The Clinical and Biochemical Implications of Pyruvate Carboxylase Deficiency DARRYL C. D E V I V O , MOREY W. HAYMOND, MARY P. LECKIE, YVONNE L. BUSSMANN, DAVID B. McDOUGAL, JR., AND ANTHONY S. PAGLIARA Edward Mallinckrodt Department of Pediatrics and the Department of Neurology and Neurosurgery (Neurology), and Pharmacology, Washington University School of Medicine and the Divisions of Neurology and Metabolism, St. Louis Children's Hospital, St. Louis, Missouri 63110 tions reflected a deficiency of hepatic pyruvate carboxylase. The apparent Km of hepatic citrate synthase for oxaloacetate was 4.6 /i.M. Calculated tissue oxaloacetate concentrations were 0.50-0.84 /i.M suggesting that tricarboxylic acid cycle activity was severely limited by the decreased availability of this substrate. An iv glucose tolerance test resulted in the paradoxical synthesis of ketone bodies. This observation, coupled with the intermittent hypercholesterolemia and the increased tissue acetyl-CoA concentrations, suggests that pyruvate carboxylase is important in modulating the fractional distribution of intracellular acetyl-CoA between the tricarboxylic acid cycle, the /3-hydroxy-^-methylglutaryl-CoA cycle (and the synthesis of cholesterol and ketone bodies), and fatty acid synthesis. Treatment in future cases might be directed toward increasing tissue concentrations of oxaloacetate. (J Clin Endocrinol Metab 45: 1281, 1977)
ABSTRACT. A 10 month old female infant was evaluated for severe lactic acidosis. Clinically she was well nourished and had a substantial amount of adipose tissue despite recurrent episodes of acidosis. Her psychomotor development was retarded, her movements were dystonic and generalized seizures punctuated her course. Metabolic abnormalities included elevated blood concentrations of lactate, pyruvate, /3-hydroxybutyrate, acetoacetate, alanine, proline and glycine, decreased blood concentrations of glutamine, aspartate, valine and citrate, and intermittent elevations of serum cholesterol. A trial on a high-fat diet worsened the clinical condition and intensified the ketoacidosis and hyperalaninemia. Analysis of hepatic tissue obtained by open biopsy revealed increased concentrations of lactate, alanine, acetyl-CoA and other short-chain acyl-CoA esters, and decreased concentrations of oxaloacetate, citrate, a-ketoglutarate, malate and aspartate. The blood and tissue metabolic perturba-
C
HRONIC infantile lactic acidosis is an unusual condition which may be associated with biochemical defects in hepatic gluconeogenesis, disturbances in pyruvate oxidation, organic acidemias and Leigh's subacute necrotizing encephalomyelopathy (SNE) (1). The controversy surrounding the causal relationship of pyruvate carboxylase deficiency to SNE has been summarized recently by Blass and Cederbaum (2). It would seem that the histopathological definition of SNE may be associated with Received February 14, 1977. Supported in part by the following grants: USPHS RR00036, AM 17904, NS 09808, HD 06355, NS 06800, Diabetes and Endocrinology Centers Grant AM-17094, and the Jerry Lewis Neuromuscular Disease Research Center Grant from the Muscular Dystrophy Association. Presented in part at the American Federation Meeting for Clinical Research, Chicago, Illinois, November 1976 (Clin Res 24: 526A, 1976). Reprint requests to: Dr. DeVivo, 500 South Kingshighway, St. Louis, Missouri 63178.
several different biochemical abnormalities, one of which is pyruvate carboxylase deficiency. Five cases of hepatic pyruvate carboxylase deficiency have been described. In 1968, Hommes et al. (3) described a one year old infant with elevated blood pyruvate and lactate concentrations who had three siblings with neuropathological lesions characteristic of SNE. An almost complete absence of pyruvate carboxylase activity was noted in tissue obtained from hepatic biopsy. A similar observation was made in a 6 month old infant by Tang et al. in 1972 (4) which also occurred in a clinical setting consistent with SNE. In that same year Grover and associates (5) reported a 38 month old child who died and exhibited brain lesions of SNE. Liver biopsy when this patient was 10 months old revealed normal pyruvate carboxylase activity, but the activity of this enzyme was absent in brain and liver tissues obtained at autopsy.
1281
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1282
D E V I V O ET
These investigators, therefore, concluded that pyruvate carboxylase was not the primary deficiency in SNE. Yoshida et al. (6) described a 10 year old mentally retarded female with hyperalaninemia and pyruvic acidemia who had a partial deficiency (20% of control activity) of hepatic pyruvate carboxylase. Finally, Brunette et al. (7) reported a female infant with hypoglycemia, pyruvic acidemia, lactic acidemia and hyperalaninemia who improved with thiamine treatment. In this patient the loss of enzyme activity was confined to the low Km component of hepatic pyruvate carboxylase (8). Scrutton and White (9) have suggested that this patient may have had a defect in pyruvic dehydrogenase activity. They suggested that the observations made in this patient may have reflected a deficit in acetyl-CoA formation from pyruvate in the liver homogenate resulting in a decrease in the steady-state concentration of acetyl-CoA in the assay system at any given pyruvate concentration. Maximal activity of pyruvate carboxylase is critically dependent on an optimal concentration of acetyl-CoA as a positive modifier for this enzyme. We are reporting our findings which have accrued from the study of a 10 month infant with chronic lactic acidosis. The circulating substrate abnormalities included increased concentrations of pyruvate, lactate, /3-hydroxybutyrate, acetoacetate, alanine, proline and glycine, and decreased concentrations of aspartate, glutamine, valine and citrate. An absolute deficiency of pyruvate carboxylase activity was demonstrated in hepatic tissue obtained by open biopsy. The results of these studies suggest that pyruvate carboxylase may play an important role in governing the tricarboxylic acid cycle rate and in influencing the fractional distribution of acetyl-CoA among the pathways involved in the synthesis of high energy phosphates, fatty acids, cholesterol and ketone bodies. Case Report The patient was a 3100 g product of a full term pregnancy of a 23 year old gravida 4, para 0,
AL.
JCE & M • 1977 Vol 45 • No 6
abortus 3, white female. The previous spontaneous abortions occurred during the first trimester of pregnancy. There was no consanguinity and the family history was negative for stillbirths or unexplained infant deaths. The patient developed respiratory distress at 8 h of age which spontaneously resolved. She subsequently did well until three months of age when she developed minor motor and generalized seizures and was admitted to a local hospital. Her evaluation at that time was unremarkable including normal electrolytes, glucose, calcium, phosphorus, urinary amino acids, electroencephalogram and skull radiographs. She was started on phenobarbital and diphenylhydantoin with subsequent improvement in her seizure disorder. At five months of age the patient was readmitted to her local hospital with stridor, dyspnea, ketonuria and metabolic acidosis. Her pH was 7.28, Pco2 22 torr, CO2 7 mEq/1, blood lactic acid 7.2 mM and uric acid 15.2 mg/dl. The patient was placed on a diet containing approximately 40% fat, 50% carbohydrate (glucose and maltose only), and 10% protein, plus 40 mEq/day of oral sodium bicarbonate. Her condition stabilized on this dietary regimen but she did not achieve normal neurological development. At 10 months of age the patient was readmitted with severe metabolic acidosis and status epilepticus and she was treated initially with large doses of iv sodium bicarbonate and anticonvulsants. She improved and was maintained on 40-60 mEq of NaHCO3 added daily to her formula. Subsequently she was transferred to St. Louis Children's Hospital. On admission she was noted to be an irritable, floppy infant with high pitched, incessant crying and Kussmaul respirations. She appeared well nourished with a length at the 3-10th percentile, weight (7.9 kg) at the 10th percentile, and a head circumference at the 40th percentile. Her abdomen was soft and there was no organomegaly. Neurologic examination revealed retarded development with poor head control, decreased muscle tone, and intermittent dystonic posturing of the upper extremities. Initial laboratory data revealed a normal complete blood count, chest x-ray, calcium, phosphorus, urea nitrogen, creatinine, ammonia, and liver function studies. The serum sodium concentration was 137 mEq/1, potassium 5.0 mEq/1, CO211.8 mEq/1, chloride 104 mEq/1, cholesterol 285 mg/dl, and uric acid 9.4 mg/dl. An 8 h fasting plasma glucose value was 75 mg/dl.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY Plasma amino acid screening revealed a high alaninc concentration (860 (AM). Marked elevations of lactate (11.2 mM), pyruvate (0.54 HIM), and ketone bodies (acetoacetate 1.4 mM, /3hydroxybutyrate 2.2 mM) were noted on arterial blood samples. There was no inhibition of the enzyme thiamine pyrophosphate-ATP phosphoryltransferase by the patient's urine performed on two separate samples (courtesy of Dr. J. H. Pincus). The electroencephalogram was abnormal due to posterior sharp waves and spike wave complexes. Because a defect in pyruvic dehydrogenase was considered, medium chain triglycerides were added to the formula to increase the fat content, and she was given thiamine 50 mg po bid. The patient's condition deteriorated and she developed further metabolic acidosis (arterial pH 7.20, serum CO, 5.5 mEq/1). Oral feedings were discontinued one week after admission and she was treated with large doses of iv sodium bicarbonate and glucose (650 mg/kg/h). Over the subsequent two days she experienced only mild improvement. The rate of glucose administration was reduced (330 mg/kg/h) and her condition improved further. She was then offered a formula with a caloric distribution of 50% CHO (maltose and glucose), 10% protein and 40% fat plus 50 mEq of sodium bicarbonate per day. The patient's metabolic responses during the first two weeks of hospitalization are detailed in the in vivo results section below. A second episode of severe metabolic acidosis occurred during an episode of enteropathic Escherichia coli diarrhea. She rapidly became acidemic (arterial pH 7.02, serum CO2 3.0 mEq/1) and developed status epilepticus. She was treated with antibiotics and anticonvulsants (Valium® and phenobarbital). As much as 280 mEq/day of iv sodium bicarbonate was required to maintain her arterial pH at 7.3 and serum CO2 > 14 mEq/1. Subsequently, she did well on a regular infant diet (50% CHO, 35% fat, 15% protein) and 90 mEq of sodium bicarbonate daily. An open muscle biopsy was performed without difficulty. However, following an open liver biopsy she again developed a metabolic acidosis (pH 7.23) which rapidly responded to large doses of iv sodium bicarbonate and dextrose. After she had made a full recovery and was 5 days postoperative, an iv glucose tolerance test was performed. By discharge at 13 months of age, she had established a normal weight gain and had some improvement in her neurologic status. She was a happy baby who could smile and coo.
1283
She had achieved good head control, and could stand with support. At home, she did well for one month. She then developed a final episode of severe metabolic acidosis, was admitted to her local hospital, and died. Examination of the central nervous system was specifically denied by the parents. Materials and Methods All studies were carried out in the Washington University Clinical Research Unit at St. Louis Children's Hospital after informed consent was obtained from the parents. In vivo studies Numerous venous cutdown procedures were performed prior to admission at St. Louis Children's Hospital precluded venous blood sampling. Two milliliters of arterial blood were obtained at each sampling. Two hundred fx\ of whole blood were immediately precipitated with an equal volume of cold 3M perchloric acid. The remaining blood was added to a tube containing EDTA and 1000 KlU/ml of Trasylol® (FBA Pharmaceuticals). The supernatant fractions, after centrifugation at 4 C, were stored at - 8 0 C until assayed. Blood lactate and pyruvate (10), /3-hydroxybutyrate and acetoacetate (11), and citrate (10) were assayed microfluorometrically on neutralized perchloric acid extracts of whole blood. Glucose (10), alanine (12), aspartate (10), glutamate and glutamine (13) were determined microfluorometrically on neutralized perchloric acid extracts of plasma. Insulin (14), glucagon (15), and growth hormone (16) were determined by radioimmunoassay and cortisol by protein binding assay (17). Quantitative amino acids were determined on a Beckman 119 amino acid analyzer utilizing standard physiologic methodology (18). An open muscle biopsy was performed on the 44th hospital day with the first sample rapidly frozen in liquid nitrogen for measurements of enzyme activities and metabolite concentrations and subsequent samples processed for enzyme histochemistry and electronmicroscopy. An open liver biopsy was performed on the 65th hospital day. The patient received only iv fluids for 14 h prior to surgery which contained 5% glucose and 60 mEq/1 sodium bicarbonate. This solution was continued intraoperatively and general anesthesia was accomplished by using a halothane-nitrous oxide-oxygen mixture. Loose sutures were placed in the liver prior to excision of the tissue.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1284
DEVIVO ET AL.
Approximately 5 sec elapsed from the time of first incision to immersion of the biopsy tissue in liquid nitrogen. A second biopsy was taken subsequently for histological and ultrastructural examination. An iv glucose tolerance test (0.75 g/kg BW) was performed through the venous cutdown catheter on the 70th hospital day, after 8 hours of fasting during which time the intravenous line was kept open by the infusion of normal saline. In vitro studies Reagents used for metabolite and enzyme assays were the highest quality commercially available. Lactic dehydrogenase and phosphotransacetylase were obtained from Worthington and Sigma Chemical Companies respectively: acetylCoA (lithium salt) from Pabst-Laboratory Biochemicals, and (1-MC) sodium pyruvate and (I4C) sodium bicarbonate from New England Nuclear Company. The frozen liver tissue was powdered under liquid nitrogen and maintained at - 8 0 C. A fraction of the powered tissue was homogenized in 9 volumes of hydrochloric acid-methanol-perchloric acid (19) for determinations of glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerol 3phosphate, pyruvate, lactate, oxaloacetate, citrate, malate, ATP, ADP, AMP, alanine, aspartate, and a-ketoglutarate according to the methods of Lowry and Passonneau (10). Tissue /3-hydroxybutyrate and acetoacetate (20), glutamate and glutamine (13) also were determined microfluorometrically. Coenzyme A and several of its esters were measured using a modification iof the phosphotransacetylase cycling method of/Stadtman, Novelli, and Lipmann (21, see also 22), coupled with methods designed to destroy selected CoA esters and free CoA specifically.1 The entire set of coenzyme A assays required only 20 to 30 mg of liver. The following enzymes were assayed in liver tissue homogenates according to the referenced methods: Lactic dehydrogenase (EC 1.1.1.27) in the direction of lactate formation (23), pyruvic dehydrogenase (EC 1.2.4.1) represented a modification (see below) of the methodology reported by several investigators (24-26), pyruvate carboxylase (EC 6.4.1.1) according to Adam and 1
McDougal, Jr., D. B., and R. Dargar, manuscript in preparation.
JCE & M • 1977 Vol 45 • No 6
Haynes (27) with minor modifications (see below), acetyl-CoA carboxylase (EC 6.4.1.2) (28), citrate synthase (EC 4.1.3.7) (29), malic dehydrogenase (EC 1.1.1.37) in the direction of oxaloacetate synthesis (30), /3-hydroxybutyric dehydrogenase (EC 1.1.1.30) in the direction of acetoacetate synthesis (31), and acetoacetyl-CoA thiolase (EC 2.3.1.9) by measuring the disappearance of the acetoacetyl-CoA-Mg++ complex (31). The particulate enzymes (except for pyruvic dehydrogenase) were solubilized by three consecutive freeze-thawing steps. This procedure did not influence significantly the activities of the soluble enzymes. The pyruvic dehydrogenase assay was miniaturized by adapting the assay to the methodology described for assaying glutamic acid decarboxylase (32). Frozen powdered liver tissue was homogenized at —IOC in 4 volumes of 20 mM potassium phosphate buffer (pH 7.0) containing 40% (v/v) glycerol using a motordriven pestle. The enzyme activity in this homogenate represented the active fraction (PDHactlve). A portion of the fresh homogenate was incubated at 37 C for 60 min with 10 mM MgCl2 and 1 mM CaCl2 to activate the enzyme (PDHtotai) fully. The inactive enzyme fraction was calculated from the difference between PDHtotal and PDH activc . The complete reaction mixture (15 /xl containing 13 fig tissue/ix\) was placed in a 5 x 60 mm microtest tube (internal diameter 3.4 mm (10)) which was maintained in a melting ice bath. The test tube was sealed with a 20 /xl ring of 2N sulfuric acid. The test tubes were transferred to a water bath at 37 C for 4 min and re-immersed in the melting ice bath to terminate the reaction. Preliminary studies demonstrated that the reaction was linear for at least 6 min. The test tubes were connected by polyethylene tubing to a second set of microtest tubes which contained 50 /xl NCS® (AmershamSearle) followed immediately thereafter by breaking the sulfuric acid ring to inactivate completely the reaction mixture. The H CO 2 was trapped in the NCS during a 120 min post-incubation at 37 C. The final concentrations of the reactants at incubation were 100 mM phosphate buffer (pH 8.0), 10 mM 2-mercaptoethanol, 6 mM NAD+, 2 mM thiamine pyrophosphate, 2 mM MgCl2, 1 mM coenzyme-A, and 1 mM (1-14C) pyruvate (specific radioactivity 0.5 //,Ci//u,mol); 15 units/ml lactic dehydrogenase and 12 units/ml phosphotransacetylase. The NCS was transferred quantitatively to a scintillation vial containing PPO-dimethyi POPOP counting fluid (32). Efficiency (90-95%) was determined by adding a known
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY amount of [14C]toluene as an internal standard. The assay was blanked by omitting coenzyme-A. The pyruvate carboxylase assay (27) was modified as follows: the frozen powdered liver was hand homogenized at 4 C in 9 volumes of 0.25M sucrose containing 10 mM Tris-HCl buffer (pH 7.4), 2 mM 2-mercaptoethanol and 0.7 mM acetylCoA; the homogenate was freeze-thawed x3 to disrupt the mitochondria; the acid stable radioactivity was counted in Aquasol® (New England Nuclear) using a known amount of [14C]toluene as an internal standard (efficiency 90-95%). The assay was blanked by omitting pyruvate. Skin biopsies were obtained from the patient, mother and father, minced and grown to confluence in petri dishes. The fibroblasts were harvested and stored in liquid nitrogen until regrown to a confluent stage for determinations of enzyme activities. The pyruvate carboxylase and acetyl-CoA carboxylase activities were determined on the fibroblast cells which had been mechanically scraped from the culture tube on the morning of assay. The preparation of the homogenates and the assay conditions were identical to the procedures described for the liver sample. Fibroblasts grown from a patient with Friedreich's ataxia were used as a disease control. Control patient data With the exception of citrate, all circulating substrate concentrations on normal controls were derived from previously published data from our laboratory (33). Blood citrate concentrations were compared to data obtained from an additional 16 disease control children following an overnight fast. The iv glucose tolerance test data were compared to a child being evaluated for ketotic hypoglycemia who had an intravenous glucose tolerance test (0.75 g/kg BW) performed after 20 h of fasting. Hepatic tissue was obtained from 2 children undergoing open liver biopsy for diagnostic purposes. One child (control #1) had partial deficiencies of hepatic phosphorylase and debrancher enzymes (a condition which has been reported previously (34)) and the other child (control #2) had idiopathic hepatic fibrosis. The former child (control #1) had normal circulating substrate concentrations and no evidence of a defect in hepatic gluconeogenesis. The liver tissue from this child with glycogen storage disease was rapidly excised and frozen in a manner identical to our patient and was utilized for the measurement of hepatic substrate concentrations and
1285
some enzymatic determinations. The other patient sample was frozen approximately 45 sec after excision and was utilized only for enzyme determinations and CoA ester determinations. Results In vivo studies Figure 1 depicts selected substrate concentrations together with the caloric distribution of consumed carbohydrate, fat and protein during the first two weeks of hospitalization. The fat content of the diet was increased progressively (30 g/day —»• 70 g/day) over the first seven days of hospitalization. During this period of study the plasma glucose (2.7-5.8 mM), blood lactate (9.4-11.9 mM), and blood pyruvate (268-534 /AM) concentrations remained relatively constant and the plasma alanine concentrations increased moderately (850-*>1200 (MM). Blood ketone bodies increased (/3-hydroxybutyrate 2.2^-5.7 mM, and acetoacetate 1.4—> 3.4 mM, B/A 1.57 -> 1.67) and intensified the metabolic acidosis (Fig. 1). During this period basal plasma glucagon concentrations were increased when compared to those of our overnight fasted children (272 vs. 134 ± 29 pg/ml, mean ± SEM), and increased further (—»421 pg/ml) with the administration of the high fat diet. However, the plasma insulin concentrations remained low ( than 98%) or exclusively localized within the mitochondrial compartment (9,48) and the enzyme has been identified in several rat tissues including high activities in liver, kidney, lactating mammary gland and adrenal gland, moderate activities in white adipose tissue, brain, heart and testes, and low activities in skeletal muscle and intestinal mucosa (49). It is the initial rate-limiting enzyme in hepatic gluconeogenesis (50) but its anaplerotic function and its role in lipogenesis and ketogenesis has been emphasized as well (46,50). We noted a significant deficiency of pyruvate carboxylase in the liver of our patient. The results of the mixing experiment (Table 5) militate against the presence of an endogenous inhibitor as an explanation for the lack of enzyme activity. Biotin was added to the homogenates for certain experiments and failed to alter the activity of the enzyme. Perhaps more meaningful in this regard was the demonstration of normal activity of hepatic and fibroblast acetyl-CoA carboxylase, also a biotin-dependent enzyme. We did not have the opportunity to determine whether pyruvate carboxylase was present in other body tissues, although the elevated muscle tissue lactate and pyruvate concentrations suggest the possibility that this enzyme was deficient in muscle also. TABLE 6. Fibroblast enzyme activity (nmol/min/g prot) Enzyme
Patient
Mother
Father
Control
Pyruvate carboxylase Acetyl CoA carboxylase
0 522
370 453
320 371
500 636
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY The possible relationship between hepatic pyruvate carboxylase deficiency in this patient and the histopathological diagnosis of SNE cannot be discussed further because the necropsy did not include examination of the central nervous system. Nevertheless, the clinical features describing our patient are not particularly consistent with the findings of SNE in this age group which have been reported in the literature (51) and two urine samples failed to contain any enzyme inhibitor (35). We were inpressed with the episodic nature of the illness, the well-nourished and chubby appearance of the infant and the intensity of the convulsions when she was acutely ill. One might speculate that the episodic nature of the illness might reflect the intracellular concentrations of oxaloacetate which presumably were dependent in large part upon the availability of alternative precursors other than pyruvate, namely aspartate, glutamate and glutamine. Whether the generalized convulsions represented a similar abnormality within the brain cannot be resolved from this case. It is worth noting, however, that pyruvate carboxylase is present within mammalian brain tissue. The generous deposits of sc fat were apparent in our patient at the time of muscle and hepatic biopsies. This observation is to be contrasted with the emaciated appearance of infants who have had lactic acidosis secondary to pyruvic dehydrogenase deficiency (52). In the latter group of patients with pyruvic dehydrogenase deficiency, it also has been speculated that pyruvate carboxylate activity may be impaired functionally because of decreased synthesis of acetyl-CoA from carbohydrate precursors (9). As has been stated earlier, the allosteric dependence of pyruvate carboxylase upon acetyl-CoA has been well established. Recently, it has been suggested that a high fat diet may have therapeutic value in patients with pyruvic dehydrogenase deficiency, a maneuver which theoretically should provide an alternative precursor for
1291
the intracellular synthesis of acetyl-CoA (52). However, in our patient with pyruvate carboxylase deficiency a high fat diet was poorly tolerated and precipitated an additional episode of severe metabolic acidemia. One might assume that mobilization of body fats during any infectious or catabolic stress also might worsen the patient's condition and explain, at least in part, the episodic nature of her clinical course. Direct measurements of the intracellular acetyl-CoA concentrations in our patient (Table 3) demonstrated a significant increase in this CoA ester. Similarly, the hepatic acetoacetyl-CoA and/or propionyl CoA and /3hydroxy-j8-methyl-glutaryl-CoA concentrations probably were elevated, although the contributions that other unidentified CoA esters make to these measurements have not been determined. The total CoA concentrations was greater in the patient than in the controls. Whether this value is outside the range of normal variation is unknown. However, when the concentrations of acetyl-CoA, other acid-soluble phosphotransacetylase reactive CoA esters, and "HMG" CoA are expressed as per cent of total CoA (Table 3), only the other acidsoluble phosphotransacetylase reactive CoA esters show a consistent, large increase in the patient. Clearly the measurements of several intracellular metabolites (Table 2) demonstrate a decrease in the tricarboxylic acid cycle intermediates, namely citrate, a-ketoglutarate and malate. Relative to the total CoA concentration, even succinyl-CoA seems somewhat reduced (Table 3). We have interpreted these observations to suggest that there was a functional impairment of citrate synthase activity consequent upon the decreased availability of oxaloacetate as a co-substrate. The major factors underlying the control of citrate synthase in vivo remain an unresolved problem (53) but certain conclusions can be derived from our study of this infant. The calculated hepatic oxaloacetate concentrations were approximately 1/10 the concentrations
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1292
D E V I V O ET
which were calculated in the control patient (Table 2) and approximately 1/10 of the apparent Km for citrate synthase (Fig. 3). The decreased availability of oxaloacetate, therefore, might be expected to impair tricarboxylic acid cycle activity by limiting the velocity of the condensing enzyme in the formation of citrate. Citrate formation would be linearly related to the limited availability of oxaloacetate at concentrations well below the apparent Km for this reaction. Furthermore, /3-oxidation of fatty acids would be expected to shift the intramitochondrial redox potential to a more reduced state and might further lower the oxaloacetate concentration by favoring malate synthesis by the malic dehydrogenase reaction (53). An additional potential factor which might contribute to decreased activity of citrate synthase is the elevation of the other acetyl-CoA esters. SuccinylCoA, acetoacetyl-CoA and propionyl-CoA each have been shown to compete with acetyl-CoA as a substrate for citrate synthase (53). In this regard and as shown in Table 3, other acid-soluble phosphotransacetylase reactive CoA esters (a category
AL.
JCE & M • 1977 Vol 45 • No 6
which includes acetoacetyl CoA and propionyl CoA) were significantly increased in an absolute and a relative sense. Despite considerable evidence for difficulties in the tricarboxylic acid cycle, ATP generation apparently was fast enough to meet the energy requirements of the liver (Table 2). Citrate has been viewed as the precursor for cytosolic acetyl-CoA, and as such, one might expect an impairment of fatty acid synthesis in the liver of our patient because of the observed decreased citrate concentration (Table 2). The generous deposits of sc fat, however, imply that additional mechanisms exist for the transfer of acetylCoA equivalents from the mitochondria to the cytosol (54—56). These references consider acetate, acetoacetate, /3-hydroxybutyrate, acetoacetyl-CoA and acetylcarnitine as potential precursors for cytoplasmic acetyl-CoA, particularly in adipose tissue. Alternatively, the pyruvate carboxylase activity may have been normal in our patient's adipocytes. Our patient appeared to shunt: a significant fraction of acetyl-CoA into the /3-hydroxy-/3-methyl-glutaryl-CoA cycle for the biosynthesis of cholesterol
10
FIG. 3. Lineweaver-Burke plot of citrate synthase activity and oxaloacetate concentration. The assay system was exactly as described by Srere (29). Ten /x\ of a 10% (w/v) homogenate was added to a cuvette at 37 C containing 2 ml final volume and the reaction was initiated with oxaloacetate.
.2
20
40
60 80 100 OXALOACETATE (/iM)
.6
500
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY and ketone bodies as measured by the persistent ketonemia and the intermittent elevations of the serum cholesterol concentrations. Similarly, the synthesis of ketone bodies following an iv glucose infusion (Fig. 2) reinforced our observation that the fractional distribution of acetyl-CoA into the /3-hydroxy-/3-methyl-glutaryl-CoA cycle was increased. We noted a gradual increase in the plasma concentrations of alanine (Fig. 1) which paralleled the increasing ketonemia when the patient was placed on a high fat diet. The plasma concentrations of insulin and glucagon remained relatively constant throughout this period. The parallel rise in alanine, /3-hydroxybutyrate and acetoacetate under these circumstances is at variance with the hypoalaninemia which has been observed in fasting adults (57) and in patients in whom ketone bodies have been infused (58). Conceivably, in our patient the high fat diet may have further expanded the intracellular acetyl-CoA pool and shifted the intramitochondrial redox potential to a more reduced state producing further inhibition of pyruvic dehydrogenase
FlG. 4. Schematic diagram depicting pertinent normal hepatic interrelationships between carbohydrate and lipid metabolism. The circled numbers indicate the enzymes which are reported in Table 4: 1) pyruvate carboxylase, 2) pyruvic dehydrogenase, 3) lactic dehydrogenase, 4) malic dehydrogenase, 5) citrate synthase, 6) /3-hydroxybutyric dehydrogenase. Acetyl-CoA is the pivotal metabolite linking oxidation, lipogenesis and ketogenesis.
1293
activity. Such a mechanism might lead to an increase in the intracellular pyruvate concentration and a parallel increase in alanine, explaining in part our observations. Pyruvate carboxylase activity has been reported casually to be present in human fibroblasts (59,60). We noted in fibroblasts about 3% of the enzyme activity found in the liver homogenates per unit protein and no activity in the patient's fibroblasts. The small amount of activity, however, in the control fibroblast homogenates makes diagnosis of this particular enzyme deficiency tenuous by this method. Hypoglycemia was conspicuously absent during the patient's life and this fact probably reflects the relative availability of alternative precursors for the synthesis of oxaloacetate, namely aspartate, glutamate and glutamine. However, she was fed at frequent intervals after 3 months of age and never was fasted for more than 8 h while under our care. Similarly, blood glucose concentrations were normal in two siblings with pyruvate carboxylase deficiency recently described by Saudubray et al. (61). In contrast, hepatic gluconeogenic defects
ACYL CoA
•-ACETOACETYL CoA ASPARTATE
TEROL
CO GLUTAMATE
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1294
D E V I V O ET
JCE & M • 1977 Vol 45 • No 6
AL.
GLUCOSE
ACYL CoA
ACETOACETYl CoA ASPARTATE
GLUTAMATE
at the level of phosphoenolpyruvate carboxykinase, fructose 1,6-diphosphatase and glucose-6-phosphatase have been demonstrated to be associated with profound hypoglycemia in the setting of lactic acidosis (62,63). It would appear, therefore, that the deficiency of pyruvate carboxylase is more crippling to the patient because of its importance as a governor of tricarboxylic acid cycle activity and as a modulator for the fractional distribution of acetyl-CoA into other metabolic pathways involved in lipogenesis and ketogenesis as schematically demonstrated in Figs. 4, 5. Our studies demonstrate that a diet relatively high in carbohydrate and protein and low in fat content is advisable for a patient with pyruvate carboxylase deficiency. The intolerance of a patient to a high fat diet, together with the abnormal plasma amino acid profile (increased alanine, glycine and proline, and decreased aspartate, glutamine and valine concentrations) and the persistent lactic and ketoacidosis should strongly suggest this diagnosis. As has been suggested by others (4), future patients with hepatic pyruvate carboxylase deficiency might benefit from appropriate
FIG. 5. Schematic diagram depicting the altered hepatic metabolism which results from a deficiency of pyruvate carboxylase. A decrease in pyruvate carboxylation (D limits the availability of oxaloacetate and is associated with a functional impairment in the activity of citrate synthase (D and decreased concentrations of tricarboxylic acid cycle metabolites. The decreased carboxylation of pyruvate increases transamination to alanine, reduction to lactate, and decarboxylation to acetyl-CoA. Increased concentrations of acetylCoA may stimulate fatty acid synthesis, and sterol and ketone body synthesis through the /3hydroxy-/3-methyl-glutaryl CoA cycle. Evidence of these perturbed metabolic sequences were observed in the blood and hepatic tissue of our patient.
dietary treatment perhaps supplemented with aspartic acid and/or glutamine in an effort to increase the intracellular concentrations of oxaloacetate. Acknowledgments We thank several people who assisted in the clinical care of the patient and in the laboratory studies which were performed: Drs. Bell, Brooke and Dodson for performing the liver, muscle and skin biopsies respectively; Dr. Nelson for ultrastructural examination of the muscle and liver biopsies; Dr. Volpe and Mrs. Marasa for culturing the fibroblasts and assisting us in setting up the acetyl-CoA carboxylase assay; Mrs. Strobel, Mrs. Brothers and Mr. Dallas for technical assistance; Mrs. Dargar for assisting in performing the CoA ester assays. The nursing personnel on the Clinical Research Unit and the pediatric housestaff were vitally important in the care of the patient. We also thank Mrs. Vicki O'Neill for her skilled secretarial assistance and Dr. Philip R. Dodge for his critical review of the manuscript.
References 1. Israels, S., J. C. Haworth, H. G. Dunn, and D. A. Applegarth, Lactic acidosis in childhood, Adv Pediatr 22: 267, 1975. 2. Blass, J. P., and S. D. Cederbaum, Biochemical abnormalities in Leigh's disease, Lancet 1: 1237, 1976.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY 3. Homines, F. A., H. A. Polman, and J. D. Reerink, Leigh's encephalopathy: an inborn error of gluconeogenesis, Arch Dis Child 43: 423, 1968. 4. Tang, T. T., T. A. Good, P. R. Dyken, S. D. Johnsen, S. R. McCreadie, S. T. Sy, H. A. Lardy, and F. B. Rudolph, Pathogenesis of Leigh's encephalopathy, J Pediatr 81: 189, 1972. 5. Grover, W. D., V. H. Auerbach, and M. S. Patel, Biochemical studies and therapy in subacute necrotizing encephalomyelopathy (Leigh's syndrome),; Pediatr 81: 39, 1972. 6. Yoshida, T., K. Tada, T. Konno, and T. Arakawa, Hyperalaninemia with pyruvicemia due to pyruvate carboxylase deficiency of the liver, TohokuJ Exp Med 99: 121, 1969. 7. Brunette, M. G., E. Delvin, B. Hazel, and C. R. Scriver, Thiamine-responsive lactic acidosis in a patient with deficient low-km pyruvate carboxylase activity in liver, Pediatrics 50: 702, 1972. 8. Delvin, E., C. R. Scriver, and J. L. Neal, Pyruvate carboxylase in human liver, Biochem Med 10: 97, 1974. 9. Scrutton, M. D., and M. D. White, Purification and properties of human liver pyruvate carboxylase, Biochem Med 9: 271, 1974. 10. Lowry, O. H., and J. V. Passonneau, Typical fluorometric procedures for metabolic assays, In A Flexible System of Enzymatic Analysis, Academic Press, New York, 1972, p. 68. 11. Cahill, Jr., G. F., M. G. Herrera, A. P. Morgan, J. Soeldner, J. Steinke, P. L. Levy, G. A. Richard, Jr., and D. M. Kipnis, Hormone-fuel inter-relationships during fasting,/ Clin Invest 45: 1751, 1966. 12. Karl, I. E., A. S. Pagliara, and D. M. Kipnis, A microfluorometric enzyme assay for the determination of alanine and pyruvate in plasma and tissue, 7 Lab Clin Med 80: 434, 1972. 13. Haymond, M. W., I. E. Karl, R. D. Feigin, D. C. DeVivo, and A. S. Pagliara, Hypoglycemia and maple syrup urine disease: Defective gluconeogenesis, Pediatr Res 7: 500, 1973. 14. Morgan, C. A., and A. Lazarow, Immunoassay of insulin: Two antibody system, Diabetes 12: 115, 1963. 15. Leichter, S. B., A. S. Pagliara, M. H. Grieder, S. Pohl, J. Rosai, and D. M. Kipnis, Uncontrolled diabetes mellitus and hyperglucagonemia associated with an islet cell carcinoma, Am J Med 48: 285, 1975. 16. Schalch, D. S., and M. L. Parker, A sensitive double antibody immunoassay for human growth hormone in plasma, Nature 203: 1141, 1964. 17. Beitins, I. Z., M. H. Shaw, A. Kowarski, and C. J. Migeon, Comparison of competitive protein binding radioassay of cortisol to double isotope dilution and Porter-Silber methods, Steroids 15: 765, 1970. 18. Dickinson, J. D., H. Rosenblum, and P. B. Hamilton, Ion exchange chromatography of free amino
19.
20.
21.
22. 23. 24.
25. 26.
27.
28.
29. 30. 31.
32.
33.
34.
1295
acids in the plasma of infants under 2500 grams at birth, Pediatrics 45: 606, 1970. Nelson, S. R., O. H. Lowry, and J. V. Passonneau, Changes in energy reserves in mouse brain associated with compressive head injury, In Caveness, W. F., and A. F. Walker (eds.), Head Injury Conference Proceedings, J. B. Lippincott Co., Philadelphia, 1966, p. 444. Williamson, D. H., J. Mellanby, and H. A. Krebs, Enzymic determination of D(-)-/3-hydroxybutyric acid and acetoacetic acid in blood, Biochem J 82: 90, 1962. Stadtman, E. R., G. D. Novelli, and F. Lipmann, Coenzyme A function in and acetyl transfer by the phosphotransacetylase system, J Biol Chem 191: 365, 1951. Novelli, G. D., Methods for determination of coenzyme A, Methods Biochem Anal 2: 189, 1955. Kornberg, A., Lactic dehydrogenase of muscle, Methods Enzymol 1: 441, 1955. Wieland, O., E. Seiss, F. H. Schulze-Wethmar, H. G. v. Funcke, and B. Winton, Active and inactive forms of pyruvate dehydrogenase in rat heart and kidney: Effects of diabetes, fasting and refeeding of pyruvate dehydrogenase interconversion, Arch Biochem Biophys 143: 593, 1971. Cremer, J. E., and H. M. Teal, The activity of pyruvate dehydrogenase in rat brain during postnatal development, FEBS Lett 39: 17, 1974. Jope, R., and J. P. Blass, A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver, Biochem J 150: 397, 1975. Adam, P. A. J., and R. C. Haynes, Jr., Control of hepatic mitochondrial CO2 fixation by glucagon, epinephrine, and cortison,/ Biol Chem 244: 6444, 1969. Volpe, J. J., and J. C. Marasa, Regulation of palmitic acid synthesis in cultured glial cells: Effects of lipid on fatty acid synthetase, acetylCoA carboxylase, fatty acid and sterol synthesis, J Neurochem 25: 330, 1975. Srere, P. A., Citrate synthase, Methods Enzymol 13: 3, 1969. Englard, S., and L. Siegel, Mitochondrial Lmalate dehydrogenase of beef heart, Methods Enzymol 13: 99, 1969. DeVivo, D. C , K. Fujimoto, M. P. Leckie, and H. C. Agrawal, Subcellular distribution of ketone body metabolizing enzymes in the rat brain, J Neurochem 26: 635, 1976. Sims, K. L., and F. N. Pitts, Jr., Brain glutamate decarboxylase: Changes in the developing rat brain J Neurochem 17: 1607, 1970. Haymond, M. W., I. E. Karl, and A. S. Pagliara, Ketotic hypoglycemia: an amino acid substrate limited disorder, J Clin Endocrinol Metab 38: 521, 1974. Hug, G., D. Harris, and J. A. Grunt, Two types of glycogenosis in the same girl: Combined de-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1296
35. 36.
37.
38.
39.
40.
41.
42. 43.
44.
45.
46.
47.
48.
49.
D E V I V O ET
ficiency of phosphorylase kinase in liver and debrancher in liver and muscle, Pediatr Res 10: 366, 1976 (Abstract). Pincus, J. H., J. R. Cooper, K. Piros, and V. Turner, Specificity of the urine inhibitor test for Leigh's disease, Neurology 24: 885, 1974. Harriman, D. G. F., and R. Reed, The incidence of lipid droplets in human skeletal muscle in neuromuscular disorders: A histochemical, electronmicroscopic and freeze-etch study, J Pathol 106: 1, 1972. Krebs, H. A., and R. L. Veech, Regulation of the redox state of the pyridine nucleotides in rat liver, In Sund, H., (ed.), Pyridine Nucleotide-Dependent Dehydrogenases, SpringerVerlag, Heidelberg, 1970, p. 413. Lovvry, O. H., N. J. Rosebrough, A. L. Fair, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. Srere, P. A., The citrate enzymes: The structures, mechanisms and biological functions, Curr Top Cell Regul 5: 229, 1972. Spector, L. B., Citrate cleavage and related enzymes, In Boyer, P. D. (ed.), The Enzymes, ed. 3, Academic Press, New York, 1972, p. 357. Utter, M. F., and D. B. Keech, Pyruvate carboxylase I: Nature of the reaction, J Biol Chem 238: 2603, 1963. Keech, D. B., and M. F. Utter, Pyruvate carboxylase II: Properties,; Biol Chem 238: 2609, 1963. McClure, W. R., H. A. Lardy, and H. P. Kneifel, Rat liver pyruvate carboxylase I. Preparation, properties and cation specificity,/ Biol Chem 246: 3569, 1971. McClure, W. R., H. A. Lardy, M. Wagner, and W. W. Cleland, Rat liver pyruvate carboxylase II. Kinetic studies of the forward reaction, J Biol Chem 246: 3579, 1971. McClure, W. R., H. A. Lardy, and W. W. Cleland, Rat liver pyruvate carboxylase III. Isotopic exchange studies of the first partial reaction, J Biol Chem 246: 3584, 1971. McClure, W. R., and H. A. Lardy, Rat liver pyruvate carboxylase IV. Factors affecting the regulation in vivo,] Biol Chem 246: 3591, 1971. Seufert, D., E. Herlemann, E. Albrecht, and W. Seubert, Purification and properties of pyruvate carboxylase from rat liver, Hoppe-Seylers Z Phtjsiol Chem 352: 459, 1971. Walter, P., Pyruvate carboxylase: Intracellular localization and regulation, In Hanson, R. W., and M. A. Mehlman (eds.), Gluconeogenesis: Its Regulation in Mammalian Species, John Wiley, New York, 1976, p. 239. Ballard, F. J., R. W. Hanson, and L. Reshef, Immunochemical studies with soluble and mito-
50.
51.
52.
53. 54.
55.
56.
57. 58.
59.
60.
61.
62.
63.
AL.
JCE & M • 1977 Vol 45 • No 6
chondrial pyruvate carboxylase activities from rat tissues, BiochemJ 119: 735, 1970. Barritt, G. J., G. L. Zander, and M. F. Utter, The regulation of pyruvate carboxylate activity in gluconeogenic tissues, In Hanson, R. W. and M. A. Mehlman (eds.), Gluconeogenesis: Its Regulation in Mammalian Species, John Wiley, New York, 1976, p. 3. Pincus, J. H., Subacute necrotizing encephalornyelopathy (Leigh's disease): a consideration of clinical features and etiology, Dev Med Child Neurol 14: 87, 1972. Falk, R. E., S. D. Cederbaum, J. P. Blass, G. E. Gibson, R. A. P. Kark, and R. E. Carrel, Ketonic diet in the management of pyruvate dehydrogenase deficiency, Pediatrics 58: 713, 1976. Weitzman, P. D. J., and M. J. Danson, Citrate synthase, Curr Top Cell Regul 10: 161, 1976. Porter, J. W., S. Kumar, and R. Dugan, Synthesis of fatty acids by enzymes of avian and mammalian species, Prog Biochem Pharmacol 6: 1, 1971. DeVivo, D. C , M. A. Fishman, and H. C. Agrawal, Preferential labelling of brain cholesterol by [3-14c]D(-)-3-hydroxybutyrate,Liptds 8: 649, 1973. Rous, S., Relative importance of acetate, acetoacetate and D-/3-OH-butyrate in the transport of acetyl CoA from the mitochondria to the cytoplasm for fatty acid synthesis in mice, Life Sci 18: 633, 1976. Sandek, C. D., and P. Felig, The metabolic events of starvation, Am J Med 60: 117, 1976, Sherwin, R. S., R. G. Hendler, and P. Felig, Effect of ketone infusions on amino acid and nitrogen metabolism in man, J Clin Invest 55: 1382, 1975. Stromme, J. H., O. Bornd, and P. J. Moe, Fatal lactic adidosis in a newborn attributable to a congenital defect of pyruvate dehydrogenase, Pediatr Res 10: 60, 1976. Willems, J. L., Discussion period, In Hommes, F. A., and C. J. Van den Berg (eds.), Normal and Pathological Development of Energy Metabolism, Academic Press, London 1975, p. 210. Saudubray, J. M., C. Marsac, C. Charpentier, L. Cathelineau, M. Besson Leaud, and J. P. Leroux, Neonatal congenital lactic acidosis with pyruvate carboxylase deficiency in two siblings, Ada Paediatr Scand 65: 717, 1976. Hommes, F. A., K. Bendien, J. D. Elema, H. J. Brenier, and I. Lombeck, Two cases of phosphoenolpyruvate carboxykinase deficiency, Ada Paediatr Scand 65: 233, 1976. Pagliara, A. S., I. E. Karl, J. P. Keating, B. I. Brown, and D. M. Kipnis, Hepatic fructose1,6-diphosphatase deficiency. A cause of lactic acidosis and hypoglycemia in infancy, J Clin Invest 51: 2115, 1972.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
ABSTRACT. A 10 month old female infant was evaluated for severe lactic acidosis. Clinically she was well nourished and had a substantial amount of adipose tissue despite recurrent episodes of acidosis. Her psychomotor development was retarded, her movements were dystonic and generalized seizures punctuated her course. Metabolic abnormalities included elevated blood concentrations of lactate, pyruvate, /3-hydroxybutyrate, acetoacetate, alanine, proline and glycine, decreased blood concentrations of glutamine, aspartate, valine and citrate, and intermittent elevations of serum cholesterol. A trial on a high-fat diet worsened the clinical condition and intensified the ketoacidosis and hyperalaninemia. Analysis of hepatic tissue obtained by open biopsy revealed increased concentrations of lactate, alanine, acetyl-CoA and other short-chain acyl-CoA esters, and decreased concentrations of oxaloacetate, citrate, a-ketoglutarate, malate and aspartate. The blood and tissue metabolic perturba-
C
HRONIC infantile lactic acidosis is an unusual condition which may be associated with biochemical defects in hepatic gluconeogenesis, disturbances in pyruvate oxidation, organic acidemias and Leigh's subacute necrotizing encephalomyelopathy (SNE) (1). The controversy surrounding the causal relationship of pyruvate carboxylase deficiency to SNE has been summarized recently by Blass and Cederbaum (2). It would seem that the histopathological definition of SNE may be associated with Received February 14, 1977. Supported in part by the following grants: USPHS RR00036, AM 17904, NS 09808, HD 06355, NS 06800, Diabetes and Endocrinology Centers Grant AM-17094, and the Jerry Lewis Neuromuscular Disease Research Center Grant from the Muscular Dystrophy Association. Presented in part at the American Federation Meeting for Clinical Research, Chicago, Illinois, November 1976 (Clin Res 24: 526A, 1976). Reprint requests to: Dr. DeVivo, 500 South Kingshighway, St. Louis, Missouri 63178.
several different biochemical abnormalities, one of which is pyruvate carboxylase deficiency. Five cases of hepatic pyruvate carboxylase deficiency have been described. In 1968, Hommes et al. (3) described a one year old infant with elevated blood pyruvate and lactate concentrations who had three siblings with neuropathological lesions characteristic of SNE. An almost complete absence of pyruvate carboxylase activity was noted in tissue obtained from hepatic biopsy. A similar observation was made in a 6 month old infant by Tang et al. in 1972 (4) which also occurred in a clinical setting consistent with SNE. In that same year Grover and associates (5) reported a 38 month old child who died and exhibited brain lesions of SNE. Liver biopsy when this patient was 10 months old revealed normal pyruvate carboxylase activity, but the activity of this enzyme was absent in brain and liver tissues obtained at autopsy.
1281
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1282
D E V I V O ET
These investigators, therefore, concluded that pyruvate carboxylase was not the primary deficiency in SNE. Yoshida et al. (6) described a 10 year old mentally retarded female with hyperalaninemia and pyruvic acidemia who had a partial deficiency (20% of control activity) of hepatic pyruvate carboxylase. Finally, Brunette et al. (7) reported a female infant with hypoglycemia, pyruvic acidemia, lactic acidemia and hyperalaninemia who improved with thiamine treatment. In this patient the loss of enzyme activity was confined to the low Km component of hepatic pyruvate carboxylase (8). Scrutton and White (9) have suggested that this patient may have had a defect in pyruvic dehydrogenase activity. They suggested that the observations made in this patient may have reflected a deficit in acetyl-CoA formation from pyruvate in the liver homogenate resulting in a decrease in the steady-state concentration of acetyl-CoA in the assay system at any given pyruvate concentration. Maximal activity of pyruvate carboxylase is critically dependent on an optimal concentration of acetyl-CoA as a positive modifier for this enzyme. We are reporting our findings which have accrued from the study of a 10 month infant with chronic lactic acidosis. The circulating substrate abnormalities included increased concentrations of pyruvate, lactate, /3-hydroxybutyrate, acetoacetate, alanine, proline and glycine, and decreased concentrations of aspartate, glutamine, valine and citrate. An absolute deficiency of pyruvate carboxylase activity was demonstrated in hepatic tissue obtained by open biopsy. The results of these studies suggest that pyruvate carboxylase may play an important role in governing the tricarboxylic acid cycle rate and in influencing the fractional distribution of acetyl-CoA among the pathways involved in the synthesis of high energy phosphates, fatty acids, cholesterol and ketone bodies. Case Report The patient was a 3100 g product of a full term pregnancy of a 23 year old gravida 4, para 0,
AL.
JCE & M • 1977 Vol 45 • No 6
abortus 3, white female. The previous spontaneous abortions occurred during the first trimester of pregnancy. There was no consanguinity and the family history was negative for stillbirths or unexplained infant deaths. The patient developed respiratory distress at 8 h of age which spontaneously resolved. She subsequently did well until three months of age when she developed minor motor and generalized seizures and was admitted to a local hospital. Her evaluation at that time was unremarkable including normal electrolytes, glucose, calcium, phosphorus, urinary amino acids, electroencephalogram and skull radiographs. She was started on phenobarbital and diphenylhydantoin with subsequent improvement in her seizure disorder. At five months of age the patient was readmitted to her local hospital with stridor, dyspnea, ketonuria and metabolic acidosis. Her pH was 7.28, Pco2 22 torr, CO2 7 mEq/1, blood lactic acid 7.2 mM and uric acid 15.2 mg/dl. The patient was placed on a diet containing approximately 40% fat, 50% carbohydrate (glucose and maltose only), and 10% protein, plus 40 mEq/day of oral sodium bicarbonate. Her condition stabilized on this dietary regimen but she did not achieve normal neurological development. At 10 months of age the patient was readmitted with severe metabolic acidosis and status epilepticus and she was treated initially with large doses of iv sodium bicarbonate and anticonvulsants. She improved and was maintained on 40-60 mEq of NaHCO3 added daily to her formula. Subsequently she was transferred to St. Louis Children's Hospital. On admission she was noted to be an irritable, floppy infant with high pitched, incessant crying and Kussmaul respirations. She appeared well nourished with a length at the 3-10th percentile, weight (7.9 kg) at the 10th percentile, and a head circumference at the 40th percentile. Her abdomen was soft and there was no organomegaly. Neurologic examination revealed retarded development with poor head control, decreased muscle tone, and intermittent dystonic posturing of the upper extremities. Initial laboratory data revealed a normal complete blood count, chest x-ray, calcium, phosphorus, urea nitrogen, creatinine, ammonia, and liver function studies. The serum sodium concentration was 137 mEq/1, potassium 5.0 mEq/1, CO211.8 mEq/1, chloride 104 mEq/1, cholesterol 285 mg/dl, and uric acid 9.4 mg/dl. An 8 h fasting plasma glucose value was 75 mg/dl.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY Plasma amino acid screening revealed a high alaninc concentration (860 (AM). Marked elevations of lactate (11.2 mM), pyruvate (0.54 HIM), and ketone bodies (acetoacetate 1.4 mM, /3hydroxybutyrate 2.2 mM) were noted on arterial blood samples. There was no inhibition of the enzyme thiamine pyrophosphate-ATP phosphoryltransferase by the patient's urine performed on two separate samples (courtesy of Dr. J. H. Pincus). The electroencephalogram was abnormal due to posterior sharp waves and spike wave complexes. Because a defect in pyruvic dehydrogenase was considered, medium chain triglycerides were added to the formula to increase the fat content, and she was given thiamine 50 mg po bid. The patient's condition deteriorated and she developed further metabolic acidosis (arterial pH 7.20, serum CO, 5.5 mEq/1). Oral feedings were discontinued one week after admission and she was treated with large doses of iv sodium bicarbonate and glucose (650 mg/kg/h). Over the subsequent two days she experienced only mild improvement. The rate of glucose administration was reduced (330 mg/kg/h) and her condition improved further. She was then offered a formula with a caloric distribution of 50% CHO (maltose and glucose), 10% protein and 40% fat plus 50 mEq of sodium bicarbonate per day. The patient's metabolic responses during the first two weeks of hospitalization are detailed in the in vivo results section below. A second episode of severe metabolic acidosis occurred during an episode of enteropathic Escherichia coli diarrhea. She rapidly became acidemic (arterial pH 7.02, serum CO2 3.0 mEq/1) and developed status epilepticus. She was treated with antibiotics and anticonvulsants (Valium® and phenobarbital). As much as 280 mEq/day of iv sodium bicarbonate was required to maintain her arterial pH at 7.3 and serum CO2 > 14 mEq/1. Subsequently, she did well on a regular infant diet (50% CHO, 35% fat, 15% protein) and 90 mEq of sodium bicarbonate daily. An open muscle biopsy was performed without difficulty. However, following an open liver biopsy she again developed a metabolic acidosis (pH 7.23) which rapidly responded to large doses of iv sodium bicarbonate and dextrose. After she had made a full recovery and was 5 days postoperative, an iv glucose tolerance test was performed. By discharge at 13 months of age, she had established a normal weight gain and had some improvement in her neurologic status. She was a happy baby who could smile and coo.
1283
She had achieved good head control, and could stand with support. At home, she did well for one month. She then developed a final episode of severe metabolic acidosis, was admitted to her local hospital, and died. Examination of the central nervous system was specifically denied by the parents. Materials and Methods All studies were carried out in the Washington University Clinical Research Unit at St. Louis Children's Hospital after informed consent was obtained from the parents. In vivo studies Numerous venous cutdown procedures were performed prior to admission at St. Louis Children's Hospital precluded venous blood sampling. Two milliliters of arterial blood were obtained at each sampling. Two hundred fx\ of whole blood were immediately precipitated with an equal volume of cold 3M perchloric acid. The remaining blood was added to a tube containing EDTA and 1000 KlU/ml of Trasylol® (FBA Pharmaceuticals). The supernatant fractions, after centrifugation at 4 C, were stored at - 8 0 C until assayed. Blood lactate and pyruvate (10), /3-hydroxybutyrate and acetoacetate (11), and citrate (10) were assayed microfluorometrically on neutralized perchloric acid extracts of whole blood. Glucose (10), alanine (12), aspartate (10), glutamate and glutamine (13) were determined microfluorometrically on neutralized perchloric acid extracts of plasma. Insulin (14), glucagon (15), and growth hormone (16) were determined by radioimmunoassay and cortisol by protein binding assay (17). Quantitative amino acids were determined on a Beckman 119 amino acid analyzer utilizing standard physiologic methodology (18). An open muscle biopsy was performed on the 44th hospital day with the first sample rapidly frozen in liquid nitrogen for measurements of enzyme activities and metabolite concentrations and subsequent samples processed for enzyme histochemistry and electronmicroscopy. An open liver biopsy was performed on the 65th hospital day. The patient received only iv fluids for 14 h prior to surgery which contained 5% glucose and 60 mEq/1 sodium bicarbonate. This solution was continued intraoperatively and general anesthesia was accomplished by using a halothane-nitrous oxide-oxygen mixture. Loose sutures were placed in the liver prior to excision of the tissue.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1284
DEVIVO ET AL.
Approximately 5 sec elapsed from the time of first incision to immersion of the biopsy tissue in liquid nitrogen. A second biopsy was taken subsequently for histological and ultrastructural examination. An iv glucose tolerance test (0.75 g/kg BW) was performed through the venous cutdown catheter on the 70th hospital day, after 8 hours of fasting during which time the intravenous line was kept open by the infusion of normal saline. In vitro studies Reagents used for metabolite and enzyme assays were the highest quality commercially available. Lactic dehydrogenase and phosphotransacetylase were obtained from Worthington and Sigma Chemical Companies respectively: acetylCoA (lithium salt) from Pabst-Laboratory Biochemicals, and (1-MC) sodium pyruvate and (I4C) sodium bicarbonate from New England Nuclear Company. The frozen liver tissue was powdered under liquid nitrogen and maintained at - 8 0 C. A fraction of the powered tissue was homogenized in 9 volumes of hydrochloric acid-methanol-perchloric acid (19) for determinations of glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerol 3phosphate, pyruvate, lactate, oxaloacetate, citrate, malate, ATP, ADP, AMP, alanine, aspartate, and a-ketoglutarate according to the methods of Lowry and Passonneau (10). Tissue /3-hydroxybutyrate and acetoacetate (20), glutamate and glutamine (13) also were determined microfluorometrically. Coenzyme A and several of its esters were measured using a modification iof the phosphotransacetylase cycling method of/Stadtman, Novelli, and Lipmann (21, see also 22), coupled with methods designed to destroy selected CoA esters and free CoA specifically.1 The entire set of coenzyme A assays required only 20 to 30 mg of liver. The following enzymes were assayed in liver tissue homogenates according to the referenced methods: Lactic dehydrogenase (EC 1.1.1.27) in the direction of lactate formation (23), pyruvic dehydrogenase (EC 1.2.4.1) represented a modification (see below) of the methodology reported by several investigators (24-26), pyruvate carboxylase (EC 6.4.1.1) according to Adam and 1
McDougal, Jr., D. B., and R. Dargar, manuscript in preparation.
JCE & M • 1977 Vol 45 • No 6
Haynes (27) with minor modifications (see below), acetyl-CoA carboxylase (EC 6.4.1.2) (28), citrate synthase (EC 4.1.3.7) (29), malic dehydrogenase (EC 1.1.1.37) in the direction of oxaloacetate synthesis (30), /3-hydroxybutyric dehydrogenase (EC 1.1.1.30) in the direction of acetoacetate synthesis (31), and acetoacetyl-CoA thiolase (EC 2.3.1.9) by measuring the disappearance of the acetoacetyl-CoA-Mg++ complex (31). The particulate enzymes (except for pyruvic dehydrogenase) were solubilized by three consecutive freeze-thawing steps. This procedure did not influence significantly the activities of the soluble enzymes. The pyruvic dehydrogenase assay was miniaturized by adapting the assay to the methodology described for assaying glutamic acid decarboxylase (32). Frozen powdered liver tissue was homogenized at —IOC in 4 volumes of 20 mM potassium phosphate buffer (pH 7.0) containing 40% (v/v) glycerol using a motordriven pestle. The enzyme activity in this homogenate represented the active fraction (PDHactlve). A portion of the fresh homogenate was incubated at 37 C for 60 min with 10 mM MgCl2 and 1 mM CaCl2 to activate the enzyme (PDHtotai) fully. The inactive enzyme fraction was calculated from the difference between PDHtotal and PDH activc . The complete reaction mixture (15 /xl containing 13 fig tissue/ix\) was placed in a 5 x 60 mm microtest tube (internal diameter 3.4 mm (10)) which was maintained in a melting ice bath. The test tube was sealed with a 20 /xl ring of 2N sulfuric acid. The test tubes were transferred to a water bath at 37 C for 4 min and re-immersed in the melting ice bath to terminate the reaction. Preliminary studies demonstrated that the reaction was linear for at least 6 min. The test tubes were connected by polyethylene tubing to a second set of microtest tubes which contained 50 /xl NCS® (AmershamSearle) followed immediately thereafter by breaking the sulfuric acid ring to inactivate completely the reaction mixture. The H CO 2 was trapped in the NCS during a 120 min post-incubation at 37 C. The final concentrations of the reactants at incubation were 100 mM phosphate buffer (pH 8.0), 10 mM 2-mercaptoethanol, 6 mM NAD+, 2 mM thiamine pyrophosphate, 2 mM MgCl2, 1 mM coenzyme-A, and 1 mM (1-14C) pyruvate (specific radioactivity 0.5 //,Ci//u,mol); 15 units/ml lactic dehydrogenase and 12 units/ml phosphotransacetylase. The NCS was transferred quantitatively to a scintillation vial containing PPO-dimethyi POPOP counting fluid (32). Efficiency (90-95%) was determined by adding a known
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY amount of [14C]toluene as an internal standard. The assay was blanked by omitting coenzyme-A. The pyruvate carboxylase assay (27) was modified as follows: the frozen powdered liver was hand homogenized at 4 C in 9 volumes of 0.25M sucrose containing 10 mM Tris-HCl buffer (pH 7.4), 2 mM 2-mercaptoethanol and 0.7 mM acetylCoA; the homogenate was freeze-thawed x3 to disrupt the mitochondria; the acid stable radioactivity was counted in Aquasol® (New England Nuclear) using a known amount of [14C]toluene as an internal standard (efficiency 90-95%). The assay was blanked by omitting pyruvate. Skin biopsies were obtained from the patient, mother and father, minced and grown to confluence in petri dishes. The fibroblasts were harvested and stored in liquid nitrogen until regrown to a confluent stage for determinations of enzyme activities. The pyruvate carboxylase and acetyl-CoA carboxylase activities were determined on the fibroblast cells which had been mechanically scraped from the culture tube on the morning of assay. The preparation of the homogenates and the assay conditions were identical to the procedures described for the liver sample. Fibroblasts grown from a patient with Friedreich's ataxia were used as a disease control. Control patient data With the exception of citrate, all circulating substrate concentrations on normal controls were derived from previously published data from our laboratory (33). Blood citrate concentrations were compared to data obtained from an additional 16 disease control children following an overnight fast. The iv glucose tolerance test data were compared to a child being evaluated for ketotic hypoglycemia who had an intravenous glucose tolerance test (0.75 g/kg BW) performed after 20 h of fasting. Hepatic tissue was obtained from 2 children undergoing open liver biopsy for diagnostic purposes. One child (control #1) had partial deficiencies of hepatic phosphorylase and debrancher enzymes (a condition which has been reported previously (34)) and the other child (control #2) had idiopathic hepatic fibrosis. The former child (control #1) had normal circulating substrate concentrations and no evidence of a defect in hepatic gluconeogenesis. The liver tissue from this child with glycogen storage disease was rapidly excised and frozen in a manner identical to our patient and was utilized for the measurement of hepatic substrate concentrations and
1285
some enzymatic determinations. The other patient sample was frozen approximately 45 sec after excision and was utilized only for enzyme determinations and CoA ester determinations. Results In vivo studies Figure 1 depicts selected substrate concentrations together with the caloric distribution of consumed carbohydrate, fat and protein during the first two weeks of hospitalization. The fat content of the diet was increased progressively (30 g/day —»• 70 g/day) over the first seven days of hospitalization. During this period of study the plasma glucose (2.7-5.8 mM), blood lactate (9.4-11.9 mM), and blood pyruvate (268-534 /AM) concentrations remained relatively constant and the plasma alanine concentrations increased moderately (850-*>1200 (MM). Blood ketone bodies increased (/3-hydroxybutyrate 2.2^-5.7 mM, and acetoacetate 1.4—> 3.4 mM, B/A 1.57 -> 1.67) and intensified the metabolic acidosis (Fig. 1). During this period basal plasma glucagon concentrations were increased when compared to those of our overnight fasted children (272 vs. 134 ± 29 pg/ml, mean ± SEM), and increased further (—»421 pg/ml) with the administration of the high fat diet. However, the plasma insulin concentrations remained low ( than 98%) or exclusively localized within the mitochondrial compartment (9,48) and the enzyme has been identified in several rat tissues including high activities in liver, kidney, lactating mammary gland and adrenal gland, moderate activities in white adipose tissue, brain, heart and testes, and low activities in skeletal muscle and intestinal mucosa (49). It is the initial rate-limiting enzyme in hepatic gluconeogenesis (50) but its anaplerotic function and its role in lipogenesis and ketogenesis has been emphasized as well (46,50). We noted a significant deficiency of pyruvate carboxylase in the liver of our patient. The results of the mixing experiment (Table 5) militate against the presence of an endogenous inhibitor as an explanation for the lack of enzyme activity. Biotin was added to the homogenates for certain experiments and failed to alter the activity of the enzyme. Perhaps more meaningful in this regard was the demonstration of normal activity of hepatic and fibroblast acetyl-CoA carboxylase, also a biotin-dependent enzyme. We did not have the opportunity to determine whether pyruvate carboxylase was present in other body tissues, although the elevated muscle tissue lactate and pyruvate concentrations suggest the possibility that this enzyme was deficient in muscle also. TABLE 6. Fibroblast enzyme activity (nmol/min/g prot) Enzyme
Patient
Mother
Father
Control
Pyruvate carboxylase Acetyl CoA carboxylase
0 522
370 453
320 371
500 636
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY The possible relationship between hepatic pyruvate carboxylase deficiency in this patient and the histopathological diagnosis of SNE cannot be discussed further because the necropsy did not include examination of the central nervous system. Nevertheless, the clinical features describing our patient are not particularly consistent with the findings of SNE in this age group which have been reported in the literature (51) and two urine samples failed to contain any enzyme inhibitor (35). We were inpressed with the episodic nature of the illness, the well-nourished and chubby appearance of the infant and the intensity of the convulsions when she was acutely ill. One might speculate that the episodic nature of the illness might reflect the intracellular concentrations of oxaloacetate which presumably were dependent in large part upon the availability of alternative precursors other than pyruvate, namely aspartate, glutamate and glutamine. Whether the generalized convulsions represented a similar abnormality within the brain cannot be resolved from this case. It is worth noting, however, that pyruvate carboxylase is present within mammalian brain tissue. The generous deposits of sc fat were apparent in our patient at the time of muscle and hepatic biopsies. This observation is to be contrasted with the emaciated appearance of infants who have had lactic acidosis secondary to pyruvic dehydrogenase deficiency (52). In the latter group of patients with pyruvic dehydrogenase deficiency, it also has been speculated that pyruvate carboxylate activity may be impaired functionally because of decreased synthesis of acetyl-CoA from carbohydrate precursors (9). As has been stated earlier, the allosteric dependence of pyruvate carboxylase upon acetyl-CoA has been well established. Recently, it has been suggested that a high fat diet may have therapeutic value in patients with pyruvic dehydrogenase deficiency, a maneuver which theoretically should provide an alternative precursor for
1291
the intracellular synthesis of acetyl-CoA (52). However, in our patient with pyruvate carboxylase deficiency a high fat diet was poorly tolerated and precipitated an additional episode of severe metabolic acidemia. One might assume that mobilization of body fats during any infectious or catabolic stress also might worsen the patient's condition and explain, at least in part, the episodic nature of her clinical course. Direct measurements of the intracellular acetyl-CoA concentrations in our patient (Table 3) demonstrated a significant increase in this CoA ester. Similarly, the hepatic acetoacetyl-CoA and/or propionyl CoA and /3hydroxy-j8-methyl-glutaryl-CoA concentrations probably were elevated, although the contributions that other unidentified CoA esters make to these measurements have not been determined. The total CoA concentrations was greater in the patient than in the controls. Whether this value is outside the range of normal variation is unknown. However, when the concentrations of acetyl-CoA, other acid-soluble phosphotransacetylase reactive CoA esters, and "HMG" CoA are expressed as per cent of total CoA (Table 3), only the other acidsoluble phosphotransacetylase reactive CoA esters show a consistent, large increase in the patient. Clearly the measurements of several intracellular metabolites (Table 2) demonstrate a decrease in the tricarboxylic acid cycle intermediates, namely citrate, a-ketoglutarate and malate. Relative to the total CoA concentration, even succinyl-CoA seems somewhat reduced (Table 3). We have interpreted these observations to suggest that there was a functional impairment of citrate synthase activity consequent upon the decreased availability of oxaloacetate as a co-substrate. The major factors underlying the control of citrate synthase in vivo remain an unresolved problem (53) but certain conclusions can be derived from our study of this infant. The calculated hepatic oxaloacetate concentrations were approximately 1/10 the concentrations
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1292
D E V I V O ET
which were calculated in the control patient (Table 2) and approximately 1/10 of the apparent Km for citrate synthase (Fig. 3). The decreased availability of oxaloacetate, therefore, might be expected to impair tricarboxylic acid cycle activity by limiting the velocity of the condensing enzyme in the formation of citrate. Citrate formation would be linearly related to the limited availability of oxaloacetate at concentrations well below the apparent Km for this reaction. Furthermore, /3-oxidation of fatty acids would be expected to shift the intramitochondrial redox potential to a more reduced state and might further lower the oxaloacetate concentration by favoring malate synthesis by the malic dehydrogenase reaction (53). An additional potential factor which might contribute to decreased activity of citrate synthase is the elevation of the other acetyl-CoA esters. SuccinylCoA, acetoacetyl-CoA and propionyl-CoA each have been shown to compete with acetyl-CoA as a substrate for citrate synthase (53). In this regard and as shown in Table 3, other acid-soluble phosphotransacetylase reactive CoA esters (a category
AL.
JCE & M • 1977 Vol 45 • No 6
which includes acetoacetyl CoA and propionyl CoA) were significantly increased in an absolute and a relative sense. Despite considerable evidence for difficulties in the tricarboxylic acid cycle, ATP generation apparently was fast enough to meet the energy requirements of the liver (Table 2). Citrate has been viewed as the precursor for cytosolic acetyl-CoA, and as such, one might expect an impairment of fatty acid synthesis in the liver of our patient because of the observed decreased citrate concentration (Table 2). The generous deposits of sc fat, however, imply that additional mechanisms exist for the transfer of acetylCoA equivalents from the mitochondria to the cytosol (54—56). These references consider acetate, acetoacetate, /3-hydroxybutyrate, acetoacetyl-CoA and acetylcarnitine as potential precursors for cytoplasmic acetyl-CoA, particularly in adipose tissue. Alternatively, the pyruvate carboxylase activity may have been normal in our patient's adipocytes. Our patient appeared to shunt: a significant fraction of acetyl-CoA into the /3-hydroxy-/3-methyl-glutaryl-CoA cycle for the biosynthesis of cholesterol
10
FIG. 3. Lineweaver-Burke plot of citrate synthase activity and oxaloacetate concentration. The assay system was exactly as described by Srere (29). Ten /x\ of a 10% (w/v) homogenate was added to a cuvette at 37 C containing 2 ml final volume and the reaction was initiated with oxaloacetate.
.2
20
40
60 80 100 OXALOACETATE (/iM)
.6
500
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY and ketone bodies as measured by the persistent ketonemia and the intermittent elevations of the serum cholesterol concentrations. Similarly, the synthesis of ketone bodies following an iv glucose infusion (Fig. 2) reinforced our observation that the fractional distribution of acetyl-CoA into the /3-hydroxy-/3-methyl-glutaryl-CoA cycle was increased. We noted a gradual increase in the plasma concentrations of alanine (Fig. 1) which paralleled the increasing ketonemia when the patient was placed on a high fat diet. The plasma concentrations of insulin and glucagon remained relatively constant throughout this period. The parallel rise in alanine, /3-hydroxybutyrate and acetoacetate under these circumstances is at variance with the hypoalaninemia which has been observed in fasting adults (57) and in patients in whom ketone bodies have been infused (58). Conceivably, in our patient the high fat diet may have further expanded the intracellular acetyl-CoA pool and shifted the intramitochondrial redox potential to a more reduced state producing further inhibition of pyruvic dehydrogenase
FlG. 4. Schematic diagram depicting pertinent normal hepatic interrelationships between carbohydrate and lipid metabolism. The circled numbers indicate the enzymes which are reported in Table 4: 1) pyruvate carboxylase, 2) pyruvic dehydrogenase, 3) lactic dehydrogenase, 4) malic dehydrogenase, 5) citrate synthase, 6) /3-hydroxybutyric dehydrogenase. Acetyl-CoA is the pivotal metabolite linking oxidation, lipogenesis and ketogenesis.
1293
activity. Such a mechanism might lead to an increase in the intracellular pyruvate concentration and a parallel increase in alanine, explaining in part our observations. Pyruvate carboxylase activity has been reported casually to be present in human fibroblasts (59,60). We noted in fibroblasts about 3% of the enzyme activity found in the liver homogenates per unit protein and no activity in the patient's fibroblasts. The small amount of activity, however, in the control fibroblast homogenates makes diagnosis of this particular enzyme deficiency tenuous by this method. Hypoglycemia was conspicuously absent during the patient's life and this fact probably reflects the relative availability of alternative precursors for the synthesis of oxaloacetate, namely aspartate, glutamate and glutamine. However, she was fed at frequent intervals after 3 months of age and never was fasted for more than 8 h while under our care. Similarly, blood glucose concentrations were normal in two siblings with pyruvate carboxylase deficiency recently described by Saudubray et al. (61). In contrast, hepatic gluconeogenic defects
ACYL CoA
•-ACETOACETYL CoA ASPARTATE
TEROL
CO GLUTAMATE
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1294
D E V I V O ET
JCE & M • 1977 Vol 45 • No 6
AL.
GLUCOSE
ACYL CoA
ACETOACETYl CoA ASPARTATE
GLUTAMATE
at the level of phosphoenolpyruvate carboxykinase, fructose 1,6-diphosphatase and glucose-6-phosphatase have been demonstrated to be associated with profound hypoglycemia in the setting of lactic acidosis (62,63). It would appear, therefore, that the deficiency of pyruvate carboxylase is more crippling to the patient because of its importance as a governor of tricarboxylic acid cycle activity and as a modulator for the fractional distribution of acetyl-CoA into other metabolic pathways involved in lipogenesis and ketogenesis as schematically demonstrated in Figs. 4, 5. Our studies demonstrate that a diet relatively high in carbohydrate and protein and low in fat content is advisable for a patient with pyruvate carboxylase deficiency. The intolerance of a patient to a high fat diet, together with the abnormal plasma amino acid profile (increased alanine, glycine and proline, and decreased aspartate, glutamine and valine concentrations) and the persistent lactic and ketoacidosis should strongly suggest this diagnosis. As has been suggested by others (4), future patients with hepatic pyruvate carboxylase deficiency might benefit from appropriate
FIG. 5. Schematic diagram depicting the altered hepatic metabolism which results from a deficiency of pyruvate carboxylase. A decrease in pyruvate carboxylation (D limits the availability of oxaloacetate and is associated with a functional impairment in the activity of citrate synthase (D and decreased concentrations of tricarboxylic acid cycle metabolites. The decreased carboxylation of pyruvate increases transamination to alanine, reduction to lactate, and decarboxylation to acetyl-CoA. Increased concentrations of acetylCoA may stimulate fatty acid synthesis, and sterol and ketone body synthesis through the /3hydroxy-/3-methyl-glutaryl CoA cycle. Evidence of these perturbed metabolic sequences were observed in the blood and hepatic tissue of our patient.
dietary treatment perhaps supplemented with aspartic acid and/or glutamine in an effort to increase the intracellular concentrations of oxaloacetate. Acknowledgments We thank several people who assisted in the clinical care of the patient and in the laboratory studies which were performed: Drs. Bell, Brooke and Dodson for performing the liver, muscle and skin biopsies respectively; Dr. Nelson for ultrastructural examination of the muscle and liver biopsies; Dr. Volpe and Mrs. Marasa for culturing the fibroblasts and assisting us in setting up the acetyl-CoA carboxylase assay; Mrs. Strobel, Mrs. Brothers and Mr. Dallas for technical assistance; Mrs. Dargar for assisting in performing the CoA ester assays. The nursing personnel on the Clinical Research Unit and the pediatric housestaff were vitally important in the care of the patient. We also thank Mrs. Vicki O'Neill for her skilled secretarial assistance and Dr. Philip R. Dodge for his critical review of the manuscript.
References 1. Israels, S., J. C. Haworth, H. G. Dunn, and D. A. Applegarth, Lactic acidosis in childhood, Adv Pediatr 22: 267, 1975. 2. Blass, J. P., and S. D. Cederbaum, Biochemical abnormalities in Leigh's disease, Lancet 1: 1237, 1976.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
PYRUVATE CARBOXYLASE DEFICIENCY 3. Homines, F. A., H. A. Polman, and J. D. Reerink, Leigh's encephalopathy: an inborn error of gluconeogenesis, Arch Dis Child 43: 423, 1968. 4. Tang, T. T., T. A. Good, P. R. Dyken, S. D. Johnsen, S. R. McCreadie, S. T. Sy, H. A. Lardy, and F. B. Rudolph, Pathogenesis of Leigh's encephalopathy, J Pediatr 81: 189, 1972. 5. Grover, W. D., V. H. Auerbach, and M. S. Patel, Biochemical studies and therapy in subacute necrotizing encephalomyelopathy (Leigh's syndrome),; Pediatr 81: 39, 1972. 6. Yoshida, T., K. Tada, T. Konno, and T. Arakawa, Hyperalaninemia with pyruvicemia due to pyruvate carboxylase deficiency of the liver, TohokuJ Exp Med 99: 121, 1969. 7. Brunette, M. G., E. Delvin, B. Hazel, and C. R. Scriver, Thiamine-responsive lactic acidosis in a patient with deficient low-km pyruvate carboxylase activity in liver, Pediatrics 50: 702, 1972. 8. Delvin, E., C. R. Scriver, and J. L. Neal, Pyruvate carboxylase in human liver, Biochem Med 10: 97, 1974. 9. Scrutton, M. D., and M. D. White, Purification and properties of human liver pyruvate carboxylase, Biochem Med 9: 271, 1974. 10. Lowry, O. H., and J. V. Passonneau, Typical fluorometric procedures for metabolic assays, In A Flexible System of Enzymatic Analysis, Academic Press, New York, 1972, p. 68. 11. Cahill, Jr., G. F., M. G. Herrera, A. P. Morgan, J. Soeldner, J. Steinke, P. L. Levy, G. A. Richard, Jr., and D. M. Kipnis, Hormone-fuel inter-relationships during fasting,/ Clin Invest 45: 1751, 1966. 12. Karl, I. E., A. S. Pagliara, and D. M. Kipnis, A microfluorometric enzyme assay for the determination of alanine and pyruvate in plasma and tissue, 7 Lab Clin Med 80: 434, 1972. 13. Haymond, M. W., I. E. Karl, R. D. Feigin, D. C. DeVivo, and A. S. Pagliara, Hypoglycemia and maple syrup urine disease: Defective gluconeogenesis, Pediatr Res 7: 500, 1973. 14. Morgan, C. A., and A. Lazarow, Immunoassay of insulin: Two antibody system, Diabetes 12: 115, 1963. 15. Leichter, S. B., A. S. Pagliara, M. H. Grieder, S. Pohl, J. Rosai, and D. M. Kipnis, Uncontrolled diabetes mellitus and hyperglucagonemia associated with an islet cell carcinoma, Am J Med 48: 285, 1975. 16. Schalch, D. S., and M. L. Parker, A sensitive double antibody immunoassay for human growth hormone in plasma, Nature 203: 1141, 1964. 17. Beitins, I. Z., M. H. Shaw, A. Kowarski, and C. J. Migeon, Comparison of competitive protein binding radioassay of cortisol to double isotope dilution and Porter-Silber methods, Steroids 15: 765, 1970. 18. Dickinson, J. D., H. Rosenblum, and P. B. Hamilton, Ion exchange chromatography of free amino
19.
20.
21.
22. 23. 24.
25. 26.
27.
28.
29. 30. 31.
32.
33.
34.
1295
acids in the plasma of infants under 2500 grams at birth, Pediatrics 45: 606, 1970. Nelson, S. R., O. H. Lowry, and J. V. Passonneau, Changes in energy reserves in mouse brain associated with compressive head injury, In Caveness, W. F., and A. F. Walker (eds.), Head Injury Conference Proceedings, J. B. Lippincott Co., Philadelphia, 1966, p. 444. Williamson, D. H., J. Mellanby, and H. A. Krebs, Enzymic determination of D(-)-/3-hydroxybutyric acid and acetoacetic acid in blood, Biochem J 82: 90, 1962. Stadtman, E. R., G. D. Novelli, and F. Lipmann, Coenzyme A function in and acetyl transfer by the phosphotransacetylase system, J Biol Chem 191: 365, 1951. Novelli, G. D., Methods for determination of coenzyme A, Methods Biochem Anal 2: 189, 1955. Kornberg, A., Lactic dehydrogenase of muscle, Methods Enzymol 1: 441, 1955. Wieland, O., E. Seiss, F. H. Schulze-Wethmar, H. G. v. Funcke, and B. Winton, Active and inactive forms of pyruvate dehydrogenase in rat heart and kidney: Effects of diabetes, fasting and refeeding of pyruvate dehydrogenase interconversion, Arch Biochem Biophys 143: 593, 1971. Cremer, J. E., and H. M. Teal, The activity of pyruvate dehydrogenase in rat brain during postnatal development, FEBS Lett 39: 17, 1974. Jope, R., and J. P. Blass, A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver, Biochem J 150: 397, 1975. Adam, P. A. J., and R. C. Haynes, Jr., Control of hepatic mitochondrial CO2 fixation by glucagon, epinephrine, and cortison,/ Biol Chem 244: 6444, 1969. Volpe, J. J., and J. C. Marasa, Regulation of palmitic acid synthesis in cultured glial cells: Effects of lipid on fatty acid synthetase, acetylCoA carboxylase, fatty acid and sterol synthesis, J Neurochem 25: 330, 1975. Srere, P. A., Citrate synthase, Methods Enzymol 13: 3, 1969. Englard, S., and L. Siegel, Mitochondrial Lmalate dehydrogenase of beef heart, Methods Enzymol 13: 99, 1969. DeVivo, D. C , K. Fujimoto, M. P. Leckie, and H. C. Agrawal, Subcellular distribution of ketone body metabolizing enzymes in the rat brain, J Neurochem 26: 635, 1976. Sims, K. L., and F. N. Pitts, Jr., Brain glutamate decarboxylase: Changes in the developing rat brain J Neurochem 17: 1607, 1970. Haymond, M. W., I. E. Karl, and A. S. Pagliara, Ketotic hypoglycemia: an amino acid substrate limited disorder, J Clin Endocrinol Metab 38: 521, 1974. Hug, G., D. Harris, and J. A. Grunt, Two types of glycogenosis in the same girl: Combined de-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
1296
35. 36.
37.
38.
39.
40.
41.
42. 43.
44.
45.
46.
47.
48.
49.
D E V I V O ET
ficiency of phosphorylase kinase in liver and debrancher in liver and muscle, Pediatr Res 10: 366, 1976 (Abstract). Pincus, J. H., J. R. Cooper, K. Piros, and V. Turner, Specificity of the urine inhibitor test for Leigh's disease, Neurology 24: 885, 1974. Harriman, D. G. F., and R. Reed, The incidence of lipid droplets in human skeletal muscle in neuromuscular disorders: A histochemical, electronmicroscopic and freeze-etch study, J Pathol 106: 1, 1972. Krebs, H. A., and R. L. Veech, Regulation of the redox state of the pyridine nucleotides in rat liver, In Sund, H., (ed.), Pyridine Nucleotide-Dependent Dehydrogenases, SpringerVerlag, Heidelberg, 1970, p. 413. Lovvry, O. H., N. J. Rosebrough, A. L. Fair, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. Srere, P. A., The citrate enzymes: The structures, mechanisms and biological functions, Curr Top Cell Regul 5: 229, 1972. Spector, L. B., Citrate cleavage and related enzymes, In Boyer, P. D. (ed.), The Enzymes, ed. 3, Academic Press, New York, 1972, p. 357. Utter, M. F., and D. B. Keech, Pyruvate carboxylase I: Nature of the reaction, J Biol Chem 238: 2603, 1963. Keech, D. B., and M. F. Utter, Pyruvate carboxylase II: Properties,; Biol Chem 238: 2609, 1963. McClure, W. R., H. A. Lardy, and H. P. Kneifel, Rat liver pyruvate carboxylase I. Preparation, properties and cation specificity,/ Biol Chem 246: 3569, 1971. McClure, W. R., H. A. Lardy, M. Wagner, and W. W. Cleland, Rat liver pyruvate carboxylase II. Kinetic studies of the forward reaction, J Biol Chem 246: 3579, 1971. McClure, W. R., H. A. Lardy, and W. W. Cleland, Rat liver pyruvate carboxylase III. Isotopic exchange studies of the first partial reaction, J Biol Chem 246: 3584, 1971. McClure, W. R., and H. A. Lardy, Rat liver pyruvate carboxylase IV. Factors affecting the regulation in vivo,] Biol Chem 246: 3591, 1971. Seufert, D., E. Herlemann, E. Albrecht, and W. Seubert, Purification and properties of pyruvate carboxylase from rat liver, Hoppe-Seylers Z Phtjsiol Chem 352: 459, 1971. Walter, P., Pyruvate carboxylase: Intracellular localization and regulation, In Hanson, R. W., and M. A. Mehlman (eds.), Gluconeogenesis: Its Regulation in Mammalian Species, John Wiley, New York, 1976, p. 239. Ballard, F. J., R. W. Hanson, and L. Reshef, Immunochemical studies with soluble and mito-
50.
51.
52.
53. 54.
55.
56.
57. 58.
59.
60.
61.
62.
63.
AL.
JCE & M • 1977 Vol 45 • No 6
chondrial pyruvate carboxylase activities from rat tissues, BiochemJ 119: 735, 1970. Barritt, G. J., G. L. Zander, and M. F. Utter, The regulation of pyruvate carboxylate activity in gluconeogenic tissues, In Hanson, R. W. and M. A. Mehlman (eds.), Gluconeogenesis: Its Regulation in Mammalian Species, John Wiley, New York, 1976, p. 3. Pincus, J. H., Subacute necrotizing encephalornyelopathy (Leigh's disease): a consideration of clinical features and etiology, Dev Med Child Neurol 14: 87, 1972. Falk, R. E., S. D. Cederbaum, J. P. Blass, G. E. Gibson, R. A. P. Kark, and R. E. Carrel, Ketonic diet in the management of pyruvate dehydrogenase deficiency, Pediatrics 58: 713, 1976. Weitzman, P. D. J., and M. J. Danson, Citrate synthase, Curr Top Cell Regul 10: 161, 1976. Porter, J. W., S. Kumar, and R. Dugan, Synthesis of fatty acids by enzymes of avian and mammalian species, Prog Biochem Pharmacol 6: 1, 1971. DeVivo, D. C , M. A. Fishman, and H. C. Agrawal, Preferential labelling of brain cholesterol by [3-14c]D(-)-3-hydroxybutyrate,Liptds 8: 649, 1973. Rous, S., Relative importance of acetate, acetoacetate and D-/3-OH-butyrate in the transport of acetyl CoA from the mitochondria to the cytoplasm for fatty acid synthesis in mice, Life Sci 18: 633, 1976. Sandek, C. D., and P. Felig, The metabolic events of starvation, Am J Med 60: 117, 1976, Sherwin, R. S., R. G. Hendler, and P. Felig, Effect of ketone infusions on amino acid and nitrogen metabolism in man, J Clin Invest 55: 1382, 1975. Stromme, J. H., O. Bornd, and P. J. Moe, Fatal lactic adidosis in a newborn attributable to a congenital defect of pyruvate dehydrogenase, Pediatr Res 10: 60, 1976. Willems, J. L., Discussion period, In Hommes, F. A., and C. J. Van den Berg (eds.), Normal and Pathological Development of Energy Metabolism, Academic Press, London 1975, p. 210. Saudubray, J. M., C. Marsac, C. Charpentier, L. Cathelineau, M. Besson Leaud, and J. P. Leroux, Neonatal congenital lactic acidosis with pyruvate carboxylase deficiency in two siblings, Ada Paediatr Scand 65: 717, 1976. Hommes, F. A., K. Bendien, J. D. Elema, H. J. Brenier, and I. Lombeck, Two cases of phosphoenolpyruvate carboxykinase deficiency, Ada Paediatr Scand 65: 233, 1976. Pagliara, A. S., I. E. Karl, J. P. Keating, B. I. Brown, and D. M. Kipnis, Hepatic fructose1,6-diphosphatase deficiency. A cause of lactic acidosis and hypoglycemia in infancy, J Clin Invest 51: 2115, 1972.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:15 For personal use only. No other uses without permission. . All rights reserved.
