- Email: [email protected]
Glutathione deficiency as a complication of methylmalonic acidemia: Response to high doses of ascorbate
The Journal of Pediatrics Volume 129, Number 3
tectomy; therefore we continue to manage asymptomatic gallstones conservatively. Contrary to the recommendations of Winter et al.,5 we believe a similar conservative policy should apply also to those with gallbladder sludge. The apparent difference between our experience and that of our American colleagues may result from a greater degree of symptomatic selection in American patients and differences in interpretation of the significance of nonspecific abdominal pain. However, it is also possible that differences in diet or therapeutic regimens contribute to real differences between the Jamaican and American populations. REFERENCES
1. Barrett-Connor E. Cholelithiasis in sickle cell anemia. Am J Med 1968;45:889-98. 2. Lachman BS, Lazerson J, Starshak RJ, Vanghters FM, Werlin SL. The prevalence of cholelithiasis in sickle cell disease as diagnosed by ultrasound and cholecystography. Pediatrics 1979; 64:601-3. 3. Samaik S, Slovis TL, Corbett DP, Emami A, Whitten CF. Incidence of cholelithiasis in sickle cell anemia using the ultrasonic gray-scale technique. J Pediatr 1980;96:1005-8. 4. Nzeh DA, Adedoyin MA. Sonographic pattern of gallbladder
Treacy et al.
5. 6.
7. 8. 9. 10. 11.
12.
13. 14.
445
disease in children with sickle cell anaemia. Pediatr Radiol 1989;19:290-2. Winter S, Kirmey TR, Ware RE. Gallbladder sludge ha children with sickle cell disease. J Pediatr 1994;125:747-9. Webb DKH, Darby JS, Dunn DT, Terry SI, Serjeant GR. Gallstones in Jamaican children with homozygous sickle cell disease. Arch Dis Child 1989;64:693-6. Lee SP, Maher K, Nicholls JF. Origin and fate ofbiliary sludge. Gastroenterology 1988;94:170-6. Paumgarmer G, Sauerbruch T. Gallstones: pathogenesis. Lancet 1991;338:1117-21. Carey MC. Pathogenesis of gallstones. Recenti Prog Med 1992;83:379-8I. Bouchier IAD. The formation of gallstones. Keio J Med 1992; 41:1-5. Bernhoft RA, Pellegrini CA, Broderick WC, Way LW. Pigment sludge and stone formation in the acutely ligated dog gallbladder. Gastroenterology 1983;85:1166-71. Everson GT, Nemeth A, Kom-ourianS, et al. Gallbladderfunction is altered in sickle hemoglobinopathy. Gastroenterology 1989;96:1307-16. Lee SP. Whither biliary sludge--Do you exist? Gastroenterology 1991;101:1758-79. Ohara N, Schaefer J. Clinical significance of biliary sludge. J Clin Gastroenterol 1990;12:291-4.
Glutathione deficiency as a complication of methylmalonic acidemia' Response to high doses of ascorbate E. Treacy, MB, FRCPC, L. ArDour, MD, FRCPC, P. Chessex, MD, FRCPC, G. Graham, MD, L. Kasprzak, BSC, K. Casey, BSC(NUTR),L. Bell, MD, FRCPC, O. Mamer, PhD, and C. R. Scriver, MD, FRCPC From the Biochemical Genetics Unit, Division of Medical Genetics, Montreal Children's Hospital, the Research Center, HOpital Sainte-Justine, Montreal, the Division of Nephro!ogy, Montreal Children's Hospital, and the Mass Spectrometry Unit, McGill University, Montreal, Quebec, Canada
A 7-year-old boy with deficient activity of methylmalonyl coenzyme A mutase (mut- methylmalonic acidemia) was seen in severe metabolic crisis. After hemodialysis and clearance of toxic metabolites, severe lactic acidosis persisted with multiorgan failure. Glutathione deficiency was noted and high-dose ascorbate therapy (120 mg/kg) commenced. Glutathione deficiency may contribute to the lactic acidosis observed during decompensation in patients with methylmaIonic acidemia. (J Pediatr 1996;129:445-8) Methylmalonic acidemia (McKusick 251000), an inborn error of metabolism affecting catabolism of four amino acids, valine, isoleucine, methionine, and threonkne, is caused by absent Submitted for publication Aug. 8, 1995; accepted April 26, 1996. Reprint requests: Eileen Treacy, Biochemical Genetics, Room A-717, Montreal Children's Hospital, 2300 Tupper St., Montreal, Quebec H3H 1P3, Canada. Copyright © 1996 by Mosby-Year Book, Inc. 0022-3476/96/$5.00 + 0 9/22/74506
(mut°) or deficient (rout-) activity of methylmalonyl coenzyme A mutase, the enzyme that catalyzes conversion of methylmalonyl CoA to succinyl CoA. 1 Propionyl CoA and derivatives accumulate in methylmalonic acidemia and are known to cause a variety of secondary metabolic disturbances.i, 2 Propionic acid is derived from three sources: (1) metabolism of the aforementioned amino acids, (2) B-oxidation of odd-numbered carbon fatty acids, and (3) propiogenic bowel flora, I The goal in treatment of methylmalonic acidemia is to reduce the production of propionyl CoA and its derivatives and
446
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The Journal of Pediatrics September 1996
to increase its renal clearance as propionyl CoA, and its metabolites are known to produce a variety of metabolic disturbances that have major effects on intermediary metabolism. 13 We describe a 7-year-old West African boy with methylmalonic acidemia (mut-) who had a fulminant decompensation after a previously benign course. 4 After hemodialysis and clearance of propionic and methylmalonate metabolites, he had chronic impairment of energy production and lactic acidosis. A deficiency of glutathione was confirmed. Pharmacologic doses of ascorbate alleviated the lactic acidosis. CASE R E P O R T
The diagnosis of methylmalonic acidemia (mut-, 10% residual activity) in this West African boy followed ascertainment by newborn screening. He had constant high urinary levels of methylmaCoA MMA
CoenzymeA Methylmalonic acid
I [
I
Ionic acid (to a maximum level of 13,371 ~tmol/mmol creatmine (normal value, <25 ~mol/mmol creatinine) and high se: rum levels of MMA (range, 101 to 610 gmol/L creatinine; normal value, <17 gmol/L). His medical history has been reported previously. 5 Compliance with therapy was imperfect; however, his cognitive development was normal at the time of presentation. He was admitted to the hospital, obtunded after a 2-day history of vomiting, anorexia, and epigastric pain. Laboratory investigations were as follows: capillary blood: pH, 6.95; partial pressure of carbon 'dioxide, 16 mm Hg; bicarbonate, 3.5 mmol/L; base excess, -28; lactate, 6.4 mmol/L; anmaonia, 201 gmol/L; urea, 18.7 mmol/L; creatinine, 134 pmol/L; sodium, 139 mmol/L; potassium, 4.3 mmol/L; amylase, 246 ILl/L; fipase, less than 4 IU/L; hemoglobin, 94 gm/L; leukocyte count, 17.3 x 109/L; platelet count, 397 x 109/L; serum MMA levels, 5.2 mmol/L; lactic acid, 32.7 mmol/L; pymvic acid, 0.48 mlnol/L; 3-hydroxy-N-valeric acid, 1.3 mmoUL; and 2-hydroxy-2-methylmalonic acid, 0.72 inmol/L. The patient was treated with a glucose infusion, insulin, and bicarbonate replacement. During the ensuing 24 hours his' condition deteriorated, with upper gastrointestinal tract bleeding and increasing hyperlactacidemia. The serum amylase and lipase activities were within normal limits after day 2. Subsequent serial assays and computed tomographic examination of the abdomen did not indicate a diagnosis of pancreatitis. An initial hemodialysis cleared the.propionic and methylmalonic metabolites but did not resolve the hyperlactacidemia (Figure). A tubulopathy documented at presentation subsequently resolved after dialysis. After hemodialysis the patient was maintained on a regimen of total parenteral nutrition (amino acid solution at 1 gm/kg per day), with camitine, metronidazole, and high-dose vitamin supplementation, including thiamine and pantothenic acid. Hepatomegaly was noted 3 days after the initial presentation with consistent hypertriglyceridemia. Hyperlactacidemia persisted (Figure), with elevation of Krebs cycle intermedi-
ates, normal lactate/pyruvate ratios, and elevation of blood alanine concentrations, suggesting inhibition of oxidative phosphorylation or the pymvate dehydrogenase complex. Hemodialysis was repeated twice after the first cycle after further decompensations with repeated viral infections, followed by intermittent peritoneal dialysis. A femoral deep venous thrombosis ensued, with disseminated intravascular coagulopathy. After the resolution of the latter, symptoms of acute liver dysfunction appeared and then progressed to a Kwashiorkor-like state (eczematous dermatitis and edema) despite intravenous and oral alimentation to meet the recommended protein requirement. During this phase the blood lactate level was as high as 23 retool/L, whereas the MMA and propionic acid intermediates remained in a low to moderate range (Figure) and were significantly lower than at presentation (<10% of presentation value). In keeping with the parents' wishes, a diagnostic liver biopsy was not performed. 5-Oxoproline (pyroglutamate) was first detected in the patient's urine (156 pmol/mmol creatinine In <77]) 2 weeks after admission. The value rose to 2145 gmol/mmol creatmine with increasing liver dysfunction and coexistent tubulopathy. Glutathione deficiency was suspected. The corresponding plasma glntathione level was 0.34 gmol/L (mean, 0.6---0.2 gmol]L for age-matched control subjects) at 2 weeks, falling to 0.0054 gmol/L. Ascorbate therapy (2 gila/ day) was given as a replacement antioxidant for glutathione and possible activator of oxidative phosphorylation. This was accompanied by a gradual decline of blood lactate concentration (Figure). Though the caloric and protein intake did not change significantly, liver function improved and peritoneal dialysis was gradually withdrawn. With improvement of the patient's general status, the protein intake was increased to 1.3 gm/kg per day. The plasma concentrations of cysteine were disproportionately low in comparison with plasma methionine concentrations and were almost undetectable, with low methionine levels, at the time of the highest detected levels of 5-oxoproline (Figure). At the time of discharge the 5-oxoprolinuria had resolved with the decrease in lactate levels, and a follow-up glutathione assay showed a level of 0.114 ~mol/L. METHODS Analytical methods. Urinary and serum organic acids and serial plasma amino acids were assayed by conventional methods. Total glutathione was measured in plasma according to the method of Griffith. 6 RESULTS Serum organic acids. Serum MMA levels were 5.2 mmol/L at presentation and 0.00941 mmol/L after hemodialysis. Lactic acid concentrations were 32.7 rmnol/L before dialysis and 23.1 after hemodialysis, and pyruvate concent-rations were 0.48 mmol/L before dialysis and undetectable after dialysis. [3-Hydroxy-n-valerate and 2-hydroxy-2-me-
The Journal of Pediatrics Volume 129, Number 3
Treacy et al.
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Figure. Results of lactate liver function tests and urinary MMA levels plotted chronologically with interventions. AST, Aspartate aminotransferase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; BILl, bilimbin. thylmalonate were detected before dialysis at concentrations of 1.30 and 0.72 mmol/L, respectively. These last two metabolites are not normally observed and were completely cleared by the hemodialysis. Urinary organic acid profiles. Lactate concentrations were 537 pmol/mmol creatinine before dialysis and 4160 pmol/mmol creatinine 4 days after dialysis. Methylmalonate
concentrations were 11,300 ~mol/mmol creatinine before dialysis and' 110 pmol/mmol creatinine afterward; pyruvate concenlxations were 294 pmol/mmol creatinine before dialysis and undetectable afterward. Four days after dialysis, fumarate and succinate were detected in the urine at concentrations of 129 and 430 pmol/mmol creatinine, respectively (normal value, <5 ~mol/mmol ereatinine). 5-Oxoproline was
448
Treacy et aL
noted in the urine at a concentration of 156 ~mol/mmol creatinine 4 days after the second hemodialysis (normal value, <77). At the commencement of ascorbate therapy, this level was 2145 gmol/mmol creatinine. After 4 weeks of treatment with ascorbate, this level was 17 gmol/mmol. DISCUSSION
Our patient manifested signs of liver impairment, tubulopathy, and hypoproteinemia, with markedly elevated urinary 5-oxoproline and plasma lactate levels and low plasma glutathione levels. He responded to high doses of ascorbate as a replacement antioxidant with resolution of the 5-oxoprolinuria, mucosal skin breakdown, jaundice, and hyperlactacidemia. Effective long-term management of methylmalonic and propionic acidemiais achieved by supplying adequate amounts of amino acids and nitrogen whereby metabolite accumulation occurs when the catabolism of essential amino acids is excessive, the critical amino acids are supplied in excess, or both. 7 We treated our patient with protein limitation, giving the recommended dally intake of protein. 5-Oxoprolinuria developed with coexistent low plasma cysteine levels. 5-Oxoprolinuria is observed in glutathione synthetase deficiency as a consequence of overproduction resulting from defective feedback inhibition of "y-glutamylcysteine synthetase.4 Glutathione, along with ascorbic acid and vitamin E, is central to the intracellular antioxidant defense system of mammalian cells as a free radical scavenger~ Cysteine is a direct precursor of glutathione, and glutathione deficiency is a feature of malnutrition and liver dysfunction, s Cysteine is a precursor of CoA. Increased requirements for CoA and glutathione in decompensated methylmalonic or propionic acidemia may therefore contribute to an overall cysteine deficit. Glutathione also functions in the transport of amino acids,4 which may have contributed to the amino acid deficit observed with apparently adequate protein supplementation.4 Heales et al.9 reported inhibition of complexes I and IV after glutathione synthetase inhibition experimentally reproduced in rats. Severe glutathione deficiency has been reported with rapid multiorgan failure, cellular damage mainly involving the mitochondria. 4' 10,11 High-dose ascotbate therapy has been shown to be effective in sparing mitochondrial glutathione levels and decreasing cellular damage in reduced glutathione-depleted animals and in a patient with hereditary glutathione synthetase deficiency. 12 Ascorbate is formed by a reaction between reduced glutathione and intracellularly derived dehydroascorbate. 11 Disorders of oxidative phosphorylation are now recognized to be associated with kidney disease, hepatic failure, hypertrophic cardiomyopathy, villous atrophy, pancreatitis, recurrent infection, and myopathy. 13,14 A number of the complications of methylmalonic acidemia may be associated with glutathione deficiency, oxidative stress, and disorders of oxidative phosphorylation.9 In this case the 5-oxoprolinuria did not become apparent until the glutathione levels
The Journal of Pediatrics September 1996
were profoundly depleted; thus glutathione deficiency may be more prevalent in these disorders than was previously considered. Ascorbate in high doses may serve as a useful adjunctive antioxidant therapy. 9 A role for cysteine supplementation to increase glutathione and CoA availability may also exist. With the usual dietary restrictions (and use of amino acid solutions), this amino acid may be limited and may require supplementation in patients with methylmalonic and propionic acidemia. We thank Liem Hoang for the computergraphics and Luc Choini~re, Robert Gigu~re,and Jean-ClaudeLavoie for organic acid analyses and glutathione assays. We also thank Dr. Jean-FranCis Lemay and the many physicians and nurses for their expert care of out"patient. REFERENCES
1. Fenton WA, Rosenberg LE. Disorders of propionate and methylmalonate metabolism. In: Scfiver CR, Beaudet AL, Sly WS, VaUe D, editors. The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill, 1995:1423-77. 2. Nakai A, Shigematsu Y, Saito M, Kikawa Y, Sudo M. Pathophysiologic study on methylmalonic acidosis: decrease in liver high-energy phosphate after propionate loading in rats. Pediatr Res 1991;30:5-10. 3. Van der Meer SB, Poggi F, Spada M, et al. Clinical outcome of long-term management of patients with vitamin B12-unresponsive methylmalonic acidemia. J Pediatr 1994;125:903-8. 4. Meister A, Larsson A. Glutathionesynthetasedeficiencyand other disordersof the-y-glutamylcycle. In: Scriver CR, Beandet AL, Sly WS, Valle E, editors. The metabolic and molecular bases of inheriteddisease.7th ed. New York: McGraw-Hill, 1995:1461-77. 5. Treacy E, Clow C, Mamer OA, Scriver CR. Methylmalonic acidemia with a severe chemical but benign clinical phenotype. J Pediatr 1993;122:428-9. 6. Griffith OW. Determination Of glutathione and glutathione disulfides using glutathione reductase and 2-vinylpyridine. Anal Biochem 1980;106:207-12. 7. Ney D, Bay R, Sandubray JM, Kelts D, Kulovich S, Sweetman L, Nyhan WL. An evaluation of protein requirements in methylmalonic acidemia. J Inher Metab Dis 1985;8:132-42. 8. Roediger WEW. New views on the pathogenesis of Kwashiorkor: methionine and other amino acids. J Pediatr Gastroenterol Nutr 1995;21:130-6. 9. Heales SJ, Davies SEC, Bates TE, Clark JB. Depletion of brain glutathione is accompanied by impaired mitochondrial function and decreased N-acetyl aspartate concentration. Neurochem Res 1995;20(1):31-8. 10. Jain A, Martennson J, Stole E, Auld PAM, Meister A. Glutathione deficiency leads to mitochondrial damage in brain. Proc Natl Acad Sci USA 1991;88:1913-7. 11. WincHer RS, Orselli SM, Rex TS. The redox couple between glutathionineand ascorbicacid: a chemicaland physiologicalperspective. Free Radical Biology and Medicine 1994;17(14): 333-49. 12. Jain A, Buist N, Kennaway N, Powell B, Auld P, Martensson J. Effect of ascorbate or N-acetylcysteine treatment in a patient with hereditary glutathione synthetase deficiency. J Pediatr 1994;124:229-33. 13. Rotig A, Goutieres F, Niaudet P, et al. Deletion of mitochondrial DNA in a patient with chronic tnhulointerstitial nephritis. J Pediatr 1995;126:597-601. 14. Kahler SG, Sherwood GW, WooIf D, et al. Pancreatitis in patients with organic acidemias. J Pediatr 1993;124:239-43.
tectomy; therefore we continue to manage asymptomatic gallstones conservatively. Contrary to the recommendations of Winter et al.,5 we believe a similar conservative policy should apply also to those with gallbladder sludge. The apparent difference between our experience and that of our American colleagues may result from a greater degree of symptomatic selection in American patients and differences in interpretation of the significance of nonspecific abdominal pain. However, it is also possible that differences in diet or therapeutic regimens contribute to real differences between the Jamaican and American populations. REFERENCES
1. Barrett-Connor E. Cholelithiasis in sickle cell anemia. Am J Med 1968;45:889-98. 2. Lachman BS, Lazerson J, Starshak RJ, Vanghters FM, Werlin SL. The prevalence of cholelithiasis in sickle cell disease as diagnosed by ultrasound and cholecystography. Pediatrics 1979; 64:601-3. 3. Samaik S, Slovis TL, Corbett DP, Emami A, Whitten CF. Incidence of cholelithiasis in sickle cell anemia using the ultrasonic gray-scale technique. J Pediatr 1980;96:1005-8. 4. Nzeh DA, Adedoyin MA. Sonographic pattern of gallbladder
Treacy et al.
5. 6.
7. 8. 9. 10. 11.
12.
13. 14.
445
disease in children with sickle cell anaemia. Pediatr Radiol 1989;19:290-2. Winter S, Kirmey TR, Ware RE. Gallbladder sludge ha children with sickle cell disease. J Pediatr 1994;125:747-9. Webb DKH, Darby JS, Dunn DT, Terry SI, Serjeant GR. Gallstones in Jamaican children with homozygous sickle cell disease. Arch Dis Child 1989;64:693-6. Lee SP, Maher K, Nicholls JF. Origin and fate ofbiliary sludge. Gastroenterology 1988;94:170-6. Paumgarmer G, Sauerbruch T. Gallstones: pathogenesis. Lancet 1991;338:1117-21. Carey MC. Pathogenesis of gallstones. Recenti Prog Med 1992;83:379-8I. Bouchier IAD. The formation of gallstones. Keio J Med 1992; 41:1-5. Bernhoft RA, Pellegrini CA, Broderick WC, Way LW. Pigment sludge and stone formation in the acutely ligated dog gallbladder. Gastroenterology 1983;85:1166-71. Everson GT, Nemeth A, Kom-ourianS, et al. Gallbladderfunction is altered in sickle hemoglobinopathy. Gastroenterology 1989;96:1307-16. Lee SP. Whither biliary sludge--Do you exist? Gastroenterology 1991;101:1758-79. Ohara N, Schaefer J. Clinical significance of biliary sludge. J Clin Gastroenterol 1990;12:291-4.
Glutathione deficiency as a complication of methylmalonic acidemia' Response to high doses of ascorbate E. Treacy, MB, FRCPC, L. ArDour, MD, FRCPC, P. Chessex, MD, FRCPC, G. Graham, MD, L. Kasprzak, BSC, K. Casey, BSC(NUTR),L. Bell, MD, FRCPC, O. Mamer, PhD, and C. R. Scriver, MD, FRCPC From the Biochemical Genetics Unit, Division of Medical Genetics, Montreal Children's Hospital, the Research Center, HOpital Sainte-Justine, Montreal, the Division of Nephro!ogy, Montreal Children's Hospital, and the Mass Spectrometry Unit, McGill University, Montreal, Quebec, Canada
A 7-year-old boy with deficient activity of methylmalonyl coenzyme A mutase (mut- methylmalonic acidemia) was seen in severe metabolic crisis. After hemodialysis and clearance of toxic metabolites, severe lactic acidosis persisted with multiorgan failure. Glutathione deficiency was noted and high-dose ascorbate therapy (120 mg/kg) commenced. Glutathione deficiency may contribute to the lactic acidosis observed during decompensation in patients with methylmaIonic acidemia. (J Pediatr 1996;129:445-8) Methylmalonic acidemia (McKusick 251000), an inborn error of metabolism affecting catabolism of four amino acids, valine, isoleucine, methionine, and threonkne, is caused by absent Submitted for publication Aug. 8, 1995; accepted April 26, 1996. Reprint requests: Eileen Treacy, Biochemical Genetics, Room A-717, Montreal Children's Hospital, 2300 Tupper St., Montreal, Quebec H3H 1P3, Canada. Copyright © 1996 by Mosby-Year Book, Inc. 0022-3476/96/$5.00 + 0 9/22/74506
(mut°) or deficient (rout-) activity of methylmalonyl coenzyme A mutase, the enzyme that catalyzes conversion of methylmalonyl CoA to succinyl CoA. 1 Propionyl CoA and derivatives accumulate in methylmalonic acidemia and are known to cause a variety of secondary metabolic disturbances.i, 2 Propionic acid is derived from three sources: (1) metabolism of the aforementioned amino acids, (2) B-oxidation of odd-numbered carbon fatty acids, and (3) propiogenic bowel flora, I The goal in treatment of methylmalonic acidemia is to reduce the production of propionyl CoA and its derivatives and
446
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The Journal of Pediatrics September 1996
to increase its renal clearance as propionyl CoA, and its metabolites are known to produce a variety of metabolic disturbances that have major effects on intermediary metabolism. 13 We describe a 7-year-old West African boy with methylmalonic acidemia (mut-) who had a fulminant decompensation after a previously benign course. 4 After hemodialysis and clearance of propionic and methylmalonate metabolites, he had chronic impairment of energy production and lactic acidosis. A deficiency of glutathione was confirmed. Pharmacologic doses of ascorbate alleviated the lactic acidosis. CASE R E P O R T
The diagnosis of methylmalonic acidemia (mut-, 10% residual activity) in this West African boy followed ascertainment by newborn screening. He had constant high urinary levels of methylmaCoA MMA
CoenzymeA Methylmalonic acid
I [
I
Ionic acid (to a maximum level of 13,371 ~tmol/mmol creatmine (normal value, <25 ~mol/mmol creatinine) and high se: rum levels of MMA (range, 101 to 610 gmol/L creatinine; normal value, <17 gmol/L). His medical history has been reported previously. 5 Compliance with therapy was imperfect; however, his cognitive development was normal at the time of presentation. He was admitted to the hospital, obtunded after a 2-day history of vomiting, anorexia, and epigastric pain. Laboratory investigations were as follows: capillary blood: pH, 6.95; partial pressure of carbon 'dioxide, 16 mm Hg; bicarbonate, 3.5 mmol/L; base excess, -28; lactate, 6.4 mmol/L; anmaonia, 201 gmol/L; urea, 18.7 mmol/L; creatinine, 134 pmol/L; sodium, 139 mmol/L; potassium, 4.3 mmol/L; amylase, 246 ILl/L; fipase, less than 4 IU/L; hemoglobin, 94 gm/L; leukocyte count, 17.3 x 109/L; platelet count, 397 x 109/L; serum MMA levels, 5.2 mmol/L; lactic acid, 32.7 mmol/L; pymvic acid, 0.48 mlnol/L; 3-hydroxy-N-valeric acid, 1.3 mmoUL; and 2-hydroxy-2-methylmalonic acid, 0.72 inmol/L. The patient was treated with a glucose infusion, insulin, and bicarbonate replacement. During the ensuing 24 hours his' condition deteriorated, with upper gastrointestinal tract bleeding and increasing hyperlactacidemia. The serum amylase and lipase activities were within normal limits after day 2. Subsequent serial assays and computed tomographic examination of the abdomen did not indicate a diagnosis of pancreatitis. An initial hemodialysis cleared the.propionic and methylmalonic metabolites but did not resolve the hyperlactacidemia (Figure). A tubulopathy documented at presentation subsequently resolved after dialysis. After hemodialysis the patient was maintained on a regimen of total parenteral nutrition (amino acid solution at 1 gm/kg per day), with camitine, metronidazole, and high-dose vitamin supplementation, including thiamine and pantothenic acid. Hepatomegaly was noted 3 days after the initial presentation with consistent hypertriglyceridemia. Hyperlactacidemia persisted (Figure), with elevation of Krebs cycle intermedi-
ates, normal lactate/pyruvate ratios, and elevation of blood alanine concentrations, suggesting inhibition of oxidative phosphorylation or the pymvate dehydrogenase complex. Hemodialysis was repeated twice after the first cycle after further decompensations with repeated viral infections, followed by intermittent peritoneal dialysis. A femoral deep venous thrombosis ensued, with disseminated intravascular coagulopathy. After the resolution of the latter, symptoms of acute liver dysfunction appeared and then progressed to a Kwashiorkor-like state (eczematous dermatitis and edema) despite intravenous and oral alimentation to meet the recommended protein requirement. During this phase the blood lactate level was as high as 23 retool/L, whereas the MMA and propionic acid intermediates remained in a low to moderate range (Figure) and were significantly lower than at presentation (<10% of presentation value). In keeping with the parents' wishes, a diagnostic liver biopsy was not performed. 5-Oxoproline (pyroglutamate) was first detected in the patient's urine (156 pmol/mmol creatinine In <77]) 2 weeks after admission. The value rose to 2145 gmol/mmol creatmine with increasing liver dysfunction and coexistent tubulopathy. Glutathione deficiency was suspected. The corresponding plasma glntathione level was 0.34 gmol/L (mean, 0.6---0.2 gmol]L for age-matched control subjects) at 2 weeks, falling to 0.0054 gmol/L. Ascorbate therapy (2 gila/ day) was given as a replacement antioxidant for glutathione and possible activator of oxidative phosphorylation. This was accompanied by a gradual decline of blood lactate concentration (Figure). Though the caloric and protein intake did not change significantly, liver function improved and peritoneal dialysis was gradually withdrawn. With improvement of the patient's general status, the protein intake was increased to 1.3 gm/kg per day. The plasma concentrations of cysteine were disproportionately low in comparison with plasma methionine concentrations and were almost undetectable, with low methionine levels, at the time of the highest detected levels of 5-oxoproline (Figure). At the time of discharge the 5-oxoprolinuria had resolved with the decrease in lactate levels, and a follow-up glutathione assay showed a level of 0.114 ~mol/L. METHODS Analytical methods. Urinary and serum organic acids and serial plasma amino acids were assayed by conventional methods. Total glutathione was measured in plasma according to the method of Griffith. 6 RESULTS Serum organic acids. Serum MMA levels were 5.2 mmol/L at presentation and 0.00941 mmol/L after hemodialysis. Lactic acid concentrations were 32.7 rmnol/L before dialysis and 23.1 after hemodialysis, and pyruvate concent-rations were 0.48 mmol/L before dialysis and undetectable after dialysis. [3-Hydroxy-n-valerate and 2-hydroxy-2-me-
The Journal of Pediatrics Volume 129, Number 3
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Figure. Results of lactate liver function tests and urinary MMA levels plotted chronologically with interventions. AST, Aspartate aminotransferase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; BILl, bilimbin. thylmalonate were detected before dialysis at concentrations of 1.30 and 0.72 mmol/L, respectively. These last two metabolites are not normally observed and were completely cleared by the hemodialysis. Urinary organic acid profiles. Lactate concentrations were 537 pmol/mmol creatinine before dialysis and 4160 pmol/mmol creatinine 4 days after dialysis. Methylmalonate
concentrations were 11,300 ~mol/mmol creatinine before dialysis and' 110 pmol/mmol creatinine afterward; pyruvate concenlxations were 294 pmol/mmol creatinine before dialysis and undetectable afterward. Four days after dialysis, fumarate and succinate were detected in the urine at concentrations of 129 and 430 pmol/mmol creatinine, respectively (normal value, <5 ~mol/mmol ereatinine). 5-Oxoproline was
448
Treacy et aL
noted in the urine at a concentration of 156 ~mol/mmol creatinine 4 days after the second hemodialysis (normal value, <77). At the commencement of ascorbate therapy, this level was 2145 gmol/mmol creatinine. After 4 weeks of treatment with ascorbate, this level was 17 gmol/mmol. DISCUSSION
Our patient manifested signs of liver impairment, tubulopathy, and hypoproteinemia, with markedly elevated urinary 5-oxoproline and plasma lactate levels and low plasma glutathione levels. He responded to high doses of ascorbate as a replacement antioxidant with resolution of the 5-oxoprolinuria, mucosal skin breakdown, jaundice, and hyperlactacidemia. Effective long-term management of methylmalonic and propionic acidemiais achieved by supplying adequate amounts of amino acids and nitrogen whereby metabolite accumulation occurs when the catabolism of essential amino acids is excessive, the critical amino acids are supplied in excess, or both. 7 We treated our patient with protein limitation, giving the recommended dally intake of protein. 5-Oxoprolinuria developed with coexistent low plasma cysteine levels. 5-Oxoprolinuria is observed in glutathione synthetase deficiency as a consequence of overproduction resulting from defective feedback inhibition of "y-glutamylcysteine synthetase.4 Glutathione, along with ascorbic acid and vitamin E, is central to the intracellular antioxidant defense system of mammalian cells as a free radical scavenger~ Cysteine is a direct precursor of glutathione, and glutathione deficiency is a feature of malnutrition and liver dysfunction, s Cysteine is a precursor of CoA. Increased requirements for CoA and glutathione in decompensated methylmalonic or propionic acidemia may therefore contribute to an overall cysteine deficit. Glutathione also functions in the transport of amino acids,4 which may have contributed to the amino acid deficit observed with apparently adequate protein supplementation.4 Heales et al.9 reported inhibition of complexes I and IV after glutathione synthetase inhibition experimentally reproduced in rats. Severe glutathione deficiency has been reported with rapid multiorgan failure, cellular damage mainly involving the mitochondria. 4' 10,11 High-dose ascotbate therapy has been shown to be effective in sparing mitochondrial glutathione levels and decreasing cellular damage in reduced glutathione-depleted animals and in a patient with hereditary glutathione synthetase deficiency. 12 Ascorbate is formed by a reaction between reduced glutathione and intracellularly derived dehydroascorbate. 11 Disorders of oxidative phosphorylation are now recognized to be associated with kidney disease, hepatic failure, hypertrophic cardiomyopathy, villous atrophy, pancreatitis, recurrent infection, and myopathy. 13,14 A number of the complications of methylmalonic acidemia may be associated with glutathione deficiency, oxidative stress, and disorders of oxidative phosphorylation.9 In this case the 5-oxoprolinuria did not become apparent until the glutathione levels
The Journal of Pediatrics September 1996
were profoundly depleted; thus glutathione deficiency may be more prevalent in these disorders than was previously considered. Ascorbate in high doses may serve as a useful adjunctive antioxidant therapy. 9 A role for cysteine supplementation to increase glutathione and CoA availability may also exist. With the usual dietary restrictions (and use of amino acid solutions), this amino acid may be limited and may require supplementation in patients with methylmalonic and propionic acidemia. We thank Liem Hoang for the computergraphics and Luc Choini~re, Robert Gigu~re,and Jean-ClaudeLavoie for organic acid analyses and glutathione assays. We also thank Dr. Jean-FranCis Lemay and the many physicians and nurses for their expert care of out"patient. REFERENCES
1. Fenton WA, Rosenberg LE. Disorders of propionate and methylmalonate metabolism. In: Scfiver CR, Beaudet AL, Sly WS, VaUe D, editors. The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill, 1995:1423-77. 2. Nakai A, Shigematsu Y, Saito M, Kikawa Y, Sudo M. Pathophysiologic study on methylmalonic acidosis: decrease in liver high-energy phosphate after propionate loading in rats. Pediatr Res 1991;30:5-10. 3. Van der Meer SB, Poggi F, Spada M, et al. Clinical outcome of long-term management of patients with vitamin B12-unresponsive methylmalonic acidemia. J Pediatr 1994;125:903-8. 4. Meister A, Larsson A. Glutathionesynthetasedeficiencyand other disordersof the-y-glutamylcycle. In: Scriver CR, Beandet AL, Sly WS, Valle E, editors. The metabolic and molecular bases of inheriteddisease.7th ed. New York: McGraw-Hill, 1995:1461-77. 5. Treacy E, Clow C, Mamer OA, Scriver CR. Methylmalonic acidemia with a severe chemical but benign clinical phenotype. J Pediatr 1993;122:428-9. 6. Griffith OW. Determination Of glutathione and glutathione disulfides using glutathione reductase and 2-vinylpyridine. Anal Biochem 1980;106:207-12. 7. Ney D, Bay R, Sandubray JM, Kelts D, Kulovich S, Sweetman L, Nyhan WL. An evaluation of protein requirements in methylmalonic acidemia. J Inher Metab Dis 1985;8:132-42. 8. Roediger WEW. New views on the pathogenesis of Kwashiorkor: methionine and other amino acids. J Pediatr Gastroenterol Nutr 1995;21:130-6. 9. Heales SJ, Davies SEC, Bates TE, Clark JB. Depletion of brain glutathione is accompanied by impaired mitochondrial function and decreased N-acetyl aspartate concentration. Neurochem Res 1995;20(1):31-8. 10. Jain A, Martennson J, Stole E, Auld PAM, Meister A. Glutathione deficiency leads to mitochondrial damage in brain. Proc Natl Acad Sci USA 1991;88:1913-7. 11. WincHer RS, Orselli SM, Rex TS. The redox couple between glutathionineand ascorbicacid: a chemicaland physiologicalperspective. Free Radical Biology and Medicine 1994;17(14): 333-49. 12. Jain A, Buist N, Kennaway N, Powell B, Auld P, Martensson J. Effect of ascorbate or N-acetylcysteine treatment in a patient with hereditary glutathione synthetase deficiency. J Pediatr 1994;124:229-33. 13. Rotig A, Goutieres F, Niaudet P, et al. Deletion of mitochondrial DNA in a patient with chronic tnhulointerstitial nephritis. J Pediatr 1995;126:597-601. 14. Kahler SG, Sherwood GW, WooIf D, et al. Pancreatitis in patients with organic acidemias. J Pediatr 1993;124:239-43.