Biotin in Human Nutrition JEAN-PIERRE BONJOUR

Department of Vitamin and Nutrition Research F. Hoffmann-La Roche & Co. Ltd.

Bade. Switzerland

INTRODUCTION

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), acetyl- CoA 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 mam- malian 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 discontinua- tion 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.’.’

OLDER FINDINGS

Seborrheic dermatitis of infancy, a self-limiting skin eruption of infants under six months of age, closely resembles skin changes produced in rats fed a diet rich in egg

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white. A large number of reports9 have shown a beneficial effect of biotin treatment in seborrheic dermatitis and in cases of Leiner’s disease, probably a generalized and greatly intensified form of seborrheic dermatitis. Therefore, these skin changes have been considered a sign of a biotin deficiency in infants.

When avidin, which strongly binds biotin rendering it thus unavailable for nutritional purposes, was added to the diet of four volunteers a fine brawny desquama- tion was observed in all subjects after 10 weeks on this diet. Furthermore, from the fifth week onward mental changes such as mild depression progressing to extreme lassitude were described.” Besides this report of experimentally induced “egg white injury” three further cases of an excessive intake of raw eggs have been observed.’ The two adults and the boy showed skin changes and the boy also showed alopecia totalis. The skin lesions in these patients and in the volunteers responded promptly to administration of biotin; in the boy new hair began to grow. The observed mental changes also disappeared on treatment with biotin. These findings confirm that biotin deficiency leads to skin changes both in children and adults. In severe deficiencies loss of hair can also occur.

Besides these cases no reports on biotin deficiency symptoms in humans have appeared in the literature. This absence of reports could be due to the fact that biotin-containing foods are numerous, although the absolute amount of biotin present in even the richest dietary sources is very Rich dietary sources for biotin are liver (100 pg biotin/100 g), egg yolk (52 pg/lOO g), and cooked whole cereals (20-30 pg/ 100 g). But not all of the dietary biotin seems to be bioavailable. While in corn all of the microbiologically determined biotin was available, in oats, milo, and barley only 2 6 3 0 % and in wheat none was found to be available to the

Another possible source of biotin for man could be the enteric synthesis of this vitamin by the microflora. During the 1940s several investigators noted that the amount of biotin excreted in urine and feces together is higher than the dietary biotin intake: changes in dietary biotin intake were clearly affecting urinary biotin excretion, whereas fecal excretion remained more or less unaffected. Trying to elucidate the contribution of the intestinally synthesized biotin to the total biotin requirement, patients were given sulfa drugs or antibiotics to destroy the microflora. But these experiments were inconclusive: Two studies reported no changes in urinary biotin concentration, suggesting that enterically synthesized biotin does not contribute to the biotin requirements. Two other studies observed a decrease in urinary biotin excretion, but this effect may have been due to the avidin included in the diet or to changes in absorption of dietary biotin caused by the high dosis of antibiotics administered. When measured, a reduction in fecal biotin content was noted? The uncertainty about human biotin requirements is expressed in the various dietary recommendations for this vitamin in different c o ~ n t r i e s . ~ For the first time estimated safe and adequate daily dietary intakes for biotin were recently established for the United state^.'^ Recom- mended daily intakes increase from 35wg for neonates to 100-200 fig for adults.

Intestinal absorption of biotin has been shown to occur in the first third to half of the rat small intestine with the molecule diffusing intact through the intestinal all.^.^ Other studies using hamster small intestine suggest a sodium activated transport mechanism with a minor diffusional component in biotin intestinal ~ p t a k e . ’ ~ Further investigations are needed to establish the exact mechanism for biotin absorption. Very little information is available on the metabolism of biotin in humans: it is only known that biotin is absorbed more extensively when given orally than instilled directly into the colon.16 Furthermore, biotin levels rapidly increase both in plasma and red blood cells when biotin is given intravenously, but 24 h later the levels returned to starting values and 100% of the dose was found to be excreted in urine.I7 In human urine large amounts of free biotin and very small quantities of biotin metabolites were determined

BONJOUR: BIOTIN IN HUMAN NUTRITION 99

by microbiological analysis. Bioautographic procedures revealed d-biotinsulfoxide and another unidentified metabolite in human urine, but no degradation of the cyclic urea ring of biotin.18 Recent investigations have shown that orally applied biotin is absorbed rapidly” and that the amount of biotin metabolites excreted in normal urine is as large as that of unchanged biotin2’; in these urines no d-biotinsulfoxide or -sulfone could be detected.20

The occurrence of biotin deficiency due to dietary restrictions is very rare. Nevertheless, population groups may be detected in which a state of marginal deficiency exists without apparent clinical manifestation. The assessment of biotin levels in body fluids may identify such groups. But any determination of biotin concentrations as well as investigations into the metabolism of biotin in animals and humans are complicated by the analytical procedures to be used for biotin. Biotin is present in only very small quantities in body fluids. Moreover, depending on the analytical method some of the biotin metabolites are estimated as biotin. For the assessment of biotin microorganisms are used which require biotin as growth factor, such as Lactobacillus plantarum or Ochromonas danica2 These microorganisms utilize biotin and its metabolites to different extents and, therefore, the results of analyses will differ accordingly. When biotin concentrations are determined using an isotope dilution assay with avidin, any compound containing the cyclic ureido group of biotin will be estimated as biotin.2 Colorimetric, gas chromatographic and polaro- graphic methods have also been proposed for the determination of biotin.2 In any method used bound biotin has to be released from the test material by acid hydrolysis with sulfuric acid or by digestion with papain. It is therefore not surprising that-using these different methods for the analysis of biotin-mean biotin concentrations have been reported ranging from 25 to 150 nmol/l in urine and from 0.5 to 7 nmol/l in b l ~ o d . ~ . ~ But even so, segments of the population were detected in which the biotin status was reduced compared with a control population and using the same method of analysis. Such groups were: children with seborrheic dermatitis and Leiner’s d i s e a ~ e , ~ . ~ children with burns and scalds? pregnant and lactating women,2*9*2’ athlete^,^.^ the e l d e r l ~ , ~ . ~ alcoholics,22 and patients with a~hlorhydr ia .~ .~ Low biotin concentrations were recently found also in epileptic^.^.^^ The clinical significance of reduced levels of biotin in humans is not yet clear.

RECENT DEVELOPMENTS

A defect in the degradative metabolism of branched-chain amino acids and in the propionate-methylmalonate pathway is associated with the accumulation of small molecular weight organic acids in blood and/or in urine. Some patients with organic acidemias and acidurias suffer from an inborn error of metabolism of the biotin- dependent c a r b o ~ y l a s e s . ~ ~ ~ ~ ~ Children with such an inborn error of metabolism of the biotin-containing carboxylases can be divided into two groups: one affected by an isolated defect of either of the carboxylases and the other group by a combined deficiency of the three mitochondria1 biotin-dependent carboxylases. Isolated defects have been observed for PCC and PC leading to propionic acidemia and to lactic acidosis, respectively. The cases reported up to now with an isolated defect of MCC resulting in 3-methylcrotonyl glycinuria have not included sufficiently extensive enzymatic investigations to exclude a combined carboxylase deficiency. No isolated defect has been observed for the cytosolic ACC.

Clinically, children suffering from a defect of biotin-dependent carboxylases present similarly with poor feeding, persistent vomiting, muscular hypotonia, lack of

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responsiveness, lethargy and coma, often ketoacidosis, and developmental retardation in older children. Differential diagnosis can therefore be established only after extensive biochemical investigations of organic acids in blood and urine suspected to occur in isolated deficiencies. It has been proposed2’ that the main diagnostic metabolites in urine for 3-methylcrotonyl glycinuria are 3-methylcrotonic acid and/or 3-methylcrotonylglycine; methylcitric and 3-hydroxypropionic acids in urine and propionic acid in blood for propionic acidemia; and lactic acid in blood and urine for lactic acidosis. Measurements of activities in cultured fibroblasts or leukocytes of all the biotin-dependent carboxylases must be included. These extensive biochemical investigations allow the identification of the defective enzyme and also of cases with a combined enzyme deficiency which in the past have often been wrongly classified as suffering from an isolated defi~iency.~’ The nature of the molecular defect in isolated carboxylase deficiencies is not yet known. But the lack of definite biotin responsiveness suggests that the defect involves the apoenzyme and is not dependent on the metabolism of biotin.”**’

Infants affected by a combiried deficiency of the biotin-dependent carbo~ylases”~~’ present clinically with the symptoms of organic acidemia and aciduria and most prominently also with skin rash and mostly alopecia. Biochemically each of the above-mentioned short-chain organic acids of an isolated enzyme defect can be found in blood and urine of such patients. Enzyme activities in cultured fibroblasts were noted to be reduced in some affected children and normal in others. But only this combined carboxylase defect has been shown unequivocally to be biotin-responsive in v i m and in vivo. This finding indicates that the basic genetic defect results either from an abnormality in absorption, transport, and/or metabolism of biotin or from a defect in the holocarboxylase synthetase, which covalently binds biotin to the apoenzymes. Such a holocarboxylase defect has been demonstrated in some ~ h i l d r e n . ~ ~ * ~ ’ Recently, a reduced biotinidase activity in serum of three children with typical symptoms of combined carboxylase deficiency has been reported” and confirmed in further patient^.'^.'' Such a deficient biotinidase activity prevents the liberation of biotin from biotin-containing compounds in the circulation and leads to the excretion of biocytin (r-N-biotinyl-lysine) in the urine of affected children.20

In a boy with a dietary defic:iency of biotin caused by excessive intake of raw eggs, metabolic consequences similar to those observed in combined carboxylase deficiency were describedz9: elevated excretion in urine of 3-methylcrotonylglycine, of 3- hydroxyisovaleric, 3-hydroxypropionic, methylcitric, and 3-hydroxybutyric acids. Activities of PCC and MCC in cultured fibroblasts were found to be reduced as were the biotin concentrations in blood and urine. Clinically, the 1 1-year-old mentally retarded boy presented with alopecia totalis and generalized erythematous, scaly eruptions most prominently on his face. Treatment with biotin and the omission of raw eggs from the diet led to a reversal of all the metabolic manifestations observed; the skin began to improve within days and there was hair growth within two weeks.

Skin lesions and loss of hair have also been reported in ~hildren’”~’ and adults3G36 receiving long-term total parenteral nutrition without biotin. When measured, biotin concentration^^^"*'^ in blood and urine were found to be reduced and in urine elevated excretion of abnormal organic acids (i.e. 3-methylcrotonylglycine, 3-hydroxypro- pionic, methylcitric, and lactic acids) were noted.’”’’ On treatment with biotin or on switching to a multivitamin preparation containing biotin, skin lesions healed and the hair began to regrow in all patients. Patients on total parenteral nutrition are known to develop depressive episodes which were thought not to be related to their alimentation. Nevertheless, mental changes have been reported in volunteers with experimentally induced biotin deficiency,” and irritability is often mentioned in children with an inborn error of biotin-dependent enzymes.*’ Treatment with biotin in these cases and in

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children” and adults on total parenteral n ~ t r i t i o n ~ ~ * ” improved their mental status. In a patient under psychiatric therapy receiving total parenteral nutrition without biotin, and who showed only slight skin changes, treatment with biotin relieved his depres- ~ i o n . ~ ’

In epileptics treated with anticonvulsants other than sodium valproate reduced biotin levels in plasma have been noted and elevated concentrations of abnormal organic acid in urine and of lactic acid in plasma have been found in contrast to epileptics treated with valproate or to controls.23

CONCLUSIONS

A reduction in the activity of the biotin-dependent carboxylases leads to the excretion of small molecular weight organic acids in ~ r i n e . ~ ~ . ~ ’ The nature of these metabolites depends on the metabolic step that is blocked in each case of an isolated carboxylase deficiency. In a combined carboxylase deficiency, where the activities of a t least the three mitochondria1 biotin-dependent carboxylases are deficient and in some cases also of the cytosolic ACC,” most of the abnormal metabolites identified in an isolated enzyme deficiency have been detected in the urine of affected patients. As the activity of these carboxylases is dependent on biotin c~ncentration,~*’*~ a reduction of circulating biotin levels, due to excessive intake of raw eggs,29 to total parenteral nutrition without biotin3G32 or to treatment with specific ant iconv~lsants ,~~ also leads to excretion of these abnormal organic acids in urine. Whether these abnormal metabolites are equally found in segments of the population in which reduced circulating biotin levels have been noted, remains to be investigated. And further studies are necessary to demonstrate whether the appearance of these abnormal organic acids in urine can be used as a functional parameter to define the requirement of biotin.

Patients suffering from a combined deficiency of the biotin-dependent carboxyl- ases present clinically with the symptoms of organic acidemia and aciduria and prominently also with skin rash and mostly alopecia. Skin lesions and loss of hair have been reported in patients with “egg white i n j ~ r y ” ~ * * ~ and in patients receiving total parenteral n~t r i t ion ,~”’~ skin lesions only in volunteers on a biotin-deficient diet.9 In humans-as in animals-biotin deficiency therefore provokes skin lesions and loss of hair, and the seborrheic dermatitis of infancy, often reported in the older literature: is then a true biotin-deficiency symptom.

Two basic genetic defects have been found to cause combined deficienc of the biotin-dependent carboxylases: a defect of the holocarboxylase synthetase’V2’ and recently a deficient biotinidase activity.28 Such a biotinidase deficiency leads to the appearance of biocytin in the urine of affected children, a compound that is not normally excreted.20 These findings indicate that in healthy persons biotin is cleaved from biocytin derived either from the catabolism of the biotin-containing carboxylases or from bound biotin in food. Whether biotin exists in food free or bound to lysine cannot be estimated as all determinations of biotin contents in food23”*’2 have been based on microbiological assays that include an acid hydrolysis step. Such hydrolysis liberates a t least part of the bound biotin, which may not be wholly bioavailable as indicated by the reduced availability of biotin in cereals in the chicken growth test.” Biocytin derived from the catabolism of carboxylases will be cleaved by the biotinidase occurring in blood and the biotin liberated. Biotin is thus recycled in healthy humans.

Whether biotin circulates bound to a biotin-binding protein remains to be elucidated. Certain findings seem to indicate that such a biotin-binding protein

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exists”: (1) the incomplete elimination of biotin from human and fetal calf serum by extensive dialysis or by treatment with avidin,” (2) the lower biotin concentrations found in plasma when levels were determined by an avidin binding method compared with the microbiological assay, which includes digestion with papain (J. Bausch, unpublished results), and (3) by the older observation of an accumulation of biotin in the a-globulin and albumin fractions of human plasma.“ This biotin-binding protein might be biotinidase, as in patients with a deficiency of this particular enzyme an elevated biotin clearance has been noted.” However, the increased renal loss of biotin could also be due to biocytin present in these patients.19 In the urine of healthy persons only biotin metabolites and excess biotin are excreted.

This endogenous recycling of biotin-and not the intestinal synthesis-may then be the reason why biotin deficiency symptoms take a long time to develop and are only rarely seen in healthy humans.

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DISCUSSION OF THE PAPER

L. MACHLIN (Hoffmann-Lo Roche, Nufley, NJO7IIO): You mentioned that in the elderly, in pregnant or lactating women, and I think in athletes the biotin levels were low. In these cases is there any correlation with excessive organic acid excretion or any other functional parameter or simply an observation that they are low?

J. P. BONJOUR: It is only an Observation that biotin levels are low in these subjects. Further investigations are necessary to show whether low circulating biotin levels lead also in healthy persons to the excretion of abnormal organic acids.

H. N. BHAGAVAN: (Hoflmann-LaRoche, Nutley, NJ 071 10) Could you comment on the optimum intake of biotin?

BONJOUR: I think the American Medical Association has stipulated 60 p g per day in total parenteral nutrition. The 1JS Food and Nutrition Board state in the last edition of the Recommended Dietary Allowances that 100-300 pg biotin daily will meet the needs of practically all healthy adults.