This patient presented with a typical clinical and metabolite profile consistent with multiple carboxylase deficiency, including the skin findings and their resolution with biotin treatment [1
]. A new symptom was the development of respiratory distress syndrome of the neonate despite a normal gestational age, which responded to surfactant treatment. Many patients have shown either tachypnea or breathing difficulties [19
]. It is unclear whether the reported respiratory distress was due to the metabolic acidosis or if neonatal respiratory distress syndrome was present.
In the literature, patients with holocarboxylase synthetase deficiency have responded to a variable degree to treatment with pharmacologic doses of biotin. Three possible mechanisms can explain the effect of biotin treatment [3
]. Many mutations in the HLCS gene increase the Km of the holocarboxylase synthetase enzyme for biotin, and higher concentrations of biotin are required for proper enzymatic function. Biotin also increases the transcription of the HLCS gene, resulting in more holocarboxylase enzyme being produced [3
]. This mechanism can explain improvement even in patients in which the Km is normal but the Vmax is decreased. It was also postulated that the holocarboxylase enzyme is not saturated a physiologic concentrations of biotin, leaving room for increased activity at higher biotin concentrations. If the causative mutation affected the Vmax of the enzyme, then limited benefit was observed from biotin treatment, but if the mutation affected the Km of the holocarboxylase synthetase for biotin, then substantial improvement to normalization of the carboxylases was observed [21
In the patient described here, the residual activity almost certainly arose from the Y663H missense mutation, since the second allele contained a nonsense mutation. Most missense mutations that affect the Km for biotin are located in the biotin-binding domain. The mutation in our patient, Y663H, is located 3′ beyond the avidin-binding domain of D562 to P609, but is still within the birA homologues region, which ranges from amino acid 448 to 701 [13
]. It is beyond the thus far most 3′ reported missense mutation, which involved D634 [22
]. The amino acid Y663 is preserved across a wide range of vertebrates indicating its probable functional significance. The effect of the mutation on the Km of the enzyme indicates that even this extreme 3′ end of the birA homologous domain influences biotin binding or biotin activity. In fibroblasts the Vmax of the enzyme was preserved but the Km was raised 40 fold to 164 nM. At physiologic biotin concentrations of 0.4 to 1.5 nM, the activity is insufficient to provide full biotinylation of the apocarboxylases, and the activities of all three enzymes tested in leucocytes were deficient.
However, at a dose of 100 mg/day, delivering a dose of 38 to 7 mg/kg/day decreasing as the child was growing up, the carboxylases corrected with all leucocyte carboxylase activities to within, or just under, the normal range. The metabolites corrected as well, although slight elevations of 3-hydroxyisovaleric and of 3-hydroxyisovalerylcarnitine persisted, as seen in other well-treated patients [3
]. A high protein intake resulted in a striking increase in metabolites, which disappeared again with a mild protein restriction, suggesting that the diet could not be completely unrestricted. A similar need for protein restriction has been reported before [5
]. At a dose of 10 mg/kg/day of biotin the plasma total avidin binding substances concentrations varied between 1800 and 4900 nM reflecting a biotin concentration of about 1080 and 2940 nM. At a biotin dose of 7 mg/kg/day a plasma concentration of 835 nM TABS, reflecting a biotin concentration of at least 500 nM was documented. These concentrations far exceed the measured Km of the enzyme in fibroblasts of 164 nM. Despite this apparent excess, not all carboxylases were always fully reconstituted in leucocytes, and slight metabolite elevations always persisted, and there remained a limitation to the protein intake that could be metabolized. These plasma biotin concentrations are similar to that seen in an infant receiving 10 mg/day and another receiving 20 mg/day, which had plasma biotin levels of 1300 and 4830 nM respectively [18
]. The plasma concentrations are higher than the plasma biotin levels of 87 to 445 nM reported in a patient taking from 5 to 40 mg/day (0.8–4 mg/kg/day) [24
], and at the upper end of that recorded in 4 patients receiving between 1.8 and 10.1 mg/kg/day of biotin in whom plasma biotin levels between 112 and 684 nM respectively were reported [25
]. This shows a substantial range of plasma biotin levels with currently employed doses of biotin ranging from 87 to 4830 nM. The lower levels would not be sufficient to saturate the Km of our mutant enzyme. Thus, our observations suggest that it may be prudent to measure the biotin plasma levels to ensure that the dosing used is indeed sufficient to saturate the Km of the mutant holocarboxylase synthetase enzyme.
The metabolic abnormalities required over two weeks to correct in our patient. Two mechanisms could be involved. First, the solubility of biotin limited the dissolved amount that could be given to less than 2 mg/day. If biotin is provided in a sufficiently large volume, consistency of the bioavailability for oral uptake will be ensured. Also, an intravenous formulation of biotin would have been helpful to overcome this obstacle during severe illness. Second, biotin induces the sodium-dependent multivitamin transporter SMVT, which is required for uptake and distribution of biotin to all non-neurological tissues. This increase is dependent on biosynthesis of biotinyl-5′-AMP, which is formed in the first half reaction of the holocarboxylase synthetase enzyme [7
], and then further via cGMP formation. This mechanism forms a positive feedback loop whereby as more biotin is taken up, through a higher expression of the SMVT more biotin will become bioavailable to the tissues [8
], resulting also in a higher expression of HLCS and thus again a higher production of biotinyl-5′-AMP. This mechanism is impeded in holocarboxylase synthetase deficiency where a high Km for biotin will limit the formation of biotinyl-5′-AMP. A slow gradual increase in the production of both SMVT and of biotinyl-5′-AMP may have prolonged the time of recovery.
Biotin is taken up in the brain via a facilitated transport by a different sodium-independent biotin transporter [26
]. Uptake is linear at concentrations above 60 nM. At pharmacological concentrations, biotin apparently passed the blood-brain-barrier and resulted in CSF concentrations about half of the blood concentration, and a thousand fold higher than physiologic concentrations. The normal CSF lactate, organic acids, and the absence of a lactate peak on MRS all provide evidence for correction of the enzyme activity behind the blood brain barrier. These pharmacologic and biochemical findings explain the excellent neurodevelopmental outcome.