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Ontogenesis of the intestinal transport of biotin in the rat
GASTROENTEROLOGY 1986;94:66-72
Ontogenesis of the Intestinal Transport of Biotin in the Rat HAMID M. SAID and REYADH REDAH
Departments of Pediatric Gastroenterology, Molecular Physiology, and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee
Developmental aspects of the intestinal transport of biotin were examined in suckling (16 day old) and weanling (24 day old) rats using the everted sac technique. The results were compared with those of adult rats previously reported by us using the same intestinal preparation. Transport of biotin was linear for 20 min of incubation in all age groups. Transport of biotin was significantly (p < 0.05) lower in the jejunum than the ileum of suckling rats but was not significantly different in the jejunum and the ileum of weanling rats. In adult rats, biotin transport was significantly (p < 0.01) higher in the jejunum than the ileum. In all age groups, transport of biotin in the jejunum was saturable at low concentrations (
B
iotin (2’-keto-3,4-imidazelido-Ztetrahydrothiophene-n-valeric acid; vitamin H) is an essential water-soluble vitamin that is required for normal cellular function, growth, and development. Biotin acts as a coenzyme in many metabolic reactions including fatty acid biosynthesis, gluconeogenesis,
and amino acid catabolism. Humans and higher mammals cannot synthesize biotin; therefore, they must obtain the vitamin by intestinal absorption. Biotin deficiency leads to serious clinical complications including growth retardation, neurologic disorders, and skin abnormalities (l-3). We have recently shown that transport of physiologic concentrations (Cl0 PM) of biotin in the intestine of adult rats is performed by a carrier-mediated system that is Na+-, energy-, and temperaturedependent and occurs mainly in the jejunum (4). Furthermore, we found that biotin crosses the intestinal tissue metabolically intact (4). No study is available, however, describing the mechanism and site of maximal transport of biotin in the intestine during maturation (i.e., during the suckling and weanling periods). Such a study is of physiologic and nutritional importance because biotin is essential for normal growth and development, processes that are most active during the early stages of life. The present study was designed to examine the mechanism and site of maximum transport of biotin during maturation in suckling and weanling rats. The results were compared with those previously reported by us in adult rats (4). Materials
and Methods
The following materials were obtained commercially: [8,9-3H]biotin (33 Ci/mmol) from New England Nuclear, Boston, Mass.; unlabeled biotin, desthiobiotin, and biotin methyl ester from Sigma Chemical Company, St. Louis, MO.; scintillation cocktail (ASC) from Amersham/Searle, Des Plaines, 111.; precoated cellulose thinlayer chromatography plates from Eastman Kodak, Rochester, N.Y. All other chemicals and reagents were of analytic quality. The radiochemical purity of [3H]biotin was found to be 97% by thin-layer chromatography using precoated cellulose chromatography plates and a solvent system of butanol, acetic acid, and water (4:1:9) (5). 0 1966 by the American Gastroenterological Association 0016-5065/86/$3.50
ONTOGENESIS OF BIOTIN INTESTINAL TRANSPORT
January 1988
Suckling and weanling Sprague-Dawley rats were purchased from Sasco, Omaha, Neb. Mothers and weanling rats were fed Purina Rat Chow [Ralston Purina, St. Louis, MO.) and tap water ad libitum. The National Council’s guidelines for the care and use of laboratory animals were followed. Transport studies were performed in suckling (16 day old) and weanling (24 day old) rats by use of the everted sac technique (6). Rats were killed by an overdose of ether. The abdomen was opened, and 20 cm of the jejunum (starting 8 and 10 cm from the pylorus of suckling and weanling rats, respectively) and the ileum (the most distal 20 cm of the small intestine) was removed and washed with ice-cold buffer. Everted sacs (4 cm in length) were prepared as described by us previously (4,7). Everted sacs were incubated in lo-ml Erlenmeyer flasks containing 5 ml of continuously oxygenated (100% 0,) Krebs-Ringer phosphate buffer (20 mM NaH2P04, 125 mM NaCl, 4.93 mM KCl, 1.23 mM MgSO*, 0.85 mM CaC12, and 10 mM glucose, pH 6.5). The serosal compartment was filled with the same buffer used for incubation. Incubation was carried out at 37°C (unless otherwise mentioned) in a shaking water bath (80 oscillations/min). At the end of incubation, the sacs were removed and washed, and the serosal medium was drained into a scintillation vial containing 5 ml of scintillation cocktail and analyzed for radioactivity. The viability of the in vitro preparation was previously demonstrated by measuring transmural potential difference and L-[3H]leucine (20 a) and [5-14C]methyltetrahydrofolate (50 nM) accumulation against a concentration gradient (7). Statistical
_L
1
/‘, 5
I
I
I
IO
15
20
Time
Figure
( min 1
1. Mucosal-to-serosal transport of biotin in suckling (A) and weanling (B) rats as a function of time. Biotin (0.1 ph4] was added to the mucosal medium of jejunal everted sacs at the beginning of the experiments. Incubation was performed at 37°C under continuous oxygenation. Each point represents the mean f SE of at least four separate experiments on 4 rats. Y = mx + b, where m = slope and b = y-intercept; r = correlation coefficient.
Analysis
Each group of data presented is the result of at least four separate experiments from 4 different rats and is expressed as mean ? SEM. Transport results are expressed in terms of initial tissue wet weight, a parameter that has been shown to be suitable for comparing transport data from the different age groups (8). Data were analyzed using the Student’s t-test and regression analysis.
Results Transport
69
With
Time
The mucosal-to-serosal transport of 0.1 PM biotin as a function of time was examined in jejunal everted sacs prepared from suckling and weanling rats to determine the linearity of the transport process (Figure 1). Transport of biotin proceeded in a linear manner for 20 min of incubation in both suckling and weanling rat intestine and occurred at a rate of 3.96 and 4.35 pmol/g initial tissue wet wt . min, respectively. Similarly, transport of 0.1 PM biotin in the intestine of adult rats was found in a previous study to be linear for a 20-min incubation and occurred at a rate of 3.07 pmol/g initial tissue wet wt . min (4). A 20-min incubation was, therefore, used as a standard incubation time in all subsequent studies.
Transport
in the Ileum
Mucosal-to-serosal transport of 0.1 PM biotin in ileal everted sacs of suckling and weanling rats was examined, and the results were compared with those of simultaneously incubated jejunal everted sacs prepared from the same animal. In suckling rats, transport of biotin in the ileum was significantly (p < 0.05) higher than transport in the jejunum [lo3 * 14 (n = 4) and 65 -+: 11 (n = 4) pmol/g initial tissue wet wt . 20 min, respectively]. In weanling rats, transport of biotin was not significantly different in the jejunum and the ileum [75 + 9.5 (n = 4) and 60 ? 6.8 (n = 4) pmol/g initial tissue wet wt . 20 min, respectively]. These results are in contrast with the previously reported results on biotin transport in adult rats (3 mo old), where transport in the jejunum was significantly (p < 0.01) higher than transport in the ileum (85 ? 6 and 36 t 6 pmol/g initial tissue wet wt . 25 min, respectively). Because biotin transport is higher in the jejunum than the ileum of weanling and adult rats, we decided to perform all our studies using jejunal tissue to allow better comparison of the data between the different age groups. Studies are required to determine the maturational
70
SAID AND REDAH
GASTROENTEROLOGY
changes of biotin transport in the rat ileum, and will be a subject of future investigation.
Table
1. Kinetic Jejunum
Parameters of Biotin Transport in the of Suckling, Weanling, and Adult Rats
Carrier-mediated
Transport
as a Function
of Concentration
Mucosal-to-serosal transport of biotin as a function of increasing substrate concentration (0.1-100 PM) was examined in jejunal everted sacs of suckling and weanling rats (Figure 2). Saturation of the transport process was observed at low concentrations of biotin (lO PM), transport of biotin was linear (r = 0.99 for both) and occurred at a rate of 143.8 and 111.6 pmol/g initial tissue wet wt . min for suckling and weanling rats, respectively. To eliminate the contribution of the linear nonsaturable process in the transport of low concentrations of biotin, we multiplied the linear rate of transport by the individual concentrations. We then subtracted the contribution
Rat group
Vol. 94, No. 1
Apparent K, (@fl
system
Nonsaturable
Vmax (pmollg initial tissue wet wt . min)
Diffusion rate (pmol/g initial tissue wet wt . min)
Suckling
0.63
18.3
143.6
Weanling Adult”
2.49 3.73
44.7
111.6
’ Data are from Reference
process
124.4
87.5
9.
of the nonsaturable process from the total transport at each concentration (Figure 2). Kinetic parameters (apparent K, and V,,,) of the transport process were then determined using the Lineweaver-Burke plot (Figure 2 insert) and regression analysis (Table 1). Apparent K, values of 0.63 and 2.49 /_LMand V,, values of 18.3 and 44.7 pmol/g initial tissue wet wt . min were obtained for suckling and weanling rats, respectively. Efiect of Naf
2
I
I
1
I
4
6
8
IO
Eiotin
Cone
(JJM)
w
2B
1
I
I
r.0.99 m.0,26 b. 0.1 I
3
2
I/V x100
’
.
I/S
2
I
2 Figure
I
4
I
I
6
8
Biotin
Cone
6
IO
I
IO (PM)
2. Mucosal-to-serosal transport of biotin in suckling (A) and weanling (B) rats as a function of concentration. Biotin (0.1-10 PM) was added to the incubation medium of jejunal everted sacs at the beginning of the experiments. Incubation was performed for 20 min at 37’C under continuous oxygenation. Transport by the carrier system was calculated as described in the text. Each insert is l/v against l/S; Y = mX + b, where m = slope, b = y-intercept; r = correlation coefficient.
The Na+ requirement of the transport system of biotin in suckling and weanling rats was examined by lowering Na+ concentration in the incubation medium from 145 mM (control) to 20 mM (osmolarity was compensated with choline or NH4+) (Table 2). The results showed that decreasing the Naf concentration causes severe (72%89%) and significant (p < 0.01) inhibition in biotin transport in both suckling and weanling rats. The inhibition was similar whether Na+ was replaced by choline or NH,+. Effect of Metabolic Temperature
Inhibitors
and
The effect of 1 mM 2,4dinitrophenol, 1 mM iodoacetate, and 10 mM azide on the mucosal-toserosal transport of 0.1 PM biotin into jejunal everted sacs was examined and the results were compared with simultaneously studied (untreated) controls. All metabolic inhibitors examined caused severe (54%-92%) and significant (p < 0.01) inhibition in biotin transport in both suckling and weanling rats (Table 3). In another experiment, the Q1,, value, i.e., the ratio of transport at 37°C to transport at 27°C of 0.1 PM biotin was determined. Values of 2.88 and 3.68 were calculated for suckling and weanling rats, respectively. Effect of Structural
Analogues
The effect of structural analogues desthiobiotin (50 PM) and biotin methyl ester (50 FM) on the
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72 SAID AND REDAH
affinity of the biotin transport system with maturation Similarly, there was a progressive increase in the V,, of biotin transport with maturation (18.3, 44.7, and 124.4 pmol/g initial tissue wet wt. min for suckling, weanling, and adult rats, respectively). This change in the V,, suggests that there is an increase in the numbers or activity, or both, of the transport carrier of biotin with maturation. Another interesting observation was the finding that maturation is associated with a progressive decrease in the rate of the nonsaturable transport of biotin across the intestinal epithelium (143.8, 111.6, and 87.5 pmol/g initial tissue wet wt . min for suckling, weanling, and adult rats, respectively). The factors responsible for these changes in biotin transport are not known, but they may include hormonal (15,18) or physiochemical changes in the enterocyte membrane (19) with maturation. The higher affinity of the biotin transport carrier and the higher nonsaturable rate of biotin transport in the suckling rats, together with the above-mentioned observation that biotin transport is efficient in the jejunum and the ileum during the suckling period, demonstrates how the intestine of the rapidly growing animal ensures efficient absorption of biotin. Transport of biotin in the developing intestine of suckling and weanling rats appeared to be similar to that of the mature adult rats in being Naf-dependent. The exact role of Na+ in biotin transport in the rat intestine is not clear, but it could be an indication of the existence of a biotin:Na+ cotransport system similar to that recently suggested by us for biotin transport in the human intestinal brush border membrane vesicles (20). Further studies with rat intestinal brush border membrane vesicles are required to clarify this issue. The transport of biotin in the intestine of suckling and weanling rats was also similar to that of adult rats (4) in being energydependent (as evident by the ability of metabolic inhibitors to inhibit biotin transport) and temperature-dependent (4). In summary, the present study shows that biotin transport undergoes clear changes with maturation. These include changes in the affinity and activity (and/or numbers) of the transport carrier, changes in the nonsaturable transport rate, and changes in the preferential site of biotin transport. Biotin transport, however, is similar in the suckling, weanling, and adult rats in being carrier-mediated and Naf-, energy-, and temperature-dependent.
GASTROENTEROLOGY Vol. 94,No. 1
References 1. Bonjour JB. Biotin in man’s nutrition
and therapy. Int J Vitam Nutr Res 1977;47:107-18. 2. Sweetman L, Nyhan WL. Inheritable biotin-treatable disorders and associated phenomena. Annu Rev Nutr 1986;6: 314-43. 3. Wolf B, Heard GS, Jefferson LG, Proud VK, Nance WI, Weissbecker KA. Clinical findings in four children with biotinidase deficiency detected through a state-wide neonatal screening program. N Engl J Med 1985;313:16-9. 4. Said HM, Redha R. A carrier-mediated system for transport of biotin in rat intestine in vitro. Am J Physiol 1987;252:G52-5. 5. Spencer RP, Brady K. Biotin transport by small intestine of rat, hamster and other species. Am J Physiol 1964;206:653-7. 6. Wilson TH, Wiseman G. The use of everted sacs of small intestine for the study of the transfer of substances from the mucosal to the serosal surface. J Physiol 1954;123:116-25. 7. Said HM, Ghishan FK, Murrell JE. Ontogenesis of intestinal transport of 5-methyltetrahydrofolate in the rat. Am J Physiol 1985;249:G567-71. 8. Younoszai MK, Komnick K. Maturation of the small intestine: absorption of L-valine in rats. Pediatr Res 1982;16:756-60. 9. Ghishan FK, Jenkins JT, Younoszai MK. Maturation of calcium transport in the rat small and large intestine. J Nutr 1980;110:1622-8. 10. Said HM, Ghishan FK, Greene HL, Hollander D. Developmental maturation of riboflavin intestinal transport in the rat. Pediatr Res 1985;19:1175-8. 11. Hennings SJ. Functional development of the gastrointestinal tract. In: Johnson LR, ed. Physiology of the gastrointestinal tract, 2nd ed. New York: Raven, 1987:282-300. 12. Koldovsky 0. In: Development of the Functions of the Small Intestine in Mammals and Man. Basel: Karger, 1969. 13. Barnard JA, Ghishan FK, Wilson FA. Ontogenesis of taurocholate transport by ileal brush border membrane vesicles. J Clin Invest 1985;75:869-73. 14. Said HM, Greene HL, Moore GC, Ghishan FK. Developmental maturation of o-glucose active transport system in rat intestine. Digestion 1987;36:195-200. 15. Little JM, Lester R. Ontogenesis of intestinal bile salt absorption in the neonatal rat. Am J Physiol 1980;239:G319-23. 16. Bamford DR, Ingham PA. Sugar absorption by fetal and neonatal rat intestine in vitro. J Physiol 1975;248:335-48. 17. Batt E, Schachter D. Developmental pattern of some intestinal transport mechanisms in newborn rats and mice. Am J Physiol 1969;216:1064-8. 18. Forstuer G, Garland G. The influence of hydrocortisone on the synthesis and turnover of microvillous membrane glycoproteins in suckling rat intestine. Can J Biochem 1976;54:224-30. 19. Schwarz SM, Hostether B, Ling S, Mone M, Watkins JB. Intestinal membrane lipid composition and fluidity during development in the rat. Am J Physiol 1985;248:G206-7. Na+ 20. Said HM, Redha R, Nylander W. A carrier-mediated, gradient-dependent transport for biotin in human intestinal brush border membrane vesicles. Am J Physiol 1987;253: G631-6. Received April 13, 1987. Accepted July 27,1987. Address requests for reprints to: Hamid M. Said, Vanderbilt University Medical Center, D-4113 MCN, Nashville, Tennessee 37232. This study was supported by National Institutes of Health grants BRSG RR 05424 and NIDDKD AM 26657.
Ontogenesis of the Intestinal Transport of Biotin in the Rat HAMID M. SAID and REYADH REDAH
Departments of Pediatric Gastroenterology, Molecular Physiology, and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee
Developmental aspects of the intestinal transport of biotin were examined in suckling (16 day old) and weanling (24 day old) rats using the everted sac technique. The results were compared with those of adult rats previously reported by us using the same intestinal preparation. Transport of biotin was linear for 20 min of incubation in all age groups. Transport of biotin was significantly (p < 0.05) lower in the jejunum than the ileum of suckling rats but was not significantly different in the jejunum and the ileum of weanling rats. In adult rats, biotin transport was significantly (p < 0.01) higher in the jejunum than the ileum. In all age groups, transport of biotin in the jejunum was saturable at low concentrations (
B
iotin (2’-keto-3,4-imidazelido-Ztetrahydrothiophene-n-valeric acid; vitamin H) is an essential water-soluble vitamin that is required for normal cellular function, growth, and development. Biotin acts as a coenzyme in many metabolic reactions including fatty acid biosynthesis, gluconeogenesis,
and amino acid catabolism. Humans and higher mammals cannot synthesize biotin; therefore, they must obtain the vitamin by intestinal absorption. Biotin deficiency leads to serious clinical complications including growth retardation, neurologic disorders, and skin abnormalities (l-3). We have recently shown that transport of physiologic concentrations (Cl0 PM) of biotin in the intestine of adult rats is performed by a carrier-mediated system that is Na+-, energy-, and temperaturedependent and occurs mainly in the jejunum (4). Furthermore, we found that biotin crosses the intestinal tissue metabolically intact (4). No study is available, however, describing the mechanism and site of maximal transport of biotin in the intestine during maturation (i.e., during the suckling and weanling periods). Such a study is of physiologic and nutritional importance because biotin is essential for normal growth and development, processes that are most active during the early stages of life. The present study was designed to examine the mechanism and site of maximum transport of biotin during maturation in suckling and weanling rats. The results were compared with those previously reported by us in adult rats (4). Materials
and Methods
The following materials were obtained commercially: [8,9-3H]biotin (33 Ci/mmol) from New England Nuclear, Boston, Mass.; unlabeled biotin, desthiobiotin, and biotin methyl ester from Sigma Chemical Company, St. Louis, MO.; scintillation cocktail (ASC) from Amersham/Searle, Des Plaines, 111.; precoated cellulose thinlayer chromatography plates from Eastman Kodak, Rochester, N.Y. All other chemicals and reagents were of analytic quality. The radiochemical purity of [3H]biotin was found to be 97% by thin-layer chromatography using precoated cellulose chromatography plates and a solvent system of butanol, acetic acid, and water (4:1:9) (5). 0 1966 by the American Gastroenterological Association 0016-5065/86/$3.50
ONTOGENESIS OF BIOTIN INTESTINAL TRANSPORT
January 1988
Suckling and weanling Sprague-Dawley rats were purchased from Sasco, Omaha, Neb. Mothers and weanling rats were fed Purina Rat Chow [Ralston Purina, St. Louis, MO.) and tap water ad libitum. The National Council’s guidelines for the care and use of laboratory animals were followed. Transport studies were performed in suckling (16 day old) and weanling (24 day old) rats by use of the everted sac technique (6). Rats were killed by an overdose of ether. The abdomen was opened, and 20 cm of the jejunum (starting 8 and 10 cm from the pylorus of suckling and weanling rats, respectively) and the ileum (the most distal 20 cm of the small intestine) was removed and washed with ice-cold buffer. Everted sacs (4 cm in length) were prepared as described by us previously (4,7). Everted sacs were incubated in lo-ml Erlenmeyer flasks containing 5 ml of continuously oxygenated (100% 0,) Krebs-Ringer phosphate buffer (20 mM NaH2P04, 125 mM NaCl, 4.93 mM KCl, 1.23 mM MgSO*, 0.85 mM CaC12, and 10 mM glucose, pH 6.5). The serosal compartment was filled with the same buffer used for incubation. Incubation was carried out at 37°C (unless otherwise mentioned) in a shaking water bath (80 oscillations/min). At the end of incubation, the sacs were removed and washed, and the serosal medium was drained into a scintillation vial containing 5 ml of scintillation cocktail and analyzed for radioactivity. The viability of the in vitro preparation was previously demonstrated by measuring transmural potential difference and L-[3H]leucine (20 a) and [5-14C]methyltetrahydrofolate (50 nM) accumulation against a concentration gradient (7). Statistical
_L
1
/‘, 5
I
I
I
IO
15
20
Time
Figure
( min 1
1. Mucosal-to-serosal transport of biotin in suckling (A) and weanling (B) rats as a function of time. Biotin (0.1 ph4] was added to the mucosal medium of jejunal everted sacs at the beginning of the experiments. Incubation was performed at 37°C under continuous oxygenation. Each point represents the mean f SE of at least four separate experiments on 4 rats. Y = mx + b, where m = slope and b = y-intercept; r = correlation coefficient.
Analysis
Each group of data presented is the result of at least four separate experiments from 4 different rats and is expressed as mean ? SEM. Transport results are expressed in terms of initial tissue wet weight, a parameter that has been shown to be suitable for comparing transport data from the different age groups (8). Data were analyzed using the Student’s t-test and regression analysis.
Results Transport
69
With
Time
The mucosal-to-serosal transport of 0.1 PM biotin as a function of time was examined in jejunal everted sacs prepared from suckling and weanling rats to determine the linearity of the transport process (Figure 1). Transport of biotin proceeded in a linear manner for 20 min of incubation in both suckling and weanling rat intestine and occurred at a rate of 3.96 and 4.35 pmol/g initial tissue wet wt . min, respectively. Similarly, transport of 0.1 PM biotin in the intestine of adult rats was found in a previous study to be linear for a 20-min incubation and occurred at a rate of 3.07 pmol/g initial tissue wet wt . min (4). A 20-min incubation was, therefore, used as a standard incubation time in all subsequent studies.
Transport
in the Ileum
Mucosal-to-serosal transport of 0.1 PM biotin in ileal everted sacs of suckling and weanling rats was examined, and the results were compared with those of simultaneously incubated jejunal everted sacs prepared from the same animal. In suckling rats, transport of biotin in the ileum was significantly (p < 0.05) higher than transport in the jejunum [lo3 * 14 (n = 4) and 65 -+: 11 (n = 4) pmol/g initial tissue wet wt . 20 min, respectively]. In weanling rats, transport of biotin was not significantly different in the jejunum and the ileum [75 + 9.5 (n = 4) and 60 ? 6.8 (n = 4) pmol/g initial tissue wet wt . 20 min, respectively]. These results are in contrast with the previously reported results on biotin transport in adult rats (3 mo old), where transport in the jejunum was significantly (p < 0.01) higher than transport in the ileum (85 ? 6 and 36 t 6 pmol/g initial tissue wet wt . 25 min, respectively). Because biotin transport is higher in the jejunum than the ileum of weanling and adult rats, we decided to perform all our studies using jejunal tissue to allow better comparison of the data between the different age groups. Studies are required to determine the maturational
70
SAID AND REDAH
GASTROENTEROLOGY
changes of biotin transport in the rat ileum, and will be a subject of future investigation.
Table
1. Kinetic Jejunum
Parameters of Biotin Transport in the of Suckling, Weanling, and Adult Rats
Carrier-mediated
Transport
as a Function
of Concentration
Mucosal-to-serosal transport of biotin as a function of increasing substrate concentration (0.1-100 PM) was examined in jejunal everted sacs of suckling and weanling rats (Figure 2). Saturation of the transport process was observed at low concentrations of biotin (
Rat group
Vol. 94, No. 1
Apparent K, (@fl
system
Nonsaturable
Vmax (pmollg initial tissue wet wt . min)
Diffusion rate (pmol/g initial tissue wet wt . min)
Suckling
0.63
18.3
143.6
Weanling Adult”
2.49 3.73
44.7
111.6
’ Data are from Reference
process
124.4
87.5
9.
of the nonsaturable process from the total transport at each concentration (Figure 2). Kinetic parameters (apparent K, and V,,,) of the transport process were then determined using the Lineweaver-Burke plot (Figure 2 insert) and regression analysis (Table 1). Apparent K, values of 0.63 and 2.49 /_LMand V,, values of 18.3 and 44.7 pmol/g initial tissue wet wt . min were obtained for suckling and weanling rats, respectively. Efiect of Naf
2
I
I
1
I
4
6
8
IO
Eiotin
Cone
(JJM)
w
2B
1
I
I
r.0.99 m.0,26 b. 0.1 I
3
2
I/V x100
’
.
I/S
2
I
2 Figure
I
4
I
I
6
8
Biotin
Cone
6
IO
I
IO (PM)
2. Mucosal-to-serosal transport of biotin in suckling (A) and weanling (B) rats as a function of concentration. Biotin (0.1-10 PM) was added to the incubation medium of jejunal everted sacs at the beginning of the experiments. Incubation was performed for 20 min at 37’C under continuous oxygenation. Transport by the carrier system was calculated as described in the text. Each insert is l/v against l/S; Y = mX + b, where m = slope, b = y-intercept; r = correlation coefficient.
The Na+ requirement of the transport system of biotin in suckling and weanling rats was examined by lowering Na+ concentration in the incubation medium from 145 mM (control) to 20 mM (osmolarity was compensated with choline or NH4+) (Table 2). The results showed that decreasing the Naf concentration causes severe (72%89%) and significant (p < 0.01) inhibition in biotin transport in both suckling and weanling rats. The inhibition was similar whether Na+ was replaced by choline or NH,+. Effect of Metabolic Temperature
Inhibitors
and
The effect of 1 mM 2,4dinitrophenol, 1 mM iodoacetate, and 10 mM azide on the mucosal-toserosal transport of 0.1 PM biotin into jejunal everted sacs was examined and the results were compared with simultaneously studied (untreated) controls. All metabolic inhibitors examined caused severe (54%-92%) and significant (p < 0.01) inhibition in biotin transport in both suckling and weanling rats (Table 3). In another experiment, the Q1,, value, i.e., the ratio of transport at 37°C to transport at 27°C of 0.1 PM biotin was determined. Values of 2.88 and 3.68 were calculated for suckling and weanling rats, respectively. Effect of Structural
Analogues
The effect of structural analogues desthiobiotin (50 PM) and biotin methyl ester (50 FM) on the
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affinity of the biotin transport system with maturation Similarly, there was a progressive increase in the V,, of biotin transport with maturation (18.3, 44.7, and 124.4 pmol/g initial tissue wet wt. min for suckling, weanling, and adult rats, respectively). This change in the V,, suggests that there is an increase in the numbers or activity, or both, of the transport carrier of biotin with maturation. Another interesting observation was the finding that maturation is associated with a progressive decrease in the rate of the nonsaturable transport of biotin across the intestinal epithelium (143.8, 111.6, and 87.5 pmol/g initial tissue wet wt . min for suckling, weanling, and adult rats, respectively). The factors responsible for these changes in biotin transport are not known, but they may include hormonal (15,18) or physiochemical changes in the enterocyte membrane (19) with maturation. The higher affinity of the biotin transport carrier and the higher nonsaturable rate of biotin transport in the suckling rats, together with the above-mentioned observation that biotin transport is efficient in the jejunum and the ileum during the suckling period, demonstrates how the intestine of the rapidly growing animal ensures efficient absorption of biotin. Transport of biotin in the developing intestine of suckling and weanling rats appeared to be similar to that of the mature adult rats in being Naf-dependent. The exact role of Na+ in biotin transport in the rat intestine is not clear, but it could be an indication of the existence of a biotin:Na+ cotransport system similar to that recently suggested by us for biotin transport in the human intestinal brush border membrane vesicles (20). Further studies with rat intestinal brush border membrane vesicles are required to clarify this issue. The transport of biotin in the intestine of suckling and weanling rats was also similar to that of adult rats (4) in being energydependent (as evident by the ability of metabolic inhibitors to inhibit biotin transport) and temperature-dependent (4). In summary, the present study shows that biotin transport undergoes clear changes with maturation. These include changes in the affinity and activity (and/or numbers) of the transport carrier, changes in the nonsaturable transport rate, and changes in the preferential site of biotin transport. Biotin transport, however, is similar in the suckling, weanling, and adult rats in being carrier-mediated and Naf-, energy-, and temperature-dependent.
GASTROENTEROLOGY Vol. 94,No. 1
References 1. Bonjour JB. Biotin in man’s nutrition
and therapy. Int J Vitam Nutr Res 1977;47:107-18. 2. Sweetman L, Nyhan WL. Inheritable biotin-treatable disorders and associated phenomena. Annu Rev Nutr 1986;6: 314-43. 3. Wolf B, Heard GS, Jefferson LG, Proud VK, Nance WI, Weissbecker KA. Clinical findings in four children with biotinidase deficiency detected through a state-wide neonatal screening program. N Engl J Med 1985;313:16-9. 4. Said HM, Redha R. A carrier-mediated system for transport of biotin in rat intestine in vitro. Am J Physiol 1987;252:G52-5. 5. Spencer RP, Brady K. Biotin transport by small intestine of rat, hamster and other species. Am J Physiol 1964;206:653-7. 6. Wilson TH, Wiseman G. The use of everted sacs of small intestine for the study of the transfer of substances from the mucosal to the serosal surface. J Physiol 1954;123:116-25. 7. Said HM, Ghishan FK, Murrell JE. Ontogenesis of intestinal transport of 5-methyltetrahydrofolate in the rat. Am J Physiol 1985;249:G567-71. 8. Younoszai MK, Komnick K. Maturation of the small intestine: absorption of L-valine in rats. Pediatr Res 1982;16:756-60. 9. Ghishan FK, Jenkins JT, Younoszai MK. Maturation of calcium transport in the rat small and large intestine. J Nutr 1980;110:1622-8. 10. Said HM, Ghishan FK, Greene HL, Hollander D. Developmental maturation of riboflavin intestinal transport in the rat. Pediatr Res 1985;19:1175-8. 11. Hennings SJ. Functional development of the gastrointestinal tract. In: Johnson LR, ed. Physiology of the gastrointestinal tract, 2nd ed. New York: Raven, 1987:282-300. 12. Koldovsky 0. In: Development of the Functions of the Small Intestine in Mammals and Man. Basel: Karger, 1969. 13. Barnard JA, Ghishan FK, Wilson FA. Ontogenesis of taurocholate transport by ileal brush border membrane vesicles. J Clin Invest 1985;75:869-73. 14. Said HM, Greene HL, Moore GC, Ghishan FK. Developmental maturation of o-glucose active transport system in rat intestine. Digestion 1987;36:195-200. 15. Little JM, Lester R. Ontogenesis of intestinal bile salt absorption in the neonatal rat. Am J Physiol 1980;239:G319-23. 16. Bamford DR, Ingham PA. Sugar absorption by fetal and neonatal rat intestine in vitro. J Physiol 1975;248:335-48. 17. Batt E, Schachter D. Developmental pattern of some intestinal transport mechanisms in newborn rats and mice. Am J Physiol 1969;216:1064-8. 18. Forstuer G, Garland G. The influence of hydrocortisone on the synthesis and turnover of microvillous membrane glycoproteins in suckling rat intestine. Can J Biochem 1976;54:224-30. 19. Schwarz SM, Hostether B, Ling S, Mone M, Watkins JB. Intestinal membrane lipid composition and fluidity during development in the rat. Am J Physiol 1985;248:G206-7. Na+ 20. Said HM, Redha R, Nylander W. A carrier-mediated, gradient-dependent transport for biotin in human intestinal brush border membrane vesicles. Am J Physiol 1987;253: G631-6. Received April 13, 1987. Accepted July 27,1987. Address requests for reprints to: Hamid M. Said, Vanderbilt University Medical Center, D-4113 MCN, Nashville, Tennessee 37232. This study was supported by National Institutes of Health grants BRSG RR 05424 and NIDDKD AM 26657.