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Home , Hyperlysinemia, Pyruvate carboxylase

Molecular Genetics and 101 (2010) 9–17

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Molecular Genetics and Metabolism

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Minireview Pyruvate carboxylase deficiency: Mechanisms, mimics and anaplerosis

Isaac Marin-Valencia a, Charles R. Roe a, Juan M. Pascual a,b,* a Department of Neurology, UT Southwestern Medical Center, USA b Departments of Physiology and Pediatrics and Division of Pediatric Neurology, UT Southwestern Medical Center, USA article info abstract

Article history: Pyruvate carboxylase (PC) is a regulated mitochondrial that catalyzes the conversion of pyruvate Received 26 May 2010 to oxaloacetate, a critical transition that replenishes intermediates and facilitates other Accepted 26 May 2010 biosynthetic reactions that drive . Its deficiency causes multiorgan metabolic imbalance that Available online 9 June 2010 predominantly manifests with lactic acidemia and neurological dysfunction at an early age. Three clinical forms of PC deficiency have been identified: an infantile form (Type A), a severe neonatal form (Type B), Keywords: and a benign form (Type C), all of which exhibit clinical or biochemical correlates of impaired anaplerosis. Pyruvate carboxylase There is no effective treatment for these patients and most, except those affected by the benign form, die in early life. We review the physiology of this enzyme and dissect the major clinical, biochemical, and Anaplerosis Citric acid cycle genetic aspects of its dysfunction, emphasizing features that distinguish PC deficiency from other causes of lactic acidemia that render PC deficiency potentially treatable using novel interventions capable of enhancing anaplerosis. Ó 2010 Elsevier Inc. All rights reserved.

Introduction or infantile period. Several dozen patients afflicted by the disorder have been described in detail, allowing for the formulation of over- Pyruvate carboxylase (PC; EC 6.4.1.1) is a -containing nu- lapping disease-associated clinical and biochemical phenotypes. PC clear genome-encoded mitochondrial enzyme discovered in 1959 deficiency has been reported more often in particular ethnic by Utter and Keech [1]. The enzymatic activity facilitates flux groups such as Algonquian-speaking Amerindians and Arabs, but through a key intermediary metabolism reaction: PC is responsible in most populations the general incidence of PC deficiency is rela- for the ATP-dependent of pyruvate, yielding oxaloac- tively low (1:250,000) [2]. However, it is plausible that ascertain- etate. This reaction constitutes the best recognized interconversion ment biases limit the recognition of hypomorphic or altogether required for the replenishment of pools of intermediates of the cit- divergent clinical syndromes. In practice, biochemical abnormali- ric acid cycle (CAC), a process named anaplerosis that restores ties assist in the differential diagnosis of PC deficiency, while enzy- losses that CAC derivative products are subject to during normal matic assay is still often required to establish the definitive metabolism. By extension, PC also participates in numerous meta- diagnosis. Current symptomatic or supportive treatments prove bolic pathways that depend on the availability of oxaloacetate such largely ineffective. Therefore, understanding alternative metabolic as , glycogen synthesis, , glycerogene- pathways that can be enhanced and associated metabolic dysregu- sis, the synthesis of amino acids and , and glu- lation potentially amenable to compensatory intervention in light cose-dependent secretion. Because PC is essential for of recent biochemical information is a prerequisite for the develop- these interrelated aspects of anabolism, inherited deficiency of this ment of new therapies. enzyme can a priori be expected to cause metabolic disturbances in numerous tissues, with a predilection for organs whose metabo- PC as an anabolic carbon flux regulator lism depends upon high CAC flux such as liver and brain. PC defi- ciency (OMIM 266150) is an uncommon autosomal recessive PC is localized within the of the cells of disorder that predominantly causes lactic acidemia (serum lactic many organs and tissues. Expression levels are highest in liver, adi- acid >5 mmol/L and bicarbonate <18 mmol/L) and neurological pose tissue, kidney, lactating mammary gland, and pancreatic dysfunction – encephalopathy – first manifested in the neonatal islets; modest in heart, brain, and adrenal gland; lowest in white blood cells and skin fibroblasts [3–5]. PC normally fulfills an ana- plerotic function by converting pyruvate into oxaloacetate in the * Corresponding author. Address: Rare Disorders Clinic, The University of Texas presence of elevated acetyl-CoA levels, a reaction that places PC Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8813, USA. Fax: +1 214 645 6238. at the gateway of multiple synthetic mechanisms. It commits pyru- E-mail address: [email protected] (J.M. Pascual). vate to gluconeogenesis by providing oxaloacetate for subsequent

1096-7192/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.05.004 10 I. Marin-Valencia et al. / Molecular Genetics and Metabolism 101 (2010) 9–17 conversion into phosphoenolpyruvate, a precursor of relevant because new therapeutic approaches for PC deficiency dis- (Fig. 1). Glycerol can also support the re-esterification of fatty acids cussed below aim to enhance ATP production, decreasing AMP/ATP (glycerogenesis). On the other hand, oxaloacetate can join acetyl- ratio and inactivating AMPK, re-establishing flux via the synthetic CoA in the to yield citrate by the action of citrate pathways. synthase (EC 2.3.3.1). Citrate can be exported from mitochondria to the cytoplasm and cleaved to form acetyl-CoA and oxaloacetate. Impact of PC dysfunction Cytoplasmic acetyl-CoA serves a building block for de novo synthesis (carried out mainly in liver and adipose tissue) A salient feature of PC deficiency is the conjunction of central and also contributes to the down-regulation of fatty acid b-oxida- nervous system (CNS) maldevelopment (hypotrophy) and degener- tion through the formation of malonyl-CoA, an intermediate of ation, which often dominate the phenotype. Neurological manifes- that inhibits the transport of fatty acids into tations are prominent in PC deficiency as the result of primary mitochondria by carnitine palmitoyltransferase-I (CPT-I). glial dysfunction. In the CNS, PC activity is robust in glia but absent During catabolic states, PC also sustains intra-mitochondrial from neurons [7]. The two major glial cell types include astrocytes production of oxaloacetate. The integrity of both pyruvate dehy- (abundant wherever neurons are located) and oligodendrocytes drogenase (which supplies acetyl-CoA derived from pyruvate) (present in white matter myelin). In astrocytes, the anaplerotic and PC is critical to maintain flux through aimed function of PC is required for gluconeogenesis and glycogen synthe- to fuel the CAC and leading to energy production via the respira- sis and to replenish a-ketoglutarate removed from the CAC for the tory chain. In fact, the most immediate role of the oxidation of synthesis of , the main neuronal precursor of both gluta- , branched-chain and other amino acids, as well as of the mate and c-aminobutyric acid (GABA) [7]. PC is also involved in b-oxidation of fatty acids is to provide carbon to the CAC. Impair- myelin lipid synthesis in oligodendrocytes, an underexplored pro- ment of these catabolic pathways by disease-related enzymatic cess in the context of developmental disorders that may underlie defects compromises the efficiency of the CAC in terms of ATP pro- the paucity of myelin and abundance of white matter lesions ob- duction and re-activation of synthetic pathways. Parallel covalent served in these patients. Additionally, PC is indirectly involved in modification mediated by nutrient sensors can also suppress syn- maintaining the highly active glutathione system by supplying oxa- thetic pathways [6]: AMP-mediated kinase (AMPK) is a ma- loacetate to counteract the loss of malate that may be required for jor regulator of catabolic flux. AMPK is activated under conditions the generation of NADPH both in microglia (a ubiquitous neural cell associated with elevated AMP/ATP ratio such as those that cause a type) and oligodendrocytes [7]. Lastly, in ependymal cells, the ana- decrease in energy. When activated, AMPK phosphorylates serine plerotic function of PC may be required for the oxidation of sub- and threonine residues present in many cytosolic . The strates such as branched-chain amino acids used for energy immediate result of these modifications is that sets of enzymes generation and needed to sustain kinocilliary activity [7,8]. Impair- that participate in synthetic pathways become inactivated, while ment of these critical roles of PC justifies the prominent brain lesions other enzymes that promote (by providing substrates that PC deficiency patients manifest, such as spongiform degenera- for the CAC) undergo stimulation such that the net result is the tion, neuronal loss, gliosis, delayed myelination, ventricular enhancement of catabolism. These considerations are particularly enlargement, and hypodevelopment of the corpus callosum [9–12].

Glucose LIVER Triglycerides G-3-P DHAP LIVER PEP Fatty acids NADH NAD Fatty acids Pyruvate Lactate Arginino- CPT-1 Malonyl-CoA succinate P5C NH Pyruvate Urea 3 Fatty acids Cycle Acetyl-CoA AcAc AcAc Acetyl- PC Citrulline NADH CoA NAD α-KG Aspartate OAA 3HB Glutamate Citrate Citrate NADH CAC OAA OAA NADH NAD Acetyl-CoA Malate Malate α-KG NAD Malonyl-CoA MITOCHONDRIA Saccharopine NADH NADH Fatty Acids NAD NAD Glutamate CYTOPLASM Glutamine Glutamate Astrocyte Myelin formation

Neuron Glutamate GABA BRAIN

Fig. 1. Schematic flux diagram illustrating metabolic abnormalities resulting from pyruvate carboxylase (PC) deficiency. CAC, citric acid cycle; OAA, oxaloacetate; a-KG, a-ketoglutarate; G-3-P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; P5-P, pyridoxal phosphate; AcAC, acetoacetate; 3HB, 3-hydroxybutyrate. I. Marin-Valencia et al. / Molecular Genetics and Metabolism 101 (2010) 9–17 11

PC plays a critical role in gluconeogenesis in both liver and kid- gnomonic, findings such as elevated lactate/pyruvate (L/P) ratio, ney. This phenomenon probably accounts for the low hydroxybutyrate/acetoacetate (H/A) ratio, hypercitrullinemia that patients can manifest during fasting, metabolic imbalance and , parameters that often remain unaltered or, paradoxically, even postprandrial states. Furthermore, in the li- in Types A and C patients (Table 1). However, phenotypic infer- ver, PC deficiency leads to decreased oxaloacetate availability, ences based on PC activity remain elusive, as there is no solid cor- resulting in impaired acetyl-CoA oxidation which can then be di- relation between clinical phenotype and enzyme assay in verted into ketone body and fatty acid synthesis (Fig. 1). This phe- fibroblasts, although it is generally postulated that PC abundance nomenon can explain the lipid droplet accumulation often found in (protein or mRNA levels) and residual enzymatic activity influence (steatosis) and associated hepatomegaly (enlarged li- the severity of each form of PC deficiency [2,17–19]. Disease sever- ver) [10,12–14]. Additionally, it has been hypothesized that gluco- ity has also been loosely correlated with the type of mutation that neogenesis from oxaloacetate is directly linked to bicarbonate the PC gene harbors such that missense mutations are often asso- reabsorption in proximal renal tubular cells [15]. This mechanism ciated with Type A, whereas truncating mutations are more preva- may also explain the relationship between bicarbonate reabsorp- lent in patients with Type B phenotype [20,21]. tion impairment and renal tubular acidosis observed in the most Type A PC deficiency is most common among North American severely affected PC deficient patients. Indians [14], particularly members of the Algonquian-speaking In pancreatic cells, PC participates in maintaining elevated ATP/ groups encompassing the Micmac, Cree, and Ojibwa tribes [2]. ADP and NADPH/NADP ratios in the cytoplasm, which are required Moderate lactic acidemia with normal L/P ratio and ketoacidosis for the secretion of insulin in response to changes in plasma glucose with normal H/A ratio are common biochemical findings (Table levels. Interestingly, there is a genetic epidemiological association 1). These patients first manifest at the age of 2–5 months usually between PC and : single nucleotide polymorphisms after a normal early development, presenting with failure to thrive, in the PC gene are associated with changes in acute insulin release, apathy, delayed mental and motor development, hypotonia, pyra- highlighting, together with the cited gluconeogenic function, the midal tract dysfunction, ataxia, nystagmus, and seizures [10,22– importance of PC in glucose homoeostasis [16]. 25]. Neurodegeneration leading to cerebral atrophy and hypomye- lination are common features. Early reports associated Type A PC Phenotypes of PC deficiency deficiency with Leigh syndrome (subacute necrotizing encephal- omyopathy) [26–30]. This association is well known but its Three forms of presentation have been identified in the PC defi- frequency remains uncertain because only a minority of PC defi- cient patients described thus far: ciency cases exhibited pathologically proven Leigh syndrome [26,28], because of a potential for the occurrence of suboptimal Type A (infantile or North American form) storage and assay conditions required to measure PC activity accu- Type B (neonatal or French form) rately and because a subsequent small case series study of Leigh Type C (benign form) syndrome patients failed to demonstrate PC deficiency [31]. Renal tubular acidosis has also been associated with the Type A pheno- These phenotypes can only be distinguished by their clinical type [10,28,32]. The prognosis is poor and most Type A patients presentation and probably constitute a continuum spanning from die during the first years of life. the most severe (Type B) to the less severe form (Type C). Biochem- Type B PC deficiency was first described in France [18] and is ical studies can assist in the distinction among phenotypes, as the more common in patients of Arab descent, with cases reported in most severely affected patients exhibit typical, although no patho- individuals of Algerian, Egyptian, and Saudi Arabian extraction

Table 1 Biochemical hallmarks of PC deficiency.

Type in blood Lactate-pyruvate Serum concentration Plasma concentration 3-Hydroxybutyrate/ Other ratio in blood of ammonia of amino acids acetoacetate ratio in plasma A (infantile form) 2–10 mmol/L [2] Normal [2] Normal benign form Increased alanine and Normal [2] Hypoglycemia proline levels. (<40 mg/dl) [53]. Normal citrulline and Ketosis [2]. Both the lysine levels [2,18,41] PC protein and mRNA are present, but in low levels [18] B (severe >10 mmol/L [2] Increased Moderately increased Increased citrulline, Low (<0.8) [2,11] Hypoglycemia [53] neonatal form) (>25) [2] (100–150 lmol/L) [33] proline, lysine, [2]. Both the PC and alanine [2], protein and mRNA are low glutamine [33]. absent in most Some reports document patients [2] decrease of alanine [33] C (benign form) 2–5 mmol/L [43,5] Normal [2] Normal [2] Elevated lysine, proline, Normal [2] Ketosis during and alanine [18,43]. metabolic stress [2]. Normal citrulline [2] Both the PC protein and mRNA are present [18] Normal Lactic acid levels L/P ratio in <30 days: 17–91 Alanine: 179–378 lmol/L. H/A ratio in Glucose: 45–110 mg/dl (normal values) in blood: blood: <25 [2] lmol/L [88]. Proline: 125–279 lmol/L. plasma: in neonates; 0.5–2 mmol/L [2] 1–12 months: Citrulline: 0.8–1 [63,89] 70–110 mg/dl in infants 15–72 lmol/L [88]. 11–35 lmol/L Lysine: and older children. 1–14 years: 121–286 lmol/L. Glutamine: Acetoacetate: 14–65 lmol/L [88] 347–786 lmol/L [6,14] 0–200 lmol/L [40]. 3-Hydroxybutyrate: 30–600 lmol/L [40] 12 I. Marin-Valencia et al. / Molecular Genetics and Metabolism 101 (2010) 9–17

[2]. Patients first manifest during the first 72 h of life by exhibiting vival (over 23 years of age) compared to other non-mosaic Type severe truncal hypotonia and tachypnea presumably due to meta- A patients. bolic acidosis. Generally, these patients do not suffer from prenatal Type B patients exhibit absent or diminished levels of PC both complications and exhibit normal birth weight, Apgar scores and protein and mRNA transcript [50] and insufficient residual PC unremarkable neonatal examinations [33]. In some cases, prenatal activity. These patients present at least one truncating mutation, ultrasonography has illustrated choroidal plexus cysts or ischemic- frequently affecting the carboxyl domain or biotin car- like brain lesions [34]. Macrocephaly has also been described [34– boxyl carrier protein [21], often in conjunction with another trun- 36]. Additional signs and symptoms include anorexia, failure to cating or missense mutation [20,21]. The Type B phenotype is thrive, hepatomegaly, abnormal limbs and ocular movements, associated with complex missense mutations, deletions, and splice myoclonic or generalized tonic–clonic seizures [33], pyramidal donor site mutations in homozygosity, compound heterozygosity tract dysfunction and severely disturbed mental and motor devel- and, possibly, also with mosaicism [17,21]. opment [11,36–39]. Biochemical studies typically demonstrate se- In Type C patients, both PC protein and mRNA are present at vere lactic acidemia with an elevated L/P ratio (reflecting a reduced higher abundance than in other types of PC deficiency [17]. The be- redox state in the cytoplasm), ketoacidosis with low H/A ratio nign course characteristic of these patients may be due to the pres- (reflecting mitochondrial oxidized redox state), and hypoglycemia ence of different transcripts encoding PC forms that differ in their accompanied by hypercitrullinemia, moderate hyperammonemia, first two exons [51]. A mutation affecting the first two exons of the and normal to low levels of glutamate and glutamine, parameters liver isoform still allows the brain isoform to be expressed nor- that are almost pathognomonic for this phenotype (Table 1). Renal mally, while the liver manifests PC deficiency [51]. Two individuals tubular acidosis has also been reported in these patients [40–42]. have been reported with the following mutations: S266A +/ (het- The prognosis is poor, and almost all affected infants die within erozygous mutation), S705X +/ (another potentially mosaic the first three months of life [33]. mutation), and T569A/L1137VfsX1170 (compound heterozygosity) Type C PC deficiency has been observed in five individuals [17]. [17,18,43–45] without clear ethnic predilection. Onset typically oc- curs during the first year of life and is characterized by episodic metabolic acidosis associated with lactic acidemia and, occasion- Indicators of PC deficiency ally, with ketoacidosis during metabolic stress. Neurological devel- opment is normal or mildly impaired. Dystonia, episodic ataxia, Several analytical observations complemented with structural dysarthria, transitory hemiparesis, and seizures have been de- brain considerations assist in the diagnosis of PC deficiency. scribed in some of these patients [17,18,43–45]. Biochemical profile Molecular genetics of PC deficiency Blood gasometry Constant or intermittent metabolic acidosis caused by elevated PC (located in 11q13.4–q13.5) is the only gene known to be asso- lactate levels is typical of PC deficiency. Ketoacidosis is also present ciated with PC deficiency [46]. PC includes 20 coding exons and four and contributes to the metabolic acidosis. Type A and B patients non-coding exons as part of the five untranslated region (50UTR), usually manifest chronic metabolic acidosis with elevated lactate spanning 105.9 kb [18,47]. All four non-coding exons are involved and ketosis, and Type C patients tend to manifest these features in alternative splicing, resulting in three tissue specific PC tran- only intermittently during metabolic stress [46]. scripts carrying the same coding region: variant 1 (NM_000920) (expressed in brain and liver), variant 2 (NM_022172) (present in liver and kidney), and variant 3 (BC011617) (abundant in brain Blood glucose and liver) [3,17,19,48]. The protein consists of a homotetramer of Because PC is directly involved in the synthesis of glucose, several polypeptides, namely, biotin carboxylase, carboxyltransfer- hypoglycemia is commonly found in the course of the disease, ase, pyruvate carboxylase tetramerization, and biotin carboxyl car- although it may not be the dominant biochemical abnormality at rier protein. Biotin is covalently linked to a specific lysine residue presentation. Hypoglycemia is more prominent in Type A and B pa- located close to the C-terminus [4]. The lack of mutations at this bio- tients after fasting, metabolic decompensation or in the postprand- tin-binding region has been associated with response to biotin rium [52], whereas in Type C patients glucose levels may be administration, presumably by allowing biotin-mediated catalytic normal [18], low [44], or elevated [43,45]. enhancement [49]. Molecular genetic testing has been performed on a research ba- sis. PC deficiency patients are usually homozygote and manifest Blood lactate and pyruvate the disorder with complete penetrance. Heterozygote carriers typ- Elevated levels of pyruvate and lactate are characteristically ically do not manifest clinical symptoms. Mosaicism has been in- found in this disorder. PC deficiency causes an increase in pyruvate voked in some patients to explain the discordance between levels, which is subsequently converted to lactate resulting in lac- tissue enzyme abundance and clinical phenotypes identified in se- tic acidemia (Fig. 1), a reaction that can be enhanced by the admin- lect cases [17]. istration of carbohydrates. The L/P ratio is a useful indicator of the Studies with skin fibroblasts of Type A patients typically dem- underlying cause of lactic acidemia. In PC deficiency, only Type B onstrate the presence of low levels of a mature biotin-containing patients exhibit an elevated L/P ratio (>25), reflecting a reduced re- PC protein of correct molecular weight [17,50]. Patients reported dox state (high NADH/NAD ratio) in cytoplasm. The NADH/NAD ra- with this type of PC deficiency consistently harbor two missense tio may also be elevated due to impairment of gluconeogenesis mutations in homozygous or compound heterozygous states associated with low levels of aspartate, malate, and oxaloacetate. [19,21,25], which are mostly located in the biotin carboxylase or These compounds are reducing equivalent vehicles that transit carboxyltransferase domains [21]. Potential somatic mosaicism from the into the mitochondria, resulting in increased was reported in one individual afflicted by Type A PC deficiency NADH levels [2,33]. The elevated NADH contributes to the conver- and genotype R62C +/, R631Q ++/ (mosaic mutation), A847V sion of pyruvate to lactate. The L/P ratio, however, is usually nor- ++/ (mosaic mutation) [17], and who exhibited prolonged sur- mal in Type A and C patients. I. Marin-Valencia et al. / Molecular Genetics and Metabolism 101 (2010) 9–17 13

Blood 3-hydroxybutyrate and acetoacetate are typical of Type A and B patients and are less often found in Type Ketonemia, detectable even in the fed state, is common in PC C patients [33,45]. deficiency. Decreased oxaloacetate availability leads to failure of hepatic acetyl-CoA oxidation, which is then diverted into ketone Cerebrospinal fluid (CSF) analysis body formation (Fig. 1), a phenomenon potentiated by high-fat Elevated levels of CSF lactate, pyruvate, alanine, proline, and diets [5,13,40,41]. The H/A ratio is also diagnostically helpful. Only low levels of glutamine have been reported, mainly in Type B pa- Type B patients manifest a low H/A ratio (<0.8), reflecting the mito- tients [35,41,61,62]. chondrial oxidized redox state (low NADH/NAD ratio). This finding probably also signifies impairment of the CAC not only leading to reduced NADH, but also to CAC intermediate abundance, which Additional analytical features is responsible for decreased transport of reducing equivalents from the cytosol to the mitochondria. Therefore, low levels of mitochon- Cholesterol drial NADH impair the conversion of acetoacetate into 3-hydroxy- An elevation of total cholesterol or its precursors (mevalonic butyrate, resulting in a decreased H/A ratio. The H/A ratio is often acid) may occur in Type A and B forms of PC deficiency [13,41]. normal in Type A and C patients. The excess of ketone body production can serve as precursor of acetyl-CoA and acetoacetyl-CoA synthesis in the cytoplasm, which Plasma amino acids can be converted into b-hydroxy-b-methyl-glutaryl-CoA (HMG- The deficit of oxaloacetate and, consequently, of aspartate, im- CoA) and lead to enhanced cholesterol biosynthesis. pairs the (Fig. 1). Aspartate is required for the synthesis of argininosuccinic acid from citrulline and its deficit causes an in- crease in citrulline levels and a decrease of argininosuccinate and Glucose tolerance test (GTT) , all of which are frequently detected in Type B patients. GTT can yield variable results depending on the phenotype and High levels of lysine may be detected in Type B and C patients the route of glucose administration. Persistently elevated levels of [2]. A central step in lysine catabolism requires the transfer of lactate after oral or intravenous (iv) glucose load in a Type B pa- the e-amino group to a-ketoglutarate through the intermediate tient was reported [40]. However, the ketone body response was saccharopine [53–55]. Low levels of a-ketoglutarate cause an quite different, showing a marked decrease of ketogenesis after impairment of lysine degradation throughout impediment of sac- iv glucose administration, which returned to high levels when glu- charopine metabolism, resulting in an increase in lysine levels. cose was administered orally. In a Type A patient, the GTT induced High concentrations of ammonia due to impairment of the urea cy- a biphasic pattern in the L/P ratio, manifested as an initial decrease cle can also cause hyperlysinemia, as ammonia competes with the of the ratio, followed by a progressive increase after 60 min after conversion of lysine into a-ketoglutarate and glutamate, the latter the glucose load [49]. In another Type A patient, an iv glucose load of which is then converted into glutamine [56]. Therefore, low lev- caused a significant increase in lactate and pyruvate levels associ- els of a-ketoglutarate can also explain the low concentrations of ated with a transient rise in ketone body levels [13]. In a Type C pa- glutamate and glutamine typical of these individuals, especially tient, this test did not result in significant changes in lactate or in Type B patients. As with other states of lactic acidemia, the con- pyruvate concentrations [18]. centration of proline is elevated in PC deficiency [57], possibly be- cause lactate can inhibit proline oxidase (EC 1.5.99.8) [57–59]. PC enzyme assay Alanine is also typically elevated in this disorder [2,18,43], proba- bly because pyruvate can be reversibly converted into alanine Assay of PC activity in fibroblasts, lymphocytes, and other tis- through alanine aminotransferase (EC 2.6.1.2), a frequent correlate sues except muscle is definitive for the diagnosis of patients with of lactic acidemia states. suspected PC deficiency. However, residual PC enzymatic activity is of limited value for the distinction among the three phenotypes Ammonia [2,17] because enzymatic analysis often yields activities below 5% Moderate hyperammonemia (100–150 mol/L) is commonly l of normal regardless of PC deficiency type [2,10,13,17,63].An observed in Type B patients, resulting from secondary impairment explanation of this phenomenon is that differences between en- of ureagenesis [33,60]. Increased levels of lysine may also impair zyme activity in vitro and in vivo may be due to the rapid loss of urea cycle flux by inhibiting arginase (EC 3.5.3.1), ornithine tran- PC activity when tissues are not immediately preserved (particu- scarbamylase (EC 2.1.3.3), and mitochondrial ornithine uptake larly liver tissue) or are improperly prepared [18]. On the other and by competing with ammonia for -ketoglutarate [60]. a hand, measurement of PC activity in fibroblasts can be useful to identify carriers within the family of a proband [10,23]. The assay, Urine analysis however, is unreliable for carrier determination in the general pop- The most constant finding is an elevated alanine level. Increased ulation due to a significant overlap in residual enzyme activity be- levels of proline, lysine, cystine, , citrulline, ornithine or tween obligate carriers and non-carriers. even branched-chain metabolites have been reported, mostly in Type B patients [35,41,42,45]. Renal tubular acidosis (RTA) has been reported in Type A and Type B patients, who man- Neuroimaging ifest generalized amino aciduria and high levels of bicarbonate, and when RTA is associated with multiple defects of the proximal tu- Brain structural abnormalities are frequently detected in Type A bule high urinary levels of urate, phosphate, lactate, and sodium and B patients by magnetic resonance imaging. The neuroradiolog- can also be detected [10,14,28,32,40–42]. ical findings reported in these patients include ischemic-like le- sions [34], ventricular dilatation, periventricular cysts (identified Urine organic acids almost invariably in Type B patients), reduced myelination Increased levels of lactate, pyruvate, 2-hydroxybutyrate, 3- [35,64], and subcortical leucodystrophy [19,65,66]. These findings hydroxybutyrate, and acetoacetate, in addition to low levels of are usually detected in symptomatic neonates or infants, although CAC intermediates such as 2-oxoglutarate, fumarate, succinate, ischemic-like lesions can be detected prenatally in Type B PC defi- and malate have been documented [18,33]. These abnormalities ciency [34]. 14 I. Marin-Valencia et al. / Molecular Genetics and Metabolism 101 (2010) 9–17

Diagnostic challenges Disorders of pyruvate metabolism and the CAC

Several inborn errors of metabolism share features of PC defi- complex (PDHC) deficiency: The ciency by causing downstream or remote impairment of metabolic majority of patients manifests severe, often fatal, neonatal or infan- processes for which PC serves as an entryway, or by inducing sec- tile lactic acidosis or exhibits a chronic neurodegenerative course ondary PC deficiency due to failure of action (Fig. 2). including demyelination, cystic degeneration, ectopic olivary nu- clei, hydrocephalus and agenesis of the corpus callosum. Most cases are caused by a deficiency of PDH-E1 a-subunit (OMIM Biotin disorders 300502) [51]. Biochemical analysis is characterized by increased lactate in plasma, urine, and CSF [65] with preserved L/P ratio, ele- Because PC is one of the five biotin-dependent carboxylases that vation of alanine in blood and, occasionally, of proline and glutamic use biotin as a cofactor to transfer carboxyl groups, disorders of acid. In PDH E3 subunit deficiency, moderate increases in , biotin metabolism can cause impairment of PC activity. The two and levels may arise from the participation of this principal genetic disorders of biotin metabolism include biotinidase subunit in the branched-chain ketoacid dehydrogenase (EC 1.2.4.4) (BTD; EC 3.5.1.12) deficiency (OMIM 253260) and holocarboxylase and a-ketoglutarate dehydrogenase complexes [71,72]. Urinary or- synthetase (HCS; EC 6.3.4.10) deficiency (OMIM 253270). BTD defi- ganic acid analysis usually reveals a significant increase of lactate, ciency typically causes late-onset multiple carboxylase deficiency pyruvate, and of the CAC intermediates 2-oxoglutarate and (first few months or years of life) [67]. Profound enzymatic activity 2-hydroxyglutarate, especially in E3 deficiency. Ketosis is a signif- defects (<10% of normal serum enzymatic activity) and clinical man- icant feature of E3 deficiency [73,74]. ifestations can be detected at 3–6 months of age (similarly to Type A Of note, in patients with intractable lactic acidemia, normal L/P phenotype) in patients afflicted by hypotonia, lethargy, seizures, ratio and normal PDHC activity, a mitochondrial pyruvate carrier rash, and laryngeal stridor. Biochemical findings include raised lac- defect may be suspected [75]. Disorders of CAC are characterized tate and pyruvate concentrations in blood or CSF, ketosis, mild by abnormal urinary organic acids with elevations of the specific hyperammonemia, and abnormal urinary organic acids with a char- metabolite metabolized by the impaired enzyme. acteristic increased 3-hydroxyisovaleric acid excretion [67]. HCS deficiency leads to early-onset multiple carboxylase deficiency. Similarly to PC deficiency Type B, it manifests during the first days Respiratory chain disorders of life with acute episodes of metabolic acidosis accompanied by lethargy, tachypnea, vomiting, hypotonia, and occasionally seizures In these disorders, the excess of NADH and lack of NAD arising and skin rash, leading to severe neurodevelopmental delay and early from respiratory chain dysfunction result in an increase of both H/ death if left untreated [68,69]. Urinary organic acid analysis can as- A (>2) and L/P (>25) ratios, a typical feature of these patients in sist with the differential diagnosis with Type B phenotype, manifest- addition to hyperlactacidemia and hyperketonemia, particularly ing typically high levels of lactate, 3-methylcrotonylglycine, 3- in the postabsorptive period [76]. Plasma amino acid analysis re- hydroxyisovalerate, 3-hydroxypropionate and methylcitrate [70]. veals only an increase of alanine and proline as the consequence

Metabolic acidosis with increased lactate

Abnormal organic Normal organic acids (in PC deficiency lactate, pyruvate, and ketoacids acids can be elevated and intermediates of citric acid cycle are usually decreased)

Dicarboxylic Methylmalonic aciduria acidemia, propionic Normal L/P ratio Elevated L-P acidemia and other (<25) ratio (≥25) organic acidemias; multiple carboxylase deficiency; biotinidase Fatty acid deficiency; respiratory oxidation chain disorders Decreased Normal or defects H/A ratio elevated H/A ratio Hypoglycemia Normoglycemia

Normal plasma Abnormal amino acids plasma amino Respiratory Gluconeogenic Pyruvate dehydrogenase acids chain disorders (Fructose deficiency; pyruvate disorders 1,6-DP deficiency) carboxylase deficiency type A Citric acid cycle and glycogen disorders (branched- and C (hypoglycemia can be chain amino acids can PC deficiency type B storage diseases present in both types) be elevated in KDC E3 deficiency)

Fig. 2. PC deficiency in the context of other causes of lactic acidemia. KDC, ketoglutarate dehydrogenase complex. I. Marin-Valencia et al. / Molecular Genetics and Metabolism 101 (2010) 9–17 15 of hyperlactacidemia and, occasionally, and In a case of Type B PC deficiency, an anaplerotic diet therapy hypocitrullinemia [76,77]. was formulated to contain 130 cal/kg/day with 30% of glucose, 30% of long-chain fatty acids, 5% of protein, and 35% of (4 g/kg/day) [82]. This diet was administered to a neonate with PC Mitochondrial fatty acid oxidation disorders deficiency Type B [62], manifesting clinical and biochemical improvement with decrease of lactic acidosis, L/P and H/A ratios, These defects often lead to hypoketotic hypoglycemia associ- progressive decrease of ammonia and citrulline levels in plasma, ated with mild lactic acidemia, affecting predominantly liver, heart rapid normalization of liver function, and increase of urinary excre- or muscle [78]. Other characteristic biochemical abnormalities in- tion of urinary CAC intermediates. After 4 months of treatment, the clude increased kinase levels, increased or decreased lev- patient manifested a progressive decrease of CAC intermediates els of free carnitine and acylcarnitines in plasma and elevated and bicarbonate associated with increased lactate and ketone levels of dicarboxylic acids in urine. bodies, which were reversed by the addition of citrate (7.5 mmol/ kg/day), 2-chloropropionate (50 mg/kg/day), restriction of glucose Gluconeogenic defects intake (30% of total calories), and normal protein intake. The pa- tient died at 6 months of age after a severe infection resulting in Disorders of gluconeogenesis, such as fructose-1,6-biphospha- a fatal acute metabolic decompensation. tase (FBPase) deficiency (OMIM 229700), present with lactic acide- Treatment with co-factors involved in pyruvate metabolism and mia and a more prominent hypoglycemia typically during fasting supplements of the CAC seem to improve mostly the biochemical states [79]. Blood lactate, pyruvate, alanine, and ketone bodies lev- disturbances typical of PC deficiency, without exerting significant els are usually elevated. impact in the neurological manifestations of the disease. A patient with PC deficiency Type A responded to oral biotin (9 mg/day), Citrin (mitochondrial aspartate-glutamate carrier) deficiency manifesting improving lactic acidosis and cerebral myelination without amelioration of neurological abnormalities [40,65]. Treat- The neonatal form, similarly to PC deficiency Type B, manifests ment with high doses of citrate (7.5 mmol/kg/day) and aspartate high levels of citrulline along with normal to mild elevation of (10 mmol/kg/day) has been used in Type B patients, resulting in ammonia, but usually without acidemia or ketonemia [80]. Dis- an increase of oxaloacetate and metabolites of the CAC and tinctive features of this disorder are raised levels of threonine improvement of lactic acidosis [41]. However, ketosis was not re- and , galactosemia, and cholestatic jaundice. The adult solved and there was no significant amelioration of neurological form is characterized by hypercitrullinemia and a more prominent abnormalities. Supplements of other amino acids, CAC intermedi- hyperammonemia than the neonatal form, increased serum argi- ates, and other compounds such as L-carnitine, pyridoxine, aspara- nine, and a significantly increased urinary excretion of arginino- gine, glutamate, glutamine, arginine, 2-oxoglutarate, citrate, and succinate [80]. succinate have been used in different combinations [42,83,84].In a patient with PC deficiency associated with renal tubular acidosis and , supplementation with , asparagine, A framework for future therapies and glutamine stabilized metabolic acidosis and neu- rological deterioration [53,83]. Thiamine (100 mg/day) and lipoic The foremost goal of therapies for PC deficiency as well those acid (100 mg/day), co-factors of PDHC, have generally been ineffec- devised for other inherited catabolic defects is the restoration of tive [41], except in anecdotal cases [30,85]. substrate flux into the CAC with the objective of suppressing Orthotopic liver transplantation was performed in an infant unmitigated catabolism and to activate synthetic pathways. Inter- with PC deficiency Type B [40]. The patient manifested a significant ventions that enhance ATP production lead to decreased AMP/ATP decrease in ketone body levels and an improvement in the renal ratio, thus inactivating AMPK, while reactivating the mammalian tubular function, but little effect on lactic acidemia and no signifi- target of rapamycin (mTOR) [81] and re-establishing flux via syn- cant improvement in neurological performance. thetic pathways. Seizure control requires the restoration of metabolic equilib- In the context of cancer therapeutic strategies, compounds that rium. Select anticonvulsants (valproic acid, barbiturates) have been activate AMPK while inactivating mTOR offer potential to diminish associated with adverse effects in patients with other mitochon- cellular growth and proliferation [81]. In persistently catabolic drial disorders [86]. Exacerbation of lactic acidosis and an increase conditions such as PC deficiency, the greatest benefit would appear of alanine, lysine, and leucine have been reported with the use of to stem from the opposite strategy. Dietary treatment for PC defi- ACTH in a Type B patient with infantile spasms [87]. ciency, however, has traditionally included limited options, since a high-fat diet increases ketoacidosis, and high-carbohydrate diets exacerbate lactic acidosis [13,62]. The use of an exogenous source Conclusion of substrate for enhancement of ATP production, such as the ana- plerotic compound triheptanoin (a triglyceride containing three PC is a crucial flux facilitator for all synthetic pathways that rely 7-carbon fatty acids (heptanoate)) may provide the necessary upon the formation of oxaloacetate. Inherited deficiency of this en- source of both oxaloacetate and acetyl-CoA, inside the mitochon- zyme causes broad disturbances that mostly reflect deranged liver drion to accomplish this goal. When ingested, triheptanoin is and brain metabolism. The three identified clinical phenotypes dis- metabolized mainly in the liver converting one molecule of trihep- play varying degrees of clinical severity in the setting of lactic aci- tanoin into one molecule of glycerol and three molecules of hepta- demia and neurological disturbances. The diagnosis still relies noic acid. Heptanoate (C7) undergoes a partial cycle of b-oxidation upon analysis of amino acids in blood and urine and of urinary or- in the liver to produce 5-carbon ketone bodies (b-ketopentanoate ganic acids and upon enzyme assay of PC because molecular genet- and b-hydroxypentanoate), which can then be exported to the ic testing is not readily available. At the present time, most blood. When enzymes of ketone body utilization are intact, 5-car- interventions have met with little benefit in these patients, but fu- bon ketone bodies serve as a substrate to the CAC in many organs ture therapeutic efforts will focus on the replenishment of CAC such as liver, muscle, kidney and heart and also penetrate into the intermediates to interrupt the hyperactive catabolic cascade asso- brain [82]. ciated with the disorder and to enhance ATP production. The use of 16 I. 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