Renal involvement in a patient with cobalamin A type (cblA) methylmalonic aciduria: a 42-year follow-up.

Chronic renal failure is a well-known long-term complication of methylmalonic aciduria (MMA-uria), occurring even under apparently optimal metabolic management. The onset of renal dysfunction seems to be dependent on the type of defect and vitamin B12-responsiveness. We report on a patient with a vitamin B12-responsive cobalamin A type (cblA) MMA-uria caused by a homozygous stop mutation (p.R145X) in the cobalamin A gene (MMAA). She was diagnosed with chronic kidney disease (CKD) stage III at the age of 12 years. Following re-evaluation, the patient received vitamin B12 (hydroxocobalamin) treatment, resulting in a significant decrease in the concentration of methylmalonic acid (MMA) in urine and plasma. Until age 29 years glomerular filtration rate remained stable probably due to hydroxocobalamin treatment slowing down progression to end-stage renal failure. Kidney biopsies showed non-specific manifestations of chronic interstitial inflammation. The patient received a renal transplant at age 35 years. Under continuous treatment with hydroxocobalamin there is no evidence of kidney damage due to MMA-uria until the last follow-up 6 years after transplantation. This case report illustrates (i) a long-term follow-up of a patient with MMA-uria due to cblA deficiency, (ii) the involvement of the kidney as a target organ and (iii) the importance of early and adequate vitamin B12 substitution in responsive patients. Further investigation will be necessary to prove the protective effect of hydroxocobalamin in the kidney in vitamin B12-responsive patients.

Coenzyme Q(10) is decreased in fibroblasts of patients with methylmalonic aciduria but not in mevalonic aciduria.

The content of coenzyme Q(10) (CoQ(10)) was examined in skin fibroblasts of 10 patients with mevalonic aciduria (MVA) and of 22 patients with methylmalonic aciduria (MMA). Patients with these inborn errors of metabolism are thought to be at risk for CoQ(10) depletion either by direct inhibition of the proximal pathway of CoQ(10) synthesis (MVA) or indirectly by inhibition of mitochondrial energy metabolism (MMA). We demonstrated that CoQ(10) concentrations were not significantly different from controls in MVA patients, suggesting that there may be upregulatory effects. On the other hand the CoQ(10) content in fibroblasts of patients with MMA was significantly reduced.

The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha subunits and smaller beta subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function.

mut0 methylmalonic acidemia: eleven novel mutations of the methylmalonyl CoA mutase including a deletion-insertion mutation.

Methylmalonic aciduria (MMA) is an autosomal-recessive disorder caused by inadequate function of methylmalonyl-CoA mutase (MCM), a nuclear-encoded, mitochondrial enzyme that uses adenosylcobalamin as a cofactor. Biochemical cell studies have delineated phenotypic variants: mut(0) phenotypes in which there is no detectable enzymatic activity and mut- phenotypes in which there is residual cobalamin-dependent activity. Mutation screening in MMA has led to the detection of 30 disease-specific mutations. In 14 patients with the mut(0) phenotype we found 11 novel mutations (K54X, A137V, F174S, 620insA, G203R, Q218H, A535P, H627R, 2085delG and 2270del4/ins5), 6 of them homozygous, consisting of 1 nonsense, 6 missense, 1 splice site, and 3 frame shift mutations. The position in relation to different functional domains in MCM allow for an interpretation of the identified mutations. Hum Mutat 16:179, 2000.

Biotinidase deficiency associated with renal loss of biocytin and biotin.

Clinical and biochemical investigations in six patients with congenital biotinidase deficiency are presented. The time course of biotin depletion in relation to carboxylase activities and clinical onset of symptoms was studied after withdrawal of biotin supplementation. Renal biotin clearance studies were performed in patients and controls. Renal loss of biocytin and biotin itself are shown to be a major cause for the increased biotin requirement in patients with congenital biotinidase deficiency.

Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood-brain barrier.

The uptake of biotin and the closely related biocytin was characterized in primary cultures of calf brain microvessel endothelial (CBME) cells. Biotin uptake was found to be Na(+)-gradient dependent and independent of changes in the membrane potential. Concentration dependence revealed a single saturation mechanism with a K(m) of 47 microM and a V(max) of 101 pmol/min/mg. Inhibition studies demonstrated dependence on metabolic energy and the necessity for a free carboxyl group for transport activity. The anticonvulsants primidone and carbamazepine had no inhibitory effect. Biotin uptake into CBME cells is a secondary active, electroneutral, saturable and specific process. Biocytin which accumulates in biotinidase deficiency, a human congenital disorder, did not inhibit biotin uptake and was not transported into these cells. The presence of human serum with normal biotinidase activity significantly reduced biotin uptake by about 50%. Further, added biocytin was hydrolyzed to biotin, which accumulated intracellularly but to a lesser extent than added free biotin. Biotin uptake after addition of plasma of biotinidase-deficient patients was not different from that in the presence of normal serum. These results indicate that the absence of biotinidase activity in serum does not reduce blood-brain barrier transport of biotin.

Rapid differential diagnosis of carboxylase deficiencies and evaluation for biotin-responsiveness in a single blood sample.

We have developed a method for rapid differential diagnosis of isolated or multiple deficiencies of the 3 mitochondrial biotin-dependent carboxylases: propionyl-CoA (PCC), 3-methylcrotonyl-CoA (MCC) and pyruvate carboxylase (PC), and for simultaneous evaluation of biotin-responsiveness using a single blood sample. Lymphocytes were isolated from heparinized blood and preincubated without and with 10(-5) mol/l biotin in medium before determination of PCC, MCC and PC activities. Plasma was used for estimation of biotin concentration and biotinidase activity. A definitive diagnosis could be made in 7 of 9 patients studied up to now: 4 patients suffered from biotin-nonresponsive isolated PCC-deficiency, and 3 patients from biotin-responsive multiple carboxylase deficiency caused by deficient biotinidase activity. In two patients, a carboxylase deficiency was excluded. These results were confirmed in studies using fibroblasts. In addition, a simple method for detection of deficiency in holocarboxylase synthesis is described.

Biotinidase determination in serum and dried blood spots--high sensitivity fluorimetric ultramicro-assay.

A miniaturized quantitative biotinidase assay has been developed using biotin 6-amidoquinoline as substrate and the 100-fold enhanced fluorescence of 6-amidoquinoline measured using apolar solvents. Amidoquinoline is measured after deproteinization by ethanol/acetone using individual standardisation and solvent resistant microtiter plates. The assay was optimized for end point determinations of biotinidase activities in serum and for newborn screening using dried blood spots. Serum activities obtained are closely correlated with values obtained using a quantitative validation method (r=0.96). Analytical sensitivity is around 2% of the mean activity (7.01+/-1.92 nmol/min/ml, mean+/-SD) of a healthy control population. With dried blood spots, a close correlation with values obtained using the Wallac-test kit (r=0.92) was found. Biotinidase activities of a healthy population of 651 newborns amount to 0.2429+/-0.07 nmol/min/ml blood. The analytical sensitivity is close to 1% of the mean activity.

Quantitative determination of biocytin in urine of patients with biotinidase deficiency using high-performance liquid chromatography (HPLC).

A specific method for the quantitative determination of biocytin from urine of biotinidase deficient patients is described using HPLC-separation and quantitative determination by an avidin binding method. Partial purification of biocytin from urine was achieved with an anion exchange resin and concentration of the eluate by lyophilization. The recovery of biocytin from urines was 95.3 +/- 5.9 (mean +/- SD). The precision of biocytin estimation in patients urines including the HPLC-sample preparation procedure varied between 5.9% and 10.5% (CV). Biocytin concentrations were measured in urine samples of 5 patients obtained during and/or before biotin therapy. Before treatment biocytin excretion ranged from 6.2-28.8 nmol/mmol creatinine. During therapy biocytin excretion increased to the 1.3 to 4-fold level in 3 out of 4 patients. However, there was no dose-related increase of biocytin excretion when pharmacological doses were administered. Apart from biocytin and biotin, patients excrete additional biotin derivatives. Some of these have been preliminary identified as bisnorbiotin and oxidation products of bisnorbiotin, biocytin and biotin.

Multiple carboxylase deficiency: inherited and acquired disorders of biotin metabolism.

Acquired biotin deficiency and the two known congenital disorders of biotin metabolism, biotinidase and holocarboxylase synthetase (HCS) deficiency, all lead to deficiency of the 4 biotin-dependent carboxylases, i.e. to multiple carboxylase deficiency (MCD). The underlying mechanism in HCS-deficiency, discovered in 1981, is decreased affinity of HCS for biotin impairing the formation of holocarboxylases at physiological biotin levels. In biotinidase deficiency, discovered in 1983, MCD results from progressive development of biotin-deficiency due to inability to liberate and recycle biotin which is lost in urine as biocytin. MCD leads to typical organic aciduria and severe life-threatening illness. Main symptoms and signs are feeding difficulties, neurologic abnormalities (hypotonia, impaired consciousness, seizures, ataxia) and cutaneous changes (rash, alopecia). However, the clinical presentation and age of onset are extremely variable, and organic aciduria may initially be absent in biotinidase deficiency. Therefore, the definitive diagnosis requires enzyme studies. MCD can be detected in lymphocytes obtained before treatment and biotinidase deficiency is confirmed or excluded by a colorimetric enzyme assay in plasma. Newborn screening for biotinidase deficiency has resulted in the detection of patients with partial deficiency (10-30% of mean normal activity) in addition to patients with profound deficiency (0-10%). Severe illness has been observed mainly in patients with O-activity or a Km-mutation, detection of which requires detailed investigation. HCS-deficiency has to be confirmed by enzyme assay in cultured cells. Both congenital disorders respond clinically and biochemically to oral biotin therapy. Whereas 10 mg/day or less is sufficient to treat profound biotinidase deficiency, the optimal biotin dose for patients with HCS-deficiency must be assessed individually. The prognosis of both disorders is good if biotin therapy is introduced early and continued throughout life. However, delayed commencement of therapy in biotinidase deficiency can result in irreversible neurological damage, and in HCS-deficiency a few patients have responded only partially even to massive biotin doses of up to 100 mg/day.

Biotin determination by three different methods: specificity and application to urine and plasma ultrafiltrates of patients with and without disorders in biotin metabolism.

A microbiological, an avidin-binding and a streptavidin-binding method for biotin determination were compared. All three methods detected biotin equally well but they exhibit different specificities for derivatives of biotin. The microbiological assay has the highest specificity and is the method of choice for biotin determination in biotinidase-deficient patients. The specificity of streptavidin-binding has not been investigated so far. Application of the three methods to urine samples of patients with and without biotin therapy indicated that only 50% of biotin equivalents measured with the avidin method correspond to authentic biotin as previously shown. The other 50% comprise mainly bisnorbiotin and biotin-d-sulfoxide. HPLC-separation of urine samples prior to assay confirmed this finding and revealed a bisnorbiotin oxidation product and an unknown compound as further biotin metabolites. The latter was measurable by all three methods and not detectable in plasma ultrafiltrate. This was the only metabolite which was able to restore deficient 3-methylcrotonyl-CoA carboxylase activity in biotin-deficient fibroblasts. The combination of the three methods together with HPLC-separation proved to be a valuable analytical tool for the identification of the main biotin metabolites in biological fluids.

Detection of biocytin in urine of children with congenital biotinidase deficiency.

The possible excretion of biocytin or biotinyl-peptides was investigated in urines of children with congenital biotinidase deficiency. Treatment of urine samples of affected children with normal plasma (as source of biotinidase) liberated additional free biotin (L. plantarum assay), indicating the presence of biotinyl-compounds. Using thin-layer chromatography the occurrence of biocytin was demonstrated in all urine samples of the affected children. In contrast, no biocytin could be detected in urines of healthy subjects.

The first case of 3-methylcrotonyl-CoA carboxylase (MCC) deficiency responsive to biotin.

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism with an autosomal recessive pattern of inheritance that results from a deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). We report on a nine-year-old boy with severe psychomotor retardation who developed infantile spasms at the age of three weeks. Urine analysis at the age of two years revealed massive 3-methylcrotonylglycinuria and 3-hydroxyisovaleric aciduria suggesting MCC deficiency. Carnitine serum levels were decreased. Biotin therapy led to a dramatic decrease in the frequency of seizures, disappearance of hypsarrhythmia, and near normalisation of organic aciduria. Four months later a protein-restricted diet was introduced in addition and the boy remained clinically and metabolically stable. However, severe psychomotor delay persisted, and the seizures partially reoccurred. Biochemical findings showed partial MCC deficiency in cultured fibroblasts. Molecular genetic studies revealed a heterozygote missense mutation, MCCA-R385S, converting arginine to serine in a highly conserved region of the MCCA gene. This is the first patient with MCC deficiency caused by a heterozygote mutation and who demonstrated a substantial and sustained clinical and biochemical response to therapeutic doses of biotin. Sadly, this patient again also demonstrates that the main determinant of the outcome of even easily treatable metabolic diseases is timely diagnosis.

Na(+)-dependent biotin transport into brush-border membrane vesicles from human kidney cortex.

Renal reabsorption of biotin was investigated in human kidney by means of the isolated brush-border membrane vesicle technique. Biotin uptake into the vesicles was sodium-dependent producing a typical overshoot when incubated under sodium-gradient conditions (external concentration greater than internal). This effect was not observed in the presence of gradients of KCl, LiCl or choline-chloride, nor in the absence of any salt. Using the K+/valinomycin voltage-clamp method biotin uptake remained uninfluenced, i.e. was electroneutral, whereas glucose uptake (which is known to be electrogenic in kidney of other species) was greatly increased. When biotin transport was investigated as a function of external sodium concentration a stoichiometic coupling factor of 1 for the Na(+)-biotin- cotransport was determined. Increasing the biotin concentration in the incubation medium up to 200 mumol/l led to saturation with the kinetic parameters of 31 mumol/l for the apparent Michaelis constant and 82 nmol g protein-1 30 s-1 for the maximal transport rate. Uptake was not saturable in the concentration range of 0.001-1 mumol/l. Inhibition of the biotin uptake (25 mumol/l) was observed in the presence of 250 mumol/l dethiobiotin, bisnorbiotin, thioctic acid, and probenecid, whereas biocytin, propionic acid, lactic acid, succinic acid, citric acid, ascorbic acid, primidone and carbamazepine had no effect. We conclude that renal biotin reabsorption in human kidney is specifically sodium-dependent, saturable and electroneutral. It therefore fulfills the requirements for a secondary active carrier-mediated transport system. The results suggest that biocytin is not an inhibitor of renal biotin reabsorption.

Propionic acidaemia: clinical, biochemical and therapeutic aspects. Experience in 30 patients.

Comprehensive data on 30 patients with propionic acidaemia, diagnosed by selective screening for inborn errors of metabolism, are presented. The most valuable diagnostic metabolites found were methylcitric-, 3-hydroxypropionic-, and 2-methyl-3-oxovaleric acids. Hyperlysinaemia and hyperlysinuria are also characteristic findings in this disease. The metabolic pattern found in propionic acidaemia is discussed extensively as are enzymatic findings. Residual activity of propionyl-CoA carboxylase is neither a predictive marker for severity nor for outcome of the disease. Propionate fixation assays were less reliable for confirmation of propionic acidaemia and of no prognostic value. Clinical presentation of the disease is discussed in detail. Besides the well-known unspecific findings (poor appetite, feeding difficulties, vomiting, dehydration, weight loss, muscular hypotonia, dyspnoea, somnolence, apathy, convulsion, coma, severe metabolic acidosis, hyperammonaemia) various skin abnormalities have been detected in about 50% of all patients. In 27% "dermatitis acidemica" was found.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase deficiency: long-term outcome in a case with neonatal onset.

UNLABELLED: A patient with early-onset 3-methylcrotonyl coenzyme A carboxylase (MCC) deficiency showing a severe clinical course is described. Abnormal eye and head movements suggestive of seizures were noticed soon after birth. Tonic convulsions at the age of 10 weeks led to admission. Urinary organic acid analysis using gas chromatography-mass spectrometry at 3 months of age revealed elevated concentrations of 3-hydroxyisovaleric acid (3HIVA) and 3-methylcrotonylglycine but normal levels of lactate, 3-hydroxypropionate and methylcitrate suggesting isolated MCC deficiency. This was confirmed by enzyme assays in lymphocytes and cultured skin fibroblasts: MCC activity was virtually undetectable whereas activities of propionyl-CoA and pyruvate carboxylases were within the normal range. A low protein (0.8-1.5 g/kg/day) diet supplemented with a leucine-free amino acid mixture resulted in a marked decrease of 3HIVA excretion. L-Carnitine and biotin administration had no effect on the clinical condition or metabolite excretion. Supplementation with glycine resulted in only a temporary fall of 3HIVA excretion and was therefore discontinued. L-Carnitine therapy was reintroduced later because of secondary carnitine deficiency. Compliance with treatment was poor until the age of 27 months resulting in a severe episode with seizures and coma. The general clinical condition of the patient was always good but his psychomotor development was delayed and seizures were not continuously under good control due to poor therapy compliance. The boy is now 10.5 years old and attending a school for children with learning handicaps. CONCLUSION: Isolated MCC deficiency of early-onset is a rare condition exhibiting a more severe clinical course than the later-onset form described in most other cases. The prognostic value of 3 HIVA measurements in CSF and serum should be evaluated in future cases.

Acute neonatal nonketotic hyperglycinemia: normal propionate and methylmalonate metabolism.

Propionyl-CoA carboxylase and combined methylmalonyl-CoA (MMA-CoA) racemase and -mutase activities were studied in liver and fibroblasts of two patients with the acute neonatal form of nonketotic hyperglycemia. In all experiments, these enzyme activities studied in tissues of the patients were within the range of healthy control subjects, whereas no propionyl-CoA carboxylase activity was measurable in the fibroblasts of a patient with propionic acidemia. Subcellular fractionation of liver and fibroblasts indicated that the normal amounts of MMA-CoA found after incubation of whole tissue homogenate were formed by propionyl-CoA carboxylase, a mitochondrial enzyme, and not be acetyl-CoA carboxylase, which theoretically could also be involved in the carboxylation of propionyl-CoA. From the above data as well as from clinical and biochemical observations in three patients, it was concluded that there exists a true nonketotic hyperglycinemia which is not related etiologically to the different disorders of the ketotic hyperglycinemia syndrome. True nonketotic hyperglycinemia is not associated with ketoacidosis even after loading with propionate- and MMA precursors. It must be distinguished by exclusion from mild forms of the ketotic hyperglycinemia syndrome which may present clinically as hyperglycinemia without ketosis.

Congenital methylmalonic acidemia: a variant of the B12 'non-responsive' form with evidence for reduced affinity of methylmalonyl-CoA mutase for its B12-coenzyme.

Methylmalonate metabolism was investigated in fibroblasts and leukocytes of two unrelated patient with a B12-nonresponsive type of congenital methylmalonic acidemia. Intact fibroblasts from both patients showed a defective metabolism of methyl-14 c-malonate to 14CO2, whereas no such defect was found in their intact peripheral leukocytes. In disrupted fibroblasts, the conversion of methylmalonyl coenzyme A to succinyl coenzyme A was markedly reduced but was completely normalized by the addition of 5'-deoxyadenosylcobalamin (AdoCb1; 10(-5) mol/l), the specific coenzyme of methylmalonyl coenzyme A mutase. Assays with decreasing concentrations of AdoCbl (10(-5)-10(-11) mol/l) suggested a reduced affinity of the mutase apoenzyme for its coenzyme, implicating yet another variant of this heterogeneous disease.

Na(+)-dependent biotin transport into brush-border membrane vesicles from rat kidney.

The mechanisms of biotin reabsorption in rat kidney cortex were investigated using isolated brush-border membrane vesicles. An inwardly directed Na+ gradient specifically stimulated a transient biotin overshoot. Biotin transport was not affected by a valinomycin-induced K(+)-diffusion potential, and biotin(-)-Na+ stoichiometry was found to be 1:1. As a function of concentration, the uptake showed saturation in the presence of a Na+ gradient with an apparent Michaelis constant (Km) of 55 microM and Vmax of 217 pmol.mg protein-1.25 s-1. Desthiobiotin, 250 microM, norbiotin, bisnorbiotin, thioctic acid, valeric acid, probenecid, and nonanoic acid inhibited the transport of 30 microM biotin, whereas other biotin derivatives, as well as biocytin and organic acids found in the urine of biotinidase-deficient patients, did not. Preloading of the vesicles with biotin, desthiobiotin, norbiotin, and thioctic acid in the presence of Na+ increased initial uptake of biotin from the incubation medium (trans-stimulation). Our results indicate that biotin absorption in rat kidney fulfills the criteria for a specific carrier-mediated and electroneutral Na(+)-biotin- cotransport in a 1:1 ratio. The results are discussed in context with congenital biotinidase deficiency in humans.