Biotin, a water-soluble vitamin belonging to the B complex, acts as a coenzyme in each of four carboxylases in humans: pyruvate carboxylase, propionyl CoA carboxylase, β-methylcrotonyl CoA carboxylase, and acetyl CoA carboxylase. These enzymes have important roles in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is derived from the diet and possibly also from the synthetic activity of gastrointestinal microflora.

Each of the carboxylases is synthesized as an inactive apoenzyme that is subsequently biotinylated through two partial reactions, each of which is catalyzed by the enzyme holocarboxylase synthetase. Acetyl CoA carboxylase functions principally in the cytosol, whereas the other three holocarboxylases function in mitochondria. Ultimately, these enzymes are degraded proteolytically, probably by the lysosomal autophagic system. The biotin-containing products of degradation, biocytin (ε-N-biotinyl-L-lysine) and biotinyl peptides, are acted on by biotinidase, which cleaves the amide bond between lysine and biotin. The liberated biotin is recycled and enters the free biotin pool. Biotinidase also has been shown, in presence of biocytin, to transfer biotin to nucleophilic acceptors, such as histones. The physiologic significance of this function remains to be determined.

There are two major defects in the cycle of biotin utilization in humans. Both disorders result in multiple carboxylase deficiency. Holocarboxylase synthetase deficiency (MIM 253270), also known as early-onset (neonatal) multiple carboxylase deficiency based on the usual age of onset of symptoms, is a disorder of biotinylation. Biotinidase deficiency (MIM 253260), also known as late-onset (juvenile) multiple carboxylase deficiency, is a disorder of biotin recycling.

Holocarboxylase synthetase deficiency has been described in about thirty children. The clinical symptoms include feeding and breathing difficulties, hypotonia, seizures, and lethargy, and there sometimes is progression to developmental delay or coma. Some children exhibit skin rash and alopecia. Affected children exhibit metabolic acidosis, organic aciduria, and mild to moderate hyperammonemia. The organic aciduria includes elevated concentrations of β-hydroxyisovalerate, β-methylcrotonylglycine, β-hydroxypropionate, methylcitrate, lactate, and tiglylglycine.

The enzyme defect has been demonstrated in lymphocytes, cultured fibroblasts, and cultured lymphoblasts from affected children. Holocarboxylase synthetase in tissues of most of the children examined have increased Km values for biotin, although at least one is described with a normal Km and a decreased Vmax.

Holocarboxylase synthetase deficiency is inherited as an autosomal recessive trait. Heterozygosity cannot be confirmed by measuring enzymatic activity in the tissues of parents but should be determined by mutation analysis.

The normal mammalian enzyme has been purified and characterized biochemically. The cDNA for human holocarboxylase synthetase has been isolated and sequenced. Multiple mutations within the synthetase gene have been identified. Some of these mutations occur within the biotin-binding domain and are likely responsible for the increased Km for biotin, explaining the biotin responsiveness that occurs in this disease. A mutation that results in an enzyme with a normal Km but decreased Vmax is speculated to increase in activity sufficiently in the presence of supplemented biotin compared with that at the biotin concentration supplied by the normal diet. A single base deletion/null mutation has always been found in the compound heterozygous state, usually together with a mutation within the biotin-binding domain, but never homozygous, which may be lethal in utero.

Children with holocarboxylase synthetase deficiency usually improve clinically following administration of 10 mg of oral biotin per day. One child, whose enzyme had the highest Km for biotin, continued to have a skin rash and excrete abnormal organic acids even while receiving 60 to 80 mg of biotin per day.

Holocarboxylase synthetase deficiency can be diagnosed prenatally by measuring the concentration of abnormal organic acids in the amniotic fluid and/or by measuring and comparing the various mitochondrial carboxylase activities in the amniocytes cultured with and without biotin. Prenatal diagnosis also should be possible by mutation analysis. Prenatal treatment has been performed during two pregnancies. The children were clinically normal at birth and did not have organic aciduria. It is not clear whether treatment of at-risk children with biotin immediately after birth is necessary.

Biotinidase deficiency has been described in over 120 symptomatic children. The clinical features commonly include seizures, hypotonia, ataxia, breathing problems, hearing loss, optic atrophy, developmental delay, skin rash, and alopecia. Other symptoms include conjunctivitis and fungal infections, which are probably due to abnormalities in immunoregulation. The clinical expression of the disorder is highly variable. The age at onset of symptoms ranges from several weeks to several years; median and mean age of onset are 3 and 5 months, respectively, but onset of symptoms has occurred as late as 10 years of age. Most, but not all, symptomatic children exhibit metabolic ketolactic acidosis and organic aciduria. The organic aciduria commonly manifests as elevated concentrations of β-hydroxyisovalerate, lactate, β-methylcrotonylglycine, β-hydroxypropionate, and methylcitrate.

Biotinidase deficiency is diagnosed by demonstrating deficient enzyme activity in serum. Most symptomatic children have profound biotinidase deficiency (<10 percent of mean normal serum activity). Newborn screening also has identified children with partial biotinidase deficiency (10–30 percent of normal activity). Symptoms of biotinidase deficiency have developed in a small number of children with partial deficiency, usually when they are exposed to a severe infection; the symptoms resolved with biotin treatment. Deficient enzyme activity also was found in other tissues from these children.

Normal serum contains at least nine isoforms of biotinidase, four major and five minor isoforms, between pH 4.5 and 4.35 on isoelectric focusing immunoblots using antibodies prepared against purified human serum biotinidase. The plasma enzyme has been purified to homogeneity. The enzyme is a glycosylated monomer. Patients with profound biotinidase deficiency can be classified into nine distinct biochemical phenotypes based on the presence or absence of cross-reacting material (CRM) to biotinidase, specific isoforms, and the distribution frequencies of the isoforms. There is no correlation between the age of onset or the severity of symptoms and the CRM status or isoform patterns of the symptomatic children. The isoform patterns of children identified by newborn screening are not different from those of symptomatic children. Children with partial biotinidase deficiency have CRM in their serum and can be classified into six distinct biochemical phenotypes. The isoform patterns observed in these children are not different from those of profoundly deficient patients who have CRM.

The cDNA for human serum biotinidase has been isolated and sequenced. The genomic structure and organization of the human enzyme also have been determined. The enzyme appears to be a housekeeping gene. Over two dozen mutations have been found in symptomatic children with profound biotinidase deficiency. There are at least two common mutations that occur among children with profound biotinidase deficiency identified by neonatal screening. Partial biotinidase deficiency is usually due to a combination of an allele with a missense mutation, which results in an enzyme having about 50 percent of mean normal activity and is found in about 4 percent of the general population, and an allele for profound biotinidase deficiency.

Individuals with biotinidase deficiency cannot recycle endogenous biotin and cannot release dietary-protein-bound biotin. The brain may be unable to recycle biotin adequately and may depend on biotin transferred across the blood-brain barrier. This may result in decreased pyruvate carboxylase activity in the brain and in the accumulation of lactate. The localized lactic acidosis may cause the early appearance of neurologic symptoms.

Biotinidase deficiency is inherited as an autosomal recessive trait. Heterozygotes can be determined in 95 percent of cases by demonstrating that enzyme activity in serum is intermediate between that of normal and deficient individuals. Prenatal diagnosis of biotinidase deficiency by enzymatic and mutation analysis is possible.

Biotinidase activity can be determined in the same blood-soaked filter paper used in most neonatal screening programs. More than 25 states in the United States and 25 countries are screening for the enzyme deficiency in their newborns. Children with enzyme deficiency identified by screening have been treated with biotin and have remained asymptomatic. The method used in newborn screening also offers a simple, rapid means for the physician to evaluate biotinidase activity in individuals suspected of having the disorder.

Children with biotinidase deficiency have been treated successfully with between 5 and 20 mg of oral biotin (in the free, unbound form) per day. All patients with the deficiency should respond to biotin therapy. If a child remains undiagnosed for a long time or experiences severe metabolic compromise, some of the neurologic problems, such as hearing loss, optic atrophy, or developmental delay, may not resolve. Doses of biotin only slightly above physiologic requirements eventually may be shown to suffice for treatment.

The multiple carboxylase deficiencies must be differentiated from other acute-onset metabolic disorders and nutritional deficiencies that result from dietary indiscretion or hyperalimentation with solutions lacking biotin. Because both holocarboxylase synthetase deficiency and biotinidase deficiency are so amenable to biotin therapy, these disorders should be considered in any child with nonspecific neurologic symptoms, especially when cutaneous abnormalities are present. Newborn screening for biotinidase deficiency, followed by prompt initiation of therapy in deficient children, appears to prevent the clinical consequences of this disorder.