Urinary Methylmalonic Acid Levels Research Articles

Variable phenotypes and outcomes associated with the MMACHC c.482G > A mutation: follow-up in a large CblC disease cohort

Abstract Background The aim of this study was to characterize the variable phenotypes and outcomes associated with the methylmalonic aciduria and homocystinuria type C protein gene (MMACHC) c.482G > A mutation in 195 Chinese cases with CblC disease. Methods We carried out a national, retrospective multicenter study of 195 Chinese patients with CblC disease attributable to the MMACHC c.482G > A variant either in a homozygous or compound heterozygous state. The control group consisted of 200 patients diagnosed with CblC disease who did not possess the c.482G > A mutation. Clinical features, including disease onset, symptoms, biochemical metabolites, gene mutation, and follow-up outcomes were reviewed and analyzed in detail. The median follow-up period spanned 3 years and 8 months, with a range of 1 year and 2 months to 12 years and 10 months. Results Among 195 patients carrying the c.482G > A variant, 125 (64.1%) cases were diagnosed by newborn screening (NBS), 60 (30.8%) cases were detected due to disease onset, and 10 (5.1%) cases were identified from sibling diagnoses. One hundred and seventeen (93.6%) individuals who were diagnosed by NBS, and nine patients who came from sibling diagnoses remained asymptomatic in this study. From 69 symptomatic patients of the c.482G > A group, more patients presented with later onset, and the top six common clinical symptoms at disease onset were developmental delay (59.4%), lower limb weakness and poor exercise tolerance (50.7%), cognitive decline (37.7%), gait instability and abnormal posture (36.2%), seizures (26.1%), and psychiatric and behavioral disturbances (24.6%). In the 159 symptomatic patients lacking c.482G > A variants, the most frequently observed clinical manifestations at disease onset included developmental delay (81.8%), lethargy and feeding difficulty (62.9%), lower limb weakness and poor exercise tolerance (54.7%), prolonged neonatal jaundice (51.6%), vomiting (47.2%), and seizures (32.7%). Before treatment, the levels of blood propionylcarnitine, propionylcarnitine/acetylcarnitine ratio, and homocysteine in the c.482G > A group were significantly lower (P < 0.05) than those in the non-c.482G > A group, while the concentration of urinary methylmalonic acid was slightly lower (P > 0.05). The degree of decline in the above metabolites after treatment in different groups significantly differed in both plasma total homocysteine values and urinary methylmalonic acid levels (P < 0.05). In patients carrying the c.482G > A variant compared with the non-c.428G > A group, there were markedly lower rates of mortality (0.5% vs. 2.0%) and developmental delay (20.5% vs. 65.5%). When compared with individuals diagnosed due to disease onset, those identified through NBS in either group exhibited a reduced proportion of disease onset (6.7% vs. 100% in the c.482G > A group, 54.4% vs. 100% in the non-c.482G > A group), lower mortality (0.0% vs. 1.7% in the c.482G > A group, 0.0% vs. 3.6% in the non-c.482G > A group), and had a higher percentage of patients exhibiting normal psychomotor and language development (99.3% vs. 33.3% in the c.482G > A group, 58.9% vs. 10.9% in the non-c.482G > A group). Conclusions The c.482G > A variant in MMACHC is associated with late-onset and milder phenotypes of CblC disease. Patients with this mutation tend to have a relatively better response to hydroxocobalamin, better metabolic control, and more favorable neurological outcomes. NBS and other appropriate pre-symptomatic treatments seem to be helpful in early diagnosis, resulting in favorable clinical outcomes.

Genetic testing is necessary for correct diagnosis and treatment in patients with isolated methylmalonic aciduria: a case report

BackgroundIsolated methylmalonic aciduria can be caused by pathogenic mutations in the gene for methylmalonyl-CoA mutase or in the genes encoding enzymes involved in the intracellular metabolism of cobalamin. Some of these mutations may be cobalamin responsive. The type of methylmalonic aciduria cannot always be assumed from clinical manifestation and the responsiveness to cobalamin has to be assessed for appropriate cobalamin administration, or to avoid unnecessary treatment. The cases presented herein highlight the importance of genetic testing in methylmalonic aciduria cases and the need for standardisation of the in vivo cobalamin-responsiveness assessment.Case presentationWe describe two patients who presented in the first week of life with rapid neurological deterioration caused by metabolic acidosis with severe hyperammonaemia requiring extracorporeal elimination in addition to protein restriction, energy support, carnitine, and vitamin B12 treatment. The severity of the clinical symptoms and high methylmalonic acid concentrations in the urine (>30,000 μmol/mmol of creatinine) without hyperhomocysteinaemia in both of our patients suggested isolated methylmalonic aciduria. Based on the neonatal manifestation and the high methylmalonic acid urine levels, we assumed the cobalamin non-responsive form. The in vivo test of responsiveness to cobalamin was performed in both patients. Patient 1 was evaluated as non-responsive; thus, intensive treatment with vitamin B12 was not used. Patient 2 was responsive to cobalamin, but the dose was decreased to 1 mg i.m. every two weeks with daily oral treatment due to non-compliance. Genetic tests revealed bi-allelic mutations in the genes MMAB and MMAA in Patient 1 and 2, respectively. Based on these results, we were able to start intensive treatment with hydroxocobalamin in both patients. After the treatment intensification, there was no acute crisis requiring hospitalisation in Patient 1, and the urine methylmalonic acid levels further decreased in Patient 2.ConclusionsDespite carrying out the in vivo test of responsiveness to cobalamin in both patients, only the results of molecular genetic tests led us to the correct diagnosis and enabled intensive treatment with hydroxocobalamin. The combination of the standardized in vivo test of cobalamin responsiveness and genetic testing is needed for accurate diagnosis and appropriate treatment of isolated methylmalonic aciduria.

Clinical Application of Liquid Chromatography Tandem Mass Spectrometry Using Dried Blood Spot as a More Rapid Method for Determination of Methylmalonic Acid, Propionylcarnitine, and Total Homocysteine

Abstract Methylmalonic acidemia (MMA) should be diagnosed in early infancy and receive appropriate management promptly after the diagnosis to prevent severe complications leading to death. At present, a newborn screening (NBS) method using tandem mass spectrometry (MS/MS) identifies suspected patients with MMA by elevated propionylcarnitine. In addition, a liquid chromatography tandem mass spectrometry (LC/MS/MS) method using dried blood spot is effective to detect some metabolites as a second-tier test, and reduces the false-positive rate in NBS. However, these tests were only used in screening, and not applied as an examination for evaluating treatment. Herein, we describe a 57-day-old girl with MMA under treatment with cobalamin who had elevated urinary methylmalonic acid levels. We applied the LC/MS/MS method with a separation column to evaluate her cobalamin responsiveness, and discovered an insufficient cobalamin dose earlier than would have been possible using other methods. Based on the current data, this method seems to be applicable for the follow-up of the treatment of MMA patients. However, this should be confirmed with more experience with a larger number of cases and a wider spectrum of disorders.

Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China

The mut methylmalonic acidemia (MMA) caused by the deficiency of methylmalonyl-CoA mutase (MCM) activity, which results from defects in the MUT gene. The aim of this study was to summarize the clinical and biochemical data, spectrum of mutations, treatment regime and follow-up of patients with mut MMA from Jan 2013 to Dec 2017 in Shandong province, China. Twenty patients were diagnosed with isolated mut MMA by elevated C3, C3/C2, and urine methylmalonic acid levels without hyperhomocysteinemia. The MUT gene was amplified and sequenced. Most patients received treatment with specific medical nutrition and oral l-carnitine after diagnosis. Metabolic parameters, clinical presentation and mental development were followed up. Among 20 patients with mut MMA, 14 had clinical presentations, and 12 presented in the neonatal period. Three patients died of metabolic crises triggered by infection. Twenty-three different mutations were detected, and four mutations (c.613G>A, c.446A>G, c.920-923delTCTT and c.1359delT) were novel. Most patients received timely treatment and had favorable metabolic responses, with reductions in C3, C3/C2 and urine MMA. We obtained 16 records of DQ/IQ assessments. Six patients exhibited normal development, but ten patients suffered from neurological symptoms of varying degrees and had low DQ/IQ scores. Our study contributes toward expanding the knowledge of the genetic basis of mut MMA. The c.914T>C was the most frequent mutation, and four novel mutations were detected. Patients diagnosed by newborn screening and treated at the presymptomatic stage may have better outcomes. However, these limited data do not allow any definitive statements on possible genotype-phenotype correlations that can influence the outcomes of mut MMA. Nonetheless, it is necessary for high-risk families to have early prenatal diagnoses.

Vitamin B12 Deficiency in Children With Infantile Spasms: A Case-Control Study.

There have been few case reports showing association of vitamin B12 deficiency with infantile spasms. We planned this study to see if there was an association of serum vitamin B12 deficiency in children with development of infantile spasms. Cases included children with infantile spasms of ages 6 months to 3 years. The controls were children in the same age group who had global developmental delay but no history of epileptic spasms. Mean serum vitamin B12, serum homocysteine, and urinary methylmalonic acid levels were measured in both groups and compared. Children with infantile spasms had lower mean serum vitamin B12 levels (354.1 pg/mL; standard deviation 234.1 pg/mL) as compared to children with global developmental delay without spasms (466.7 pg/mL; standard deviation 285.5 pg/mL) ( P value < .05). Mean serum homocysteine level (13.9 vs 7.8 μmol/L, P = .02) and mean urinary methylmalonic acid level (68.1 mmol/mol of creatinine vs 26.1 mmol/mol of creatinine, P = .03) were elevated in children with infantile spasms than in controls. Fourteen children (35.0%) with infantile spasms were vitamin B12 deficient compared with 3 (7.50%) controls ( P = .005). Thus, vitamin B12 deficiency may have an association with infantile spasms. More studies are needed before recommending routine measurement of serum B12 levels in children with infantile spasms.

Validation of an HPLC method for total homocysteine quantification in plasma

IntroductionHomocysteine (Hcy) is a nonessential amino acid which links the methionine and the folate cycles. Hcy levels are increased in genetic disorders, such as classic homocystinuria and methylmalonic aciduria combined with homocystinuria. Other monogenic, multifactorial diseases and physiological conditions are also associated with high Hcy levels. The aim of this study is to validate a method to quantify Hcy in plasma. Material and methodsA method to quantify Hcy in plasma by HPLC was validated, by determining the following parameters: specificity, linearity, precision, accuracy, and detection and quantification limits according ICH and EMEA guidelines. Homocysteine was measured in 43 healthy individuals, 2 patients with high levels of methylmalonic acid, and a previously diagnosed patient with classic homocystinuria. ResultsThe method was able to identify and quantify Hcy without interferences. A linear behavior was observed in a range of 6–100μM with r2=0.9967. The precision and accuracy studies showed variation coefficients under 6%. The limits of detection and quantification were 3.12μM and 6.25μM, respectively. The 43 healthy individuals studied had normal Hcy levels. The patient with elevated urinary methylmalonic acid levels and the homocystinuria patient showed high Hcy levels. ConclusionThe method is valid for the quantification of Hcy in plasma as it fulfills the requirements for validation of analytical methods. Its introduction into diagnostic and follow up algorithms is important in genetic diseases where this amino acid is increased.

Impact of HIV Infection and Zidovudine Therapy on RBC Parameters and Urine Methylmalonic Acid Levels.

Background. Anaemia is a common complication of human immunodeficiency virus (HIV) infection. The aim of this study was to investigate the impact of HIV infection and zidovudine on red blood cells (RBC) parameters and urine methylmalonic acid (UMMA) levels in patients with HIV infection. Material and Methods. A cross-sectional study involving 114 subjects, 94 of which are HIV-infected nonanaemic and 20 HIV negative subjects (Cg) as control. Full blood count parameters and urine methylmalonic acid (UMMA) level of each subject were determined. Associations were determined by Chi-square test and logistic regression statistics where appropriate. Results. Subjects on zidovudine-based ART had mean MCV (93 fL) higher than that of control group (82.9 fL) and ART-naïve (85.9 fL) subjects and the highest mean RDW. Mean UMMA level, which reflects vitamin B12 level status, was high in all HIV-infected groups but was significantly higher in ART-naïve subjects than in ART-experienced subjects. Conclusion. Although non-zidovudine therapy may be associated with macrocytosis (MCV > 95 fL), zidovudine therapy and ART naivety may not. Suboptimal level of vitamin B12 as measured by high UMMA though highest in ART-naïve subjects was common in all HIV-infected subjects.

Methylmalonic Aciduria Secondary to Selective Cobalamin Malabsorption in a Yorkshire Terrier

An 8 wk old male Yorkshire terrier was presented with a 2 wk history of recurrent hypoglycemia, lethargy, and seizures. Investigations revealed a marked increase in blood ammonia, low serum cobalamin, and increased levels of urinary methylmalonic acid (MMA) excretion. No liver vascular abnormality was detected. The patient was diagnosed with methylmalonic aciduria due to cobalamin malabsorption. The patient responded well to parenteral cobalamin administration, and the urinary MMA levels normalized rapidly following instigation of treatment. Due to the suspected hereditary nature of selective cobalamin deficiency, one sibling of this dog was screened and found to be normal. This is the first reported case of MMA secondary to hypocobalaminemia in Yorkshire terriers, and the second report of this disease in a dog in the United Kingdom. Given the fact that clinical signs of MMA are similar to those seen in dogs with portosystemic shunts and that Yorkshire terriers are predisposed to liver vascular abnormalities, this case report adds important clinical information to the current available literature.

Correction of Methylmalonic AciduriaIn VivoUsing a Codon-Optimized Lentiviral Vector

Methylmalonic aciduria is a rare disorder of organic acid metabolism with limited therapeutic options, resulting in high morbidity and mortality. Positive results from combined liver/kidney transplantation suggest, however, that metabolic sink therapy may be efficacious. Gene therapy offers a more accessible approach for the treatment of methylmalonic aciduria than organ transplantation. Accordingly, we have evaluated a lentiviral vector-mediated gene transfer approach in an in vivo mouse model of methylmalonic aciduria. A mouse model of methylmalonic aciduria (Mut(-/-)MUT(h2)) was injected intravenously at 8 weeks of age with a lentiviral vector that expressed a codon-optimized human methylmalonyl coenzyme A mutase transgene, HIV-1SDmEF1αmurSigHutMCM. Untreated Mut(-/-)MUT(h2) and normal mice were used as controls. HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for age, and Western blot analysis demonstrated significant methylmalonyl coenzyme A enzyme expression in their livers. Normalization of liver methylmalonyl coenzyme A enzyme activity in the treated group was associated with a reduction in plasma and urine methylmalonic acid levels, and a reduction in the hepatic methylmalonic acid concentration. Administration of the HIV-1SDmEF1αmurSigHutMCM vector provided significant, although incomplete, biochemical correction of methylmalonic aciduria in a mouse model, suggesting that gene therapy is a potential treatment for this disorder.

To ascertain the utility of urinary methyl malonic Acid as a potential marker of ischemic heart failure.

Methyl Malonic Acid (MMA) is known to be an integral component of the cascade of events in mitochondrial energy metabolism and since heart failure involves energy pathways, it is probable that levels of MMA could be used as a reliable biomarker to objectively identify the disease during the early stages and help in prognostication. The present study was envisaged to evaluate the relation between urinary MMA levels in patients with Ischemic heart disease and in those progressing to failure. The relationship between the severity of the disease and the level of MMA in urine were also evaluated. Analysis of urinary MMA was done by Gas Chromatography-Mass Spectrometry (GC-MS) using stable isotope dilution. Twenty patients each with Ischemic Heart Disease, heart failure and controls were recruited in this pilot study. The mean value of MMA in patients with IHD was 126.71(±66.3) pmol/L and those with IHF was 390.76 (±97.99) pmol/L with the difference being statistically significant (p<0.001). Co-existing B12 deficiency was excluded as that was a potential confounder. Levels of MMA were significantly higher in patients with heart failure compared to those with IHF, which were significantly higher than controls. Evaluation of the correlation between MMA levels and the stage of the disease did not reach statistical significance; but this requires adequately powered studies to support this preliminary finding.

A novel mutation in LMBRD1 causes the cblF defect of vitamin B 12 metabolism in a Turkish patient

In the cblF defect of vitamin B(12) (cobalamin) metabolism, cobalamin is trapped in lysosomes. Consequently, cobalamin coenzyme synthesis is blocked, and cofactors for methionine synthase and methylmalonyl-coenzyme A (CoA) mutase are deficient. We recently identified LMBRD1 as the causative gene located on chromosome 6q13 and showed that 18 out of 24 alleles in unrelated patients carried the deletion c.1056delG (p.L352fsX18) (Rutsch et al. (Nat Genet 41:234-239, 2009). LMBRD1 encodes the lysosomal membrane protein LMBD1, which presumably facilitates lysosomal cobalamin export. Our patient is the second child of consanguineous Turkish parents. He presented on the second day of life with cerebral seizures due to intraventricular hemorrhage. Plasma homocysteine and urinary methylmalonic acid levels were elevated, and serum cobalamin level was decreased. Synthesis of both cobalamin coenzymes was deficient in cultured skin fibroblasts. The cblF defect was confirmed by somatic complementation analysis. Sequencing of LMBRD1 revealed the novel deletion c.1405delG (p.D469fsX38) on both alleles. Real-time polymerase chain reaction (PCR) revealed reduced messenger RNA (mRNA) levels in patient fibroblasts compared with controls. Transfection of patient fibroblasts with the LMBD1 wild-type complement DNA (cDNA) rescued coenzyme synthesis and function, confirming this new deletion as an additional cause of the cblF defect. This case adds to the spectrum of clinical presentations and mutations of this rare disorder of lysosomal transport.

Urinary methylmalonic acid levels in patients with acute ischemic stroke

Objectives Vitamin B-12 and folate deficiency are common, especially in people aged 55 or over, and accompanied by elevated methylmalonic acid (MMA) and homocysteine concentrations. The aims of the study were to investigate the relationship between serum vitamin B-12, homocysteine, folate, erythrocyte folate and urinary MMA in patients with ischemic stroke, and to develop a simple screening HPLC method for the measurement of urinary MMA. Design and methods Twenty-eight patients aged 55 years and over with ischemic stroke and 23 age- and sex- matched healthy controls were included in the study. Serum vitamin B-12 and folate were measured by immunoassay; serum total homocysteine and urinary MMA concentrations by HPLC. Results There was no significant difference in vitamin B-12, folate and homocysteine concentrations between the patient and control groups. Urinary MMA concentrations and erythrocyte folate levels were significantly higher in patients than controls. There was a significantly negative correlation between vitamin B-12 and MMA. Conclusions Increased urinary MMA excretion is associated with ischemic stroke and it may more robustly reflect vitamin B-12 deficiency in patients with ischemic stroke. The method used in this study is eligible for routine urinary MMA measurements.

Efficacy of Living Donor Liver Transplantation for Patients With Methylmalonic Acidemia

Application of liver transplantation to methylmalonic acidemia (MMAemia) is controversial because MMAemia is caused by a systemic defect of methylmalonyl-CoA mutase. The clinical courses of seven pediatric patients with MMAemia undergoing living donor liver transplantation (LDLT) were reviewed. Serum and urinary methylmalonic acid (MMA) levels were found to be significantly decreased after LDLT, whereas serum and urinary MMA levels did not return to normal in any patient. One patient died of sepsis 44 days after LDLT. The other six patients are currently doing well. All patients had preoperative history of acute metabolic decompensation and/or metabolic stroke. However, no episode of acute metabolic decompensation or metabolic stroke was observed postoperatively in any surviving patients. In the preoperative period, all patients showed lethargy and cognitive deficit, both of which were eradicated after LDLT in all surviving patients. Preoperatively, all patients were subjected to dietary protein intake restriction and tube feeding, and were administered several metabolism-correcting medications. The metabolism-correcting medications being administered remained mostly unchanged after LDLT, whereas protein restriction was liberalized and tube feeding became unnecessary in all surviving patients. In addition, physical and neurodevelopmental growth delay remained in all surviving patients during the observation period, which ranged from 4 to 21 months with a median of 10.5 months.

Vitamin B12, Folate, Homocysteine and Urinary Methylmalonic Acid Levels in Infants

Serum vitamin B12 and folate, and their functional markers, plasma homocysteine and urinary methylmalonate (uMMA) were measured in 204 healthy, term infants at birth, and at 2 and 6 months. Compared with infants receiving formula food, those fed mother's milk had lower vitamin B12 and folate at 2 and 6 months. In infants receiving mother's milk, vitamin B12 levels were similar at birth (238 pg/ml) and 2 months (243 pg/ml), whereas with formula milk the level was significantly higher at 2 months (558 pg/ml) than at birth (257 pg/ml). Vitamin B12 was negatively correlated with homocysteine at birth and 6 months. The level of uMMA (mmol/mol creatinine) was higher at 2 (mother's milk, 25.5; formula, 23.97) and 6 months (19.77; 15) than at birth (11.97; 10.88), and was not correlated with vitamin B12 levels. Homocysteine may be a reliable marker of vitamin B12 status in neonates and infants; however, uMMA is not suitable as a marker of vitamin B12 status.

Use of fasting urinary methylmalonic acid to screen for metabolic vitamin B12 deficiency in older persons

Objectives We sought to determine the cutoffs of fasting urinary methylmalonic acid (MMA) indicating elevated and mildly elevated serum MMA concentrations in older persons. Methods We studied 113 female Chinese vegetarians older than 55 y with normal renal function. Fasting serum samples were obtained for measurement of vitamin B12, MMA, and folate and tests of renal function. A fasting urine sample was collected for MMA measurement by the stable-isotope dilution method. The correlation between serum and urinary MMA levels was examined. The optimal cutoffs of urinary MMA for predicting elevated and mildly elevated serum MMA were estimated by use of receiver operating characteristic curves. Results Fasting urinary and serum MMA levels were linearly correlated. Pearson's correlation coefficient was 0.94. The cutoff of fasting urinary MMA of 2 μM/mM of creatinine had a sensitivity of 79%, a specificity of 85%, and a positive predictive value of 93% for elevated serum MMA (>0.4 μM/L). A cutoff of 1.5 μM/mM of creatinine had a sensitivity of 86%, a specificity of 85%, and a positive predictive value of 95% for mildly elevated serum MMA (>0.3 μM/L). Both cutoffs had high positive predictive values for subnormal vitamin B12 concentrations. Conclusion Overnight fasting urinary MMA concentrations have a strong linear relation to serum MMA in older vegetarians without renal impairment. Urinary MMA is potentially useful as a screening tool for metabolic vitamin B12 deficiency in older persons.

Metabolic Vitamin B12 Status on a Mostly Raw Vegan Diet with Follow-Up Using Tablets, Nutritional Yeast, or Probiotic Supplements

Background: Pure vegetarian diets might cause cobalamin deficiency due to lack of dietary intake. It was hypothesized that a population following a vegan diet consuming mostly raw fruits and vegetables, carrot juice, and dehydrated barley grass juice would be able to avoid vitamin B₁₂ deficiency naturally. Methods: Subjects were recruited at a health ministers’ reunion based on adherence to the Hallelujah diet for at least 2 years. Serum cobalamin and urinary methylmalonic acid (MMA) assays were performed. Follow-up with sublingual tablets, nutritional yeast, or probiotic supplements was carried out on subjects with abnormal MMA results. Results: 49 subjects were tested. Most subjects (10th to 90th percentile) had followed this diet 23–49 months. 6 subjects had serum B₁₂ concentrations <147 pmol/l (200 pg/ml). 37 subjects (76%) had serum B₁₂ concentrations <221 pmol/l (300 pg/ml). 23 subjects (47%) had abnormal urinary MMA concentrations above or equal to 4.0 µg/mg creatinine. Sublingual cyanocobalamin and nutritional yeast, but not probiotic supplements, significantly reduced group mean MMA concentrations (tablet p < 0.01; yeast p < 0.05, probiotic > 0.20). Conclusions: The urinary MMA assay is effective for identifying early metabolic cobalamin deficiency. People following the Hallelujah diet and other raw-food vegetarian diets should regularly monitor their urinary MMA levels, consume a sublingual cobalamin supplement, or consume cobalamin in their food.

Reply

Editors note: The above letter was referred to the authors of the original paper, and their reply follows. In reply: We appreciate Dr. Norman's feedback on our article.1However, after reviewing the available evidence,2-6 we found that there is no‘gold standard' test at this time that will identify true vitamin B12tissue deficiency. The most commonly used standard is serum methylmalonic acid and/or homocysteine elevation greater than 2 standard deviations, as was utilized in our study1 and as has been utilized in several other clinical studies.7, 10There are limitations to these measurements, as pointed out by Dr. Norman. However, we find no evidence of consensus on a gold standard for measuring tissue vitamin B12deficiency. If anything, the leaning is toward serum assessments.” A low (22%) positive predictive value of below normal serum vitamin B12level to identify vitamin B12deficiency was reported by Matchar2 when the diagnosis of B12deficiency was made by clinical criteria. This argues that a low serum B12level, is, by itself, not sufficient to make the diagnosis of vitamin B12deficiency. Dr. Norman has reported B12deficiency, defined by urinary methylmalonic acid (MMA) >5 /μ.g/mg creatinine, using a spot urine specimen.3However, these values of MMA were not correlated with clinical manifestations of true B12 deficiency. In the same study, five of 35 individuals (14.2%) had elevated urinary MMA levels despite normal serum B12levels. This evidence contradicts the claim that urinary MMA/creatinine ratio is the gold standard for true vitamin B12tissue deficiency. One can anticipate several limitations to the use of a spot urinary MMA/creatinine ratio in determining vitamin B12deficiency. Chanarin4, 5 showed evidence 30 years ago that all B12deficient patients had low serum B12levels and most, but not all, had elevated urinary MMA levels by 24-hour urine collection, but not by a spot urine sample. Rasmussen12 reported that a protein meal caused a significant stepwise increase in the urinary excretion of MMA. The urinary concentrations of MMA varied as measured on multiple consecutive 3-hour urine collections, apparently because of variations in diuresis during the day. These findings suggest that a complete 18- to 24-hour urine collection is necessary for valid estimation of urinary excretion of MMA.13In clinical practice, however, 24-h urine collection is difficult. As pointed out by Rasumssen,13 there are several points in favor of serum assays of MMA for an indication of vitamin B12deficiency. These include: 1 there is no need for an additional assay for creatinine; 2 the dietary influences in serum in normal persons are far less marked than in urine12; and 3 measurements can be done on stored serum samples drawn at the time of B12level determination. Matchar2 comments that there is no gold standard for coalmine deficiency and recommends the use of clinical criteria to classify patients as coalmine-deficient or nondefi-cient in the absence of a single perfect diagnostic test. We concur, and we suggest that further double-blind, prospective randomized studies are necessary to determine the gold standard test for identifying true B12tissue deficiency.

Chronic Administration of Methylmalonic Acid (MMA) to Rats Causes Proteinuria and Renal Tubular Injury • 1815

Children with methylmalonic acidemia frequently develop chronic renal insufficiency, for reasons unknown. We administered MMA chronicaally to rats to test the hypothesis that MMA is nephrotoxic. Methods: 3-wk-old Sprague-Dawley rats received MMA (pH 7.2-7.4), 20mg/100g BW daily by SQ injection X 6 wks (N=6), then in drinking water (40mg/100g BW/day) for 6 more wks. Two rats received MMA in drinking water from the outset (20mg/100g BW/day X 4 wks, 40mg/100g BW/day X 8 wks). Five rats received saline injections twice daily X 6 wks. Urine and blood were collected every 2 wks. Renal biopsy was performed at 6 wks and necropsy at 12 wks. Urinary MMA was measured by stable isotope dilution gas chromatography- mass spectometry. Results: MMA-treated rats and controls were similar in food and water intake, weight gain, serum creatinine and BUN. SQ or oral MMA produced high urinary MMA levels (e.g., 1646 μM/mM creatinine at 6 wks (mean), vs. 8.26 in controls). Six of 8 MMA-treated rats had proteinuria, that was first detected at 2 wks and thereafter increased (Table). After 12 wks of MMA, glomeruli appeared normal but tubules of proteinuric rats were variably dilated (especially in the renal medulla) with proteinaceous casts and flattened epithelium. By EM, tubular epithelium showed variable mitochondrial swelling and disorganization of cristae, dilated smooth endoplasmic reticulum, dilatation of basolateral intercellular spaces, and more secondary lysosomes than controls. Conclusions: Chronic administration of MMA rats causes renal injury, which histologically appears to predominantly affect tubular epithelial cells. These findings suggest that chronic renal insufficiency associated with methylmalonic acidemia results from nephrotoxicity of MMA.

Biochemical and clinical response to hydroxocobalamin versus cyanocobalamin treatment in patients with methylmalonic acidemia and homocystinuria (cblC)

Objective: To compare the therapeutic effectiveness of hydroxocobalamin and cyanocobalamin in patients with combined methylmalonic acidemia and homocystinuria. Study design: Analysis of urine methylmalonic acid, plasma homocystine, and growth of two unrelated patients with cobalamin C disease who were initially receiving cyanocobalamin and were subsequently switched to hydroxocobalamin. Results: Each patient had a significant decrease in urine methylmalonic acid excretion while receiving cyanocobalamin, but levels remained at least 10 times normal. Cyanocobalamin treatment resulted in a decrease of plasma homocystine to near normal in one patient but had no effect on plasma homocystine in the second patient. Each patient was switched to hydroxocobalamin and urine methylmalonic acid levels decreased to the limit of detection. Plasma homocystine values while taking hydroxocobalamin remained < 5 nmol/ml in both patients. In patient 1, who continued to receive cyanocobalamin therapy for more than 1 year, growth rates (height, weight, and head circumference) were very poor. After initiation of hydroxocobalamin, growth parameters normalized with growth rates above normal. Conclusion: Intramuscular cyanocobalamin treatment is inadequate in the treatment of patients with cobalamin C disease. Appropriate management of cobalamin C disease should include only the hydroxocobalamin form of cobalamin. (J Pediatr 1998;132:121-4)

Screening elderly populations for cobalamin (vitamin B 12) deficiency using the urinary methylmalonic acid assay by gas chromatography mass spectrometry

purpose: This study assesses the value of the urinary methylmalonic acid (MMA) assay by gas chromatography mass spectrometry as a screening procedure for detection of cobalamin (Cbl) deficiency and estimates the prevalence of undetected Cbl deficiency in elderly populations. subjects and methods: A total of 809 elderly individuals over age 65 were screened using random spot urine specimens from 4 different sites: a health fair, retirement apartments, a hospital-based elderly assessment center, and a nursing home. Follow-up tests included serum total Cbl, serum MMA, and normalization of urinary and serum MMA levels with Cbl intramuscular (IM) therapy. results: The prevalence of elevated urinary MMA varied across population groups, from 3.0% in elderly visiting a health fair to 5.1% in elderly residing in retirement apartments. Follow-up on 35 of 36 subjects with elevated urinary MMA levels showed that 18 had low serum total Cbl (less than 180 pg/mL at Hospital 1 or less than 200 pg/mL at Hospital 2), 12 had lownormal Cbl (180 or 200 pg/mL to 350 pg/mL), and 5 had normal Cbl. Of the 12 subjects with low-normal Cbl on retesting, further assessment was performed in 7, and all 7 of these subjects had evidence of Cbl deficiency. Cbl IM therapy was initiated for 23 subjects; 16 were seen for follow-up and all had normal urinary MMA. conclusion: The relatively high prevalence of undetected Cbl deficiency identified in the seniors warrants additional studies of elderly populations. The sensitivity, convenience, and noninvasive nature of the urinary MMA assay by gas chromatography mass spectrometry make it a practical screening test.