Clin 8ioche.m, Vol. 24, pp. 331-336, 1991

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Lactic Acidosis and Mitochondrial Disorders DOUGLAS S. KERR Center for Inherited Disorders of Energy Metabolism, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, OH 44106, USA Characterization of the biochemical basis of various inherited disorders associated with lactic acidosis has increased dramatically in recent years. These include defects of enzymes of gluconeogenesis, pyruvate oxidation, and electron transport. Clinical manifestations of these disorders show great variation and overlap, frequently involving the central nervous system as well as skeletal and cardiac muscle. Several of these enzymes are large complexes of subunits encoded by multiple genes; the electron transport chain complexes include subunits encoded by both nuclear and mitochondrial genes. This great complexity complicates analysis of specific mutations, despite considerable progress in defining the primary structure of component proteins and their genes. With few exceptions, treatment of disorders associated with congenital lactic acidosis remains unsatisfactory.

KEY WORDS: lactic acidosis; pyruvate dehydrogenase complex; pyruvate carboxylase; phosphoenolpyruvate carboxykinase; fumarate hydratase; NADH dehydrogenase; cytochrome oxidase; mitochondria. O v e r v i e w of i n h e r i t e d d i s o r d e r s associated with lactic a c i d e m i a ccumulation of lactic acid in blood reflects a A disturbance of normal energy metabolism, usually due to incomplete oxidation of pyruvate. In clinical situations, lactate accumulation is most commonly the result of transient inadequate peripheral oxygenation but, if persistent, may be the initial laboratory clue to a group of inherited disorders which have the common effect of interfering with intracellular energy production. These disorders, referred to collectively as inherited disorders of energy metabolism, are characterized by accumulation of intermediates of carbohydrate, fat, or amino acid metabolism and result in a variety of heterogeneous clinical syndromes associated with central nervous system, skeletal muscle, cardiac muscle, or systemic dysfunction. Recognition and characterization of these disorders have greatly accelerated in recent years due to application of established biochemical and molecular laboratory

Correspondence: Douglas S. Kerr, M.D., Ph.D., Center for Inherited Disorders of Energy Metabolism, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, OH 44106, USA. Manuscript received August 16, 1990; accepted April 10, 1991. CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991

methods to clinical diagnosis of these previously obscure diseases (1). Unraveling of these metabolic puzzles has led to new unresolved questions concerning why such defects are not lethal, why they may be expressed in a tissue-specific manner or only at certain stages of development, how they are inherited, and what are the possibilities for treatment. Complete oxidation of one mole glucose to C02 results in the potential formation of 36 mole-equivalents of ATP, whereas its anaerobic conversion to lactate produces only 2 equivalents of ATP. Hence, transformation of almost all the potential energy of carbohydrate fuel into useful intracellular energy depends on intramitochondrial oxidation of pyruvate to acetyl-CoA, oxidation of acetyl-CoA in the Krebs cycle, and oxidation of the resultant reduced pyridine nucleotides or flavoproteins via the electron transport chain. Pyruvate which is not oxidized (from red blood cells which lack mitochondria or from skeletal muscle during strenuous exercise) is reduced to lactate, or transaminated to alanine, both of which are returned to the liver or kidney for utilization as gluconeogenic substrates. These general metabolic alternatives provide a functional basis for broad classification of disorders associated with congenital lactic acidosis into defects of gluconeogenesis or defects of glucose oxidation. Defects of glucose oxidation can be further subdivided into defects of oxidation of specific substrates (pyruvate or the Krebs cycle intermediates) or defects of the electron transport chain (which are common to all oxidative pathways). Correlation of clinical laboratory observations with variations of specific metabolic products in relation to diet and physiological state are useful first steps in distinguishing these subcategories of metabolic defects. Before embarking on specific enzyme assays to establish a biochemical basis for congenital lactic acidemia, accurate, repeated measurements should be made of blood lactate and pyruvate (lactate/pyruvate ratio), CSF lactate and pyruvate, plasma alanine and other amino acids, urine organic acids (including keto-acids), and plasma and urine carnitine (free and acyl-) under different conditions. Although this recommendation may appear too inconvenient or expensive, it is important to appreciate that at the present time 331

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definitive laboratory identification of the specific biochemical basis of congenital lactic acidosis is the exception rather than the rule. Available reference laboratory facilities for performing these assays are still quite limited, and interpretation of the metabolic significance of a laboratory finding must always be put in the clinical context of the individual patient if its significance and potential benefit in planning therapy are to be realized. The probability of making a specific laboratory diagnosis is greater if the lactic acidemia is severe (>3 mmol/L for venous blood), persistent or reproducible under certain conditions, and accompanied by other well-documented specific metabolic and functional abnormalities. The alternative possibility of acquired lactic acidemia secondary to inadequate peripheral oxygenation, sometimes unrecognized in focal anatomical regions (especially CNS), must always be excluded. Biochemical classification o f specific disorders DEFECTS OF GLUCONEOGENESIS

Gluconeogenic defects are typically associated with hepatic dysfunction and fasting hypoglycemia and lactic acidemia. A significant diagnostic clue (and a major principle of treatment) is that the lactic acidosis improves with feeding or parenteral administration of glucose and is worsened by fasting. Specific defects are known to affect each of the four unique enzymes of gluconeogenesis: glucose-6phosphatase, fructose-l,6-diphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase. Glucose-6-phosphatase deficiency (von Gierke's hepato-renal disease or type I glycogen storage disease), is of special historical significance because it was the first inherited disorder for which a specific enzyme deficiency was described (2). This relatively rare disorder (actually a group of disorders) continues to attract a disproportionate share of interest from clinical investigators, partly because of its unique combination of impaired growth, hyperlipidemia, platelet dysfunction, hepatomas, and glomerulonephropathy (3). It is one of the few causes of lactic acidosis for which a beneficial therapy has been established (4). Although biochemical dissection of the microsomal organization of the glucose-6-phosphatase system accounts for the clinical heterogeneity of this disorder (3,5), little progress has been reported in molecular characterization of the responsible genes. Fructose-l,6-diphosphatase deficiency has somewhat similar clinical manifestations, but is not associated with storage of glycogen (6), and appears to be less common. Only a very few cases of phosphoenolpyruvate carboxykinase deficiency have been reported, and the clinical features do not appear to be unique (7,8). A great deal of information is available about the molecular biology of 332

this enzyme; separate genes encode for distinctive mitochondrial and cytosolic forms of the enzyme (9). Pyruvate carboxylase deficiency is relatively more common and has been associated with various clinical syndromes. In the more severe neonatal "French" form, immunologically cross-reactive protein is absent (CRM-), whereas in the less severe childhood "North American" form, pyruvate carboxylase protein is present but enzymatically inactive (CRM+) (10,11). A cDNA clone for human pyruvate carboxylase has been isolated and used to map the gene to chromosome 11 (12). Prenatal diagnosis of this disorder has been successful (13). Since pyruvate carboxylase is a biotin enzyme, its activity is impaired in defects of biotin metabolism, including biotinidase deficiency and holocarboxylase synthase deficiency, both of which are causes of multiple carboxylase deficiency with a characteristic pattern of excretion of urinary organic acids (13). Early recognition and treatment of biotinidase deficiency represents the most effective intervention available for any of the various causes of congenital lactic acidosis, and assay of serum biotinidase should be a standard diagnostic test in undifferentiated cases (14). P Y R U V A T E DEHYDROGENASE COMPLEX DEFICIENCY

Defects of the pyruvate dehydrogenase complex (PDC) are currently the most commonly defined biochemical cause of congenital lactic acidosis, but account for only some 10-15% of this group of disorders. PDC is a large, multimeric enzyme complex comprised entirely of nuclear-encoded proteins. The activity is physiologically regulated by phosphorylation (inactivation) and dephosphorylation (activation), catalysed by a specific kinase and phosphatase, respectively, which are integral to the complex (15). Flux of pyruvate to acetyl-CoA leading to oxidation in the Krebs cycle or to fatty acid synthesis is increased after absorption of carbohydrate, and PDC is predominantly in the active (dephospho-) form in the insulin-dependent state. In contrast to defects of gluconeogenesis, lactic acidemia in patients with PDC deficiency is therefore characteristically increased after ingestion of carbohydrate. In contrast to electron transport chain defects, the ratio of lactate-to-pyruvate remains normal in patients with PDC deficiency because the ratio of NAD/NADH (which determines the equilibrium of lactate dehydrogenase) is normal. Plasma alanine is also increased, and lactic and pyruvic acids are usually the only urinary organic acids increased. The clinical manifestations of PDC deficiency are almost exclusively limited to the central nervous system (16,17), but are quite variable in terms of severity and age of onset. Some cases of PDC deficiency may manifest the characteristic neuropathology of Leigh's disease (subacute necrotizing encephalomyelopathy); this neuropathological syndrome is not specific to PDC deficiency but may also be associated with certain CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991

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defects of the electron transport chain (18,19). The primary structures of the 4 proteins included in the 3 main catalytic components of human PDC (EI~, EI~, E2, and E3), have been fully defined in recent years by characterization of their specific cDNAs (15). Utilization of specific component activity assays, immunoblotting, mRNA analysis, and cDNA sequencing, has made it possible to define the molecular pathology of PDC deficiency (20,21), including recent definition of several mutations of the EI~ component (22-25). The gene for the Elc~ peptide has been located on the short arm of the X-chromosome (26). Nonrandom inactivation of the X-chromosome as well as other unexplained factors play a role in variable expression of PDC deficiency, which can be diagnostically frustrating but might help explain some of the paradoxical clinical heterogeneity of these disorders (21,27). Although PDC deficiency has been typically defined in cultured skin fibroblasts, patients have been described with systemic PDC deficiency in whom the skin fibroblast activity was normal (27). To date, attempts at prenatal diagnosis of E 1 deficiency have not been successful. With respect to treatment, there is no convincing evidence that administration of thiamine has been beneficial, although this is a thiamine pyrophosphate-dependent enzyme. As expected, dietary restriction of carbohydrate may be financial in reducing the severity of accumulation of blood lactate (28). Alternatively, administration of dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase which might help activate any residual enzyme, has been shown to lower blood lactate in some PDC deficient patients (29,30). A few cases of PDC deficiency appear to be due to lack of pyruvate dehydrogenase phosphatase, resulting in failure to activate the enzyme (29,31); such cases might also be expected to benefit from dichloroacetate. DEFECTS OF THE KREBS CYCLE

The third component of PDC (E 3) is a flavoprotein which is common to other analogous enzymes, including alpha-ketoglutarate dehydrogenase and the branched-chain alpha-ketoacid dehydrogenase. Defects of this enzyme, therefore, characteristically result in accumulation of lactic, pyruvic, alpha-ketoglutaric, and branched-chain alpha-ketoacids in the urine and of the corresponding amino acids in plasma. The gene for this protein is located on chromosome 7 (15,32), and this disorder is inherited in an autosomal recessive manner (33). Deficiency of fumarase, the only established human defect of an enzyme which is specific to the Krebs cycle, has been described in several patients who were found to have accumulation of succinic and fumaric acid in the urine and lactate in blood (34,35). Combined loss of the mitochondrial and cytosolic activities of fumarase in 2 patients with this deficiency are consistent with the conclusion CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991

that both forms are encoded by the same nuclear gene. DEFECTS OF THE ELECTRON TRANSPORT CHAIN

Probably the largest and most heterogeneous group of defects of oxidative metabolism associated with lactic acidosis involve the electron transport (or respiratory) chain (ETC). Technical problems with assay of activity of the ETC complexes in readily available cells and tissues has impeded definition of the actual frequency of these disorders. The four enzyme complexes of this chain are NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II), ubiquinol-cytochrome c reductase (complex IH), and cytochrome c oxidase (complex IV). These large mitochondrial protein complexes are made up of multiple protein subunits, most of which are encoded by nuclear genes and some of which are encoded by mitochondrial DNA. The specific functions of many of these subunits are unknown, and they can only be detected by immunoassay. Several of the nuclear subunits are species specific or have tissue or developmentally regulated isoforms, and loss of a particular subunit may lead to loss of additional subunits from the ETC complexes, complicating the investigation of these very complex disorders. Deficiency of a particular ETC complex may result in quite different forms of disease associated with what appears enzymatically to be the same biochemical defect, e.g., isolated myopathy, encephalopathy, or cardiomyopathy may be associated with complex IV deficiency (36). Conversely, defects of different complexes paradoxically may be associated with very similar clinical and pathological findings, e.g., Leigh's disease, which can be associated with PDC deficiency, also may occur with deficiency of either complex I or complex IV (3739). A transient defect in a developmentally regulated isoform of an ETC subunit may result in what initially appears to be a life-threatening congenital infantile myopathy which later unexpectedly improves (40). Diagnosis of these disorders requires careful organization of clinical, metabolic, genetic, and biochemical analyses. Since ETC defects in general interfere with oxidation of NADH, the ratio of NADH/NAD and hence the blood lactate/pyruvate ratio are characteristically increased. In addition to increased lactic acid, urine organic acid analysis may show increased Krebs cycle intermediates and dicarboxylic products of incompletely oxidized fatty acids, depending on dietary intake and the physiological state. Muscle biopsies are usually required in these disorders to examine mitochondrial structure and number and to provide tissue for biochemical analyses. Ideally, muscle biopsies should be performed at a facility equipped to prepare mitochondria to remove contaminating nonmitochondrial enzymes and to permit dissection of res333

KERR piratory function in the intact fresh mitochondria with a series of distinctive substrates; the isolated mitochondria can then be frozen for subsequent enzyme, immunological, or molecular analyses. Morphologically, proliferation of the number of mitochondria and the amount of the inner cristae within mitochondria may produce the characteristic "ragged-red" appearance of the Gomori stain or striking abnormalities apparent in the electron microscopic ultrastructure. F a t droplets and glycogen granules frequently accumulate. Specialized histochemical staining, immunocytochemistry, and immunoblotting with specific antibodies may facilitate biochemical analysis of affected complexes and protein subunits. In some disorders, especially Kearns-Sayre syndrome, Southern analysis of mitochondrial DNA has detected major deletions (41). Sequence analysis of mitochondrial DNA from patients with Leber's hereditary optic neuropathy has demonstrated a characteristic point mutation in one of the mitochondrially encoded subunits of complex I (42). Although cDNAs defining the primary structure for virtually all of the subunits of Complex IV (cytochrome c oxidase) have now been isolated (43,44), definition of mutations affecting the majority of nuclear-encoded subunits seems likely to remain a difficult process for the near future, since it is generally not obvious which subunit harbors the responsible mutation in patients with proven Complex IV deficiency. Prenatal diagnosis has not been reported for ETC defects. Effective therapy for these frequently devastating conditions is not available.

Summary and perspectives The recognition and diagnosis of increasing numbers of patients with inherited biochemical disorders associated with lactic acidosis presents a complex challenge to clinicians, pathologists, and research investigators. Clinical and routine metabolic characterization of these patients is usually insufficient to define the site of the defect. In some cases, specific syndromes have been associated with particular enzyme defects, but in other situations the same clinical syndrome has been associated with different biochemical defects or, conversely, deficiency of the same enzyme has been associated with very different clinical syndromes. Although definitive therapy is not available for many of these severe conditions, in certain cases specific treatment can be extremely effective. Specific diagnoses can also permit appropriate genetic counseling. A well-coordinated, broad-based diagnostic approach is necessary for sorting out most of these confusing disorders. The first step is through clinical assessment of skeletal muscle, cardiac, hepatic, and neurological function. This should be accompanied by routine laboratory screening tests to measure blood and CSF lactate and pyruvate repetitively under conditions of feeding and fasting, 334

as well as determination of plasma amino acids, urinary organic acids, and plasma and urinary carnitine. Abnormalities of these clinical and laboratory screening tests should be followed by assay of specific enzymes in cultured skin fibroblasts, lymphocytes, muscle biopsies, and serum (biotinidase). These tests are typically performed in specialized referral laboratories. At present, effective treatment strategies are lacking for most of these disorders. Therapeutic approaches which occasionally have shown definite or limited benefit in some patients include dietary modification to select fuels that circumvent the rate-limiting metabolic process (e.g., low carbohydrate diet), addition of specific enzyme cofactors that may eliminate enzyme activity (e.g., biotin) or bypass the block (e.g., vitamin K and ascorbate), and use of pharmacological agents that act as metabolic regulators (e.g., dichloroacetate). Future investigation of inherited disorders associated with lactic acidosis will require continued definition and correlation of the underlying functional, biochemical, and molecular pathophysiology. Once specific mutations are defined, simplified techniques (e.g., use of distinctive oligonucleotide probes) will be useful for rapidly screening suspected cases. In some cases, such as defects of the ETC components, development of more efficient techniques will be required for characterization of mutations. The long-range possibility of intervention by gene replacement would be facilitated by discovery or development of animal models of these disorders. In the short-term, it appears more likely that a gene-transfer strategy will be pursued with in vitro experiments using defective cells in culture.

Acknowledgements This work was supported in part by grants from the National Institutes of Health (AM-20478) and the Bureau of Maternal and Child Health Resources Development (MCJ-009122). The author is grateful to his colleagues and staff in the Center for Inherited Disorders of Energy Metabolism, especially Dr. Arthur Zinn, whose comments were very helpful during preparation of this manuscript, as well as Drs. Charles Hoppel, Mulchand Patel, Kou-Yi Tserng, and Isaiah Wexler, whose collaborative investigations have been critical in the development of a better understanding of these disorders.

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