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Article

Riboflavin Phosphorylation Is the Crucial Event in Riboflavin Transport by Isolated Rat Enterocytes¹

Giulia Gastaldi, Giuseppina Ferrari, Anna Verri, Donatella Casirola^*, Maria Novella Orsenigo and Umberto Laforenza²

Institute of Human Physiology, University of Pavia, 27100 Pavia, Italy; ^* Department of Pharmacology and Physiology, UMD, New Jersey Medical School, Newark, NJ 07103; and Department of General Physiology and Biochemistry, University of Milan, 23100 Milan, Italy

²To whom correspondence and reprint requests should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In isolated rat enterocytes, both normoenergized (normal) and de-energized with rotenone, riboflavin intracellular metabolic processes, operating in association with a membrane-specific transport mechanism, were investigated. The contents of unlabeled (endogenous) and labeled (exogenous) flavins [riboflavin (RF), flavin mononucleotide (FMN), flavin adenindinucleotide (FAD)] were determined by HPLC before and after incubation with tritiated RF . In normoenergized enterocytes, total labeled RF content (i.e., total uptake, the sum of RF membrane transport and intracellular metabolism) increased steadily to a plateau after 20 min incubation; FMN and FAD contents reached a plateau between 3 and 20 min, whereas free RF content increased constantly. The phosphorylated forms prevailed over the free form (60% of total flavins). In de-energized enterocytes, RF total uptake was significantly lower than in normoenergized enterocytes and reached a plateau after only 3 min incubation. FMN and FAD contents were significantly lower than in normoenergized enterocytes, and free RF represented the prevailing form of flavins (70% of total RF ). In both normoenergized and de-energized enterocytes, the contents of unlabeled total RF, FMN and FAD decreased significantly after 20 min incubation, whereas free RF increased significantly only in normoenergized enterocytes. After 20 min incubation, the RF structural analog 8-dimethyl-amino-8-demethyl-RF caused a significant decrease of all flavin contents, whereas 5'-deoxy-RF decreased only the total and free RF contents. Results directly confirmed the leading role of metabolic processes such as phosphorylation in RF transport by isolated small intestinal enterocytes.

KEY WORDS: • riboflavin • isolated enterocytes • intestinal absorption • riboflavin metabolism • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In several animal species, including humans, intestinal transport of physiologic concentrations of riboflavin (RF)³ is a carrier-mediated (saturable) process, associated with intracellular phosphorylation and dephosphorylation of RF (McCormick, 1989 , Yoshimine, 1984 ). During absorption, flavin mononucleotide (FMN) and flavin adenindinucleotide (FAD), the main sources of dietary RF, are rapidly hydrolyzed to free RF by nonspecific phosphatases of the small intestinal brush border membranes (Akiyama et al. 1982 , Daniel et al. 1983 ). Free RF, which is the only absorbable form (Yoshimine, 1984 ), after crossing the small intestinal brush border membrane (Casirola et al. 1993 , Daniel and Rehner, 1992 , Said and Arianas, 1991 , Said et al. 1993 ), is again transformed into the coenzymes FMN and FAD by cytoplasmic enzymes. RF intracellular metabolism is an energy-dependent event in which RF is transformed into FMN by flavokinase (ATP: RF-5'-phosphoryltransferase, EC 2.7.1.26) and FMN into FAD by FAD-synthetase (ATP: FMN adenylyltransferase, EC 2.7.7.2) (Bowers-Komro et al. 1989 , Kasai et al. 1990 , Nakano and McCormick 1991 , Yamada et al. 1990 ). In addition, aspecific phosphatases are involved in the intracellular dephosphorylation of flavin coenzymes to free RF (McCormick, 1989 ). Thus the RF formed can cross the basolateral membrane, completing the absorption process.

The possibility that RF uptake and RF intracellular metabolism (the so-called "metabolic trapping") are coupled through a phosphorylation process has been suggested by several authors (Aw et al. 1983 , Bowers-Komro and McCormick, 1987 , Bowman et al. 1989 , McCormick et al. 1987 ). However, a strict interdependence between RF metabolism and uptake has not been directly demonstrated in isolated cells.

Uptake and metabolism of RF were investigated in different isolated cells such as hepatocytes (Aw et al. 1983 , Joseph and McCormick, 1995 ), guinea pig enterocytes (Hegazy and Schwenk, 1983 ), Caco-2 human intestinal epithelial cells (Said and Ma, 1994 ) and HK-2 human renal proximal tubule epithelial cells (Kumar et al. 1998 ).

After incubation with physiologic concentrations (0.014–0.3 µmol/L) of labeled RF, 80% of RF taken up by intestinal and renal epithelial cells is found in a free form and only 20% as FMN and FAD (Hegazy and Schwenk 1983 , Kumar et al. 1998 , Said and Ma 1994 ). In everted small intestinal sacs (Yoshimine 1984 ), incubation with labeled RF causes time-dependent increases of tissue FMN content ranging from 50 to 80% of total RF, with the remainder as free RF. In isolated hepatocytes (Joseph and McCormick 1995 ), incubation with labeled RF induces time-dependent increases of free RF and FMN, whereas FAD is detected in small amounts only after 30 min incubation. Overall, the percentages of the labeled flavins found are 80% for free RF, 16% for FMN and 4% for FAD of total RF content (sum of RF and its coenzymatic forms) (Joseph and McCormick 1995 ).

The aim of this investigation was to study the time course of RF intracellular metabolism in isolated enterocytes; during RF intestinal absorption, the process operates in association with the membrane-specific transport characterized by high binding of RF to the microvillous membrane (Casirola et al. 1993 and 1994 ). As noted previously (Gastaldi et al. 1999 ), the use of two populations of isolated enterocytes, normal (normoenergized, NE) and rotenone-de-energized (DE) as a cellular model allows parallel study of membrane transport and intracellular metabolism. In both cells, the time course of the content of unlabeled (endogenous) and labeled (exogenous) flavins (RF, FMN, FAD) was determined by HPLC before and after incubation with a physiologic concentration of labeled RF. This allowed the study not only of RF uptake, but also of the stages of RF intracellular metabolism involving both phosphorylation and dephosphorylation. The simultaneous determination of the unlabeled flavins was used to highlight the features of the whole metabolic process. Use of DE allowed the effects of energy depletion on the initial (3 min) and long-term (10–20 min) events to be assessed (uptake and metabolism, respectively). In addition, in both NE and DE, the inhibitory effect of two RF structural analogs on the contents of labeled and unlabeled flavins after a long incubation time (20 min) was investigated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chemicals.

Labeled RF ([³H]-RF, with specific activity 37 GBq/mmol) was purchased from Moravek Biochemicals (Brea, CA). It was periodically analyzed by HPLC and found to be 95% pure. [¹⁴C]-carboxyl-dextran (specific activity, 30 MBq/mmol) was from DuPont NEN (Boston, MA). Unlabeled RF was obtained from Prodotti Roche (Milan, Italy) and unlabeled FMN and FAD from Flucka Chimica (Milan, Italy). The RF analogs 5'-deoxy-RF (5'-DORF) and 8-dimethyl-amino-8-demethyl-RF (roseoflavin, 8-ROF) were a generous gift, custom-synthesized by Kasai et al. (1988 and 1990) . All other chemicals were of analytical grade and supplied by Sigma Chemical (St Louis, MO) and by BDH (Poole, UK).

Animals.

Wistar albino rats of both sexes (300–400 g body weight) from our own colony were used throughout. They were fed a standard pellet diet containing 12 mg RF/kg diet (Randoin and Causeret 1947 ), supplied by Laboratorio Dottori Piccioni (Gessate, Italy). All rats were deprived of food for 12 h before the start of the experiments, with free access to water. Animal care was in accordance with NIH guidelines (NRC 1985).

Solutions.

Medium A contained the following (mmol/L): 96 NaCl, 1.5 KCl, 27 Na citrate, 0.2 phenyl-methyl-sulfonyl-fluoride, 5.6 K₂HPO₄/KH₂PO₄, pH 7.3. Medium B contained (mmol/L): 140 NaCl, 1.5 EDTA, 0.5 dithiothreitol, 0.2 phenyl-methyl-sulfonyl-fluoride, 16 K₂HPO₄/KH₂PO₄, pH 7.3; and Medium C contained (mmol/L): 137 NaCl, 5.2 KCl, 0.6 CaCl₂, 0.8 MgSO₄, 10 D-glucose, 5 glutamine, 0.2 phenyl-methyl-sulfonyl-fluoride, 3 K₂HPO₄/KH₂PO₄, pH 7.3.

Enterocyte preparation.

Enterocytes were isolated from the small intestine using the method of Rindi and Laforenza (1997) with minor modifications. In each experiment, one rat was killed by cervical dislocation. The entire excised small intestine was rinsed with oxygenated saline, then filled with and incubated in Medium A at 37°C for 10 min under oxygenation in a thermostatic shaker (90 oscillations/min). The intraluminal content was discarded and the intestine was both filled and incubated with Medium B as previously described. This treatment was repeated twice. The intestine was then gently fingered for 2 min and the intraluminal fluid, containing enterocytes, was filtered through 250- and 100-µm mesh nylon filters, successively. The filtrate was collected in 50 mL of Medium C; the isolated enterocytes were then washed three times with Medium C and centrifuged each time at 50 x g for 2 min in a Beckman TJ-6 centrifuge (TH-4 rotor; Beckman, Fullerton, CA). Cellular protein content was measured according to Lowry et al. (1951) with bovine serum albumin as a standard.

Preincubation.

Enterocytes were preincubated as follows: 4 mL of final enterocyte suspension (16 mg protein) was placed in plastic tubes and preincubated at 37°C in a thermostatic shaker (90 oscillations/min) for 10 min using Medium C for NE. Medium C containing 25 µmol/L rotenone was used for DE.

Incubation.

The procedures of incubation, homogenization and HPLC determination were performed under the light of a sodium lamp in a dark room to avoid photodecomposition of flavins.

Time course of total and single flavin contents.

For both NE and DE, incubation at 37°C was started by adding 40 µL of Medium C, containing 0.25 µmol/L (final concentration) of [³H]-RF. Incubation was stopped by adding 35 mL ice-cold Medium C.

Effect of structural analogs on equilibrium flavin contents.

Cell suspension (4 mL) was preincubated as described above. Incubation was started by adding 40 µL of Medium C containing the following: 0.25 µmol/L [³H]-RF (control) or 0.25 µmol/L [³H]-RF plus 2.5 µmol/L 5'-DORF or 8-ROF. The incubation time was 20 min.

Flavin contents.

Flavins were determined fluorometrically, immediately after cell preparation (initial contents), in both NE and DE after 10 min preincubation in Medium C ([³H]-RF free) as well as at varying incubation times in the presence of [³H]-RF. At each time, the contents of unlabeled flavins were calculated by subtracting labeled flavins, measured radiometrically, from unlabeled plus labeled flavins, measured fluorometrically. Total flavin content was the sum of RF, FMN and FAD.

Before and after incubation with 0.25 µmol/L [³H]-RF, the cells were quickly centrifuged by using a Beckman TJ-6 centrifuge (Beckman) at 480 x g for 1 min at 4°C, and the supernatants were discarded. The pellets were gently resuspended with 2.5 mL of ice-cold Medium C and centrifuged again as described above. The final pellets were homogenized with 1.5 mL of ice-cold 30% trichloroacetic acid (TCA) and centrifuged at 17000 x g for 5 min. The supernatants were separated and extracted with diethyl ether to eliminate TCA. As determined earlier with standard FAD solutions, TCA treatment did not cause any significant hydrolysis (data not shown). The samples were then lyophilized and stored at -20°C until used, when the lyophilized extracts were resuspended with 200 µL distilled water.

The content of a single flavin was determined by HPLC of TCA extract following the method of Kasai et al. (1990) , using a Varian model 5000 LC (Walnut Creek, CA). Enterocyte extract (20 µL) was injected into a reverse-phase column (Supelcosil LC-18, 5 µm, 4.6 x 150 mm, Supelco, Bellefonte, PA). The mobile phase, containing a 50 mmol/L phosphate buffer (pH 6.25), 0.5% pyridine and 9% ethanol (v/v), was freshly prepared, filtered through 0.45-µm filters and degassed under a vacuum. The flow rate was 1.4 mL/min. Fluorescence was measured with a spectrofluorometer (Perkin-Elmer LS-30, Beaconfield, UK) at the following wavelengths: _ex = 450 nm; _em = 530 nm. Before each set of analyses, a standard solution containing a mixture of RF, FMN and FAD in concentrations of 0.6, 0.45 and 2.5 µmol/L, respectively, was processed. Retention times of the three flavins were as follows (mean ± SEM, n = 128): 14.56 ± 0.1 min for RF, 5.25 ± 0.04 min for FMN and 4.53 ± 0.04 min for FAD. For each flavin, the peak areas were determined by a computing integrator (CDS 401 Varian, Walnut Creek, CA).

For the determination of labeled flavins, the chromatographic fractions were collected automatically and their radioactivity measured by a Packard Tri-Carb 2000 CA liquid scintillation counter (Packard Instruments, Downers Grove, IL). The final results were expressed as pmol/mg protein after correction for adherent water, which was evaluated in each experiment by using [¹⁴C]-carboxyl-dextran and separate flasks according to Wilson and Treanor (1975) .

Statistical methods.

All values given are means ± SEM of triplicate determinations for each of five different preparations of isolated enterocytes. The significance of the differences between the means at different incubation times and in inhibition experiments was evaluated by using ANOVA, followed by Newman-Keuls’s Q test. Student’s t test was utilized for the comparison between NE with DE. All statistical tests were carried out by using the computer program PRIMER, version 1 (Glantz 1988 ).

	RESULTS

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NE viability, metabolic state and transport efficiency were tested routinely as previously described (Gastaldi et al. 1999 ) and showed their suitability for transport studies. The addition of 25 µmol/L rotenone (DE) drastically reduced both ATP content and functional properties [see Ricci and Rindi (1992) ]. The amount of [³H]-RF present in adherent water was 2% of cell total radioactivity. Cell protein concentration was 4 ± 0.15 g/L cellular suspension (n = 6).

Unlabeled flavin contents.

Before preincubation, the main flavin of NE was FAD; its concentration (166.2 ± 7.4 pmol/mg protein) represented 86.5% of the total unlabeled RF content, whereas FMN and RF concentrations were 24.4 ± 1.2 (12.7%) and 1.63 ± 0.16 pmol/mg protein (0.85%), respectively. After 10 min preincubation, the flavin contents in NE were virtually unchanged and not different from those of DE.

Time course of labeled flavin contents.

In NE, the contents of total labeled flavins (RF total uptake is the sum of RF membrane transport and intracellular metabolism) and FAD increased rapidly up to 10 min incubation, reaching a steady state at 10–20 min. FMN reached a plateau between 3 and 20 min, whereas RF increased constantly even after 20 min incubation (Fig. 1 ). At 3, 10 and 20 min incubations, the FMN/FAD ratio decreased from 3 to 1 and to 0.7, respectively, suggesting a rapid transformation of FMN into FAD.

View larger version (18K): [in this window] [in a new window]   Figure 1. Time course of labeled total and single flavin (RF, riboflavin; FMN, flavin mononucleotide; FAD, flavin adenindinucleotide) contents in isolated rat enterocytes incubated with [3H]-RF. Values are means ± SEM, n = 5. When not shown, SEM was within the symbol area. [3H]-RF final concentration in the incubation medium was 0.25 µmol/L. For each curve, means with different letters are significantly different (P < 0.05; ANOVA followed by Newman-Keuls’s Q test). For the comparison between normal and de-energized enterocytes, means with lowercase letters are significantly lower (P < 0.001; Student’s t test).

Assuming a normal enterocyte water content of 3.7 µL/mg protein (Baur et al. 1975 ), from the data in Figure 1 , the approximate intracellular concentrations of free [³H]-RF as well as the ratios between intracellular and extracellular concentrations for [³H]-RF at each incubation time could be calculated. The concentration ratios were similar in both NE and DE, and their values were > 1 at each incubation time, with a range from 1.7 to 2.3. Because the ratios were > 1, enterocytes therefore seem to accumulate free RF both in the presence and absence of energy.

Time course of unlabeled flavin contents.

The time course of unlabeled total and single flavin content did not differ in NE and DE (Fig. 2 ). The content of total RF and phosphorylated compounds was virtually unmodified after 3 min incubation; then it decreased, showing significant reductions as follows: in NE, for total RF, FAD and FMN contents at 20 min vs. other incubation times; in DE, for total RF and FMN at 20 min vs. other times; and for FAD at 20 vs. 0 and 3 min (Fig. 2) . In NE, free RF content, after a significant decrease at 3–10 min incubation (disappearance from the enterocyte), regained its initial value at 20 min (dephosphorylation of FMN and FAD) (Fig. 2) . In DE, free RF content remained virtually unmodified. Moreover, in DE, no significant differences in the contents of all flavins were found compared with NE.

View larger version (18K): [in this window] [in a new window]   Figure 2. Time course of unlabeled total and single flavin (RF, riboflavin; FMN, flavin mononucleotide; FAD, flavin adenindinucleotide) contents in isolated rat enterocytes incubated with [3H]-RF. Values and [3H]-RF concentration as in Figure 1 . For each curve, means with different letters are significantly different (P < 0.05; ANOVA followed by Newman-Keuls’s Q test).

RF structural analogs and flavin contents.

In NE and DE, 5'-DORF significantly lowered the contents of total labeled flavins and labeled free RF, without modifying those of FMN and FAD (Fig. 3 ). Hence, 5'-DORF reduced only RF membrane uptake. In contrast, 8-ROF significantly lowered all labeled flavins in both types of cells. Moreover, 8-ROF was a more effective inhibitor than 5'-DORF, with significant differences in total and phosphorylated flavin contents observed (Fig. 3) .

View larger version (23K): [in this window] [in a new window]   Figure 3. Inhibitory effects of riboflavin structural analogs on labeled total and single flavin (RF, riboflavin; FMN, flavin mononucleotide; FAD, flavin adenindinucleotide) contents in isolated rat enterocytes at equilibrium (20 min). Normal and de-energized enterocytes were incubated at 37°C for 20 min in the presence of 0.25 µmol/L [3H]-RF (control) or 0.25 µmol/L [3H]-RF plus 2.5 µmol/L 5'-deoxy-riboflavin (5'-DORF) or 8-dimethyl-amino-8-demethyl-riboflavin (8-ROF, roseoflavin), respectively. Values are means ± SEM, n = 5. For each group of bars, means with different letters are significantly different (P < 0.02; ANOVA followed by Newman-Keuls’s Q test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The distribution of the flavins in NE was similar to that found in rat intestinal tissue and other organs by previous authors (Cooperman and Lopez, 1984 , Friedrich 1988 , Yagi 1954 ). In isolated cells, FAD was the main flavin, whereas FMN was present in small amounts with RF only in minute amounts.

In NE, labeled RF underwent three main events, i.e., membrane crossing, intracellular phosphorylation to FMN and conversion to FAD; thus, in 20 min, total labeled RF content (i.e., total RF uptake) increased steadily up to a plateau (Fig. 1) . As for single RF compounds, free RF increased constantly during 20 min incubation, whereas FMN and FAD reached a plateau at 3–20 min. As a whole, the phosphorylated forms of RF prevailed over the free form (60% of total flavins), indicating rapid intracellular transformation of RF. These results are similar to those reported by Yoshimine (1984) in intestinal tissue but quite different from those reported by previous authors in isolated cells (Hegazy and Schwenk 1983 , Joseph and McCormick 1994, Kumar et al. 1998 . Said and Ma 1994 ), who found that only 10–30% of total labeled RF taken up by the cells was transformed into FMN and FAD after 20–45 min incubation.

In DE, all three of the events indicated above occurred, but at different rates. Total labeled RF content (total uptake) was much lower and had already reached a plateau at 3 min. Free RF content was similar to that found in NE, and remained unchanged throughout the incubation. FMN was much lower than in NE, and FAD was almost absent. The lower concentration in total [³H]-flavin contents observed in DE compared with NE was due to a reduction in phosphorylated flavins. Energy depletion decreased the intracellular concentrations of FMN and FAD by 50 and 94% respectively. In DE, at all incubation times, RF was the prevailing flavin form (70% of total RF contents). These results confirm the dependence of RF total uptake on energy (Aw et al. 1983 , Hegazy and Schwenk 1983 , Kumar et al. 1998 , Said and Ma 1994 ) and the importance of RF phosphorylation for RF total uptake in isolated cells (metabolic trapping) (Aw et al. 1983 , Bowers-Komro and McCormick 1987 , Bowman et al. 1989 , McCormick et al. 1987 , McCormick 1989 ). Therefore, energy depletion greatly affected the intracellular transformation of RF but not its entry into the enterocyte, as observed previously (Gastaldi et al. 1999 ) (Fig. 1) . A rapid uptake followed by phosphorylation (metabolic trapping) has been reported for other water-soluble vitamins such as nicotinamide and thiamin in isolated enterocytes from chicks and rats (Ricci and Rindi 1992 , Schuette and Rose 1983 ) and pyridoxine (PN)·HCl and nicotinic acid in rat intestinal everted sacs (Middleton 1978 , Stein et al. 1994 ).

In NE and DE, unlabeled flavin coenzymes were largely dephosphorylated. At 20 min incubation, the contents of total flavins, FMN and FAD had decreased significantly (Fig. 2) . Only in NE was free RF significantly increased, possibly as a consequence of the dephosphorylation of flavin coenzymes which, at this time, reached their maximum (25 and 30% for FMN and FAD, respectively). Moreover, the fact that in NE the bulk of dephosphorylation was achieved during a steady state of labeled RF phosphorylation (metabolism) could indicate a relationship between phosphorylation and dephosphorylation (Figs. 1 , 2) .

The 8-ROF and 5'-DORF analogs decreased labeled flavin contents, after 20 min incubation, by inhibiting both RF uptake and metabolism (Fig. 3) . 8-ROF, which is known to inhibit the saturable component of RF uptake (Casirola et al. 1993 , Gastaldi et al. 1999 ) and to be a good substrate for flavokinase (Kasai et al. 1990 ), significantly lowered the content of total and single flavins both in NE and DE. It was therefore able to inhibit both the uptake process, which is energy independent, and the metabolism, which is energy dependent. In contrast, 5'-DORF, which is not a substrate for flavokinase (Kasai at al. 1990 ) and significantly inhibits the saturable component of RF uptake only in NE (Gastaldi et al. 1999 ) but not in brush border membrane vesicles (Casirola et al. 1993 ), significantly lowered the contents of total RF and then only of free RF, in both NE and DE. These results are in keeping with previous observations of Gastaldi et al. (1999) and suggest that the inhibitory effect of 5'-DORF on RF uptake could be due to its binding to intracellular proteins or basolateral membrane.

Previously, Gastaldi et al. (1999) had maintained that, over a short time period (3 min), RF transport is primarily an energy-independent membrane event, whereas over long time periods (10 and 20 min), it is an intracellular energy-dependent event related to RF metabolism. The present results confirm the energy dependence over long time periods; in addition, they show that over a short time period, in NE, both of the processes (RF uptake and metabolism) involved in RF transport were active because energy depletion significantly decreased phosphorylation of labeled RF to FMN and FAD.

Furthermore, our results show that FAD synthesis is not simply an isotope exchange because the incorporation of [³H]-label from [³H]-RF into FAD was not instantaneous, but that a significant amount of [³H]-label was first present as FMN.

Finally, we found that NE accumulated labeled RF in both phosphorylated and free forms, whereas DE did so almost exclusively in free form. This indicates that RF intracellular accumulation involves two processes, i.e., an energy-dependent process, present only in NE, and an energy-independent process present in both types of cells. The apparent accumulation of RF in enterocytes, observed even under a condition of energy depletion, may account at least in part for the amount of [³H]-RF bound to enterocyte structures, particularly in brush border membranes (Casirola et al. 1993 ). In addition, these authors hypothesized that the specific RF binding sites in the cellular membrane are protein in nature (Casirola et al. 1994 ). At present, RF binding has been reported in rat intestine in vivo, in intestinal brush border membrane vesicles [Elbert 1987 , quoted by Feder et al. (1991) ] and in human placental microvillous membrane (Moe et al. 1994 ). Moreover the presence of proteins involved in RF binding was suggested by Aw et al. (1983) and demonstrated by Nokubo et al. (1989) in liver cell plasma membranes.

In conclusion, these results show that in isolated rat enterocytes as well, RF phosphorylation is the limiting step of total RF uptake. Because both RF uptake and dephosphorylation depend on RF phosphorylation, phosphorylation must be considered a crucial event in RF intestinal absorption.

	ACKNOWLEDGMENTS

 
We wish to express our gratitude to G. Rindi for his invaluable help and useful criticism in the development of this research and in the drafting of the manuscript. The authors also thank S. Kasai, Osaka City University (Japan), for his invaluable advice.

	FOOTNOTES

 
¹ Preliminary data appeared in abstract form [Gastaldi, G., Laforenza, U. & Ferrari, G. (1994) Riboflavin metabolism in rat isolated enterocytes. Pfluegers Arch. 428: R 7 (abs.)].

³ Abbreviations used: DE, rotenone-de-energized enterocytes; 5'-DORF, 5'-deoxy-riboflavin; FAD, flavin adenindinucleotide; FMN, flavin mononucleotide; NE, normoenergized enterocytes; RF, riboflavin; 8-ROF, 8-dimethyl-amino-8-demethyl-riboflavin (roseoflavin); TCA, trichloroacetic acid.

Manuscript received March 8, 2000. Initial review completed April 4, 2000. Revision accepted June 1, 2000.

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