenzyme. The crystal structure of human isovaleryl-coA dehydrogenase reveals that the C terminus is involved in tetramer stability. The residue Arg387 has an important role of in anchoring the substrate. The three-dimensional structure of human isovaleryl-coA dehydrogenase has been solved at 2.6 A (Rhead and Tanaka 1980; Tiffany et al. 1997). In vitro studies suggest that isovaleryl-coA dehydrogenase might use valproyl-coA as a substrate (Luis et al. 2011). The human gene for isovaleryl-coA dehydrogenase maps to chromosome 15q14-q15 (Ensenauer et al. 2004).

Congenital deficiency of isovaleryl-coA dehydrogenase (isovaleric acidemia)

Congenital deficiency of isovaleryl-coA dehydrogenase produces isovaleric acidemia, a disease first described in 1966 (Tanaka et al. 1966). The frequency of isovaleric acidemia ranges from 1:250,000 births in the US and 1:62,500 births in Germany. A patient with co-existence of Angelman syndrome and isovaleric acidemia has been reported, due to paternal uniparental isodisomy of chromosome 15 in which the proband inherited two paternal copies of a mutation in the isovaleryl-coA dehydrogenase gene (Lambrecht et al. 2015).

Isovaleric acidemia usually presents in the neonatal period, but it may appear later in life (Grunert et al. 2012a, b). This disorder typically manifests as acute recurrent episodes of vomiting, lethargy and coma usually precipitated by infections (Tanaka et al. 1966; Grunert et al. 2012a, b). Neurological involvement may occur including developmental delay, motor dysfunction, and cognitive deficits (Ensenauer et al. 2004; Grunert et al. 2012a, b). Pancytopenia has been documented. In post-mortem examination, myelodysplasia of the bone marrow with arrest of the myeloid series at the promyelocytic stage resembling promyelocytic leukemia has been described (Gilbert-Barness and Barness 1999) (Table 2). The severity of the disease is variable, ranging from asymptomatic individuals to death in the neonatal period. Mortality has been estimated in 33% of the patients with neonatal onset (Grunert et al. 2012a, b).

Table 2 Congenital deficiencies of enzymes involved in branched-chain amino acid metabolism: clinical phenotype

Patients with deficiency of isovaleryl-coA dehydrogenase fail to oxidize isovaleryl-coA into 3-methylcrotonyl-coA causing accumulation of isovaleryl-coA (Tables 3, 4). Accumulation of isovaleryl-coA promotes isovaleryl-carnitine formation and secondary reduction of free carnitine in plasma. Newborn screening by tandem mass spectrometry (MS/MS) detects elevated C5-carnitine in dried blood spots (Ensenauer et al. 2004). C5-carnitine species includes two isomers, isovaleryl-carnitine and 2-methylbutyryl-carnitine (Table 5). Isovaleryl-carnitine accumulates in patients with isovaleryl-coA dehydrogenase deficiency while 2-methylbutyryl-carnitine accumulates in patients with short branched-chain acyl-coA dehydrogenase deficiency, a disorder of isoleucine metabolism. Neonatal screening by tandem mass spectrometry does not discriminate between these two isomers (Mels et al. 2011). In addition, a false-positive identification of elevated C5-carnitine in newborn screening due to maternal therapy with antibiotics containing pivalic acid has been reported, suggesting the convenience of a second-tier test to avoid a false-positive result (Cloppenborg et al. 2014). A method for detecting 3-methylcrotonyl-coA, the product of the isovaleryl-coA dehydrogenase reaction, has been developed. Determination of isovaleryl-coA dehydrogenase activity by this method may be used as confirmatory test for diagnosis of isovaleric acidemia in cases detected through neonatal screening with tandem mass spectrometry (Tajima et al. 2005). Patients with isovaleric acidemia show elevated baseline urinary excretion of isovaleryl-glycine and glycine administration increases the urinary excretion of this metabolite (Tajima et al. 2005). Isovaleric acidemia is characterized by recurrent episodes of metabolic acidosis and ketoacidosis. Plasma level of isovalerate is elevated and the urinary excretion of isovalerate and 3-hydroxy-isovalerate is increased (Tanaka et al. 1966; Rhead and Tanaka 1980). Hyperglycemia associated with ketoacidosis may occur suggesting a mistaken diagnosis of diabetic ketoacidosis (Erdem et al. 2010).

Table 3 Congenital deficiencies of enzymes involved in branched-chain amino acid metabolism: biochemical phenotype

Table 4 Congenital deficiencies of enzymes involved in branched-chain amino acid metabolism: accumulated intermediates

Table 5 C4 and C5 acyl-carnitine isomers

Treatment of isovaleric acidemia includes a leucine-restricted diet and supplementation with l-carnitine and glycine, which promote the formation of isovaleryl-carnitine and isovaleryl-glycine, respectively, reducing the concentration of isovaleryl-coA (Ensenauer et al. 2004).

Leucine pathway: 3-methylcrotonyl-coA carboxylase

3-Methylcrotonyl-coA carboxylase catalyzes the carboxylation of 3-methylcrotonyl-coA to 3-methylglutaconyl-coA, the fourth step in the leucine catabolic pathway.

3-Methylcrotonyl-coA carboxylase, propionyl-coA carboxylase, pyruvate carboxylase, and acetyl-coA carboxylase are human biotin-dependent carboxylases. They consist of three domains: the biotin carrier domain, the biotin carboxylation domain that catalyzes the carboxylation of biotin, and the carboxyl-transferase domain that catalyzes the transfer of a carboxyl group from carboxy-biotin to the organic substrate specific for each carboxylase. The reaction uses ATP and bicarbonate and is reversible. 3-Methylcrotonyl-coA carboxylase consists of a larger α subunit and a smaller β subunit assembled into a α6β6 dodecamer. The larger α subunit harbors the biotin carrier domain and the biotin carboxylase domain while the smaller β subunit contains the carboxyl-transferase domain (Baumgartner et al. 2001; Grunert et al. 2012a, b). Subjects with biotin deficiency show increased urinary excretion of 3-hydroxy-isovalerate and 3-hydroxy-isovaleryl-carnitine in response to a leucine challenge, suggesting that biotin deficiency inhibits 3-methylcrotonyl-coA carboxylase activity (Mock et al. 2011). In vitro studies suggest that valproyl-coA might inhibit 3-methylcrotonyl-coA carboxylase activity (Luis et al. 2011).

The α and β subunits of 3-methylcrotonyl-coA carboxylase are encoded by separate genes. The MCCA (MCC1) gene is located to 3q25-q27 and encodes the α subunit being while the MCCB (MCC2) gene maps to chromosome 5q12-q13.1 and encodes the β subunit (Baumgartner et al. 2001).

Congenital deficiency of 3-methylcrotonyl-coA carboxylase (3-methylcrotonyl glycinuria)

Mutations in either MCCA gene or MCCB gene cause 3-methylcrotonyl-coA carboxylase deficiency (3-methylcrotonyl glycinuria), a disease first recognized in 1970. 3-Methylcrotonyl glycinuria is a frequent disorder, detected in newborn screening by tandem mass spectrometry with a frequency of approximately 1 in 50,000. In the Faroe Islands, the prevalence is the highest reported worldwide, approximately 1 in 2400, the population being genetically homogeneous with all patients carrying the homozygous mutation c.1526delG in the MCC1 gene (Holzinger et al. 2001).

The clinical onset of 3-methylcrotonyl glycinuria occurs at variable age. Some patients present during the neonatal period or infancy while others are diagnosed in adulthood. Asymptomatic mothers with 3-methylcrotonyl glycinuria have been identified by detection of abnormal neonatal screening performed in their healthy offspring, having passed the abnormal metabolites to their infants through the placenta (Grunert et al. 2012a, b; Forsyth et al. 2016). It has been estimated that 10–15% of affected individuals develop symptoms of the disease. Therefore, 3-methylcrotonyl-coA carboxylase deficiency may be considered a genetic disorder with low penetrance (Grunert et al. 2012a, b; Forsyth et al. 2016). Neurological involvement is prominent in the clinical picture including seizures (atonic seizure, status epilepticus) and other involuntary movements, mental retardation, psychomotor retardation, speech retardation, and muscle symptoms including pain, hypotonia and weakness. Some patients present with acute episodes of vomiting and impaired consciousness (Holzinger et al. 2001; Grunert et al. 2012a, b). Clinical severity of 3-methylcrotonyl-coA carboxylase deficiency is variable, from neonatal death to asymptomatic adults with only biochemical manifestations. It has been estimated that less than 1–2% have a risk for severe adverse outcome (Grunert et al. 2012a, b; Forsyth et al. 2016).

Deficiency of 3-methylcrotonyl-coA carboxylase leads to accumulation of its substrate, 3-methylcrotonyl-coA. The build-up of 3-methylcrotonyl-coA induces the formation of 3-methylcrotonyl-glycine, but there is no formation of 3-methylcrotonyl-carnitine. Instead, 3-hydroxy-isovalerate produces 3-hydroxy-isovaleryl-carnitine (a hydroxy C5-carnitine species) (Roschinger et al. 1995). Patients with 3-methylcrotonyl-coA carboxylase deficiency show elevated blood and urine levels of 3-hydroxy-isovaleryl-carnitine and secondary deficiency of free carnitine. The content of free carnitine in skeletal muscle has been reported low (Roschinger et al. 1995; Grunert et al. 2012a, b; Forsyth et al. 2016). Newborn screening with tandem mass spectrometry in dried blood spots detects elevated concentration of hydroxy C5-carnitine (OH-C5-carnitine) (Grunert et al. 2012a, b). Hydroxy-C5-carnitine species include two isomers, 3-hydroxy-isovaleryl-carnitine and 2-methyl-3-hydroxybutyryl-carnitine. Elevation of 3-hydroxy-isovaleryl-carnitine occurs in patients with 3-methylcrotonyl-coA carboxylase deficiency. Accumulation of 2-methyl-3-hydroxybutyryl-carnitine is associated with deficiency of two enzymes involved in isoleucine catabolism, 17-β-hydroxy-steroid dehydrogenase type 10 and 3-ketothiolase (Forsyth et al. 2016). A second-tier method using UHPLC-MS/MS may be utilized for quantification of acyl-carnitine esters in patients with 3-methylcrotonyl carboxylase deficiency (Minkler et al. 2015). The urinary excretion of 3-methylcrotonyl-glycine is increased in patients with 3-methylcrotonyl-coA carboxylase deficiency (3-methylcrotonyl glycinuria) (Holzinger et al. 2001; Grunert et al. 2012a, b). Patients with 3-methylcrotonyl-coA carboxylase deficiency show increased urinary excretion of 3-hydroxy-isovalerate (Holzinger et al. 2001; Grunert et al. 2012a, b; Forsyth et al. 2016). Some patients with 3-methylcrotonyl-coA carboxylase deficiency develop episodes of metabolic acidosis (ketoacidosis and lactic acidosis), hypoglycemia, and hyperammonemia particularly following infections (Forsyth et al. 2016; Grunert et al. 2012a, b; Holzinger et al. 2001).

Therapeutic approaches for patients with 3-methylcrotonyl-coA carboxylase deficiency include dietary leucine restriction and supplementation with oral l-carnitine, but the efficacy of these approaches remains unproven (Forsyth et al. 2016; Grunert et al. 2012a, b).

Leucine pathway: 3-methylglutaconyl-coA hydratase (AUH)

3-Methylglutaconyl-coA hydratase catalyzes the reversible hydration of 3-methylglutaconyl-coA to 3-hydroxy-3-methylglutaryl-coA, the fifth step in the leucine degradation pathway (Mack et al. 2006). In 1995, it was shown that the RNA-binding protein called AUH had enoyl-coA hydratase activity, catalyzing the hydration of crotonyl-coA (Nakagawa et al. 1995). In 2002, it was noted that AUH had high hydratase activity toward 3-methylglutaconyl-coA (Ijlst et al. 2002). Later investigation on substrate specificity of human AUH confirmed that the best substrates for the enzyme are 3-methylglutaconyl-coA and glutaconyl-coA (Mack et al. 2006). The crystal structure of human AUH at 2.2 A resolution has been determined, revealing that the enzyme adopts the form of a hexamer, as a dimer of trimers (Kurimoto et al. 2001). The AUH gene located on chromosome 9q22.31 encodes 3-methylglutaconyl-coA hydratase (Ijlst et al. 2002).

Congenital deficiency of 3-methylglutaconyl-coA hydratase (3-methyl-glutaconic aciduria type 1)

Mutations in the AUH gene leading to 3-methylglutaconyl-coA hydratase deficiency cause 3-methyl-glutaconic aciduria type 1 (Ijlst et al. 2002). Secondary forms of 3-methyl-glutaconic aciduria have been described in which the activity of 3-methylglutaconyl-coA hydratase is normal and there are no mutations in the AUH gene. Among them are Barth syndrome (3-methyl-glutaconic aciduria type II), Costeff syndrome (3-methyl-glutaconic aciduria type III) and “unspecified” 3-methyl-glutaconic aciduria (type IV). In these forms of secondary 3-methyl-glutaconic aciduria, the increased urinary excretion of 3-methyl-glutaconate and 3-methylglutarate is due to unknown causes (Ijlst et al. 2002; Wortmann et al. 2014). Leucine loading increases the urinary excretion of 3-methyl-glutaconate in patients with AUH deficiency (3-methyl-glutaconic aciduria type 1), unlike patients with secondary forms of 3-methyl-glutaconic aciduria. Leucine loading may be used to discriminate between primary and secondary forms of 3-methyl-glutaconic aciduria (Wortmann et al. 2014). 3-Methyl-glutaconic aciduria type 1 is a rare disease that has been reported in a few patients (Ijlst et al. 2002).

3-Methyl-glutaconic aciduria type 1 usually appears during infancy or childhood. Clinical features are primarily neurological and include leukoencephalopathy, delayed speech development, learning disability, seizures, cerebellar abnormalities, and severe psychomotor retardation with neurological handicap. Clinical severity of 3-methyl-glutaconic aciduria type 1 is variable, from severe neurological involvement to asymptomatic patients detected by neonatal screening (Ijlst et al. 2002).

Deficiency of 3-methylglutaconyl-coA hydratase leads to accumulation of its substrate, 3-methylglutaconyl-coA. No characteristic acyl-carnitine or acyl-glycine profiles have been reported. Patients with 3-methyl-glutaconic aciduria type 1 show elevated urinary excretion of 3-methyl-glutaconate, 3-methylglutarate, and 3-hydroxy-isovalerate (Ijlst et al. 2002; Mack et al. 2006).

Leucine pathway: 3-hydroxy-3-methylglutaryl-coA lyase

3-Hydroxy-3-methylglutaryl-coA lyase catalyzes the irreversible cleavage of 3-hydroxy-3-methylglutaryl-coA to form acetyl-coA + acetoacetate. This reaction represents the sixth and last step in leucine catabolism and accounts for the formation of ketone bodies (acetoacetate and 3-hydroxybutyrate) (Mitchell et al. 1993). The substrate of the enzyme (3-hydroxy-3-methylglutaryl-coA) is produced both in the leucine catabolic pathway and in the ketogenesis pathway (through the condensation of acetyl-coA and acetoacetyl-coA, catalyzed by the enzyme 3-hydroxy-3-methylglutaryl-coA synthase) (Fukao et al. 2014). 3-Hydroxy-3-methylglutaryl-coA lyase cleaves 3-hydroxy-3-methylglutaryl-coA from either pathway into acetyl-coA+ acetoacetate. Acetoacetate may be reduced to 3-hydroxybutyrate (β-hydroxybutyrate) (Fukao et al. 2014). In human liver, 3-hydroxy-3-methylglutaryl-coA lyase exists predominantly in the mitochondrial network, but a small amount (5.6%) is identified in the peroxisomal fraction. The crystal structure at 2.1 A resolution of the recombinant human mitochondrial 3-hydroxy-3-methylglutaryl-coA lyase has been determined. The enzyme action requires the presence of a divalent cation such as Mg²⁺ or Mn²⁺ (Fu et al. 2006). The human 3-hydroxy-3-methylglutaryl-coA lyase locus (HMGCL) maps to 1p36.1 (Aoyama et al. 2015).

Congenital deficiency of 3-hydroxy-3-methylglutaryl-coA lyase (3-hydroxy-3-methylglutaric aciduria)

3-Hydroxy-3-methylglutaric aciduria was first reported in 1976 (Leung et al. 2009). The analysis of 33 members of a four-generation family supports an autosomal recessive mode of inheritance (Barash et al. 1990). Congenital 3-hydroxy-3-methylglutaryl-coA lyase deficiency may be caused by paternal uniparental isodisomy of chromosome 1 (Aoyama et al. 2015).

The disease usually presents during the neonatal period or infancy, but adult presentation has been reported. 3-Hydroxy-3-methylglutaryl-coA lyase deficiency typically produces acute episodes of intractable vomiting and lethargy triggered generally by infections. Neurological manifestations including seizures, macrocephaly, and leukoencephalopathy have been documented (Roe et al. 1986). Dilated cardiomyopathy and left ventricular noncompaction may occur (Leung et al. 2009).

Deficiency of 3-hydroxy-3-methylglutaryl-coA lyase leads to accumulation of its substrate, 3-hydroxy-3-methylglutaryl-coA. There is no formation of the respective carnitine ester (3-hydroxy-3-methylglutaryl-carnitine), but the production of 3-methylglutaryl-carnitine is increased and patients with 3-hydroxy-3-methylglutaryl-coA lyase deficiency show increased urinary excretion of 3-methylglutaryl-carnitine and secondary free carnitine deficiency (Roe et al. 1986). There is elevated urinary excretion of 3-hydroxy-3-methylglutarate, 3-methylglutarate, 3-methyl-glutaconate, and 3-hydroxy-isovalerate in the urinary organic acid analysis (Roe et al. 1986; Gibson et al. 1988; Leung et al. 2009). In patients with 3-hydroxy-3-methylglutaryl-coA lyase deficiency, the generation acetoacetate and acetyl-coA (the products of the reaction) is inhibited and ketogenesis from both fatty acid oxidation and leucine catabolism is impaired (Fukao et al. 2014). Serum 3-hydroxybutyrate is undetectable (Leung et al. 2009). 3-Hydroxy-3-methylglutaryl-coA lyase deficiency may cause recurrent episodes of nonketotic hypoglycemia, metabolic acidosis, and hyperammonemia. Plasma concentration of lactic acid may be elevated during acute illness (Roe et al. 1986; Gibson et al. 1988).

Enzymes involved in isoleucine catabolism

In the isoleucine catabolic pathway, BCAT catalyzes the transamination of isoleucine into 2-keto-3-methylvalerate. The BCKD complex catalyzes the oxidative decarboxylation of 2-keto-3-methylvalerate to 2-methylbutyryl-coA. Short branched-chain acyl-coA dehydrogenase converts 2-methylbutyryl-coA into tiglyl-coA, which is transformed into 2-methyl-3-hydroxybutyryl-coA by an unidentified enzyme. 17-β-Hydroxy-steroid dehydrogenase type 10 catalyzes the conversion of 2-methyl-3-hydroxybutyryl-coA into 2-methyl-acetoacetyl-coA. In the last step, 3-ketothiolase produces propionyl-coA and acetyl-coA from 2-methyl-acetoacetyl-coA (Fig. 5).

Fig. 5

Isoleucine catabolic pathway

Isoleucine pathway: short branched-chain acyl-coA dehydrogenase

Human short branched-chain acyl-coA dehydrogenase is a homotetramer that catalyzes the irreversible conversion of 2-methylbutyryl-coA into tiglyl-coA (C5:1), the third step in the isoleucine degradation pathway (Alfardan et al. 2010). Investigations on the substrate specificity of human short branched-chain acyl-coA dehydrogenase reveal highest activity with the substrates 2-methylbutyryl-coA and butyryl-coA whereas this enzyme shows no activity towards isobutyryl-coA, an intermediate in the valine breakdown pathway (Andresen et al. 2000). In vitro studies show that human short branched-chain acyl-coA dehydrogenase might use valproyl-coA as a substrate (Luis et al. 2011). The ACADSB gene, located to chromosome 10q25-q26, encodes human short branched-chain acyl-coA dehydrogenase (Andresen et al. 2000).

Congenital deficiency of short branched-chain acyl-coA dehydrogenase (2-methylbutyryl-glycinuria)

Congenital deficiency of short branched-chain acyl-coA dehydrogenase was first reported in 2000. The prevalence of neonatal screening positive for 2-methylbutyryl-glycinuria is approximately 1 in 540,780 births while the prevalence of the disease among the Hmong ethnic group is 1 in 131 births. The higher prevalence in the Hmong population is due to a specific mutation possibly explained by a founder effect in this ethnic group (Van Calcar et al. 2013).

The age of presentation is variable, ranging from the neonatal period to adulthood. Some affected individuals remain asymptomatic. Clinical manifestations are predominantly neurological and include mental retardation, psychomotor delay, seizures, hypotonia, and muscular atrophy (Andresen et al. 2000). Autism has been reported (Kanavin et al. 2007). Long-term outcome of the disease is poorly defined (Alfardan et al. 2010). Short branched-chain acyl-coA dehydrogenase deficiency may sometimes represent a harmless metabolic variant, as some affected individuals detected during routine newborn screening remain free of clinical symptoms (Sass et al. 2008).

Short branched-chain acyl-coA dehydrogenase deficiency leads to accumulation of its substrate, 2-methylbutyryl-coA (Van Calcar et al. 2013). Affected patients show elevated concentration of C5-carnitine in blood spots analyzed by tandem mass spectrometry. C5-carnitine may represent two isomers, 2-methylbutyryl-carnitine and isovaleryl-carnitine. Accumulation of C5-carnitine may be due to deficiency of short branched-chain acyl-coA dehydrogenase (elevated 2-methylbutyryl-carnitine) or isovaleric acidemia (elevated isovaleryl-carnitine), a defect in leucine catabolism. Newborn screening by tandem mass spectrometry in blood spots does not distinguish between them (Van Calcar et al. 2013). 2-Methylbutyryl-carnitine concentration in blood and urine is increased among patients with short branched-chain acyl-coA dehydrogenase deficiency. The urinary excretion of 2-methylbutyryl-glycine is also increased (2-methylbutyryl-glycinuria) in these patients (Andresen et al. 2000; Kanavin et al. 2007; Alfardan et al. 2010; Van Calcar et al. 2013).

The need for and type of therapy in patients with short branched-chain acyl-coA dehydrogenase deficiency remains unclear, as the disorder may be benign (Sass et al. 2008; Alfardan et al. 2010). No beneficial effect has been detected after 5 months with a low-protein diet (Kanavin et al. 2007).

Isoleucine pathway: unidentified enzyme

The human enzyme responsible for the hydration of tiglyl-coA into 2-methyl-3-hydroxybutyryl-coA, the fourth step in the isoleucine degradation pathway, has not been identified and no deficient state has been reported so far. It was thought that short-chain enoyl-coA hydratase (crotonase) might catalyze this enzymatic step, but it has been shown that human short-chain enoyl-coA hydratase (crotonase) has a very limited activity on tiglyl-coA. By contrast, this enzyme catalyzes the hydration of methyl-acrylyl-coA to produce 3-hydroxy-isobutyryl-coA in the valine catabolic pathway. Metabolic consequences of short-chain enoyl hydratase (crotonase) deficiency are restricted to valine catabolism and to some extent to short-chain fatty acid oxidation whereas isoleucine catabolism remains intact (Peters et al. 2014; Ferdinandusse et al. 2015; Haack et al. 2015; Yamada et al. 2015).

Isoleucine pathway: 17-β-hydroxy-steroid dehydrogenase type 10 (2-methyl-3-hydroxybutyryl-coA dehydrogenase)

17-β-Hydroxy-steroid dehydrogenase type 10 catalyzes the reversible oxidation of 2-methyl-3-hydroxybutyryl-coA into 2-methyl-acetoacetyl-coA, the fifth step in the isoleucine degradation pathway. This enzyme is a NAD⁺-dependent multifunctional homotetramer active with diverse substrates such as steroids, fatty acids, bile acids, and xenobiotics, being involved in isoleucine catabolism and steroid metabolism, among other functions (Yang et al. 2007). In 2003, the human gene coding 17-β-hydroxy-steroid dehydrogenase type 10 was located to Xp11.2 and named HADH2 (Ofman et al. 2003). In 2007, HADH2 gene was re-named HSD17B10 (Korman and Yang 2007).

Congenital deficiency of 2-methyl-3-hydroxybutyryl-coA dehydrogenase (2-methyl-3-hydroxy-butyric aciduria)

In 2003, mutations in the HSD17B10 gene were identified as the cause of congenital deficiency of 17-β-hydroxy-steroid dehydrogenase type 10 (2-methyl-3-hydroxy-butyric aciduria), a rare disorder identified in a few families and inherited as a X-chromosomal trait (Ofman et al. 2003). Deficiency of 17-β-hydroxy-steroid dehydrogenase type 10 usually presents during infancy or childhood. Patients with this disorder suffer progressive neurodegenerative disease, although atypical forms without neurological regression have been recognized (Akagawa et al. 2016). Clinical features include mental retardation, loss of motor skills, retinal degeneration, ataxia, spastic diplegia, seizures, and abnormal movements such as choreoathetosis (Zschocke et al. 2000; Ensenauer et al. 2002; Seaver et al. 2011). Hypertrophic cardiomyopathy has been documented (Ensenauer et al. 2002). Heterozygous females usually show milder clinical phenotype with non-progressive developmental delay and intellectual disability but they may be clinically normal (Zschocke et al. 2000). Brain MRI may show brain atrophy, occipital periventricular white matter lesions, and alterations of the basal ganglia, but could be normal (Seaver et al. 2011).

Deficiency of 17-β-hydroxy-steroid dehydrogenase type 10 leads to accumulation of its substrate, 2-methyl-3-hydroxybutyryl-coA. Newborn screening with tandem mass spectrometry in dried blood spots detects elevated concentration of hydroxy C5-carnitine. Hydroxy-C5-carnitine species include two isomers, 2-methyl-3-hydroxybutyryl-carnitine and 3-hydroxy-isovaleryl-carnitine. Elevation of 3-hydroxy-isovaleryl-carnitine occurs in patients with 3-methylcrotonyl-coA carboxylase deficiency, a disorder of the leucine pathway. Accumulation of 2-methyl-3-hydroxybutyryl-carnitine is associated with deficiency of two enzymes involved in isoleucine catabolism, 17-β-hydroxy-steroid dehydrogenase type 10 and 3-ketothiolase (Forsyth et al. 2016). In patients with 17-β-hydroxy-steroid dehydrogenase type 10 deficiency, the increase in plasma level of 2-methyl-3-hydroxybutyryl-carnitine may be intermittent or absent (Akagawa et al. 2016). The plasma level of tiglyl-carnitine (C5:1-carnitine) and the urinary excretion of tiglyl-glycine may be increased both in 17-β-hydroxy-steroid dehydrogenase type 10 deficiency and in 3-ketothiolase deficiency. In patients with 17-β-hydroxy-steroid dehydrogenase type 10 deficiency, the urinary excretion of 2-methyl-3-hydroxybutyrate is increased while the urinary excretion of 2-methyl-acetoacetate is normal (Zschocke et al. 2000; Ensenauer et al. 2002; Seaver et al. 2011; Akagawa et al. 2016). In patients with deficiency of 17-β-hydroxy-steroid dehydrogenase type 10, an oral isoleucine load results in markedly elevated excretion of 2-methyl-3-hydroxybutyrate and tiglyl-glycine, and a sustained elevation of plasma isoleucine level compared to controls (Zschocke et al. 2000). Patients with 17-β-hydroxy-steroid dehydrogenase type 10 deficiency show lactic acidosis, hyperammonemia, and hypoglycemia (Zschocke et al. 2000; Seaver et al. 2011; Akagawa et al. 2016).

There is no effective treatment for 17-β-hydroxy-steroid dehydrogenase type 10 deficiency.

An isoleucine-restricted diet has been implemented with no long-term improvement in neurological outcome (Zschocke et al. 2000).

Isoleucine pathway: 3-ketothiolase (β-ketothiolase, β-oxo-thiolase, 3-oxo-thiolase, mitochondrial acetoacetyl-coA thiolase, acetyl-coA acetyltransferase-1, T2)

3-Ketothiolase is involved both in the isoleucine degradation pathway and in ketolysis (ketone body utilization). In isoleucine catabolism, 3-ketothiolase catalyzes the thiolysis of 2-methyl-acetoacetyl-coA into propionyl-coA and acetyl-coA. In the metabolism of ketone bodies, 3-ketothiolase catalyzes the thiolytic cleavage of acetoacetyl-coA to yield two molecules of acetyl-coA. This is the last step of ketolysis that allows the utilization of ketone bodies in extrahepatic tissues (Robinson et al. 1979; Fukao et al. 2010a, b). The ACAT1 gene located on 11q22.3-q23.1 encodes 3-ketothiolase (Tilbrook et al. 2008).

Congenital deficiency of 3-ketothiolase

Mutations in the ACAT1 gene cause 3-ketothiolase deficiency, a disorder first reported in 1971 (Tilbrook et al. 2008). Clinical presentation of 3-ketothiolase deficiency tends to occur during infancy or childhood, the median age at onset being 15 months (Fukao et al. 2001, 2010a, b; Tilbrook et al. 2008). Intermittent episodes of vomiting and lethargy that may progress to coma generally precipitated by infections are the typical clinical picture of 3-ketothiolase deficiency. Patients are usually asymptomatic during episodes. Other clinical features include cardiomyopathy and prolonged QT interval. Long-term outcome is poorly documented. Fatalities have been reported (Fukao et al. 2001, 2010a, b; Tilbrook et al. 2008).

3-Ketothiolase deficiency leads to accumulation of its two substrates, 2-methyl-acetoacetyl-coA (isoleucine catabolic pathway) and acetoacetyl-coA (ketolysis). In 3-ketothiolase deficiency, acyl-carnitine analysis from dried blood spots shows elevated tiglyl-carnitine and a hydroxy-C5-carnitine species representing 2-methyl-3-hydroxybutyryl-carnitine (Fukao et al. 2010a, b; Catanzano et al. 2010). The urinary excretion of tiglyl-glycine, 2-methyl-acetoacetate, and 2-methyl-3-hydroxybutyrate is usually increased and the amount of these metabolites increases after a dietary load of isoleucine (Daum et al. 1973). However, the acyl-carnitine profile in blood spots and the urinary excretion of tiglyl-glycine, 2-methyl-acetoacetate, and 2-methyl-3-hydroxybutyrate may be normal in patients with 3-ketothiolase deficiency, even during an acute crisis (Fukao et al. 2010a, b; Catanzano et al. 2010). The absence of 2-methyl-acetoacetate in urine may be attributed to the instability of this ketoacid, as it undergoes spontaneous decarboxylation to 2-butanone (Catanzano et al. 2010). The urinary excretion of butanone is elevated in patients with 3-ketothiolase deficiency (Catanzano et al. 2010; Daum et al. 1973). 3-Ketothiolase deficiency causes recurrent episodes of ketoacidosis generally triggered by infections. During the episodes, the plasma level and urinary excretion of ketone bodies (acetoacetate and 3-hydroxybutyrate) is elevated because they cannot be utilized (Fukao et al. 2010a, b). The high plasma level of acetoacetate may produce a false-positive reaction for salicylate suggesting an erroneous diagnosis of salicylate intoxication in patients with 3-ketothiolase deficiency (Robinson et al. 1979; Tilbrook et al. 2008).

Biochemical consequences of 3-ketothiolase deficiency and 17-β-hydroxy-steroid dehydrogenase type 10 deficiency have commonalities. Both of them share increased blood tiglyl-carnitine level and increased urinary excretion of tiglyl-glycine and 2-methyl-3-hydroxybutyrate. However, the urinary excretion of 2-methyl-acetoacetate is elevated in 3-ketothiolase deficiency while this metabolite is absent in the urine of patients with 17-β-hydroxy-steroid dehydrogenase type 10 deficiency (Zschocke et al. 2000). A UHPLC-MS/MS second-tier assay able to separate carnitine isomers has been described and can be used in patients with 3-ketothiolase deficiency (Minkler et al. 2015).

The management of an acute crisis in 3-ketothiolase deficiency is supportive with infusion of glucose to ameliorate the catabolic state. Long-term management consists of avoidance of fasting and protein restriction.

Enzymes involved in valine catabolism

In valine catabolism, BCAT catalyzes the reversible transamination between valine and 2-keto-isovalerate. The BCKD complex catalyzes the irreversible oxidative decarboxylation of 2-keto-isovalerate into isobutyryl-coA. Isobutyryl-coA dehydrogenase converts isobutyryl-coA in methyl-acrylyl-coA. Short-chain enoyl-coA hydratase generates 3-hydroxy-isobutyryl-coA from methyl-acrylyl-coA. 3-Hydroxy-isobutyryl-coA hydrolase converts 3-hydroxy-isobutyryl-coA into 3-hydroxy-isobutyrate, which is transformed into methylmalonate semialdehyde by an unknown enzyme. Methylmalonate semialdehyde dehydrogenase generates propionyl-coA from methylmalonate semialdehyde (Fig. 6).

Fig. 6

Valine catabolic pathway

Valine pathway: isobutyryl-coA dehydrogenase (ACAD8)

Isobutyryl-coA dehydrogenase catalyzes the conversion of isobutyryl-coA into methyl-acrylyl-coA, the third step in valine catabolism (Roe et al. 1998a, b). Isobutyryl-coA dehydrogenase is a tetramer with very high activity toward isobutyryl-coA while no activity is detectable when butyryl-coA, valeryl-coA or isovaleryl-coA is used as substrates (Nguyen et al. 2002). The crystal structure of human isobutyryl-coA dehydrogenase has been determined (Battaile et al. 2004). Acetyl-salicylic acid might inhibit the activity of this enzyme, as the administration of acetyl-salicylic acid to healthy humans increases the urinary excretion of isobutyryl-carnitine. However, only in some of the subjects the urinary excretion of isobutyryl-carnitine reaches the level observed in patients with isobutyryl-coA dehydrogenase deficiency (Mels et al. 2011). In vitro studies suggest that isobutyryl-coA dehydrogenase activity is not affected by valproyl-coA (Luis et al. 2011). The human gene coding isobutyryl-coA dehydrogenase (ACD8) maps to chromosome 11q25 (Nguyen et al. 2002).

Congenital deficiency of isobutyryl-coA dehydrogenase

Isobutyryl-coA dehydrogenase deficiency is a rare disorder detected in few individuals (Yoo et al. 2007). The disease was first reported in 1998 in a female who presented at 12 months of age with dilated cardiomyopathy (Roe et al. 1998a, b). Subsequent genetic investigation identified mutations in the gene encoding isobutyryl-coA dehydrogenase as the cause of the disease (Nguyen et al. 2002). The clinical phenotype of patients with isobutyryl-coA deficiency is not well defined. In a follow-up evaluation of patients identified through newborn screening, the majority of them have normal growth and development (Pena et al. 2012).

Isobutyryl-coA dehydrogenase deficiency leads to accumulation of its substrate, isobutyryl-coA, which promotes the formation of isobutyryl-carnitine C4-carnitine specie) and isobutyryl-glycine. This disorder is usually diagnosed on newborn screening with tandem mass spectrometry by an elevation of C4-carnitine concentration in dried blood spots (Yoo et al. 2007; Yun et al. 2015). C4-carnitine species include two isomers, butyryl-carnitine and isobutyryl-carnitine. Butyryl-coA is generated during the β-oxidation of fatty acids and then oxidized into crotonyl-coA (C4:1-coA) by short-chain acyl-coA dehydrogenase. Deficiency of this enzyme leads to accumulation of butyryl-coA and formation of butyryl-carnitine and butyryl-glycine. By contrast, isobutyryl-coA accumulates in patients with isobutyryl-coA dehydrogenase deficiency, promoting the formation of isobutyryl-carnitine, which increases in blood and urine (Roe et al. 1998a, b; Yoo et al. 2007). Newborn screening with tandem mass spectrometry does not discriminate between these two C4-carnitine species (butyryl-carnitine and isobutyryl-carnitine) and, therefore, cannot differentiate patients with isobutyryl-coA dehydrogenase deficiency and short-chain acyl-coA dehydrogenase deficiency (Yun et al. 2015). A second-tier method using UHPLC–MS/MS may separate and quantify acyl-carnitine isomers in patients with these disorders (Minkler et al. 2015). The urinary concentration of isobutyryl-glycine is elevated in patients with isobutyryl-coA dehydrogenase deficiency while the urinary excretion of butyryl-glycine is normal (Yoo et al. 2007; Yun et al. 2015). The urinary excretion of ethylmalonate is normal in patients with isobutyryl-coA dehydrogenase deficiency while is increased in patients with short-chain acyl-coA dehydrogenase deficiency (Oglesbee et al. 2007).

Valine pathway: short-chain enoyl-coA hydratase (crotonase)

In the human valine catabolic pathway, short-chain enoyl-coA hydratase catalyzes the hydration of methyl-acrylyl-coA to produce 3-hydroxy-isobutyryl-coA by adding a water molecule to the double bond of methyl-acrylyl-coA (Peters et al. 2014). The substrate specificity of the human enzyme for crotonyl-coA, 3-methylcrotonyl-coA, acrylyl-coA, methyl-acrylyl-coA, and tiglyl-coA has been determined. Human short-chain enoyl-coA hydratase shows the highest specificity for crotonyl-coA and the lowest specificity for tiglyl-coA. The enzyme binds to tiglyl-coA but hydrates only a small amount of this substrate. Human short-chain enoyl-coA hydratase has moderate specificity for methyl-acrylyl-coA, acrylyl-coA, and 3-methylcrotonyl-coA (Yamada et al. 2015). Although these studies suggest that human short-chain enoyl-coA hydratase might act on several substrates and metabolic pathways, the biochemical consequences of its deficiency appear to be restricted to the valine catabolic pathway. Isoleucine and leucine catabolism are unaffected by deficiency of short-chain enoyl-coA hydratase and the role of this enzyme in the β-oxidation of short-chain fatty acids seems to be limited. Results of studies investigating the contribution of short-chain enoyl-coA hydratase to short-chain fatty acid β-oxidation are inconclusive. Some patients with short-chain enoyl-coA hydratase deficiency show elevated blood butyryl-carnitine level (Ganetzky et al. 2016) and increased urinary excretion of ethylmalonate that may reflect a disturbance in the β-oxidation of short-chain fatty acids, as both alterations are typically observed in deficiency of short-chain acyl-coA dehydrogenase (Peters et al. 2014; Haack et al. 2015; Ganetzky et al. 2016). However, there is no accumulation of crotonyl-carnitine and crotonyl-glycine in patients with short-chain enoyl-coA hydratase deficiency, suggesting that short-chain fatty acid catabolism is unaltered (Peters et al. 2014). In vitro palmitate loading on fibroblasts that may expose a defect in β-oxidation of fatty acids has yielded conflicting results. Some patients with short-chain enoyl-coA hydratase deficiency show normal responses to palmitate loading test in fibroblasts, suggesting that fatty acid β-oxidation is unaffected (Ferdinandusse et al. 2015). However, other patients experience a mild increase in butyryl-carnitine level after palmitate loading, uncovering a defect in mitochondrial β-oxidation of short-chain fatty acids (Haack et al. 2015). The human ECHS1 gene, assigned to chromosome 10q26.2-q26.3, encodes short-chain enoyl-coA hydratase (Janssen et al. 1997).

Congenital deficiency of short-chain enoyl-coA hydratase

Short-chain enoyl-coA hydratase deficiency is a rare congenital disorder that usually presents during the neonatal period or infancy. Clinical manifestations are predominantly neurological, including hypotonia, epilepsy, developmental delay, deafness, and optic atrophy. Cardiomyopathy and dysmorphic features have been documented. Skeletal muscle biopsy shows no abnormality (Peters et al. 2014; Haack et al. 2015; Ganetzky et al. 2016). The clinical course is generally severe with death during the first year of life, but some patients survive into adulthood (Haack et al. 2015). Brain MRI may show generalized atrophy, bilateral reduced myelination of the basal ganglia (bilateral T2 hyperintensities in the basal ganglia), and vacuolating leukoencephalopathy (Haack et al. 2015; Ferdinandusse et al. 2015; Peters et al. 2014).

Short-chain enoyl-coA hydratase deficiency causes accumulation of its substrate in the valine catabolism, methyl-acrylyl-coA (Peters et al. 2014; Yamada et al. 2015). Cysteine and cysteamine conjugates of methyl-acrylyl-coA such as S-(2-carboxypropyl)-cysteine and S-(2-carboxypropyl)-cysteamine are elevated in urine screening performed by tandem mass spectrometry (Loupatty et al. 2007; Peters et al. 2014; Yamada et al. 2015). The urinary excretion of 2-methyl-2,3-dihydroxybutyrate is also increased, likely reflecting accumulation and subsequent metabolism of methyl-acrylyl-coA, as 2-methyl-2,3-dihydroxybutyrate is a potential derivative of acrylyl-coA, although the enzymatic steps leading to its formation are unclear (Peters et al. 2014; Haack et al. 2015; Yamada et al. 2015). Organic acid analysis in urine reveals slightly elevated concentration of ethylmalonate (Haack et al. 2015). Some patients with short-chain enoyl-coA hydratase deficiency show elevated blood level of butyryl-carnitine, but there is no build-up of crotonyl-carnitine and crotonyl-glycine (Peters et al. 2014; Haack et al. 2015; Ganetzky et al. 2016). Tiglyl-glycine is not formed in patients with deficiency of short-chain enoyl-coA hydratase confirming that the enzyme has very limited activity toward tiglyl-coA (Yamada et al. 2015). The activity of the PDH complex is reduced in skeletal muscle from patients with short-chain enoyl-coA hydratase deficiency and consequently serum lactate level is elevated. The underlying mechanism of the reduction in PDH activity is unknown, but it may contribute to the clinical presentation of short-chain enoyl-coA hydratase deficiency (Peters et al. 2014; Ferdinandusse et al. 2015; Haack et al. 2015; Ganetzky et al. 2016).

Valine pathway: 3-hydroxy-isobutyryl-coA hydrolase

3-Hydroxy-isobutyryl-coA hydrolase catalyzes the conversion of 3-hydroxy-isobutyryl-coA to 3-hydroxy-isobutyrate, the fifth step of valine catabolism (Ferdinandusse et al. 2013). Some single nucleotide polymorphisms in the 3-hydroxy-isobutyryl-coA hydrolase gene have been associated with increased expression of 3-hydroxy-isobutyryl-coA hydrolase mRNA and elevated plasma methylmalonate level (Molloy et al. 2016).

Congenital deficiency of 3-hydroxy-isobutyryl-coA hydrolase

3-Hydroxy-isobutyryl-coA hydrolase deficiency is a rare inborn error of valine catabolism, first described in 1982, reported in a few patients (Loupatty et al. 2007). Clinical onset of 3-hydroxy-isobutyryl-coA hydrolase deficiency occurs usually during infancy, but adult presentation has been documented (Schottmann et al. 2016). 3-Hydroxy-isobutyryl-coA hydrolase deficiency is generally a severe disease, with progressive neurological deterioration in infancy and high mortality rate, although a milder course may occur when the disease appears later in life (Schottmann et al. 2016). The clinical picture of 3-hydroxy-isobutyryl-coA hydrolase deficiency is predominantly neurological, with hypotonia, delay in motor milestones, ataxia, dystonia, seizures, and neurological regression with progressive deterioration in neurological function (Loupatty et al. 2007; Ferdinandusse et al. 2013). Other clinical manifestations include dysmorphic facial features and episodes of ketoacidosis (Loupatty et al. 2007). In one patient, post-mortem examination has revealed tetralogy of Fallot, vertebral anomalies and agenesis of the cingulate gyrus and corpus callosum (Loupatty et al. 2007). In patients with 3-hydroxy-isobutyryl-coA hydrolase deficiency, brain MRI usually shows progressive white matter atrophy and bilateral symmetrical hyperintensities of the basal ganglia (Loupatty et al. 2007; Ferdinandusse et al. 2013).

Deficiency of 3-hydroxy-isobutyryl-coA hydrolase causes accumulation of its substrate, 3-hydroxy-isobutyryl-coA and upstream accumulation of methyl-acrylyl-coA, suggesting that the action of short-chain enoyl-coA hydratase is reversible. Patients with 3-hydroxy-isobutyryl-coA hydrolase deficiency show increased level of hydroxy-C4-carnitine (OH-C4-carnitine) in blood spots, although the elevation may be intermittent (Ferdinandusse et al. 2013; Loupatty et al. 2007; Stiles et al. 2015). Hydroxy-C4-carnitine may represent two isomers, 3-hydroxybutyryl-carnitine and 3-hydroxy-isobutyryl-carnitine. Elevation of 3-hydroxybutyryl-carnitine occurs in patients with disorders of fatty acid oxidation (medium- and short-chain 3-hydroxy-acyl-coA dehydrogenase deficiency) (Stiles et al. 2015). Elevation of 3-hydroxy-isobutyryl-carnitine in blood and urine occurs in patients with 3-hydroxy-isobutyryl-coA hydrolase deficiency (Stiles et al. 2015; Peters et al. 2015).

Patients with 3-hydroxy-isobutyryl-coA hydrolase deficiency show upstream accumulation of methyl-acrylyl-coA. Increased urinary excretion of cysteine and cysteamine conjugates of methyl-acrylyl-coA, such as S-2-carboxypropyl-cysteamine and S-2-carboxypropyl-cysteine, may be detected by tandem mass spectrometry. These conjugates accumulate in multiple tissues, particularly liver, kidney, and brain (Loupatty et al. 2007; Ferdinandusse et al. 2013; Stiles et al. 2015). Elevated urinary excretion of 2-methyl-2,3-dihydroxybutyrate is detected by gas chromatography–mass spectrometry, likely reflecting accumulation and subsequent metabolism of methyl-acrylyl-coA (Stiles et al. 2015).

Patients with 3-hydroxy-isobutyryl-coA hydrolase deficiency show reduction in PDH activity and respiratory chain dysfunction and elevated blood lactate. The pathogenic mechanism underlying these alterations remains to be elucidated (Ferdinandusse et al. 2013; Schottmann et al. 2016).

There is considerable overlap in the biochemical features of short-chain enoyl-coA hydratase and 3-hydroxy-isobutyryl-coA hydrolase deficiencies. Both diseases show reduction in the activity of PDH complex and accumulation of methyl-acrylyl-coA and its conjugates. However, the urinary excretion of 3-hydroxy-isobutyryl-carnitine is elevated in 3-hydroxy-isobutyryl-coA hydrolase-deficient patients unlike patients with short-chain enoyl-coA hydratase deficiency (Stiles et al. 2015).

Valine pathway: 3-hydroxy-isobutyrate dehydrogenase

The enzyme that catalyzes the conversion of 3-hydroxy-isobutyrate into methylmalonate semialdehyde, the sixth step in valine catabolism has not been identified. The HIBADH gene located on chromosome 7p15.2 encodes human 3-hydroxy-isobutyrate dehydrogenase, but the role of this enzyme in valine catabolism is unclear, as no pathogenic mutations in the HIBADH gene have been reported (Loupatty et al. 2006).

3-Hydroxy-isobutyric aciduria

Accumulation of 3-hydroxybutyrate with increased urinary excretion of this metabolite has been named 3-hydroxy-isobutyric aciduria. In patients with this condition, the activity of 3-hydroxy-isobutyrate dehydrogenase in fibroblasts is normal and no mutations in the HIBADH gene have been found. By contrast, mutations in the ALDH6A1 gene that encodes methylmalonate semialdehyde dehydrogenase have been identified in patients with 3-hydroxy-isobutyric aciduria (Loupatty et al. 2006; Sass et al. 2012).

3-Hydroxy-isobutyric aciduria is a rare disorder described in very few patients. It usually manifests in the neonatal period and its mortality rate is high. Clinical manifestations include microcephaly, dysmorphic features, delayed motor development, mental retardation, seizures, ventricular hypertrophy, and episodes of vomiting and lethargy (Ko et al. 1991; Loupatty et al. 2006; Sass et al. 2012). Skeletal muscle biopsy reveals increased amounts of glycogen and mitochondrial proliferation and ragged red fibers suggestive of a respiratory chain defect (Ko et al. 1991; Loupatty et al. 2006; Sass et al. 2012).

In patients with 3-hydroxy-isobutyric aciduria, there is elevated urinary excretion of 3-hydroxy-isobutyrate and the excretion of this metabolite in urine increases after the administration of valine (Ko et al. 1991; Loupatty et al. 2006; Sass et al. 2012). Plasma alanine and lactate levels are elevated, suggesting PDH dysfunction (Ko et al. 1991). Methyl-acrylyl-coA conjugates such as S-(2-carboxylpropyl)cysteine and S-(2-carboxypropyl)cysteamine are not found (Ko et al. 1991).

Valine pathway: methylmalonate semialdehyde dehydrogenase

Methylmalonate semialdehyde dehydrogenase catalyzes the oxidative decarboxylation of methylmalonate semialdehyde into propionyl-coA, being involved in the catabolic breakdown of both valine and thymine. In the valine catabolic pathway, methylmalonate semialdehyde dehydrogenase catalyzes the oxidative decarboxylation of (S)-methylmalonate semialdehyde into propionyl-coA, the seventh step of valine catabolism. Thymine metabolism generates (R)-aminoisobutyric acid, which is deaminated to (R)-methylmalonate semialdehyde. This enantiomer of methylmalonate semialdehyde is also a substrate for methylmalonate semialdehyde dehydrogenase that catalyzes its oxidative decarboxylation to propionyl-coA (Marcadier et al. 2013). The human gene ALDH6A1 encodes methylmalonate semialdehyde dehydrogenase (Chambliss et al. 2000). Disease-causing mutations in the ALDH6A1 gene have been identified in patients with 3-hydroxy-isobutyric aciduria and in patients with methylmalonate semialdehyde dehydrogenase deficiency. Therefore, 3-hydroxy-isobutyric aciduria and methylmalonate semialdehyde dehydrogenase deficiency are likely the same disease, due to deficiency of methylmalonate semialdehyde dehydrogenase associated with mutations in the ALDH6A1 gene.

Congenital deficiency of methylmalonate semialdehyde dehydrogenase

Very few patients with methylmalonate semialdehyde dehydrogenase deficiency have been identified. The clinical picture of methylmalonate semialdehyde dehydrogenase deficiency is characterized by developmental delay and dysmorphic features without metabolic acidosis (Roe et al. 1998a, b; Sass et al. 2012; Marcadier et al. 2013). Patients with methylmalonate semialdehyde dehydrogenase deficiency show increased urinary concentration of 3-hydroxy-isobutyrate with normal activity of 3-hydroxy-isobutyrate dehydrogenase in fibroblasts (Marcadier et al. 2013; Sass et al. 2012). The urinary excretion of methylmalonate is increased (Roe et al. 1998a, b; Marcadier et al. 2013). Plasma lactate is elevated (Marcadier et al. 2013).

Summary

Branched-chain aminotransferase catalyzes the first step in the catabolic pathway of leucine, isoleucine and valine, the reversible conversion of branched-chain amino acids to branched-chain ketoacids. At the same time, glutamate is converted into 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the second step, the irreversible oxidative decarboxylation of the branched-chain ketoacids, producing isovaleryl-coA, 2-methylbutyryl-coA, and isobutyryl-coA from leucine, isoleucine and valine, respectively. Next steps are different for each one of the branched-chain amino acids. In the leucine catabolic pathway, isovaleryl-coA sequentially produces 3-methylcrotonyl-coA, 3-methylglutaconyl-coA, 3-hydroxy-3-methylglutaryl-coA and ultimately acetyl-coA and acetoacetate. In the isoleucine degradation pathway, 2-methylbutyryl-coA is sequentially converted into tiglyl-coA, 2-methyl-3-hydroxybutyryl-coA, 2-methyl-acetoacetyl-coA and finally propionyl-coA and acetyl-coA. In valine catabolism, isobutyryl-coA is successively converted in methyl-acrylyl-coA, 3-hydroxy-isobutyryl-coA, 3-hydroxy-isobutyrate, methylmalonate semialdehyde, and propionyl-coA. Congenital deficiency of the enzymes involved in branched-chain amino acid metabolism causes clinical phenotypes with predominant neurological manifestations, such as mental retardation and seizures, suggesting that intermediates in the catabolic pathways have significant roles in the function of central nervous system. Acute metabolic crises with vomiting and lethargy induced by infections also occur in most enzyme deficiencies and are typically present in congenital deficiency of 3-ketothiolase. Plasma level of branched-chain amino acids is elevated in obesity and diabetes mellitus, although the clinical repercussions are uncertain.