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Previous Article | Next Article 
Molecular and Cellular Biology, February 2007, p. 1247-1253, Vol. 27, No. 4
0270-7306/07/$08.00+0 doi:10.1128/MCB.01621-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Johan W. Jonker,1,
Hilde Rosing,2
Jos H. Beijnen,2 and
Alfred H. Schinkel1*
Division of Experimental Therapy, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands,1 Department of Pharmacy and Pharmacology, Slotervaart Hospital, 1066 EC Amsterdam, The Netherlands2
Received 31 August 2006/ Returned for modification 13 November 2006/ Accepted 27 November 2006
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ABSTRACT |
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INTRODUCTION |
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One of the important vitamins occurring in milk is riboflavin (vitamin B2). Milk is a primary dietary source of riboflavin, and vitamin B2 deficiency is often endemic in human populations that subsist on diets poor in dairy products and meat (17). Riboflavin is converted to the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which participate as electron carriers in many key enzymatic redox reactions in the body (Fig. 1) (17). Riboflavin cannot be synthesized by mammals, and there is a only limited, short-term storage capacity for this vitamin in the liver (8, 17). Humans are vulnerable to develop a vitamin B2 deficiency during periods of dietary deprivation or stress, and this may lead to a variety of clinical abnormalities, including growth retardation, anemia, skin lesions, and degenerative changes in the nervous system (7). Therefore, this water-soluble vitamin should be present in the diet on a daily basis.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals. [3H]riboflavin (20 Ci mmol–1), riboflavin, FMN, and FAD were from Sigma Chemical Co. (St. Louis, MO). D-[carbonyl-14C]biotin (58.0 mCi mmol–1) was from Amersham Biosciences (Little Chalfont, Buckinghamshire, United Kingdom). Methoxyflurane (Metofane) was from Medical Developments Australia Pty. Ltd. (Springvale, Victoria, Australia).
In vitro transport assays. Transwell transport assays were carried out as previously described (18). Vectorial transport of [3H]riboflavin (10 nM and 500 nM, in addition to an estimated 200 nM in Optimem culture medium) across the monolayer was determined, either with or without the Bcrp1 inhibitor Ko143 (1). Vesicular transport assays were performed as described at pH 7.4 (4), using vesicles from Sf9 cells infected with human BCRP-expressing or wild-type baculovirus in the presence or absence of 4 mM ATP.
Western analysis. Milk retrieved from wild-type or Bcrp1–/– mice was loaded on a polyacrylamide gel in dilution series. Blots were probed with horseradish peroxidase-labeled antibody against murine immunoglobulin G (IgG) or IgA (sc-2005 and sc-3791; Santa Cruz Laboratories, CA). Equal loading across the lanes was confirmed with total protein staining (Ponceau S and India ink).
Hematological analysis of plasma. Standard hematological analyses on plasma were performed on a Beckman Coulter Ac T diff analyzer (Beckman Coulter, Mijdrecht, The Netherlands) to determine white and red blood cell counts, hemoglobin, hematocrit, and platelets.
Pharmacokinetic experiments.
For the determination of radiolabeled riboflavin or biotin in plasma, milk, and organs, riboflavin or biotin was administered by intravenous (i.v.) injection into the tail veins of mice lightly anesthetized with methoxyflurane. Riboflavin and biotin were dissolved in phosphate-buffered saline. Plasma of adult males, virgin females, lactating females, and pups for measurement of endogenous levels of riboflavin, FMN and FAD, or radiolabeled riboflavin or biotin was obtained by cardiac puncture under methoxyflurane anesthesia. Mice were sacrificed by cervical dislocation or, in the case of pups, decapitation. Organs were removed and solubilized in Solvable (Perkin-Elmer, MA). Intestinal contents were separated from intestinal tissue. Milk was collected from lactating wild-type or Bcrp1–/– females with pups of 
10 days old. Approximately 10 min prior to milk sampling, mice received oxytocin (200 µl of a 1-IU/ml solution) subcutaneously to stimulate milk secretion. Milk samples were collected bilaterally from all mammary glands by gentle vacuum suction, and blood samples were taken by cardiac puncture under methoxyflurane anesthesia. Levels of radioactivity in plasma, milk, feces, and tissue homogenates were determined by liquid scintillation counting. Endogenous levels of riboflavin, FMN, and FAD were quantified by high-pressure liquid chromatography (HPLC).
HPLC analysis of riboflavin, FMN, and FAD. Riboflavin, FMN, and FAD were determined as described previously (22), with modifications. Samples were protected from light. Methanol (four times the sample volume) was added to plasma and milk samples to precipitate proteins. After mixing, samples were centrifuged for 10 min at 10,500 x g. Supernatants were evaporated to dryness at 40°C under a stream of nitrogen. Samples were reconstituted in 100 µl of methanol-50 mM ammonium acetate (pH 5) (30:70, vol/vol), and 25-µl aliquots were injected into the HPLC system. The chromatographic system consisted of a Perkin-Elmer 200 series pump and ISS 200 autosampler (Perkin-Elmer, Norwalk, CT). Chromatographic separation was performed on a Zorbax SB C18 column (150 by 4.6 mm [inside diameter]; 3.5-µm particle size) (Rockland Technologies Inc., Newport, DE). The mobile phase consisted of a mixture of 50 mM ammonium acetate (pH 5) and methanol, and the following gradient was used: 0 to 5 min with 30% to 90% methanol, 5 to 7 min with 90% methanol, 7 to 8 min with 90% to 30% methanol, and 8 to 12 min with 30% methanol. The flow rate was 1.0 ml/min, and the detection was performed fluorimetrically using a FP920 Intelligent fluorescence detector (Jasco International Co. Ltd., Tokyo, Japan) with excitation and emission wavelengths set at 372 and 520 nm, respectively, and a 40-nm bandwidth. The capacity of the flow cell of the fluorescence detector was 16 µl. Riboflavin, FMN, and FAD eluted after approximately 5.3, 3.9, and 2.5 min, respectively. Processed samples were stable in the autosampler for at least 24 h. Calibration concentrations were between 0.001 and 10 µg/ml. Calibration curves were calculated by least-squares linear regression using a weighting factor of the reciprocal of the concentration. The lower limit of quantification was 0.01 µg/ml.
Measurements of vitamins in milk. Milk samples retrieved from wild-type or Bcrp1–/– lactating dams were pooled (n = 3), and the vitamins listed in Table 2 were measured (by TNO, Zeist, The Netherlands) by HPLC (vitamins A, B1, B6, C, E, and K1), competitive protein binding (vitamin B12), or microbiological turbidity assay (vitamin B9 and biotin). Biotin was measured in separate milk samples of wild-type and Bcrp1–/– mice (n = 3).
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RESULTS |
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Bcrp1 secretes [3H]riboflavin into milk. To investigate the role of Bcrp1 in the secretion of riboflavin into milk, we administered a trace amount of [3H]riboflavin (1.67 nmol/kg) i.v. to lactating wild-type and Bcrp1–/– mice and collected blood and milk at 30 min after administration. We observed a 13-fold lower concentration of [3H]riboflavin in milk from Bcrp1–/– versus wild-type mice (Fig. 2A). After correction for plasma [3H]riboflavin concentrations, which were 1.7-fold lower in wild-type than in Bcrp1–/– mice, we observed a 22-fold difference in the milk-to-plasma ratio of [3H]riboflavin (Fig. 2A). These data indicate that Bcrp1 plays a substantial role in the secretion of riboflavin into milk.
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The [3H]riboflavin tissue distribution was determined at 30 min after i.v. administration (1.67 nmol/kg). There were 1.8-fold-higher plasma and liver [3H]riboflavin levels in Bcrp1–/– than in wild-type mice (Table 1). Levels of [3H]riboflavin in most other organs were similarly increased. The percentage of the dose of [3H]riboflavin retrieved from the contents of small intestine was 6.2-fold lower in Bcrp1–/– than in wild-type mice (Fig. 2C), indicating substantial Bcrp1-mediated hepatobiliary or direct intestinal excretion of [3H]riboflavin into the lumen of this organ. The same appeared to occur in colon (Fig. 2C). Bcrp1 thus has a marked influence on riboflavin availability in plasma and tissues and on its excretion into the gut lumen.
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0.05 nmol per mouse) is comparatively low, as substantially more endogenous riboflavin is present in the blood compartment alone (in male wild-type mice, 0.2 nmol). At 30 min after a considerably higher i.v. dose (664 nmol/kg, or 20 nmol per mouse [i.e., a 100-fold excess over the blood riboflavin pool]), there was again a twofold-higher plasma [3H]riboflavin level in Bcrp1–/– than in wild-type mice (72 ± 8.8 versus 37 ± 8.5 nM; P < 0.01), and liver accumulation of [3H]riboflavin was 1.5-fold higher in Bcrp1–/– than in wild-type mice (9.5% ± 0.5 versus 6.2% ± 1.9; P < 0.05). Bcrp1 thus has a marked effect on riboflavin pharmacokinetics at both low and high exposure levels. Riboflavin transport by murine Bcrp1 and human BCRP in vitro. Riboflavin transport by Bcrp1 in vitro was tested using the polarized canine kidney cell line MDCK-II and a subclone transduced with murine Bcrp1 cDNA. Cell lines were grown to confluent polarized monolayers on porous membrane filters, and vectorial transport of [3H]riboflavin across the monolayer was determined. Parental MDCK-II cells displayed a high basolaterally directed translocation of [3H]riboflavin, whereas apically directed translocation was very low, indicating the presence of an active transepithelial absorptive riboflavin transport process (Fig. 3A). This is in concordance with the accumulation of riboflavin in basolateral spaces and fluid-filled "domes" of MDCK-II cells (15) and may reflect the mechanism(s) responsible for reabsorption of riboflavin in the kidney and possibly in the intestine (9, 20). Bcrp1 is apically located and should transport its substrates to the apical side of the monolayer, possibly counteracting the endogenous absorptive process (19). Indeed, in Bcrp1-transduced MDCK-II cells the basolaterally directed translocation was markedly decreased (5.2-fold) and the apically directed translocation was increased (3.3-fold), resulting in net apical transport of riboflavin (Fig. 3B). Similar results were obtained with 10 or 500 nM riboflavin. The apical riboflavin transport by Bcrp1 was completely inhibited by Ko143, a selective Bcrp1 inhibitor (Fig. 3C and D) (1). Qualitatively similar results were obtained with MDCK-II cells expressing human BCRP (not shown). MDCK-II or polarized pig kidney LLC-PK1 cells expressing human multidrug resistance protein 2, human MDR1, or murine Mdr1a did not display apically directed transport of riboflavin (not shown).
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Endogenous plasma riboflavin levels display Bcrp1-dependent gender-specific differences. Endogenous riboflavin and its cofactors FMN and FAD were measured by HPLC in plasma from adult wild-type and Bcrp1–/– mice fed a standard chow, which contains 12 to 14 mg/kg riboflavin (Fig. 4). Both sexes were tested, as males have higher hepatic levels of Bcrp1 than females, resulting in gender-specific pharmacokinetic differences (16). Whereas FMN levels did not differ between wild-type and Bcrp1–/– mice or between sexes, riboflavin levels in male Bcrp1–/– mice were 1.5-fold higher than those in male wild-type mice. In contrast, females did not display differences in endogenous plasma riboflavin levels, and these also were not different from the levels in Bcrp1–/– males (Fig. 4). Endogenous plasma riboflavin levels thus display Bcrp1-dependent gender-specific differences. Differences in FAD levels between wild-type and Bcrp1–/– mice were minor in both sexes (Fig. 4).
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6,000 nM riboflavin (Bcrp1–/–, 96 nM), on 6-mg/kg and 1-mg/kg riboflavin diets this was decreased to 416 ± 60 and 20 ± 27 nM, respectively. Maternal intake of riboflavin thus has a profound effect on Bcrp1-dependent riboflavin secretion into milk. We investigated whether oral supplementation of riboflavin to the lactating mother could increase the riboflavin content of the milk. Indeed, a single oral dose of riboflavin (1 mg/kg, equivalent to the daily intake on standard chow) given to dams which had been on chow without added riboflavin for 5 days partly restored milk riboflavin secretion 1 hour after administration, and this was Bcrp1 dependent (Fig. 5B). The milk riboflavin concentration after supplementation in these dams was still far lower than that in dams on standard chow (containing 12 to 14 mg/kg riboflavin), probably due to the depleted riboflavin state of the dams on deficient chow (compare Fig. 5A and B).
Riboflavin equivalents in pups from Bcrp1–/– mothers are due to cofactor FAD in milk. To assess the consequences of riboflavin deficiency in milk for pups, we analyzed the riboflavin status of Bcrp1+/– pups nurtured by wild-type and Bcrp1–/– mothers. This avoids confounding effects from different handling of riboflavin by Bcrp1+/+ and Bcrp1–/– pups. Due to the almost complete absence of riboflavin in the milk of Bcrp1–/– lactating dams, plasma riboflavin levels in 7- to 12-day-old exclusively milk-fed pups with Bcrp1–/– dams were 1.6-fold decreased compared to those in pups with wild-type dams (84 ± 19 versus 134 ± 27 nM; P < 0.01 [dams were on standard chow]). This is a very modest reduction considering the 63-fold difference in milk riboflavin secretion (Fig. 5A and C). Moreover, despite negligible amounts of riboflavin in Bcrp1–/– milk, pups nurtured by Bcrp1–/– dams thrive (12, 19), contradicting a severe riboflavin shortage.
Riboflavin can also be derived from dietary FMN and FAD, which are broken down in the intestinal lumen to riboflavin before absorption (8). When we tested milk from mothers on standard chow, FMN levels were quite low and ca. sixfold reduced in Bcrp1–/– milk, but FAD levels were substantial and unchanged in Bcrp1–/– milk (Fig. 5A). Assuming complete conversion of FMN and FAD to riboflavin, Bcrp1–/– milk still contained 26% of riboflavin equivalents compared to wild-type milk, which is apparently more than enough to salvage riboflavin delivery to the pups. Indeed, in 7- to 12-day-old milk-fed pups (Bcrp1+/–) nurtured by wild-type or Bcrp1–/– dams, the riboflavin derivative FAD was present in comparable amounts in plasma (Fig. 5C). This suggests that there is sufficient riboflavin equivalent availability in pups drinking milk from Bcrp1–/– dams on standard chow.
When the transfer of total vitamin B2 equivalents (riboflavin, FAD, and FMN) was limited in milk of wild-type and Bcrp1–/– lactating mothers by replacing their standard chow with a diet without riboflavin supplementation, all pups suffered from vitamin B2 deficiency (lack of growth, fatty degeneration of liver, and severe anemia) (not shown). Intraperitoneal supplementation of riboflavin to the mothers salvaged these deficiencies in the suckling pups, however, without any obvious difference between pups with wild-type or Bcrp1–/– mothers. This is presumably due to the conversion to and subsequent secretion of FAD into milk of the wild-type and Bcrp1–/– mothers (not shown). Clearly, there are at least two independent pathways involved in secretion of vitamin B2 equivalents (riboflavin, FMN, and FAD) into milk. We note that the secretion of FAD into milk is likely also active, as the milk-to-plasma ratio is 16 ± 5.2, indicating a concentrative process.
Bcrp1 deficiency reduces biotin (vitamin H) levels in milk. As Bcrp1 is a broad-specificity transporter, riboflavin might not be the only vitamin transported into milk by Bcrp1. Indeed, when we tested a range of other vitamins in pooled wild-type versus Bcrp1–/– milk samples, we observed a threefold-lower level of biotin (vitamin H) in Bcrp1–/– milk. None of the other vitamins tested was substantially lower in Bcrp1–/– milk (Table 2). Analysis of separate milk samples (n = 3) revealed a 3.5-fold-lower concentration of biotin in milk of Bcrp1–/– compared to wild-type mice (47 ± 24 versus 163 ± 32 µg/100 ml; P < 0.01). Additionally, when we administered [14C]biotin (690 nmol/kg) i.v. to lactating wild-type and Bcrp1–/– mice and collected blood and milk at 30 min after administration, we observed a 2.3-fold-lower concentration of [14C]biotin in milk from Bcrp1–/– versus wild-type mice (322 ± 73 versus 779 ± 246 nM; P < 0.05). When corrected for plasma [14C]biotin concentrations, which were 1.2-fold higher in Bcrp1–/– than in wild-type mice, we observed a 2.6-fold difference in the milk-to-plasma ratio of [14C]biotin (2.2 ± 1.0 versus 5.8 ± 1.8; P < 0.05). This indicates that in addition to reducing riboflavin levels, Bcrp1 deficiency also reduces biotin levels in milk, albeit not as profoundly.
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DISCUSSION |
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BCRP has broad substrate specificity and actively extrudes a wide variety of drugs, carcinogens, and dietary toxins from cells. Due to its expression in intestine, liver, placenta, and blood-brain barrier, it has an important role in protection of the organism from these noxious compounds (5, 19). Surprisingly, rather than evolving a dedicated transporter, the mammary gland has recruited this broad-specificity transporter to pump riboflavin into milk. The tissue distribution of BCRP/Bcrp1 combined with the dual utilization of BCRP/Bcrp1 as a vitamin and a xenotoxin transporter potentially compromises essential biological functions of the respective organs in which it is expressed, i.e., in the breast, production of safe, xenotoxin-free milk and in the intestine, liver, placenta, brain, etc., efficient systemic and tissue uptake of dietary riboflavin. In spite of this paradox, the dual utilization of BCRP/Bcrp1 is evolutionarily conserved, at least between humans and mice, but presumably also in cows and other mammals (13). Riboflavin must be provided in the diet of mammals or synthesized by bacteria present in the intestinal lumen. In either case it must be absorbed from the intestine. We demonstrated that Bcrp1 can reduce the systemic and tissue levels of riboflavin in mice, but the overall effects were modest. Apparently, the efficient riboflavin uptake systems present in the intestine and in other blood-tissue barriers and cells, combined with subsequent metabolic trapping of riboflavin by conversion to FAD, are sufficiently effective to offset the riboflavin extrusion capacity of BCRP/Bcrp1 and thus avoid overall vitamin B2 deficiency.
Vitamin B2 is essential for many key enzymatic reactions in the body. However, despite negligible amounts of riboflavin in Bcrp1–/– milk, pups nurtured by Bcrp1–/– dams did not show any signs of malnourishment or growth retardation (13, 19; this study), contradicting a severe vitamin B2 shortage. Indeed, although pup plasma riboflavin levels were 1.6-fold decreased in Bcrp1–/– versus wild-type milk (Fig. 5), plasma levels of the cofactor FAD did not differ. This indicates that under the favorable conditions of our animal facility, with an ad libitum supply of food containing ample riboflavin for nursing dams, pups of Bcrp1–/– dams still received sufficient vitamin B2 equivalents. Clearly, in addition to the transport of riboflavin by BCRP, there is at least one other mechanism to deliver vitamin B2 equivalents to the young, in the form of FAD. We do not know the nature of the mechanism(s) responsible for FAD secretion into milk. However, we hypothesize that this redundancy may be important to ensure vitamin B2 supply to pups under different environmental circumstances, as there may be conditions where either FAD or riboflavin secretion into milk is compromised. We showed earlier that a BCRP inhibitor could nearly abrogate Bcrp1-mediated secretion into milk (13), and other conditions might affect the FAD secretion process. Under more natural conditions than in a mouse facility, an erratic food supply of variable content for nursing dams will be common. Moreover, (intestinal) infections and other stresses for the pups may result in reduced dietary intake or intestinal uptake capacity for riboflavin or in increased physiological riboflavin demand. Under such circumstances, the ability to secrete large amounts of vitamin B2 equivalents into milk and the possibility of utilizing several independent secretion mechanisms may be essential for survival of the pups.
Suboptimal BCRP activity may also be caused by genetic polymorphisms. In humans, BCRP activity can vary extensively due to known genetic polymorphisms (3, 11, 21) or due to intentional or coincidental use of drugs that inhibit BCRP (5). This might compromise riboflavin secretion into milk and increase dependence on the alternative FAD secretion pathway. It will be of interest to test whether reduced BCRP activity in humans also diminishes riboflavin secretion into human milk. In Holstein cattle, a correlation was recently found between a polymorphism in BCRP (Y581S) and modestly altered fat and protein contents and quantity of milk production (6). Whether the activity or proper membrane localization of BCRP is affected by this amino acid substitution remains to be determined, but it will be interesting to test whether this polymorphism affects riboflavin transfer into milk.
We considered the possibility that riboflavin secretion into milk might not be a primary function of BCRP in the mammary gland, as there was no immediate deficiency seen in the pups. However, if secretion of other vitamins or nutrients (in addition to the ones we tested) was the primary function of BCRP in the mammary gland, the question still arises of why there is no obvious deficiency phenotype in pups. An entirely different hypothesis proposes that xenotoxin secretion by BCRP into milk might be biologically useful. By supplying milk with modest amounts of xenotoxins to which the mother is exposed, BCRP could help start the induction of appropriate xenotoxin detoxification proteins in the pups during lactation. As a consequence, by the time that the pups have to switch from milk to solid food (likely identical to the food ingested by the mother), they are already prepared for exposure to the xenotoxins present in this food. However, while we consider this a hypothesis worth testing in the future, it still would not explain why riboflavin transport is evolutionarily conserved in BCRP. If xenotoxin transport was the only relevant function of BCRP, mice (and humans) would be better off with a BCRP that does not transport riboflavin, thus avoiding the complications of reduced systemic riboflavin availability. Of course, it is possible that both functions of BCRP in the mammary gland are biologically important, which would resolve most of the paradox in our findings.
The transport of riboflavin by BCRP/Bcrp1 can explain earlier findings. For instance, the apically directed transport of riboflavin observed in Caco-2 cells and rat ileum (9) could be explained by the expression of Bcrp1 at the apical membrane in small and large intestinal epithelial cells; we demonstrated a more-than-sixfold reduction in excretion of riboflavin into the intestinal lumen due to the absence of Bcrp1 in mice. Yanagawa et al. (20) observed that riboflavin can be secreted by proximal tubules in the kidney, and this might be explained by the apical expression of Bcrp1 in this organ (12). Similarly, the observed competition for transport in mouse leukemia cells between the anticancer drug methotrexate and riboflavin, both Bcrp1 substrates, could be due to Bcrp1 expression in these cells (14).
We note that efficient riboflavin extrusion by BCRP and Bcrp1 might cause problems during cell culture of highly BCRP-expressing cells, since cells need riboflavin for survival. Analogous to the situation observed earlier with the BCRP substrate folate (10), high BCRP expression might result in the need for higher vitamin (folate or riboflavin) concentrations in the medium for optimal cell growth.
While this study establishes the principle that an ABC transporter can actively concentrate a vitamin into milk, it gives rise to many questions for future studies. For instance, it will be of interest to investigate what mechanism is responsible for the active secretion of FAD into milk and to establish what the respective roles of the free riboflavin and FAD secretion processes are in supplying the infant with vitamin B2 equivalents. It will also be interesting to establish whether other vitamins and nutrients are also transported into milk by ABC transporters.
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ACKNOWLEDGMENTS |
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This work was supported by the Dutch Cancer Society.
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FOOTNOTES |
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Published ahead of print on 4 December 2006.
Present address: Department of Physiology, Faculty of Veterinary Medicine, University of Leon, Campus de Vegazana s/n, 24071 León, Spain.
Present address: The Salk Institute for Biological Studies, Howard Hughes Medical Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037.
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, translocation from the basolateral to the apical compartment; •, translocation from the apical to the basolateral compartment. Error bars (often smaller than the symbols) indicate SD. (E) BCRP-mediated uptake of [3H]riboflavin (0.25 µM) in membrane vesicles from Sf9 cells infected with BCRP-expressing or wild-type baculovirus. Data represent the mean uptake ± standard error at various time points (n = 6 to 9; **, P < 0.01). Uptake of [3H]riboflavin in Sf9-BCRP vesicles in the presence of ATP was significantly higher than uptake in the absence of ATP in these vesicles or uptake in Sf9-wild-type vesicles in the presence or absence of ATP, at all time points (P < 0.01).
