Intestinal absorption of biotin and biocytin in the rat

Bioscience Reports, Vol. 7, No. 8, 1987

Intestinal Absorption of Biotin and Biocytin in the Rat K. DakshinamurtP, J. Chauhan and H. Ebrahim Received August 12, 1987

KEY WORDS: biotin; biocytin;biotinidase; intestinal absorption. The uptake of biotin and biocytin was investigated in rat intestine using the everted sac technique. It has been shown that at biotin and biocytin concentrations !ess than 40 and 50 nM respectively, absorption proceeds by a saturable process, whereas at higher concentrations uptake by passive diffusion predominates. Fractionation of solublized brush border preparations indicates that biotinidase is the only protein which binds biotin in this preparation.

INTRODUCTION There are conflicting reports on intestinal absorption of biotin, with suggestions that the mode of uptake of biotin differs among the species. Earlier investigations using the everted sac technique (1-2) reported that the absorption of biotin in the rat occurred by passive diffusion. Spencer and Brody (2) observed that the hamster intestine transported biotin against a concentration gradient. In later work Berger et al. (3) presented evidence for a sodium-dependent saturable process for the uptake of biotin by the proximal part of the hamster intestine. However the K~, given for biotin transport (1.0mM) was in the unphysiological range. The uptake of biotin by human cell lines was reported by us (4-6) to be saturable. Based on the binding of avidin and avidin-biotin complex to rat liver plasma membrane (7), we suggested that avidin in these systems was mimicking a natural biotin-binding carrier involved in biotin transport (8). This was corroborated by Cohen and Thomas (9) in their study using fully differentiated 3T3-L1 cells. BowersKomro and McCormick (10) have indicated that biotin was transported in Department of Biochemistry,Universityof Manitoba, Winnipeg, Canada, R3E 0W3. To whom correspondenceshould be addressed.

667 0144-8463/87/0800-0667505.00/0 9 1987 Plenum Publishing Corporation

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Dakshinamurti, Chauhan and Ebrahim

hepatocytes by a sodium-dependent process but was not associated with a definitive saturable process. Gore et aI. (11), who have used isolated rat mucosal celIs in their study, concluded that at physiological concentrations the uptake of biotin by these ceils was a passive phenomenon. However, both Bowman et al. (12) and Said and Redha (13) have concluded that at concentrations less than 5 #M absorption of biotin by rat jejunal segments proceeded largely by a saturable process. Although a carrier has been proposed for biotin uptake no attempt has been made to identify this carrier. Most of the biotin in foods such as meat and cereals is protein bound (14-16). The enzymatic hydrolysis in the gastrointestinal tract would result in the release of biocytin (or biotinyl peptides) rather than of free biotin. There is no report on the intestinal absorption of biocytin. We report here on the intestinal uptake of biotin and biocytin and suggest that biotinidase [EC 3.1.1.12] is a carrier protein in intestinal transport of biotin and biocytin. MATERIALS AND METHODS

Materials Silica gel thin layer chromatography plates were purchased from Fisher Scientific Company. [3H]biotin (10 Ci/mmol) was a gift from Hoffman La Roche (Nutley, NJ). NCS tissue solublizer and OCS were obtained from New England Nuclear. All other chemicals used were of reagent grade and were obtained from standard commercial sources.

Synthesis and Purification of [3H]Biocytin [3H]biocytin was synthesised from [3H]biotin by the method of Bodanzky and Fagan (17). Traces of free biotin were removed on Bio Rad AG1-X2 200M00 mesh resin in the acetate form as previously described (18). It was further purified on a preparative TLC silica gel plate using ethanol/water (1:1, v/v) as solvent system. The area corresponding to the migration of authentic biocytin was scrapped and packed into a column. The material was eluted with distilled water and the sample reduced to a small volume by freeze drying. The TLC plate did not show any free biotin contamination in [3H]biocytin. The specific radioactivity of [3H]biocytin as determined by avidin-binding assay (18) was 0.1 Ci/mmol.

Biotinidase Activity The biotinidase activity was determined using N-(d-biotinyl)-p-aminobenzoate substrate as described previously (19,20),

Biotin Binding Activity This assay relies on strong non-covalent binding of biotin to biotin-binding proteins. Biotin-binding activity for biotin-binding proteins was determined as described for biotin-holocarboxylase synthetase (21) and used by us for biotinidase (22).

Intestinal transport of biotin and biocytin

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Preparation of Everted Jejunal Segments Male Sprague-Oawley rats (200-250g) were sacrified by decapitation and a midline incision made. The duodenal and the ileocecal end were identified, cut and discarded. The entire piece of the proximal jejunum was flushed with 40 ml of ice cold 0.9 % saline. The jejunum was everted by catching on end on a glass rod (1.5 mm diameter) and everting the whole length. The whole procedure upto this point took three minutes. The everted jejunum was washed with 0.9 % saline and cut into pieces of 2.0 cm length. These jejunum segments were used immediately for studies on the uptake of [3H]biotin or [3H]biocytin.

Uptake of Biotin in Jejunum Segments The uptake studies were performed in 20 ml plastic scintillation vials containing 12ml of oxygenated Krebs Ringer phosphate buffer pH 7.4, and 11 mM glucose. Varying amounts of unlabelled biotin upto the desired concentration were added. The amount of [3H]biotin added was such that the final specific radioactivity of 1.29 Ci/mmol remained constant at each biotin concentration. The uptake studies were done at 37~ in a shaking Dubnoff water bath at 90 oscillations per minute. The reaction was started by adding the 2.0 cm everted jejunal segments at timed intervals. After 10 minutes of incubation the jejunum segment was removed and placed on a single layer of cheese cloth held in place by a rubber band over a wide neck flask. The piece of tissue was washed with 5 ml ice cold Krebs-Ringer phosphate buffer, pH 7.4 containing 1 mM biotin and the excess fluid drained off. The segments were weighed wet. Each piece of tissue was transferred into a plastic scintillation vial and solublized with 1 ml NCS. To the solublized sample 15 ml OCS was added and the radioactivity determined in a Beckman S-2000 liquid scintillation spectrometer.

Uptake of Bioeytin in Jejunal Segments The uptake of biocytin was studied as described for biotin. The concentration of biocytin used in this study was between 7.8 nM to 200 nM. The radioactive biocytin (0.1 Ci/mmol) was diluted to give the required concentration of biocytin in the experiments.

Preparation and Fractionation of Cytosol and Brush Border Membrane Vesicles The method of Kessler et al. (23) was used for preparing jejunal brush border membrane vesicles and cytosol fraction. Brush border membranes were solublized in 0.3 ~o sodium deoxycholate on ice for half an hour. The solubilized fraction was obtained after centrifuging at 35,000 x g for half an hour. The supernatant fraction containing all the biotin-binding activity was dialyzed overnight against 50raM phosphate buffer, pH 7.0, containing 1 mM EDTA, and 1 mM 2-mercaptoethanol. The dialyzed material was concentrated to 1.0 ml and used for fractionation on sucrose density gradient. The method of Martin and Ames (24) was used for fractionation of cytosol and solubilized brush border fraction.

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RESULTS AND DISCUSSION There are conflicting reports as to the origin of biotin for its intestinal absorption. It has been assumed that biotin synthesized by the colonic microflora makes a contribution to the host nutrition. However various studies (12-13) show that the transport of biotin was higher in the jejunum than the ileum and was minimal in the colon. Along with the earlier observation (25) that biotin was better absorbed when given orally than when instilled into the colon, this would suggest that although biotin is synthesized by colonic microflora it might not be a significant source of biotin nutrition for the host. The present study examined the transport of biotin and biocytin in rat intestine using the everted sac method. Transport was linear with time during 20 minutes incubation (data not shown here). However a 10 minute incubation period was chosen so as to maintain functional integrity of rat intestinal mucosa (26). The uptake of biotin in jejunal segments is shown in Fig. 1(a). The range of biotin concentration studied was between 0.05 to 3.0/~M. The relationship between the biotin concentration and the 10 minute uptake appears to be essentially linear. However, at the lower biotin concentration (less than 0.25/~M) there appeared to be some deviation from this linearity. Furthermore, when the range was narrowed down to 2.5 to 60 nM biotin uptake proceeded by a process which showed saturation kinetics (Fig. 1(b)). In general our results show carrier mediated uptake of biotin upto 40 nM (Kin = 17 nM, Vmax= 6 pmol/g wet wt/10 rain). Beyond this concentration biotin transport proceeded by passive diffusion as indicated by the linear relationship between uptake and

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Fig. 1. Uptake of biotin in jejunal segments. The uptake of biotin in 2.0cm jejunal segments was determined as a function of the concentration of biotin. The data shown is a mean _+SE of three experiments. Biotin ranges used were (a) 0.05 to 3.0#M and (b) 2.5 to 60 nM.


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concentration of biotin. The biphasic nature of the uptake of biotin indicated by this study is in agreement with other results of uptake of biotin in rat jejunum (12-13). However Km of 17 nM observed in the present study is much lower than Km = 9.6 #M (12) and Kt = 3.75/~M (13) reported for rat intestine. Taking into account the food intake and gastric emptying time one would expect the biotin concentration in the jejunal lumen to reach only the nanomolar rather than the micromolar concentration range. Hence, any study on the transport of biotin in the micromolar range will not represent a physiological situation. Figure 2 shows the uptake of biocytin by the jejunal segments. The amount of biocytin transported appears to plateau at about 50 nM biocytin concentration (Kin = 17 nM, Vmax= 18 pmol/g wet wt/10 rain); however the uptake of biocytin above 50 nM is essentially a direct function of the biocytin concentration. The concentration of biocytin used in this experiment was between 7.8 nM and 200 nM. Biocytin is taken up by the jejunal segments and its transport was three fold higher in comparison to the transport of biotin. The higher uptake of biocytin observed here would indicate that biocytin itself can be transported and that biotinidase would liberate free biotin inside the mucosal cells. High biotinidase activities have been reported in rat intestinal homogenate (27-28). It is also possible that biocytin in the lumen is hydrolyzed to

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biotin and lysine before biotin enters the mucosal cell. This would be possible if the intestinal secretion contained biotinidase. There is no information in the literature regarding this. The biphasic transport of biotin and biocytin in the rat small intestine observed in the present investigation suggests that when the biotin concentration in the gut is below 50 nM, the saturable uptake mechanism would operate so that enough biotin is made available to the animal. If such a system indeed operated in other mammals, including man, it would have tremendous advantage in the context of a fluctuating amounts of biotin ingested in the diet. This is born out by the rarity of primary biotin deficiency in humans. The late-onset type of multiple carboxylase deficiency was shown to be due to deficiency of biotinidase (29). It is suggested in certain cases of lateonset type multiple carboxylase deficiency that these patients lack the system for absorbing biotin in nanomolar range (30). Furthermore it has been shown that these patients only respond to pharmacological doses of biotin (31) indicating that only the saturable portion of biotin transport system is defective in these patients. In order to identify the nature of the biotin-binding carrier protein in rat intestine we fractionated solublized brush border membrane and cytosol by sucrose density gradient centrifugation. Figure 3 shows the biotinidase activity and biotin-binding profiles of the separated proteins from the two preparations. In both fractions biotinidase activity migrated slightly ahead of albumin (68 K). The biotin-binding activity in cytosol and brush border preparations migrated to an identical position to the biotinidase peak and there was good correlation at each point between two

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Fig. 3. Analysis of cytosol and solublized brush border membrane on sucrose density gradient. Brush border membrane was prepared as described under Materials and Methods. (a) Cytosol and (b) the detergent treated, dialyzed preparation of brush border proteins were separated by sucrose density gradient centrifugation. 0.3 ml of cytosol and solublized brush border proteins were layered in separate tubes on top ofa 5 to 2 0 ~ gradient of sucrose (12.5 ml) containing 0.1 M potassium phosphate buffer, pH 6.0, 1 mM 2-mercaptoethanol and I mM EDTA. Centrifugation was at 3~ for 22 h at 35,000 rpm in the Beckman SW 40 Ti rotor. At the end of the run, fractions were collected from the bottom of the gradient and analyzed for biotinidase ( O - - - - O ) and biotin-binding activity ( m - - - m ) .


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activities. F u r t h e r m o r e , w h e n we used n - b r o m o s u c c i n i m i d e a n d p - c h l o r o m e r c u r y b e n z o a t e (inhibitors of b i o t i n i d a s e activity (20,22,32)) b o t h b i o t i n i d a s e a n d b i o t i n b i n d i n g activities were inhibited suggesting the role of t r y p t o p h a n a n d cysteine residues for b i o t i n - b i n d i n g activity. The results of the present investigation indicate t h a t b i o t i n i d a s e is the only p r o t e i n in the b r u s h b o r d e r m e m b r a n e which binds biotin. Based o n this p r e l i m i n a r y in v i t r o s t u d y it is suggested t h a t b i o t i n i d a s e in vivo m a y have a role in the t r a n s p o r t of biotin.

ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y the M e d i c a l Research C o u n c i l of C a n a d a .

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