Jean-Pierre Bonjour

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.

Biotin status of epileptics.

Microbiologically determined plasma biotin levels in 404 epileptics under long-term treatment with anticonvulsants were markedly lower than in 112 controls (p less than 0.0005). Patients with partial epilepsy had lower biotin levels and higher average daily intake of AC than those with generalized epilepsy. Epileptics treated with valproate sodium in monotherapy showed considerably higher biotin levels than epileptics with monotherapy of primidone (PRM), carbamazepine (CBZ), phenytoin (PHT) or phenobarbital (PB). The group of epileptics with high average daily dose of anticonvulsants had lower biotin levels than the group with low dose. In three patients with newly recognized epilepsy biotin levels were normal before starting anticonvulsant medication, increased during the first week and fell under the starting level in the following weeks. Four epileptics treated with PHT, PB, PRM or CBZ had an increased urinary excretion of organic acids, as found in patients with a deficiency of biotin-dependent carboxylases. In 37 epileptics undergoing long-term treatment plasma lactate concentrations were determined; they had a higher mean concentration than that found in controls. Our results suggest, that the lowering of biotin in epileptics is caused by intake of anticonvulsants and has a biochemical effect in these patients. It is discussed, whether this could be a factor in the mode of action of anticonvulsants.

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.

Biotinidase Deficiency: Factors Responsible for the Increased Biotin Requirement

Inability to recycle biotin from endogenous biocytin in congenital biotinidase deficiency is associated with increased requirement of exogenous free biotin. We have observed that severe biotin depletion with clinical and biochemical consequences occurs within 12 days after birth in a newborn patient and within 15–20 days after withdrawal of biotin supplementation in four other patients. Our studies have shown that:(1)Urinary loss of biotin and biocytin are major causes for this rapid biotin depletion.(2)Intestinal absorption of biotin seems to be normal at least at the loading dose of 1.5 µg/kg.(3)At normal or subnormal plasma biotin concentrations biocytin is found in low concentrations (below 1 nmoll−1) in plasma of patients but at much higher concentrations in urine (100–600 nmoll−1).(4)An oral load of biocytin results in patients in unchanged biotin levels but in a marked rise of biocytin in plasma followed by rapid renal excretion of biocytin whereas in controls biotin levels in plasma increase rapidly and biocytin remains below detection levels.

Biotin in Human Nutrition

Biotin serves as prosthetic group in a number of enzymes four of which occur in animal and human tissues namely: pyruvate carboxylase (EC 6.4.1.1) (PC), acetylCoA carboxylase (EC 6.4.1.2) (ACC), propionyl-CoA carboxylase (EC 6.4.1.3) (PCC), and 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) (MCC).’*2*’ Biotin is linked in these enzymes covalently to the c amino group of a lysine residue in the apoenzyme; the linkage is established by holoenzyme synthetase. This enzyme has a high specificity for d-biotin and a broad one for the apoenzyme, suggesting that the environment of the lysine residue to which the biotin is attached may be similar in the various apoenzymes. All biotin-containing enzymes are concerned with the transfer of a carbonyl group. The mechanism of these reactions has been el~cidated.’.~.‘ The four biotin-containing carboxylases are involved in carbon-chain elongation steps in mammalian metabolism: in gluconeogenesis (PC), fatty acid synthesis (ACC), propionate metabolism (PCC), and in the catabolism of leucine (MCC).’.’ The activities of these carboxylases were found to decrease progressively in different tissues of animals when fed a biotin-deficient diet, and to be rapidly restored on administration of biotin although at different rates for the various tissues.’.’ In humans biotin administration enhanced PCC,”6 MCCSV6 and also PC’ activities in leukocytes and after discontinuation of biotin PCC and MCC activities returned to normal: whereas the ACC activity remained unaffected.’ The glycoprotein avidin, which is a component of raw egg white, strongly binds biotin and also compounds structurally related to biotin containing an intact “ureido” function in cis c~nfiguration.~.’ Feeding raw egg white induces biotin deficiency in most animals. The animals cease to grow and start to develop characteristic pathological symptoms affecting mainly the skin (seborrheic dermatitis) and the growth of hair (alopecia) and on treatment with biotin all symptoms disappear.*-’.’ Besides this experimental “egg white injury,” spontaneous outbreaks of biotin deficiency which respond to biotin treatment have occurred in animals under field conditions. Lesions were similar to those produced under experimental conditions. The reason for these outbreaks is not known but might be due to poor bioavailability of biotin in the feed used. Furthermore, biotin-responsive disease conditions have been noted, such as the Fatty Liver and Kidney Syndrome (FLKS) in broilers, which is due to a suboptimal biotin content in the rations coupled with certain nutritional and environmental stress factors.’.’

Biotin-responsive multiple carboxylase deficiency (MCD): deficient biotinidase activity associated with renal loss of biotin.

Biotin-responsive multiple carboxylase deficiencies (MCD) are inherited disorders characterized biochemically by the accumulation of a typical pattern of organic acids, caused by decreased activity of the three mitochondrial biotin-containing enzymes: propionyl CoA carboxylase (PCC., EC 6.4.1.3), 3-methylcrotonyl CoA carboxylase (MCC., EC 6.4.1.4) and pyruvate carboxylase (PC., EC 6.4.1.1). Clinically this disorder exists in at least two forms, namely an early-onset (neonatal) and a late-onset (juvenile) form. Both types of the disorder respond to high doses of biotin. In most instances, the neonatal form appears to be caused by deficient holocarboxylase synthetase activity due to an elevated K m for biotin (Burri et al., 1981), while in some patients with late-onset MCD the primary biochemical defect has recently been found to be a deficiency in the activity of biotinidase, the enzyme regenerating biotin from endogenous biocytin (Wolf et al., 1983).

Intestinal absorption and renal excretion of biotin in patients with biotinidase deficiency

We have investigated four patients from three unrelated families with typical clinical and biochemical features of “late-onset” multiple carboxylase deficiency. All patients suffered from biotinidase deficiency (plasma biotinidase activities 1.4%–3% of normal). Intestinal absorption of biotin, measured in three of the patients using a single load of 1.5 μg/kg, was found to be normal. Deficient activities of the mitochondrial biotin-dependent carboxylases in lymphocytes of one of these patients increased from 25% of mean basal control values to 33%–36% within 45 min and to 46%–47% within 2 h of the 1.5 μg/kg biotin load. After a high biotin load of 100 μg/kg, the values normalised within 45 min in all three patients studied. These results indicate normal cellular transport of biotin and normal holocarboxylase synthesis. After cessation of biotin supplementation, the plasma and urinary biotin in patients decreased to subnormal levels. In one patient, available for more detailed studies, both plasma and urinary biotin declined about twice as fast as in controls (apparent half-life 12–14 h in the patient and 26 h in controls). These results point to increased excretion of free biotin in our patient. Renal loss of biotin is one of the factors contributing to the high biotin requirement observed in patients with biotinidase deficiency.