- Email: [email protected]
Therapeutic approaches to cobalamin-C methylmalonic acidemia and homocystinuria
Therapeutic approaches to cobalamin-C methylmalonic acidemia and homocystinuria Dennis W. B a r t h o l o m e w , MD, Mark L. Batshaw, MD, Robert H. Allen, MD, Charles R. Roe, MD, D a v i d Rosenblatt, MD, D a v i d L. V a l l e , MD, a n d Clair A. F r a n c o m a n o , MD From the Department of Pediatrics and the John F. Kennedy Institute, The Johns Hopkins UniversitySchool of Medicine, Baltimore; the Department of Medicine, Universityof Colorado School of Medicine, Denver; the Department of Genetics, Duke UniversitySchool of Medicine, Durham, North Carolina; and the Departments of Medicine, Pediatrics, and Biology, MRC Genetics Group, McGill University,Montreal The use of h y d r o x o c o b a l a m i n (OH-B~2), betaine, carnitine, and folinic a c i d were studied in two children with the c o b a l a m i n C form of m e t h y l m a l o n i c a c i d e m i a and homocystinuria. When d a i l y injections of I mg OH-BI2 were discontinued for 3 weeks, there was no significant c h a n g e in total plasma homocysteine or methionine levels and only a modest increase in m e t h y l m a l o n a t e . Orally administered OH-BI2 1 m g / d in one patient was associated with a n increase in plasma homocystine and a decrease in methionine within 1 month. Withdrawal of betaine 250 m g / k g / d was also associated with a rise in plasma homocystine and a fall in methionine levels. Carnitine 100 m g / k g / d led to an increase in urinary excretion of propionylcarnitine, but did not affect plasma methylmaIonate levels. No beneficial b i o c h e m i c a l effect of folinic acid could be d o c u m e n t e d at a dose of 25 m g / d . Our results suggest that d a i l y injections of OH-B42 are not necessary to maintain m e t a b o l i c control and that orally administered OH-BI2 is unlikely to be effective. Betaine appears to a c t synergistically with OH-B4= and should be part of the treatment regimen. Although there are theoretical reasons for using L-carnitine and folinic acid, we could not document their effectiveness in these two patients. (J PEDIATR1988;112:32-9)
The cobalamin C B~ activation defect resulting in methylmalon!c acidemia and homocystinuria (McKusick 27740) ~ is characterized by neurodevelopmental delay, failure to thrive, megaloblastic anemia, and pigmentary retinopathy. 2 The cellular uptake and processing of cobalamin are impaired, and the subsequent production of t h e active cofactors for homocysteine and methylmalonyl-CoA catabolism (methylcobalamin and adenosylcobalamin) is significantly reduced. Although intramuscular hydroxoco-
Supported by Grant MO1-00052 from the National Institutes of Health. Submitted for publication June 15, 1987; accepted Aug. 10, 1987. Reprint requests: Mark L. Batshaw, MD, John F. Kennedy Institute, 707 N. Broadway, Baltimore, MD 21205.
32
balamin does not completely correct the biochemical defect, it does lead to marked clinical and biochemical improvement? .4 Treatment regimens in this disease have
Ado-B~2 CbI-C CH3B~2 CN-B~ CoA OH-B~z THF
Adenosylcobalamin Cobalamin C Methylcobalamin Cyanocobalamin Coenzyme A Hydroxocobalamin Tetrahydrofolate
varied widely, and optimum therapy has yet to be established. Our study was undertaken to evaluate a number of approaches to therapy: (1) the possible efficacy of orally
Volume 112 Number 1
Cbl-C methylmalonic acidemia and homocystinuria
33
Table h Cobalamin processing in fibroblasts
18-Hour uptake Propionate (nmol/mg protein) q-OH-Blz Patient 1 Patient 2 Control 1 Control 2
2.7 3.9 10.0 7.3
-OH-B12 1.2 (14%) 1.8 (21%) 10.0 6.8
Methyl-THF (nmol/mg protein) +OH-B;2 0.048 0.052 0.198 0.471
Radiolabeled CN-B12
-OH-B~2
Uptake (pg/10 6 cells)
0.025 (28%) 0.035 (39%) 0.080 0.100
0.5 (15%) 0.4 (12%) 2.0 4.7
Distribution %CH3-Bt2 5.1 1.5 41.5 73.8
%Ado-B~2 10.2 5.7 19.8 10.5
Other 84.7 92.8 38.7 15.7
No complementationwas observedafter fusion with known CbI-C fibroblasts. Percent of control in parenthesis.
administered OH-B~2, (2) the frequency of OH-B12 injections necessary to maintain homeostasis, and (3) the potential value of adjunctive therapy with L-carnitine, betaine, and folinic acid. CASE R E P O R T S
Patient 1. This 15-month-old white male infant was the 3480 g product of an unremarkable 43-week gestation in a 27-year-old gravida 1 woman. Apgar scores were 8 at 1 minute and 9 at 5 minutes. There were no neonatal complications, and the infant was discharged to home at 5 days of age with breast-feeding. He fed well and had normal weight gain until 3 months of age, when h e was found to be lethargic and irritable, with tachypnea and hypothermia. Examination revealed right upper lobe pneumonia and moderate hypotonia. Admission laboratory data included white blood cell count 5100/mm 3 with 15% neutrophils, hemoglobin 10.2 g/dL, platelet count 97,000/mm 3, and mean corpuscular volume 99 fL (normal range 70 to 86 fL). Plasma ammonium level was normal. Plasma amino acids and urine organic acids were studied because of continued hypotonia and neutropenia. Gas chromatography of the urine revealed a large peak consistent with methylmalonic acid, and amino acid column chromatography was remarkable for an elevated homocystine level of 32 #mol/L (normal, not detectable) and a low methionine level of 2 ~mol/L (normal 15 to 49 ~mol/L). A tentative diagnosis of B~2 activation defect was made after determining that the s e r u m B12 level was normal. Therapy was begun with OH-B~2 (alphaRedisol, Merck Sharpe & Dohme, Rahway, N.J.) 1 mg/d intramuscularly and betaine free base (Sigma Chemical Co., St. Louis) 250 mg/kg/d orally. At the time of diagnosis, the child did not fixate or follow visually. Ophthalmologic evaluation revealed macular mottling with a pale optic disc. The electroretinogram flicker or cone response was diminished to <50/xV (normal 60 to 100 ttV). Four months after initiation of OH-B~2/betaine theapy, the ERG response improved to 77 #V, despite the development of macular "bull's-eye" lesions and horizontal and rotatory nystagmus. The patient was able to follow visually and reach for objects. At 15 months, he continues to show normal growth (height, 50th percentile; weight, 75th percentile) and has had no recurrence of clinical symptoms or neutropenia. Despite marked initial
developmental delay (1 month level at 3 months), his functioning is at the 12-month level, equivalent to a developmental quotient of 80. Metabolic control, as measured by column chromatography of plasma methionine (15 to 30 ~mol/L) and homocystine (0 to 4 /zmol/L) has been excellent. Patient 2. This 4-year-old white girl is distantly related to patient 1 through a common ancestor. She was the 2330 g product of a 41-week gestation in a 36-year-old gravida 2 woman who had scant first-trimester bleeding and decreased fetal movements. The infant was born via emergency cesarean section because of abnormal decelerations of the fetal heart rate during labor induction. Apgar scores were 2 at 1 minute and 6 at 5 minutes. She required brief ventilatory support. The only subsequent abnormal finding on physical examination was a heart murmur; echocardiography showed a mild Ebstein anomaly. The infant breast-fed well, and was discharged to home at 5 days of age. However, she failed to thrive, became increasingly lethargic and irritable, and had loose stools and low-grade fever. She was readmitted to the hospital at 3 weeks of age; hemoglobin was 15.6 g/dL, white blood cell count 3900/mm 3with 30% neutrophils, and platelet count 49,000/mm 3. She was intolerant of feedings, and vomited intermittently, necessitating intravenous alimentation. She was discharged after 2 months, only to be readmitted within 1 week with recurrent vomiting, dehydration, and acidosis. Metabolic evaluation revealed a plasma homocystine level of 37 ~zmol/L and methionine 9 /zmol/L. The serum B~2 level was normal. A B12 activation defect was postulated. Ophthalmologic examination showed horizontal and rotatory nystagmus with a granular macula that subsequently developed into pigmentary retinopathy. Serial ERGs since 14 months of age have consistently shown reduced scotopic or rod response of about 150 ~V (normal 300 to 400 pV) and flicker response of about 50 #V. Good metabolic control has been maintained with daily injections of OH-B~z 1 mg intramuscularly plus betaine 250 mg/kg/d and oral folic acid 1 mg/d orally. She remains below the 5th percentile for age in height and weight and continues to lag somewhat behind her peers in expressive language and visuomotor skills. Her developmental quotient was 61 at 3 months of age. At 54 months she had a Stanford-Binet Verbal IQ of 77 and Visual-Perceptual IQ of 54. Good biochemical control has been maintained, with plasma homocystine levels ranging from 0 to 4
34
Bartholomew et al.
The Journal of Pediatrics January 1988
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Fig. t. Treatment course in patient l. After diagnosis at 3 months of age, hydroxocobalamin (OH-B~2) 1 mg/d IM and betaine 250 mg/kg/d therapy resulted in fall in plasma methionine (Met) and homocystine (Hey) levels within 24 hours (days 2 to 3). Cessation of orally administered betaine for 1 month (beginning day 90) led to fall in Met and rise in Hey levels, which was corrected when betaine was resumed (Day 119). Later trial of OH-B~2orally (day 150) similarly resulted in fall in Met and rise in Hey levels. Free Hey was measured by amino acid chromatography.
#mol/L and methionine levels 18 to 30 ~mol/L, as measured by column chromatography. METHODS Plasma carnitine and urinary long- and short-chain acylcarnitine levels were determined using a radioenzymatic assay? Further characterization of urinary acylcarnitine derivatives was done With fast-atom bombardment mass spectroscopy and constant base/excess ratio-linked scanning. 6,7 Plasma methylmalonate was assayed using capillary gas chromatography-mass spectroscopy? Serum B~2 levels were determined by a competitive protein binding assay? The unusually high cobalamin levels necessitated serial dilutions of the serum before assay. Heparinized blood was obtained for measurement of plasma amino acids. The blood was spun down and run immediately by amino acid chromatography using a Beckman System 6300 high-performance amino acid analyzer (Beckman Instruments Inc., Brea, Calif.). This method measures homocystine disulfides, a measure of free homocystine. In a portion of the study, total homocysteine was quantified by gas chromatography-mass spectroscopy after reduction of all plasma mixed disulfides.1~ Studies of cobalamin processing in fibroblasts and complementation studies were performed as previously described. "~3 Dietary protein and methionine intakes were comparable during the various phases of the study. Informed consent was obtained from the parents before each phase of the study.
RESULTS
Cobalamin processing in fibroblasts. Markedly decreased processing of propionate (14% to 21% of normal) and of methyltetrahydrofolate (28% to 39% of normal) was demonstrated in cultured fibroblasts in the absence of OH-B12 (Table I). Processing of propionate in C b l - C cells increased when OH-B12 was added to the media. There was a minimal increase of methyl-THF processing in CbI-C cells with supplemental OH-B12. Uptake of cyanocobalamin by fibroblasts was reduced to 12% to 15% of control. The percentage of total cobalamin represented by Ado-B~2 and CH3-B12, the active cofactors for propionate and homocysteine metabolism, was reduced. There was no complementation after fusion with known Cbl-C fibroblasts. These studies establish the diagnosis of Cbl-C mutant form of methylmalonic acidemia and homocystinuria. Efficacy of oral OH-B12. OH-B~2 1 mg orally given to two adult volunteers resulted in a less than twofold rise in serum B~2 levels 4 hours after ingestion, with a return to baseline by 48 hours (data not shown). Further, a trial of OH-B~2 1 mg orally given three times a day for 1 week did not lead to a significant rise in serum B~2 levels compared with baseline in the control subjects (1015 pg/mL vs 1145 pg/mL). In patient 1, given OH-B~2 1 m g / d orally for 1 month (Fig. 1), there was a loss of metabolic control and an increase in homocystine (10/xmol/L vs 1 /xmol/L) and decrease in methionine (21 #mol/L vs 40 ~zmol/L). These
Volume 112 Number 1
Cbl-C methylmalonic acidemia and homocystinuria
PATIENT
35
2
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Fig. 9. Metabolic measurements in patient 2. Blood levelsof B]2,methylmalonate (MMA), reduced homocysteine(Hcy), and methionine (Met) during 3 weeks after single intramuscular injection of 1 mg OH-B12 either with (solid lines) or without (dashed lines) supplements of folate and carnitine. Betaine was continued throughout the study. Total Hcy was measured by gas chromatography-mass spectroscopy. (See Fig. 3 for similar information on patient 1.)
levels reverted toward normal when OH-B12 intramuscularly was restarted (Days 150 to 180). There were no clinical symptoms during this time. In this study, homocystine levels reflect only the homocystine disulfides as measured by amino acid column chromatography. Required frequency of OH-B~2injections. In both control subjects and affected children, serum B12 levels rose approximately 20-fold 24 hours after a 1 mg intramuscular injection of OH-B~ 2. The half-life of OH-B12 in the serum
of adult male control subjects was approximately 24 hours, whereas the half-life in the two affected children was 62 hours. In the children, plasma B~2levels decreased from the 24-hour peak of approximately 200,000 pg/mL to < 10,000 pg/mL, during a 19-day follow-up (Figs. 2 and 3). There was no significant increase in total plasma homocysteine in either patient over the 19 days; the mean baseline value for homocysteine was 78 gmol/L vs 77 #mol/L 19 days after injection. A control standard of pooled normal human
36
Bartholomew et al.
The Journal of Pediatrics January 1988
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Fig. 3. Metabolic measurements in patient 1. See legend presented with Fig. 2 for explanation of illustration.
serum for homocysteine was 11 umol/L. Plasma methionine levels were 15 #mol/L at the beginning of the study and 23 ~mol/L at its conclusion. Plasma methylmalonate levels varied from 39 #mol/L to 183 #mol/L, with a mean value of 56 #mol/L in patient 1 and 139 tzmol/L in patient 2 (normal 0.2 to 0.7 ~mol/L). In sum, there was some day-to-day variability in the metabolic measurements, but no consistent alterations occurred over the 19 days after OH-B~2 injection. There were no adverse clinical symptoms during the course of this study. Effect of carnitine therapy. In patient 1, pretreatment
free and total carnitine levels were normal, but short-chain acylcarnitine esters were increased. In patient 2, there were decreased levels of both free and total carnitine (Table II). The predominant short-chain acylcarnitine esters in both patients were propionylcarnitine and methylmalonylcarnitine. After an oral loading dose of L-carnitine 100 mg/kg (Sigma-Tau), the urinary acylcarnitine profiles in both patients showed excretion of a mixture of free carnitine and acetyl-, propionyl-, and octenoyl-carnitine esters. Daily supplementation with 100 mg/kg L-carnitine resulted in
Volume 112 Number 1
Cbl-C rnethylmalonic acidemia and homoeystinuria
37
T a b l e II. Plasma carnitine and methylmalonate (MMA) levels (#tool/L) before and during
L-carnitine supplementation (100 mg/kg/d) Plasma carnitine Condition
Patient 1 Baseline Treated Patient 2 Baseline Treated Normal*
Acylcarnitine esters
Total
Free
Short chain
Long chain
60 99
42 73
16 22
2 4
23 84 46 +__10
15 54 37 _+ 8
6 25 6 _+ 4
2 5 4+ 2
Plasma MMA
52 56 147 140 0.4 _+ 0.1
*Mean -+ SD.
Table III. Effect of calcium leucovorin on red cells and on plasma methylmalonate, homocystine, and methionine levels Patient 4
Hgb (g/dL) MCV (fL) MCFI (pg) Morphology MMA (umol/L) Hey (/~mol/L) Met (gmol/L)
Patient 2
Pre
Post
Pre
Post
Normal
12.9 88.8 30.9 Normal 34.6 69 12
12.3 93.7 31.0 Normal 38.6 64 13
12.5 92.5 30.6 Normal 64 64 30
12.1 91.5 29.5 Normal 68.3 68.3 22.3
11.7-13.8 75-87 24-30 Normal 0.2-0.7 7-20 12-33
MCV, meancorpuscularvolume;MCH, meancorpuscularhemoglobin;MMA,methylmalonate;Hey, homocysteine;Met, methionine.
a marked increase in both total carnitine and in these short-chain acylcarnitine esters (Table II). However, removing the L-carnitine supplement from the treatment regimen for 3 weeks did not result in a change in plasma homocysteine or methylmalonate levels (Fig. 2). Because of the presence of octenoyl (C8)-earnitine esters, the possibility of an abnormality in peroxisomal fatty acid oxidation was considered, because very-longchain fatty acids are normally oxidized down to the C8 length in peroxisomes. This was tested by measuring levels of very-long-chain fatty acid levels in plasma. The ratio of very-long-chain fatty acids (C26/C22) was found to be normal, 0.016 (normal <0.030) in the two children, suggesting normal fatty acid oxidation. Effect of betaine. Betaine was deleted from the treatment regimen was patient 1 for 1 month between days 90 and 119 (Fig. 1). The child continued to receive daily injections of 1 mg OH-B~2. During this time there was a modest increase in homocystine levels and a decrease in
methionine levels, which was reversed (between days 120 and 150) by reintroducing betaine supplements of 250 mg/kg/d. On the other hand, the continued use of betaine between days 150 and 180, when OH-Blz orally was substituted for intramuscular injection, did not prevent an increase in plasma homocystine and a decrease in methionine. Effect of folinic acid. Serum folate levels in both children were normal, >20 ng/mL, and neither patient had anemia at the time of study. Mean corpuscular volume was at the upper limits of normal, but there was no apparent hypersegmentation of the nuclei in neutrophils. Calcium leueovorin (Wellcovin, Wellcome Diagnostics, Research Triangle Park, N.C.) 25 mg/d was administered orally in two divided doses for 14 days. OH-B12, betaine, and L-carnitine were continued according to the above treatment regimen. Hemoglobin, hematocrit, red blood cell indices, plasma methylmatonate, homocysteine, and methionine were assessed for 2 weeks before the study to
38
Bartholomew et al.
determine baseline values (Table III). No differences were seen in pretreatment versus treatment levels. DISCUSSION Fourteen patients with CbI-C B~2 activation defect have been described?,4,t4-22 As found in our children, plasma methylmalonate levels are at least 100 times normal, but do not approach the 200- to 2000-fold increase in patients with methylmalonyl-CoA mutase deficiency or Cbl-B deficiency.23 Homocystine levels are also elevated, and methionine levels are subnormal. Therapy of the Cbl-C defect has consisted primarily of injections of vitamin BlZ. Frequency of injections has varied from every 1 to 28 days. Despite clinical improvement, B1z supplements do not fully correct the underlying biochemical defect, and it is unknown whether residual methylmalonic acidemia and homocystinemia are detrimental to the child's physical or intellectual development. The intellectual development in our children is in the borderline normal range, and has been stable or improved since onset of therapy. OH-B~ appears to be more effective than CN-BI2 in treating this disorder? OH-B~2 remains at the site of injection longer than does CN-B12, 24 and there is a more -sustained rise in serum B~2 levels and less immediate urinary excretion of cobalamin metabolites after OH-B~2 intramuscular injections as compared with CN-B12. 25 Fibroblasts from patients with Cbl-C defect have only 30% of control activity of the methyltransferase holoenzyme in the presence of CN-B~z, but have up to 100% of control activity in the presence of OH-BIE,26 For these reasons, we did not attempt therapy with CN-Bx2. We initially studied whether orally administered OHBi2 might be an appropriate substitute for intramuscular OH-B~2 injections. Our studies in adult control subjects demonstrated that even massive doses of OH-BI2 given orally (1000 times the recommended daily allowance of 3 #g for an adult 27) were inadequate to raise serum B~2 levels significantly. Further, we noted that orally given OH-Ba2 1 mg/d in one patient resulted in a fall in plasma methionine with a concurrent rise in homoeystine over a 1-month period. For these reasons we did not pursue oral OH-B~2 therapy in our patients. Betaine is a methyl donor that has been used successfully in treating B6-unresponsive homocystinuria caused by cystathionine B synthetase deficiency.28The mechanism of action of betaine involves the conversion of homocysteine to methionine via betaine homocysteine methyltransferase, thus bypassing the BxE-dependent cytosolic 5-methyl-tetrahydrofolate homocysteine methyltransferase. This reaction does not require CH3Baz as a cofactor. 28The possible utility of betaine in Cbl-C mutants is suggested by our pilot study
The Journal of Pediatrics January 1988
in whch the removal of this medication was associated with a lower plasma methionine and higher plasma homocystine level despite continuation of daily intramuscular injections of OH-B12. On the other hand, betaine did not normalize methionine levels in the absence of OH-B12. Thus betaine and OH-B~2 may have a synergistic effect. Subnormal plasma carnitine levels, found in one of our children, have been noted in other organic acidemias. 29 This is t h e presumed result of a limited protein intake combined with increased urinary excretion of carnitine esters. Carnitine could increase excretion of propionate and methylmalonate as carnitine esters and restore the acyl-CoA/free CoA ratio in the mitochondria.3~Carnitine therapy did increase propionylcarnitine excretion in the urine but did not have a significant effect on plasma methylmalonate levels. Folic acid or its activated form, formyl-THF (folinic acid or leucovorin) has also been recommended as a therapeutic adjunct in this disorder because of a postulated deficiency of intracellular reduced folate polyglutamates as a result of a "methylfolate trap. ''3~ Pharmacologic doses of folinic acid provide a means of bypassing the methylfolate trap. 32 However, the mild macrocytic anemia and relative hypomethioninemia in our patients was unaltered by pharmacologic doses of folinie acid. In summary, we found that OH-B12 1 mg intramuscularly plus betaine 250 mg/kg/d orally was effective in the treatment of Cbl-C defect in two children. Orally administered OH-B~2 appeared to be ineffective. Daily OH-B12 injections did not seem to be required in our patients, Long-term supplementation with L-carnitine 100 to 200 mg/kg/d increased propionylcarnitine excretion and may therefore be useful, but we did not demonstrate a consistent decrease in plasma methylmalonate during carnitine supplemention. Although there are theoretical advantages to the use of folinic acid supplements, we could not document their value in our short-term study. We thank Dr. David Watkins for performing the complementation studies in our patients; Ann Moser for measuring verylong-chain fatty acid levels; and Ms. Bernadine Peters and the nurses of the Pediatric Clinical Research Unit. REFERENCES
1. McKusick VM. Mendelian inheritance in man, 7th ed. Baltimore: Johns Hopkins University Press, 1986:1297. 2. Robb RM, Dowton SB, Fulton AB, Levy HL. Retinal degeneration in vitamin B12disorder associated with methylmalonic aciduria and sulfur amino acid abnormalities. Am J Ophthalmol 1984; 97:691-6. 3. Mitchell GA, Watkins D, Melancon SB, et al. Clinical heterogeneity in cobalamin C variant of combined homocystinuria and methylmalonicaciduria. J PEDIATR1986;108:4105.
Volume 112 Number 1 4. Carmel R, Bedros AA, Mace JW, Goodman SI. Congenital methylmalonic aciduria: homocystinuria with megaloblastic anemia. Observations on response to hydroxocobalamin and on the effect of homocysteine and methionine on the deoxyuridine suppression test. Blood 1980;55:570-9. 5. Brass EP, Hoppel CL. Carnitine metabolism in the fasting rat. J Biol Chem 1978;253:2688-93. 6. Millington DS, Smith JA. Fragmentation patterns by fastlinked electric and magnetic field scanning. Organic Mass Spectrom 1977;12:264. 7. Barber M, Bordoli RS, Sedgwick RD, Tyler AN. Fast atom bombardment of solids (FAB): a new ion source for mass spectrometry. JCS Chem Comm 1981;325. 8. Stabler SP, Marcell PD, Podell ER, Allen RH, Lindenbaum J. Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatographymass spectrometry. J Clin Invest 1986;77:1606-12. 9. Rothenberg SP. Assay of serum vitamin Bt2 concentration using Co57-B12 and intrinsic factor. Proc Soc Exp Biol Med 1961;108:45-8. 10. Stabler SP, Marcell PD, Podell ER, Allen RH, Lindenbaum J. Homocysteine is an accurate indicator of cobalamin or folate deficiency [Abstract]. Blood 1985;60(suppl 1):50A. 11. Rosenblatt DS, Cooper BA, Pottier A, Lue-Shing H, Matiaszuk N, Grauer K. Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis. J Clin Invest 1984i74:2149-56. 12. Watkins D, Rosenblatt DS. Failure of lysosomal release of vitamin B~2: a new complementation group causing methylmalonic aciduria (cblF). Am J Hum Genet 1986;39:404-8. 13. Willard HF, Mellman IS, Rosenberg LE. Genetic complementation among inherited deficiencies of methylmalonylCoA mutase activity: evidence for a new class of human cobalamin mutant. Am J Hum Genet 1978;30:1-13. 14. Levy HL, Mudd SH, Schulman JD, Dreyfus PM, Abeles RH. A derangement in B12 metabolism associated with homocystinemia, cystathioninemia, hypomethioninemia and methylmalonic aciduria. Am J Med 1970;48:390-7. 15. Goodman SI, Moe PG, Hammond KB, Mudd SH, Uhlendorf BW. Homocystinuria with methylmalonic aciduria: two cases in a sibship. Biochem Med 1970;4:500-15. 16. Dillon M J, England JM, Gompertz D, et al. Mental retardation, megaloblastic anaemia, methylmalonic aciduria and abnormal homocysteine metabolism due to an inborn error in vitamin B~2 metabolism. Clin Sci Mol Med 1974;47:43-61. 17. Anthony M, McLeay AC. A unique case of derangement of vitamin B12 metabolism. Proc Aust Assoc Neurol 1976;13: 61-5. 18. Baumgartner ER, Wick H, Maurer R, Egli N, Steinmann B. Congenital defect in intracellular cobalamin metabolism resulting in homocystinuria and methylmalonic aciduria. I. Case report and histopathology. Heir Paediatr Acta 1979; 34:465-82.
Cbl-C methylmalonic acidemia and homocystinuria
39
19. Linnell JC~ Miranda B, Bhatt HR, et al. Abnormal cobalamin metabolism in a megaloblastic child with homocystinuria, cystathioninuria and methylmalonic aciduria. J Inher Metab Dis 1983;6:13%9. 20. Ribes A, Vilaseca MA, Briones P, et al. Methylmalonic aciduria with homocystinuria. J Inher Metab Dis t984; 7(suppl 2):129-30. 21. Shinnar S, Singer HS. Cobalamin C mutation (methylmalonic aciduria and homocystinuria) in adolescence: a treatable cause of dementia and myelopathy. N Engl J Med 1984; 311:451-4. 22. Dayan AD, Ramsey RB. An inborn error of vitamin B12 metabolism associated with cellular deficiency of coenzyme forms of the vitamin: pathological and neurochemical findings in one case. J Neurol Sci 1974;23:117-28. 23. Rosenberg LE. Disorders' of propionate and methylmalonate metabolism. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited disease, 5th ed. New York: McGraw-Hill, 1983: 491. 24. Boddy K, King P, Mervyn L, Macleod A, Adams JF. Retention of cyanocobalamin, hydroxocobalamin and coenzyme Blz after parenteral administration. Lancet 1968;2:7102. 25. Chalmers JN, Shinton NK. Comparison of hydroxocobalamin and cyanocobalamin in the treatment of pernicious anemia. Lancet 1965;2:1305. 26. Fenton WA, Rosenberg LE. Genetic and biochemical analysis of human cobalamin mutants in cell culture. Annu Rev Genet 1978;12:223-48. 27. Seetharam B, Alpers DH. Absorption and transport of cobalamin (vitamin B~2). Annu Rev Nutr 1982;2:343-69. 28. Smolin LA, Benevenga N J, Berlow S. The use of betaine for the treatment of homocystinuria. J PEDIATR 1981;99:46772. 29. Engel AG, Rebouche CJ. Carnitine metabolism and inborn errors. J Inher Metab Dis 1984;7(suppt 1):38-43. 30. Chalmers RA, Stacey TE, Tracey BM, et al. L-Carnitine insufficiency in disorders of organic acid metabolism: response to L-carnitine by patients with methylmalonic aciduria and 3-hydroxy-3-methylglutaric aciduria. J Inher Metab Dis 1984;7(suppl 2):109-10. 31. Ravindranath Y, Buck S, Krieger I. Beneficial effect of folic acid in addition to vitamin B12 in congenital methylmalonic aciduria and homocystinuria: results of deoxyuridine suppression test [Abstract]. Pediatr Res 1986;20:270A. 32. Mehta BM, Gisolfi AL, Hutchison D J, Nirenberg A, Kellick MG, Rosen G. Serum distribution of citrovorum factor and 5-methyltetrahydrofolate following oral and IM administration of calcium leucovorin in normal adults. Cancer Treat Rep 1978;62:345-50.
The cobalamin C B~ activation defect resulting in methylmalon!c acidemia and homocystinuria (McKusick 27740) ~ is characterized by neurodevelopmental delay, failure to thrive, megaloblastic anemia, and pigmentary retinopathy. 2 The cellular uptake and processing of cobalamin are impaired, and the subsequent production of t h e active cofactors for homocysteine and methylmalonyl-CoA catabolism (methylcobalamin and adenosylcobalamin) is significantly reduced. Although intramuscular hydroxoco-
Supported by Grant MO1-00052 from the National Institutes of Health. Submitted for publication June 15, 1987; accepted Aug. 10, 1987. Reprint requests: Mark L. Batshaw, MD, John F. Kennedy Institute, 707 N. Broadway, Baltimore, MD 21205.
32
balamin does not completely correct the biochemical defect, it does lead to marked clinical and biochemical improvement? .4 Treatment regimens in this disease have
Ado-B~2 CbI-C CH3B~2 CN-B~ CoA OH-B~z THF
Adenosylcobalamin Cobalamin C Methylcobalamin Cyanocobalamin Coenzyme A Hydroxocobalamin Tetrahydrofolate
varied widely, and optimum therapy has yet to be established. Our study was undertaken to evaluate a number of approaches to therapy: (1) the possible efficacy of orally
Volume 112 Number 1
Cbl-C methylmalonic acidemia and homocystinuria
33
Table h Cobalamin processing in fibroblasts
18-Hour uptake Propionate (nmol/mg protein) q-OH-Blz Patient 1 Patient 2 Control 1 Control 2
2.7 3.9 10.0 7.3
-OH-B12 1.2 (14%) 1.8 (21%) 10.0 6.8
Methyl-THF (nmol/mg protein) +OH-B;2 0.048 0.052 0.198 0.471
Radiolabeled CN-B12
-OH-B~2
Uptake (pg/10 6 cells)
0.025 (28%) 0.035 (39%) 0.080 0.100
0.5 (15%) 0.4 (12%) 2.0 4.7
Distribution %CH3-Bt2 5.1 1.5 41.5 73.8
%Ado-B~2 10.2 5.7 19.8 10.5
Other 84.7 92.8 38.7 15.7
No complementationwas observedafter fusion with known CbI-C fibroblasts. Percent of control in parenthesis.
administered OH-B~2, (2) the frequency of OH-B12 injections necessary to maintain homeostasis, and (3) the potential value of adjunctive therapy with L-carnitine, betaine, and folinic acid. CASE R E P O R T S
Patient 1. This 15-month-old white male infant was the 3480 g product of an unremarkable 43-week gestation in a 27-year-old gravida 1 woman. Apgar scores were 8 at 1 minute and 9 at 5 minutes. There were no neonatal complications, and the infant was discharged to home at 5 days of age with breast-feeding. He fed well and had normal weight gain until 3 months of age, when h e was found to be lethargic and irritable, with tachypnea and hypothermia. Examination revealed right upper lobe pneumonia and moderate hypotonia. Admission laboratory data included white blood cell count 5100/mm 3 with 15% neutrophils, hemoglobin 10.2 g/dL, platelet count 97,000/mm 3, and mean corpuscular volume 99 fL (normal range 70 to 86 fL). Plasma ammonium level was normal. Plasma amino acids and urine organic acids were studied because of continued hypotonia and neutropenia. Gas chromatography of the urine revealed a large peak consistent with methylmalonic acid, and amino acid column chromatography was remarkable for an elevated homocystine level of 32 #mol/L (normal, not detectable) and a low methionine level of 2 ~mol/L (normal 15 to 49 ~mol/L). A tentative diagnosis of B~2 activation defect was made after determining that the s e r u m B12 level was normal. Therapy was begun with OH-B~2 (alphaRedisol, Merck Sharpe & Dohme, Rahway, N.J.) 1 mg/d intramuscularly and betaine free base (Sigma Chemical Co., St. Louis) 250 mg/kg/d orally. At the time of diagnosis, the child did not fixate or follow visually. Ophthalmologic evaluation revealed macular mottling with a pale optic disc. The electroretinogram flicker or cone response was diminished to <50/xV (normal 60 to 100 ttV). Four months after initiation of OH-B~2/betaine theapy, the ERG response improved to 77 #V, despite the development of macular "bull's-eye" lesions and horizontal and rotatory nystagmus. The patient was able to follow visually and reach for objects. At 15 months, he continues to show normal growth (height, 50th percentile; weight, 75th percentile) and has had no recurrence of clinical symptoms or neutropenia. Despite marked initial
developmental delay (1 month level at 3 months), his functioning is at the 12-month level, equivalent to a developmental quotient of 80. Metabolic control, as measured by column chromatography of plasma methionine (15 to 30 ~mol/L) and homocystine (0 to 4 /zmol/L) has been excellent. Patient 2. This 4-year-old white girl is distantly related to patient 1 through a common ancestor. She was the 2330 g product of a 41-week gestation in a 36-year-old gravida 2 woman who had scant first-trimester bleeding and decreased fetal movements. The infant was born via emergency cesarean section because of abnormal decelerations of the fetal heart rate during labor induction. Apgar scores were 2 at 1 minute and 6 at 5 minutes. She required brief ventilatory support. The only subsequent abnormal finding on physical examination was a heart murmur; echocardiography showed a mild Ebstein anomaly. The infant breast-fed well, and was discharged to home at 5 days of age. However, she failed to thrive, became increasingly lethargic and irritable, and had loose stools and low-grade fever. She was readmitted to the hospital at 3 weeks of age; hemoglobin was 15.6 g/dL, white blood cell count 3900/mm 3with 30% neutrophils, and platelet count 49,000/mm 3. She was intolerant of feedings, and vomited intermittently, necessitating intravenous alimentation. She was discharged after 2 months, only to be readmitted within 1 week with recurrent vomiting, dehydration, and acidosis. Metabolic evaluation revealed a plasma homocystine level of 37 ~zmol/L and methionine 9 /zmol/L. The serum B~2 level was normal. A B12 activation defect was postulated. Ophthalmologic examination showed horizontal and rotatory nystagmus with a granular macula that subsequently developed into pigmentary retinopathy. Serial ERGs since 14 months of age have consistently shown reduced scotopic or rod response of about 150 ~V (normal 300 to 400 pV) and flicker response of about 50 #V. Good metabolic control has been maintained with daily injections of OH-B~z 1 mg intramuscularly plus betaine 250 mg/kg/d and oral folic acid 1 mg/d orally. She remains below the 5th percentile for age in height and weight and continues to lag somewhat behind her peers in expressive language and visuomotor skills. Her developmental quotient was 61 at 3 months of age. At 54 months she had a Stanford-Binet Verbal IQ of 77 and Visual-Perceptual IQ of 54. Good biochemical control has been maintained, with plasma homocystine levels ranging from 0 to 4
34
Bartholomew et al.
The Journal of Pediatrics January 1988
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Fig. t. Treatment course in patient l. After diagnosis at 3 months of age, hydroxocobalamin (OH-B~2) 1 mg/d IM and betaine 250 mg/kg/d therapy resulted in fall in plasma methionine (Met) and homocystine (Hey) levels within 24 hours (days 2 to 3). Cessation of orally administered betaine for 1 month (beginning day 90) led to fall in Met and rise in Hey levels, which was corrected when betaine was resumed (Day 119). Later trial of OH-B~2orally (day 150) similarly resulted in fall in Met and rise in Hey levels. Free Hey was measured by amino acid chromatography.
#mol/L and methionine levels 18 to 30 ~mol/L, as measured by column chromatography. METHODS Plasma carnitine and urinary long- and short-chain acylcarnitine levels were determined using a radioenzymatic assay? Further characterization of urinary acylcarnitine derivatives was done With fast-atom bombardment mass spectroscopy and constant base/excess ratio-linked scanning. 6,7 Plasma methylmalonate was assayed using capillary gas chromatography-mass spectroscopy? Serum B~2 levels were determined by a competitive protein binding assay? The unusually high cobalamin levels necessitated serial dilutions of the serum before assay. Heparinized blood was obtained for measurement of plasma amino acids. The blood was spun down and run immediately by amino acid chromatography using a Beckman System 6300 high-performance amino acid analyzer (Beckman Instruments Inc., Brea, Calif.). This method measures homocystine disulfides, a measure of free homocystine. In a portion of the study, total homocysteine was quantified by gas chromatography-mass spectroscopy after reduction of all plasma mixed disulfides.1~ Studies of cobalamin processing in fibroblasts and complementation studies were performed as previously described. "~3 Dietary protein and methionine intakes were comparable during the various phases of the study. Informed consent was obtained from the parents before each phase of the study.
RESULTS
Cobalamin processing in fibroblasts. Markedly decreased processing of propionate (14% to 21% of normal) and of methyltetrahydrofolate (28% to 39% of normal) was demonstrated in cultured fibroblasts in the absence of OH-B12 (Table I). Processing of propionate in C b l - C cells increased when OH-B12 was added to the media. There was a minimal increase of methyl-THF processing in CbI-C cells with supplemental OH-B12. Uptake of cyanocobalamin by fibroblasts was reduced to 12% to 15% of control. The percentage of total cobalamin represented by Ado-B~2 and CH3-B12, the active cofactors for propionate and homocysteine metabolism, was reduced. There was no complementation after fusion with known Cbl-C fibroblasts. These studies establish the diagnosis of Cbl-C mutant form of methylmalonic acidemia and homocystinuria. Efficacy of oral OH-B12. OH-B~2 1 mg orally given to two adult volunteers resulted in a less than twofold rise in serum B~2 levels 4 hours after ingestion, with a return to baseline by 48 hours (data not shown). Further, a trial of OH-B~2 1 mg orally given three times a day for 1 week did not lead to a significant rise in serum B~2 levels compared with baseline in the control subjects (1015 pg/mL vs 1145 pg/mL). In patient 1, given OH-B~2 1 m g / d orally for 1 month (Fig. 1), there was a loss of metabolic control and an increase in homocystine (10/xmol/L vs 1 /xmol/L) and decrease in methionine (21 #mol/L vs 40 ~zmol/L). These
Volume 112 Number 1
Cbl-C methylmalonic acidemia and homocystinuria
PATIENT
35
2
200~000 100,000
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Fig. 9. Metabolic measurements in patient 2. Blood levelsof B]2,methylmalonate (MMA), reduced homocysteine(Hcy), and methionine (Met) during 3 weeks after single intramuscular injection of 1 mg OH-B12 either with (solid lines) or without (dashed lines) supplements of folate and carnitine. Betaine was continued throughout the study. Total Hcy was measured by gas chromatography-mass spectroscopy. (See Fig. 3 for similar information on patient 1.)
levels reverted toward normal when OH-B12 intramuscularly was restarted (Days 150 to 180). There were no clinical symptoms during this time. In this study, homocystine levels reflect only the homocystine disulfides as measured by amino acid column chromatography. Required frequency of OH-B~2injections. In both control subjects and affected children, serum B12 levels rose approximately 20-fold 24 hours after a 1 mg intramuscular injection of OH-B~ 2. The half-life of OH-B12 in the serum
of adult male control subjects was approximately 24 hours, whereas the half-life in the two affected children was 62 hours. In the children, plasma B~2levels decreased from the 24-hour peak of approximately 200,000 pg/mL to < 10,000 pg/mL, during a 19-day follow-up (Figs. 2 and 3). There was no significant increase in total plasma homocysteine in either patient over the 19 days; the mean baseline value for homocysteine was 78 gmol/L vs 77 #mol/L 19 days after injection. A control standard of pooled normal human
36
Bartholomew et al.
The Journal of Pediatrics January 1988
PATIENT
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Fig. 3. Metabolic measurements in patient 1. See legend presented with Fig. 2 for explanation of illustration.
serum for homocysteine was 11 umol/L. Plasma methionine levels were 15 #mol/L at the beginning of the study and 23 ~mol/L at its conclusion. Plasma methylmalonate levels varied from 39 #mol/L to 183 #mol/L, with a mean value of 56 #mol/L in patient 1 and 139 tzmol/L in patient 2 (normal 0.2 to 0.7 ~mol/L). In sum, there was some day-to-day variability in the metabolic measurements, but no consistent alterations occurred over the 19 days after OH-B~2 injection. There were no adverse clinical symptoms during the course of this study. Effect of carnitine therapy. In patient 1, pretreatment
free and total carnitine levels were normal, but short-chain acylcarnitine esters were increased. In patient 2, there were decreased levels of both free and total carnitine (Table II). The predominant short-chain acylcarnitine esters in both patients were propionylcarnitine and methylmalonylcarnitine. After an oral loading dose of L-carnitine 100 mg/kg (Sigma-Tau), the urinary acylcarnitine profiles in both patients showed excretion of a mixture of free carnitine and acetyl-, propionyl-, and octenoyl-carnitine esters. Daily supplementation with 100 mg/kg L-carnitine resulted in
Volume 112 Number 1
Cbl-C rnethylmalonic acidemia and homoeystinuria
37
T a b l e II. Plasma carnitine and methylmalonate (MMA) levels (#tool/L) before and during
L-carnitine supplementation (100 mg/kg/d) Plasma carnitine Condition
Patient 1 Baseline Treated Patient 2 Baseline Treated Normal*
Acylcarnitine esters
Total
Free
Short chain
Long chain
60 99
42 73
16 22
2 4
23 84 46 +__10
15 54 37 _+ 8
6 25 6 _+ 4
2 5 4+ 2
Plasma MMA
52 56 147 140 0.4 _+ 0.1
*Mean -+ SD.
Table III. Effect of calcium leucovorin on red cells and on plasma methylmalonate, homocystine, and methionine levels Patient 4
Hgb (g/dL) MCV (fL) MCFI (pg) Morphology MMA (umol/L) Hey (/~mol/L) Met (gmol/L)
Patient 2
Pre
Post
Pre
Post
Normal
12.9 88.8 30.9 Normal 34.6 69 12
12.3 93.7 31.0 Normal 38.6 64 13
12.5 92.5 30.6 Normal 64 64 30
12.1 91.5 29.5 Normal 68.3 68.3 22.3
11.7-13.8 75-87 24-30 Normal 0.2-0.7 7-20 12-33
MCV, meancorpuscularvolume;MCH, meancorpuscularhemoglobin;MMA,methylmalonate;Hey, homocysteine;Met, methionine.
a marked increase in both total carnitine and in these short-chain acylcarnitine esters (Table II). However, removing the L-carnitine supplement from the treatment regimen for 3 weeks did not result in a change in plasma homocysteine or methylmalonate levels (Fig. 2). Because of the presence of octenoyl (C8)-earnitine esters, the possibility of an abnormality in peroxisomal fatty acid oxidation was considered, because very-longchain fatty acids are normally oxidized down to the C8 length in peroxisomes. This was tested by measuring levels of very-long-chain fatty acid levels in plasma. The ratio of very-long-chain fatty acids (C26/C22) was found to be normal, 0.016 (normal <0.030) in the two children, suggesting normal fatty acid oxidation. Effect of betaine. Betaine was deleted from the treatment regimen was patient 1 for 1 month between days 90 and 119 (Fig. 1). The child continued to receive daily injections of 1 mg OH-B~2. During this time there was a modest increase in homocystine levels and a decrease in
methionine levels, which was reversed (between days 120 and 150) by reintroducing betaine supplements of 250 mg/kg/d. On the other hand, the continued use of betaine between days 150 and 180, when OH-Blz orally was substituted for intramuscular injection, did not prevent an increase in plasma homocystine and a decrease in methionine. Effect of folinic acid. Serum folate levels in both children were normal, >20 ng/mL, and neither patient had anemia at the time of study. Mean corpuscular volume was at the upper limits of normal, but there was no apparent hypersegmentation of the nuclei in neutrophils. Calcium leueovorin (Wellcovin, Wellcome Diagnostics, Research Triangle Park, N.C.) 25 mg/d was administered orally in two divided doses for 14 days. OH-B12, betaine, and L-carnitine were continued according to the above treatment regimen. Hemoglobin, hematocrit, red blood cell indices, plasma methylmatonate, homocysteine, and methionine were assessed for 2 weeks before the study to
38
Bartholomew et al.
determine baseline values (Table III). No differences were seen in pretreatment versus treatment levels. DISCUSSION Fourteen patients with CbI-C B~2 activation defect have been described?,4,t4-22 As found in our children, plasma methylmalonate levels are at least 100 times normal, but do not approach the 200- to 2000-fold increase in patients with methylmalonyl-CoA mutase deficiency or Cbl-B deficiency.23 Homocystine levels are also elevated, and methionine levels are subnormal. Therapy of the Cbl-C defect has consisted primarily of injections of vitamin BlZ. Frequency of injections has varied from every 1 to 28 days. Despite clinical improvement, B1z supplements do not fully correct the underlying biochemical defect, and it is unknown whether residual methylmalonic acidemia and homocystinemia are detrimental to the child's physical or intellectual development. The intellectual development in our children is in the borderline normal range, and has been stable or improved since onset of therapy. OH-B~ appears to be more effective than CN-BI2 in treating this disorder? OH-B~2 remains at the site of injection longer than does CN-B12, 24 and there is a more -sustained rise in serum B~2 levels and less immediate urinary excretion of cobalamin metabolites after OH-B~2 intramuscular injections as compared with CN-B12. 25 Fibroblasts from patients with Cbl-C defect have only 30% of control activity of the methyltransferase holoenzyme in the presence of CN-B~z, but have up to 100% of control activity in the presence of OH-BIE,26 For these reasons, we did not attempt therapy with CN-Bx2. We initially studied whether orally administered OHBi2 might be an appropriate substitute for intramuscular OH-B~2 injections. Our studies in adult control subjects demonstrated that even massive doses of OH-BI2 given orally (1000 times the recommended daily allowance of 3 #g for an adult 27) were inadequate to raise serum B~2 levels significantly. Further, we noted that orally given OH-Ba2 1 mg/d in one patient resulted in a fall in plasma methionine with a concurrent rise in homoeystine over a 1-month period. For these reasons we did not pursue oral OH-B~2 therapy in our patients. Betaine is a methyl donor that has been used successfully in treating B6-unresponsive homocystinuria caused by cystathionine B synthetase deficiency.28The mechanism of action of betaine involves the conversion of homocysteine to methionine via betaine homocysteine methyltransferase, thus bypassing the BxE-dependent cytosolic 5-methyl-tetrahydrofolate homocysteine methyltransferase. This reaction does not require CH3Baz as a cofactor. 28The possible utility of betaine in Cbl-C mutants is suggested by our pilot study
The Journal of Pediatrics January 1988
in whch the removal of this medication was associated with a lower plasma methionine and higher plasma homocystine level despite continuation of daily intramuscular injections of OH-B12. On the other hand, betaine did not normalize methionine levels in the absence of OH-B12. Thus betaine and OH-B~2 may have a synergistic effect. Subnormal plasma carnitine levels, found in one of our children, have been noted in other organic acidemias. 29 This is t h e presumed result of a limited protein intake combined with increased urinary excretion of carnitine esters. Carnitine could increase excretion of propionate and methylmalonate as carnitine esters and restore the acyl-CoA/free CoA ratio in the mitochondria.3~Carnitine therapy did increase propionylcarnitine excretion in the urine but did not have a significant effect on plasma methylmalonate levels. Folic acid or its activated form, formyl-THF (folinic acid or leucovorin) has also been recommended as a therapeutic adjunct in this disorder because of a postulated deficiency of intracellular reduced folate polyglutamates as a result of a "methylfolate trap. ''3~ Pharmacologic doses of folinic acid provide a means of bypassing the methylfolate trap. 32 However, the mild macrocytic anemia and relative hypomethioninemia in our patients was unaltered by pharmacologic doses of folinie acid. In summary, we found that OH-B12 1 mg intramuscularly plus betaine 250 mg/kg/d orally was effective in the treatment of Cbl-C defect in two children. Orally administered OH-B~2 appeared to be ineffective. Daily OH-B12 injections did not seem to be required in our patients, Long-term supplementation with L-carnitine 100 to 200 mg/kg/d increased propionylcarnitine excretion and may therefore be useful, but we did not demonstrate a consistent decrease in plasma methylmalonate during carnitine supplemention. Although there are theoretical advantages to the use of folinic acid supplements, we could not document their value in our short-term study. We thank Dr. David Watkins for performing the complementation studies in our patients; Ann Moser for measuring verylong-chain fatty acid levels; and Ms. Bernadine Peters and the nurses of the Pediatric Clinical Research Unit. REFERENCES
1. McKusick VM. Mendelian inheritance in man, 7th ed. Baltimore: Johns Hopkins University Press, 1986:1297. 2. Robb RM, Dowton SB, Fulton AB, Levy HL. Retinal degeneration in vitamin B12disorder associated with methylmalonic aciduria and sulfur amino acid abnormalities. Am J Ophthalmol 1984; 97:691-6. 3. Mitchell GA, Watkins D, Melancon SB, et al. Clinical heterogeneity in cobalamin C variant of combined homocystinuria and methylmalonicaciduria. J PEDIATR1986;108:4105.
Volume 112 Number 1 4. Carmel R, Bedros AA, Mace JW, Goodman SI. Congenital methylmalonic aciduria: homocystinuria with megaloblastic anemia. Observations on response to hydroxocobalamin and on the effect of homocysteine and methionine on the deoxyuridine suppression test. Blood 1980;55:570-9. 5. Brass EP, Hoppel CL. Carnitine metabolism in the fasting rat. J Biol Chem 1978;253:2688-93. 6. Millington DS, Smith JA. Fragmentation patterns by fastlinked electric and magnetic field scanning. Organic Mass Spectrom 1977;12:264. 7. Barber M, Bordoli RS, Sedgwick RD, Tyler AN. Fast atom bombardment of solids (FAB): a new ion source for mass spectrometry. JCS Chem Comm 1981;325. 8. Stabler SP, Marcell PD, Podell ER, Allen RH, Lindenbaum J. Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatographymass spectrometry. J Clin Invest 1986;77:1606-12. 9. Rothenberg SP. Assay of serum vitamin Bt2 concentration using Co57-B12 and intrinsic factor. Proc Soc Exp Biol Med 1961;108:45-8. 10. Stabler SP, Marcell PD, Podell ER, Allen RH, Lindenbaum J. Homocysteine is an accurate indicator of cobalamin or folate deficiency [Abstract]. Blood 1985;60(suppl 1):50A. 11. Rosenblatt DS, Cooper BA, Pottier A, Lue-Shing H, Matiaszuk N, Grauer K. Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis. J Clin Invest 1984i74:2149-56. 12. Watkins D, Rosenblatt DS. Failure of lysosomal release of vitamin B~2: a new complementation group causing methylmalonic aciduria (cblF). Am J Hum Genet 1986;39:404-8. 13. Willard HF, Mellman IS, Rosenberg LE. Genetic complementation among inherited deficiencies of methylmalonylCoA mutase activity: evidence for a new class of human cobalamin mutant. Am J Hum Genet 1978;30:1-13. 14. Levy HL, Mudd SH, Schulman JD, Dreyfus PM, Abeles RH. A derangement in B12 metabolism associated with homocystinemia, cystathioninemia, hypomethioninemia and methylmalonic aciduria. Am J Med 1970;48:390-7. 15. Goodman SI, Moe PG, Hammond KB, Mudd SH, Uhlendorf BW. Homocystinuria with methylmalonic aciduria: two cases in a sibship. Biochem Med 1970;4:500-15. 16. Dillon M J, England JM, Gompertz D, et al. Mental retardation, megaloblastic anaemia, methylmalonic aciduria and abnormal homocysteine metabolism due to an inborn error in vitamin B~2 metabolism. Clin Sci Mol Med 1974;47:43-61. 17. Anthony M, McLeay AC. A unique case of derangement of vitamin B12 metabolism. Proc Aust Assoc Neurol 1976;13: 61-5. 18. Baumgartner ER, Wick H, Maurer R, Egli N, Steinmann B. Congenital defect in intracellular cobalamin metabolism resulting in homocystinuria and methylmalonic aciduria. I. Case report and histopathology. Heir Paediatr Acta 1979; 34:465-82.
Cbl-C methylmalonic acidemia and homocystinuria
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