in the Rat1 ,2
J. A. TILLOTSON ANDM. S. KARCZ, JR. Department of Nutrition, Letterman Army Institute of Research, Presidio of San Francisco, California 94129 ABSTRACT Male rats were fed a riboflavin-deficient diet for 25 days and then fed [2-14C]riboflavin to replete their riboflavin pool(s). During a second depletion period, urine and feces were collected and 14Cexcretion monitored. The urine was chromatographed on R-15 resorcinol resin and 14C was measured in all fractions. The 14C compounds in the individual fractions were shown to be different by thin layer chromatography. The amount of 14C recovered as riboflavin increased after acid hydrolysis of the urine indicating the presence of riboflavin in conjugated forms. All R-15 fractions were found to support growth in a microbiological (L. casei) assay for riboflavin. This suggested that other derivatives, in addition to riboflavin and flavin nucleotides, are biologically active. However, the conjugated compounds were apparently less active, since acid hydrolysis of the urine enhanced its growth-promoting ability of L. casei. One 14C metabolite was identified as urea, thus providing evidence for degradation of the riboflavin molecule. This study has shown that the metabolic fates of riboflavin in the rat include conjugation and extensive degradation. J. Nutr. 107: 1269-1276,1977. INDEXING rats
KEY WORDS
riboflavin
The metabolic fate of riboflavin in the mammalian system is not clearly under stood. Although it is generally believed that the vitamin is not significantly de graded in mammals (1, 2), several groups have provided evidence to the contrary. Christensen (3) isolated five unidentified urinary 14C-riboflavin metabolites from normal rats fed excess riboflavin as one dose. Only one metabolite was fluorescent and none were conjugated. The 14Clabeled compounds were not stored in the tissues. Shen 3 isolated two fluorescent me tabolites from rat urine and tentatively identified them as flavinyl glucosides. Flavin glucosides have been isolated from rat (4) and cat (5) liver. Faulker and Lambooy (6) fed normal rats [2-14C]ribo flavin and recovered more 14C than could be accounted for by microbiological assay for flavins. Fazekas et al. (7) reported urinary compounds which were bound by riboflavin binding protein in chicken egg
riboflavin metabolites
whites which did not support growth of Tetrahymena pyriformis. Bessey et al. (8) observed that riboflavin fed in excess of 50 mg to rats could not be recovered as ribo flavin in urine. Owen et al. (9) identified a riboflavin metabolite in goat urine which had the properties of lumiflavin, but pos sessed no biological activity when assayed with L. casei. Riboflavin metabolism has usually been investigated in animals which were given excess riboflavin. Riboflavin in excess of Received for publication February 6, 1976. 1The opinions or assertion«contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. *In the conducting the research In this for re port, investigators adhered described to the "Guide Laboratory Animal Facilities and Care" as promul gated by the committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Labora tory Animal Resources, National Academy of Sciences —National Research Council. a Shen, Y. : M.S. thesis, 1967. Riboflavin metabolites in rat urine. Iowa State University.
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Urinary Riboflavin Metabolites
1270
J. A. TILLOTSON
AND M. S. KARCZ, JR.
ductase (EGR) (10). On day 26, the rats were placed in individual metal metab olism cages away from direct light. Each rat was fed orally twice daily, for 8 days, 100 ¿u.! of aqueous solution containing 40 fig of riboflavin and [2-14CJriboflavin (10). During the repletion period, each rat re ceived a total of 6.0 /¿Ci[2-14C]riboflavin. On the 8th day of the repletion period, the AC of the EGR of individual rats was in the normal range, and it was assumed that the body pools were saturated in respect to riboflavin (10). The rats were tailcupped to prevent coprophagy throughout the 2nd depletion period and fed the ribo flavin free diet for 10 days. The rats were killed with an overdose of ether on the llth day of the 2nd depletion period. The MATERIALS AND METHODS tissues were excised immediately, frozen Equipment and reagents. All reagents on dry ice and stored at minus 70°until were analytical reagent grade. The [2-14C]- analyzed. riboflavin4 had a specific activity of 23.4 Urine and feces. The urine was collected mCi/mMole and was >9Q% riboflavin. daily in brown bottles containing 0.5 ml Radioactivity in column effluents was mon l N HC1 during the repletion and 2nd de itored with a scintillation spectrophotompletion period. The urine volumes were eter 5 with a 1 ml anthracene flow cell. Fif measured daily, aliquots counted for 14C teen ml of scintillation fluid °was added to activity and samples frozen until analyzed. each 1 ml sample and the samples were The lower half of each cage was rinsed counted in a liquid scintillation spectro- daily with water, an aliquot of the wash photometerT with an absolute activity ings counted for 14Cand the washings dis analyzer. Fecal samples were oxidized.8 carded. The total urinary 14Cwas the com bined 14C in the urine and cage washings. Precautions were taken to prevent photo chemical reactions during all procedures The feces were collected during the re by protecting the samples from light. pletion and 2nd depletion period, and Animals. Twelve growing male rats9 stored frozen. At the end of the study pe (50 to 60 g) (Group 1) and 11 adult male riod, the feces were lyophilized, weighed, rats (200-210 g) (Group 2) were fed a mixed, and aliquot weighed, oxidized, and riboflavin free diet10 ad libitum for 25 the 14C determined. The urine of four rats days. Partial riboflavin deficiency of the from each group was combined for days rats was ascertained by weight loss and 2, 3, and 4 (samples 1 and 3) and days 8, significant increase of the activity coeffi 9, and 10 (samples 2 and 4) of the 2nd cient (AC) of erythrocyte glutathione re- depletion period for Chromatographie sep aration (table 1). TABLE 1 A portion of each of three urine sam Percentage of urinary ltC released as CO¡from the ples was made 0.2 N in respect to HC1 and urine samples incubated with urease1
sample11 Urine
» Amersham/Searle, Arlington Heights, Illinois. .Model 338, Downers Grove, 8Bio Sol 3 In toluene, Beckman Instruments, Fullerton, California. 7Packard Instruments, Model 544, Downers Grove, Illinois. 8Packard Instruments, Model 305, Downers Grove, Illinois.
released(net collection(2nd of 5Packard Instruments, % total)4.83.90.08.7 of Illinois. depletion period)2-48-102-48-10"CO,
rats)2 (Growing rats)3 (Growing rats)4 (Mature (Mature rats)Days 1The reaction mixtures which contained 0.5 ml urine, 4.5 ml HiO, and 20 mg urea.se were incubated 3 hours at 37°. 1 Pooled samples from four growing or four mature rata.
• Charles River Breeding Laboratories, Massachusetts.
Wilmington,
10The basal diet contained (In %) : glucose, 64.5; vitamin free casein, 20.0 ; corn oil, 6.8 ; salt mixture, 3.0 ; L-cystine, 0.2 ; cellulose, 3.0 ; riboflavin free vita min mixture, 2.5.
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the daily requirement [16 /¿g/dayfor max imal growth (2, 8)] is excreted in the urine (8) and interferes with detection and assay of riboflavin metabolites. The purpose of the present investigation was to study the fate of [2-14C]riboflavin in growing (weanling) and mature rats. The riboflavin pool(s) were repleted with [2-I4C]riboflavin following partial riboflavin depletion by dietary control. Urinary 14C-metabolites were investigated during the early phase of a second riboflavin de pletion period. Thus, the presence of ex cess riboflavin was eliminated and the use of the isotope provided a means of moni toring metabolites which could not other wise be detected.
RIBOFLAVINMETABOLITES
sheet with periodic acid (25 mg/24 ml methanol) (12) and its R, established in the different solvent systems. Aliquots of the water, 10% acetone and 50% acetone in water fractions from the R-15 chroma tography were concentrated and a sample of each was applied to the chromatogram until a noticeable concentration of salt was visible. The salt fractions from the R-15 chromatography were not investi gated by TLC as the salts caused streak ing. The chromatograms were developed in the solvents as outlined. Fluorescent areas were detected under UV light, marked, and each chromatogram cut into 1 cm strips. Each strip was placed in a scintillation vial, 1 ml water and 15 ml scintillation fluid °were added. The 14C was measured and the 14C TLC profile of the urinary metabolites determined. Urease incubation. The percentage of 14C-urea in the four urine samples were determined by measurement of 14CO2 re leased during the incubation of urine with urease. Twenty-five ml Erlenmeyer flasks were capped with rubber septum caps with hanging center wells.12 Optimal 14CO2 release was obtained with 0.5 ml urine, 4.5 ml water and 20 mg urease. The samples (duplicates) were incubated for 3 hours at 37°. Duplicate blanks were prepared for each sample and subtracted from the sample 14C. Following the incubation, 0.2 ml alkaline solution13 was added to the center well to absorb the 14CO2; 0.4 ml 60% citric acid was added to the incuba tion mixture. The flasks were shaken for 2 hours at 37°.The hanging center wells were carefully removed, placed in scintil lation vials and 10 ml scintillation fluid 14 added to each. The samples were vigor ously shaken and radioactivity measured. No inhibition was seen in the presence of excess urease. Microbiological assays. Urine samples ( 1, 2, and 4 ) fractions, untreated and acid treated, and all R-15 fractions were as sayed with L. casei for riboflavin (13). The urinary and fecal 14C during the repletion period were analyzed by non"Bakerflex silica gel 1-B, J. T. Baker Chemical Co., Phillipsburg, New Jersey. «Kontes, Vineland, New Jersey. " Hyamine hydroxide, Packard Instruments, Down ers Grove, Illinois. 14Aguasol, New England Nuclear, Boston, Massa chusetts.
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autoclaved 20 minutes at 1.1 kg/cm2 to hydrolyze any conjugated flavin com pounds. If the pH was above 5, more acid was added and the procedure repeated. R-15 resorcinol resin chromatography. The R-15 resin was prepared according to the method of Koziolowa and Koziol (11) and stored in water at 4°.The columns (7 X 1 cm) were poured, prewashed with 30 ml 0.5 N NaOH, water, 30 ml 3 N HC1 and water. The six urine samples (three untreated and three acid treated) were added to individual columns. The columns were eluted stepwise with 50 ml 0.05 M ammonium sulfate, 100 ml water, 50 ml 10% acetone in water and 50 ml 50% ace tone in water, gravity flow ( 1 to 2 ml/min ute). The flavin nucleotides were eluted with 10% acetone in water and riboflavin in the 50% acetone fraction. Each fraction was collected and the 14C measured. Indi vidual fractions were concentrated on a flash evaporator at 40°,made to known volume and aliquots removed for 14C measurement, microbiological assays and TLC. Urine sample 3 (untreated) was chromatographed on R-15 by combined stepwise and gradient elution procedures. The columns were eluted with 125 ml 0.05 M ammonium sulfate, 250 ml water and a linear gradient of 250 ml each 10% ace tone in water and 60% acetone in water. The 14C was monitored during the pro cedure and 2 ml fractions collected. The ammonium sulfate fractions, the water fractions, the major peak of the acetone gradient and the remainder of the acetone fraction of sample 3 (fig. 2) were com bined and the 14C determined for each combined sample. The salt fraction (un treated and acid treated) and the com bined acetone fractions ( untreated and acid treated) were rechromatographed on R-15 columns by stepwise elution. Thin layer chromatography (TLC). Com mercial thin layer chromatogramsll and solvent systems described by Treadwell and Metzler (12), were used for TLC. Flavin mononucleotide, flavin dinucleotide and riboflavin were standards. Lumichrome was obtained by the photochemical re action of riboflavin on the TLC sheet (12). Formylmethyl flavin was obtained by spraying the riboflavin spot on the TLC
1271
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J. A. TILLOTSON
AND M. S. KARCZ, JR.
analyses of variance were performed with a computer program, BMDO8V ( 15).
24 »10 -
••C-Bj-REPlETIOm -
10 12 14 16 18 SECOND DEPLETION —
TIME (DAYS)
Fig. 1 Average daily urinary 14C (dpm) ex cretion of Group 1 (growing rats, N-12) and Group 2 (mature rats, N-ll) during [2-"C]riboflavin repletion and subsequent depletion by re moval of dietary riboflavin. Each rat was fed 6.0 /id [2-"C]riboflavin.
paired i-tests (14). An analyses of vari ance determined the difference between growing and mature rats during the 2nd depletion period. The design employed a 2 factor design with repeated measures as one factor. To facilitate computation, one rat was randomly deleted from the grow ing group for urine and fecal 14C. The
Figure 1 shows the average daily uri nary excretion of 14C by the growing and mature rats during the experimental pe riod. Groups 1 and 2 respectively excreted (mean ±SEM) 13.3% ±3.4% and 16.0% ±5.0% of the total dose in the urine dur ing the repletion period and an additional 0.5% ±0.13% and 0.7% ±0.14% during the subsequent depletion period. These numbers were significantly different (P < 0.001) during depletion, but not during repletion (P < 0.05). The growing rats ex creted more fecal 14C (P < 0.001) during the repletion period than did the mature rats (42.9% ±13.3% vs 23.9% ±8.1%). However, there was no difference (P < 0.05) between the groups during the sub sequent depletion period (1.3% ±1.2% vs. 1.6% ±1.1% of the dose). Four to 9% of the urinary 14C was re covered as CO2 after incubation of the combined urine samples with urease (table 1). Less than 1% of the urinary 14C was soluble in chloroform. Table 2 summarizes the 14C distribution observed when the urine samples were chromatographed on the R-15 columns. Radioactivity was measured in all frac-
TABLE 2 Distribution of urinary "C compounds after R-16 chromatography Distribution
of "C1
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RESULTS
recovery Acetone Acetone of urinary "C83.071.971.2—76.182.668.1——92.87 Sample11lA22A44A3Peak fraction40.120.931.936.146.353.039.996.698.71.95.6Water fraction17.459.129.232.417.023.011.0——21.272.9Total fraction13.46.48.68.313.38.314.53.41.314.37.510% fraction27.113.630.323.123.515.534.6——62.614.050%
3-13-1APeak 3-23-2ASalt 1Percentage of that recovered. 2 Sample 1, 2, and 4 (table 1) [untreated and acid treated (A)] were eluted by the stepwise procedure. Sample 3 (untreated) was eluted by combined gradient and stepwise pro cedure and the salt (3-1) and acetone fraction (3-2) [untreated and acid treated (A)] were rechromatographed by the stepwise procedure.
1273
RIBOFLAVIN METABOLITES 25,
20
40
60
100 120 -H20-
-0.05N(NH4)2$O4—
FRACTIONS
140
160 -*-10-6055 180 200ACETONE 220 240 GRADIENT-» 260 280
( tub« number )
Fig. 2 "C elution profile (cpm) of urine (sample 3) from an R-15 column by stepwise and gradient elution ( 150 ml 0.05 M ammonium sulfate, 250 ml water and a linear gradient formed with 250 ml each of 10 and 60% acetone in water).
tions. The total 14C recovery from the columns was somewhat disappointing and less than 1% of the 14C was eluted with 100% acetone. Ninety-eight percent of the [2-14C]riboflavin added to rat urine was eluted from an R-15 column in the 50% acetone fraction and identified as riboflavin by TLC. Figure 2 shows the R-15 14Celution pro file of urine sample 3 chromatographed by
WMBC
the combined stepwise and gradient elu tion procedure. The majority of the radio activity was eluted either in the salt frac tion or the early part of the acetone gra dient. When the combined salt and acetone fractions (untreated and acid treated) were chromatographed on R-15 resin, 96% of the 14C in the initial salt fraction (un treated and acid treated) was again eluted in the salt fraction. The acid treatment of the acetone fraction caused a marked shift of 14C from the 10% to the 50% acetone eluant (table 2). Figures 3 and 4 illustrate the TLC sepa ration of the "C and fluorescent com pounds in the 3 R-15 fractions of sample 2 (untreated and acid treated) in three solvent systems. The 14C TLC profiles of the four urine samples were similar. Fol-
BAW TLC SAMPLE NUMBER
Fig. 3 The "C
and fluorescent (A)
profiléof TLC separations of individual R-15 fractions [Sample ¿—untreated and acid treated (A)]; 3—water fraction; 4—10% acetone frac tion, and 5—50% acetone fraction. Solvents: WMBC—water: Methanol: n butyl alcohol: chloroform (2:2:8:1.5 (v/v); BAW—n butyl alcohol: acetic acid: water [5:1:2.2 (v/v)]. (a) Flavin mononucleotide; (b) Flavin dinucleotide; (c) riboflavin.
WMBC TLC SAMPLE NUMBER
Fig. i-is. i4 The *..- "C ~ v() and fluorescent (A) TLC profile of R-15 urinary fractions [S-4—10% acetone fraction (untreated)]; [S-4A—50% ace tone fraction (acid treated)]. Solvents: WMBC and water. Standards—see figure 3.
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0
1274
J. A. TILLOTSON
AND M. S. KARCZ, JR.
part the large amounts of 14C excreted in the feces during the repletion phase. The urinary and fecal 14C excretion suggests that the mature rat will absorb more ribo f118272090263020181423Sample32195014711755229141641867171006711150592429 flavin during riboflavin repletion and me SpecificUrineUrine tabolize the tissue riboflavin faster during depletion than the growing rat. The uri nary 14C excretion during depletion be tween the two groups suggest parallel re sponse of tissue metabolism differing only (A)R-15(NH4), in magnitude. No differences were seen between the fecal 14C of the two groups SO«(NH«)2 during the 2nd depletion period. The fecal (A)H.OH,OSO« 14C in both groups decreased significantly (A)10% with time as would be expected. The Acetone10% fecal 14C during depletion most likely (A)50% Acetone represent mucosal sloughing and bile se Acetone50% cretion (17). Tailcups prevented coproAcetone (A)Activity1 Ehagy, however, reabsorption of the la1 (A) = acid treated. 2Specific activity eled compounds from the intestines can = DPM/ng riboflavin. 3Samples as listed in not be ignored. Fecal 14Ccompounds were table 1. not investigated as intestinal microflora would alter all or part of the [2-l4C]ribolowing the periodic acid treatment, a com pound^) in the acetone fractions had an flavin or its metabolites. Degradation of the isoalloxazine ring was Rf similar to formyl methyl flavin. Other ascertained by the isolation of 14C-urea solvent systems (12) were also used to from urine. Our values were higher than further demonstrate qualitatively the dif ferences between the 14C compounds in Christensen reported (3); however, they the individual R-15 fractions. The 14C represent a percentage of the urinary 14C in the absence of excess di areas on the TLC plates were shown to be metabolites etary 14C-riboflavin. distinct bands by autoradiography. In previous studies, riboflavin was gen Riboflavin was measured microbiologically by measuring growth of L. casei in erally fed in excess of the daily mainte urine samples 1, 2, and 4 (untreated and nance requirement of the rat and any ex treated) and in the R-15 fractions. The cess riboflavin was excreted in the urine. This riboflavin would mask the small biological activity of the unidentified compounds was compared with that of amounts of flavin metabolites, thus prevent riboflavin. The biological activity of the ing detection and identification. Only min l4C-metabolites was increased by acid imal increases in the tissue flavin concen tration were measured in normal rats fed treatment, table 3. An increase in I/specific activity X IO3 indicates increased L. casei high levels of riboflavin (18), therefore, adequate time should be allowed durifig growth. Preliminary extraction of 14Ccompounds labeling experiments for the turnover of the 14C-riboflavin with tissue flavins. In in selected tissues has demonstrated re tention of 14C riboflavin 10 days after re the present study, the tissue flavins were partially depleted of riboflavin by dietary moval of riboflavin from the diet. These restriction. Thus, the uptake of l4C-riboresults will be reported in a separate flavin was enhanced. During the 2nd ribo paper. flavin depletion period, the tissue 14CDISCUSSION flavins were utilized by the rat to satisfy its riboflavin requirements and the 14C Optimal riboflavin absorption is ob urinary compounds reflected only ribo tained only when dietary riboflavin is in flavin metabolism. The urinary 14C-comcorporated into the food ( 16). Our facili ties prevented incorporation of the 14C- pounds were true flavin metabolites or riboflavin in the diet, thus explaining in catabolites since 14C-riboflavin added to TABLE 3 Microbiological assays of urine and R-16 resin fractions
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RIBOFLAVIN
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We have shown that the tissue of rats, partially depleted of riboflavin, will ab sorb and retain [2-"C]riboflavin or com pounds containing the riboflavin. After removal of dietary riboflavin, the tissue 14C-flavin compounds were utilized to sat isfy the rats riboflavin requirements. These compounds were metabolized and/or chemically degraded and at least 10 14C flavin related compounds were isolated from urine by the R-15 and thin layer chromatography. This metabolism or degradation of riboflavin explain in part the requirement of the rat and probably other mammals for a constant dietary source of riboflavin. LITERATURE
CITED
1. Yagi, K., Nagatsu, T., Nagatsu-Ishibashi I. & Ohashi, A. (1966) Migration of injected "C labeled riboflavin into rat tissues. J. Biochem. 59, 313-315. 2. Yang, C. & McCormick, D. B. (1967) Degradation and excretion of riboflavin in the rat. J. Nutr. 93, 445-453. 3. Christensen, S. (1971) Studies of ribo flavin metabolism in the rat. No. 6 Properties of radioactive metabolites excreted after ad ministration of 1*C-2-riboflavin. Acta Pharmacol. Toxicol. 30, 177-184. 4. Whitby, L. G. (1952) Riboflavinyl glucoside: a new derivative of riboflavin. Biochem. J. 50, 433-438. 5. Kasai, S., Isemura, S. & Masuoka, Matsui, K. (1972) Identification of riboflavinyl o D glucósido in cat liver. J. Vitaminology IS, 17-23. 6. Faulker, R. D. & Lambooy, J. P. (1961) Intestinal synthesis of riboflavin in the rat. J. Nutr. 75, 373-378. 7. Fazekas, A. G., Mendendez, G. E. & Rivlin, R. S. (1974) A competitive protein bind ing assay for urinary riboflavin. Biochem. Med. 9, 167-176. 8. Bessey, O. A., Lowry, O. H., Davis, E. B. & Dorn, J. L. (1958) The riboflavin economy of the rat. J. Nutr. 64, 185-202. 9. Owen, E. C., Montgomery, J. P. & Proudfoot, R. (1962) Properties of a metabolite of riboflavin. Biochem. J. 82, 8 p. 10. Tillotson, J. A. & Sauberlich, H. E. (1971) Effect of riboflavin depletion and repletion on the erythrocyte glutathione reducÃ-ase in the rat. J. Nutr. 101, 1459-1466. 11. Koziolowa, A. & Koziol, J. (1968) A new method of isolation of natural flavins using phenol type resins. J. Chromatog. 34, 216221. 12. Treadwell, G. E., Jr. & Metzler, D. E. ( 1972) Development and application of methods for the study of free flavins in plant tissues. Anal. Biochem. 46, 261-275.
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rat urine was recovered and unchanged following R-15 and thin-layer chromatography. This study has demonstrated that prior to fluorometric assays or TLC, riboflavin and its metabolites should be isolated from nonflavin compounds in urine or biologi cal extracts. The R-15 resin quantitatively absorbs flavins (11), and it has been as sumed that the ammonium sulfate and water washes remove only proteins and nonflavin compounds from resin. How ever, 14C was measured in all R-15 frac tions and the 14C compounds in three fractions were shown to be different by TLC. Several of the unidentified "C com pounds had similar Rt values in one or more solvent systems. This similarity would prevent the identification and sepa ration of some compounds when urine is chromatographed directly on PC or TLC. Conjugation of one or more 14C com pounds derived from [2-14C]riboflavin was shown by the shift of a percentage of the 14C in the 10% acetone fraction to the 50% acetone fraction following acid hy drolysis. Further, conjugation of one or more flavin compounds was shown by in creased growth of L. casei in the 50% acetone fraction as well as the original urine samples following acid hydrolysis. Measurement of microbiological growth in all R-15 fractions suggest that other com pounds as well as riboflavin and the flavin nucleotides are biologically active. Also, the conjugated forms have less biological activity. Tentative identification of formylmethyl flavin in the acetone fractions after the periodic acid treatment imply that the ribityl group remains intact (12). Figures 3 and 4 show that measurement of fluorescence fail to detect all 14C com pounds containing or derived from [2-1JC]riboflavin. Also, not all fluorescent areas contain 14C. Treadwell and Metzler (12) obtained similar findings with plant flavins. Therefore, until the riboflavin me tabolites are identified and new assays de veloped, labeling experiments with 14C compounds is the only reliable method of detection of the flavin metabolites. Excess 14C-riboflavin when present should be sep arated from the metabolites by R-15 resin chromatography.
METABOLITES
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J. A. TILLOTSON
AND M. S. KARCZ, JR. 16. Levy, G. & Jusko, W. J. (1966) Factors affeeling the absorption of riboflavin in man. J. Pharma. Sci. 55, 285-289. 17. Christensen, S. (1969) Studies of riboflavin metabolism in the rat; #1—urinary and fecal excretion after oral administration of FMN. Acta. Pharmacol. Toxicol. 27, 27-33. 18. Rivlin, R. S. (1970) Riboflavin metabolism. New Eng. J. Med. 283, 463-472.
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13. Pearson, W. N. (1967) Riboflavin in the Vitamins edited by P. Gyorgy and W. N. Pearson, Academic Press, New York, Vol. 7, 99-136. 14. Winer, B. J. (1971) Statistical principle in experimental design. McGraw-Hill, New York. 15. Dixon, W. J. (1973) Biomedicai Computer Programs. University of California Press, Los Angeles.
J. A. TILLOTSON ANDM. S. KARCZ, JR. Department of Nutrition, Letterman Army Institute of Research, Presidio of San Francisco, California 94129 ABSTRACT Male rats were fed a riboflavin-deficient diet for 25 days and then fed [2-14C]riboflavin to replete their riboflavin pool(s). During a second depletion period, urine and feces were collected and 14Cexcretion monitored. The urine was chromatographed on R-15 resorcinol resin and 14C was measured in all fractions. The 14C compounds in the individual fractions were shown to be different by thin layer chromatography. The amount of 14C recovered as riboflavin increased after acid hydrolysis of the urine indicating the presence of riboflavin in conjugated forms. All R-15 fractions were found to support growth in a microbiological (L. casei) assay for riboflavin. This suggested that other derivatives, in addition to riboflavin and flavin nucleotides, are biologically active. However, the conjugated compounds were apparently less active, since acid hydrolysis of the urine enhanced its growth-promoting ability of L. casei. One 14C metabolite was identified as urea, thus providing evidence for degradation of the riboflavin molecule. This study has shown that the metabolic fates of riboflavin in the rat include conjugation and extensive degradation. J. Nutr. 107: 1269-1276,1977. INDEXING rats
KEY WORDS
riboflavin
The metabolic fate of riboflavin in the mammalian system is not clearly under stood. Although it is generally believed that the vitamin is not significantly de graded in mammals (1, 2), several groups have provided evidence to the contrary. Christensen (3) isolated five unidentified urinary 14C-riboflavin metabolites from normal rats fed excess riboflavin as one dose. Only one metabolite was fluorescent and none were conjugated. The 14Clabeled compounds were not stored in the tissues. Shen 3 isolated two fluorescent me tabolites from rat urine and tentatively identified them as flavinyl glucosides. Flavin glucosides have been isolated from rat (4) and cat (5) liver. Faulker and Lambooy (6) fed normal rats [2-14C]ribo flavin and recovered more 14C than could be accounted for by microbiological assay for flavins. Fazekas et al. (7) reported urinary compounds which were bound by riboflavin binding protein in chicken egg
riboflavin metabolites
whites which did not support growth of Tetrahymena pyriformis. Bessey et al. (8) observed that riboflavin fed in excess of 50 mg to rats could not be recovered as ribo flavin in urine. Owen et al. (9) identified a riboflavin metabolite in goat urine which had the properties of lumiflavin, but pos sessed no biological activity when assayed with L. casei. Riboflavin metabolism has usually been investigated in animals which were given excess riboflavin. Riboflavin in excess of Received for publication February 6, 1976. 1The opinions or assertion«contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. *In the conducting the research In this for re port, investigators adhered described to the "Guide Laboratory Animal Facilities and Care" as promul gated by the committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Labora tory Animal Resources, National Academy of Sciences —National Research Council. a Shen, Y. : M.S. thesis, 1967. Riboflavin metabolites in rat urine. Iowa State University.
1269
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Urinary Riboflavin Metabolites
1270
J. A. TILLOTSON
AND M. S. KARCZ, JR.
ductase (EGR) (10). On day 26, the rats were placed in individual metal metab olism cages away from direct light. Each rat was fed orally twice daily, for 8 days, 100 ¿u.! of aqueous solution containing 40 fig of riboflavin and [2-14CJriboflavin (10). During the repletion period, each rat re ceived a total of 6.0 /¿Ci[2-14C]riboflavin. On the 8th day of the repletion period, the AC of the EGR of individual rats was in the normal range, and it was assumed that the body pools were saturated in respect to riboflavin (10). The rats were tailcupped to prevent coprophagy throughout the 2nd depletion period and fed the ribo flavin free diet for 10 days. The rats were killed with an overdose of ether on the llth day of the 2nd depletion period. The MATERIALS AND METHODS tissues were excised immediately, frozen Equipment and reagents. All reagents on dry ice and stored at minus 70°until were analytical reagent grade. The [2-14C]- analyzed. riboflavin4 had a specific activity of 23.4 Urine and feces. The urine was collected mCi/mMole and was >9Q% riboflavin. daily in brown bottles containing 0.5 ml Radioactivity in column effluents was mon l N HC1 during the repletion and 2nd de itored with a scintillation spectrophotompletion period. The urine volumes were eter 5 with a 1 ml anthracene flow cell. Fif measured daily, aliquots counted for 14C teen ml of scintillation fluid °was added to activity and samples frozen until analyzed. each 1 ml sample and the samples were The lower half of each cage was rinsed counted in a liquid scintillation spectro- daily with water, an aliquot of the wash photometerT with an absolute activity ings counted for 14Cand the washings dis analyzer. Fecal samples were oxidized.8 carded. The total urinary 14Cwas the com bined 14C in the urine and cage washings. Precautions were taken to prevent photo chemical reactions during all procedures The feces were collected during the re by protecting the samples from light. pletion and 2nd depletion period, and Animals. Twelve growing male rats9 stored frozen. At the end of the study pe (50 to 60 g) (Group 1) and 11 adult male riod, the feces were lyophilized, weighed, rats (200-210 g) (Group 2) were fed a mixed, and aliquot weighed, oxidized, and riboflavin free diet10 ad libitum for 25 the 14C determined. The urine of four rats days. Partial riboflavin deficiency of the from each group was combined for days rats was ascertained by weight loss and 2, 3, and 4 (samples 1 and 3) and days 8, significant increase of the activity coeffi 9, and 10 (samples 2 and 4) of the 2nd cient (AC) of erythrocyte glutathione re- depletion period for Chromatographie sep aration (table 1). TABLE 1 A portion of each of three urine sam Percentage of urinary ltC released as CO¡from the ples was made 0.2 N in respect to HC1 and urine samples incubated with urease1
sample11 Urine
» Amersham/Searle, Arlington Heights, Illinois. .Model 338, Downers Grove, 8Bio Sol 3 In toluene, Beckman Instruments, Fullerton, California. 7Packard Instruments, Model 544, Downers Grove, Illinois. 8Packard Instruments, Model 305, Downers Grove, Illinois.
released(net collection(2nd of 5Packard Instruments, % total)4.83.90.08.7 of Illinois. depletion period)2-48-102-48-10"CO,
rats)2 (Growing rats)3 (Growing rats)4 (Mature (Mature rats)Days 1The reaction mixtures which contained 0.5 ml urine, 4.5 ml HiO, and 20 mg urea.se were incubated 3 hours at 37°. 1 Pooled samples from four growing or four mature rata.
• Charles River Breeding Laboratories, Massachusetts.
Wilmington,
10The basal diet contained (In %) : glucose, 64.5; vitamin free casein, 20.0 ; corn oil, 6.8 ; salt mixture, 3.0 ; L-cystine, 0.2 ; cellulose, 3.0 ; riboflavin free vita min mixture, 2.5.
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the daily requirement [16 /¿g/dayfor max imal growth (2, 8)] is excreted in the urine (8) and interferes with detection and assay of riboflavin metabolites. The purpose of the present investigation was to study the fate of [2-14C]riboflavin in growing (weanling) and mature rats. The riboflavin pool(s) were repleted with [2-I4C]riboflavin following partial riboflavin depletion by dietary control. Urinary 14C-metabolites were investigated during the early phase of a second riboflavin de pletion period. Thus, the presence of ex cess riboflavin was eliminated and the use of the isotope provided a means of moni toring metabolites which could not other wise be detected.
RIBOFLAVINMETABOLITES
sheet with periodic acid (25 mg/24 ml methanol) (12) and its R, established in the different solvent systems. Aliquots of the water, 10% acetone and 50% acetone in water fractions from the R-15 chroma tography were concentrated and a sample of each was applied to the chromatogram until a noticeable concentration of salt was visible. The salt fractions from the R-15 chromatography were not investi gated by TLC as the salts caused streak ing. The chromatograms were developed in the solvents as outlined. Fluorescent areas were detected under UV light, marked, and each chromatogram cut into 1 cm strips. Each strip was placed in a scintillation vial, 1 ml water and 15 ml scintillation fluid °were added. The 14C was measured and the 14C TLC profile of the urinary metabolites determined. Urease incubation. The percentage of 14C-urea in the four urine samples were determined by measurement of 14CO2 re leased during the incubation of urine with urease. Twenty-five ml Erlenmeyer flasks were capped with rubber septum caps with hanging center wells.12 Optimal 14CO2 release was obtained with 0.5 ml urine, 4.5 ml water and 20 mg urease. The samples (duplicates) were incubated for 3 hours at 37°. Duplicate blanks were prepared for each sample and subtracted from the sample 14C. Following the incubation, 0.2 ml alkaline solution13 was added to the center well to absorb the 14CO2; 0.4 ml 60% citric acid was added to the incuba tion mixture. The flasks were shaken for 2 hours at 37°.The hanging center wells were carefully removed, placed in scintil lation vials and 10 ml scintillation fluid 14 added to each. The samples were vigor ously shaken and radioactivity measured. No inhibition was seen in the presence of excess urease. Microbiological assays. Urine samples ( 1, 2, and 4 ) fractions, untreated and acid treated, and all R-15 fractions were as sayed with L. casei for riboflavin (13). The urinary and fecal 14C during the repletion period were analyzed by non"Bakerflex silica gel 1-B, J. T. Baker Chemical Co., Phillipsburg, New Jersey. «Kontes, Vineland, New Jersey. " Hyamine hydroxide, Packard Instruments, Down ers Grove, Illinois. 14Aguasol, New England Nuclear, Boston, Massa chusetts.
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autoclaved 20 minutes at 1.1 kg/cm2 to hydrolyze any conjugated flavin com pounds. If the pH was above 5, more acid was added and the procedure repeated. R-15 resorcinol resin chromatography. The R-15 resin was prepared according to the method of Koziolowa and Koziol (11) and stored in water at 4°.The columns (7 X 1 cm) were poured, prewashed with 30 ml 0.5 N NaOH, water, 30 ml 3 N HC1 and water. The six urine samples (three untreated and three acid treated) were added to individual columns. The columns were eluted stepwise with 50 ml 0.05 M ammonium sulfate, 100 ml water, 50 ml 10% acetone in water and 50 ml 50% ace tone in water, gravity flow ( 1 to 2 ml/min ute). The flavin nucleotides were eluted with 10% acetone in water and riboflavin in the 50% acetone fraction. Each fraction was collected and the 14C measured. Indi vidual fractions were concentrated on a flash evaporator at 40°,made to known volume and aliquots removed for 14C measurement, microbiological assays and TLC. Urine sample 3 (untreated) was chromatographed on R-15 by combined stepwise and gradient elution procedures. The columns were eluted with 125 ml 0.05 M ammonium sulfate, 250 ml water and a linear gradient of 250 ml each 10% ace tone in water and 60% acetone in water. The 14C was monitored during the pro cedure and 2 ml fractions collected. The ammonium sulfate fractions, the water fractions, the major peak of the acetone gradient and the remainder of the acetone fraction of sample 3 (fig. 2) were com bined and the 14C determined for each combined sample. The salt fraction (un treated and acid treated) and the com bined acetone fractions ( untreated and acid treated) were rechromatographed on R-15 columns by stepwise elution. Thin layer chromatography (TLC). Com mercial thin layer chromatogramsll and solvent systems described by Treadwell and Metzler (12), were used for TLC. Flavin mononucleotide, flavin dinucleotide and riboflavin were standards. Lumichrome was obtained by the photochemical re action of riboflavin on the TLC sheet (12). Formylmethyl flavin was obtained by spraying the riboflavin spot on the TLC
1271
1272
J. A. TILLOTSON
AND M. S. KARCZ, JR.
analyses of variance were performed with a computer program, BMDO8V ( 15).
24 »10 -
••C-Bj-REPlETIOm -
10 12 14 16 18 SECOND DEPLETION —
TIME (DAYS)
Fig. 1 Average daily urinary 14C (dpm) ex cretion of Group 1 (growing rats, N-12) and Group 2 (mature rats, N-ll) during [2-"C]riboflavin repletion and subsequent depletion by re moval of dietary riboflavin. Each rat was fed 6.0 /id [2-"C]riboflavin.
paired i-tests (14). An analyses of vari ance determined the difference between growing and mature rats during the 2nd depletion period. The design employed a 2 factor design with repeated measures as one factor. To facilitate computation, one rat was randomly deleted from the grow ing group for urine and fecal 14C. The
Figure 1 shows the average daily uri nary excretion of 14C by the growing and mature rats during the experimental pe riod. Groups 1 and 2 respectively excreted (mean ±SEM) 13.3% ±3.4% and 16.0% ±5.0% of the total dose in the urine dur ing the repletion period and an additional 0.5% ±0.13% and 0.7% ±0.14% during the subsequent depletion period. These numbers were significantly different (P < 0.001) during depletion, but not during repletion (P < 0.05). The growing rats ex creted more fecal 14C (P < 0.001) during the repletion period than did the mature rats (42.9% ±13.3% vs 23.9% ±8.1%). However, there was no difference (P < 0.05) between the groups during the sub sequent depletion period (1.3% ±1.2% vs. 1.6% ±1.1% of the dose). Four to 9% of the urinary 14C was re covered as CO2 after incubation of the combined urine samples with urease (table 1). Less than 1% of the urinary 14C was soluble in chloroform. Table 2 summarizes the 14C distribution observed when the urine samples were chromatographed on the R-15 columns. Radioactivity was measured in all frac-
TABLE 2 Distribution of urinary "C compounds after R-16 chromatography Distribution
of "C1
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RESULTS
recovery Acetone Acetone of urinary "C83.071.971.2—76.182.668.1——92.87 Sample11lA22A44A3Peak fraction40.120.931.936.146.353.039.996.698.71.95.6Water fraction17.459.129.232.417.023.011.0——21.272.9Total fraction13.46.48.68.313.38.314.53.41.314.37.510% fraction27.113.630.323.123.515.534.6——62.614.050%
3-13-1APeak 3-23-2ASalt 1Percentage of that recovered. 2 Sample 1, 2, and 4 (table 1) [untreated and acid treated (A)] were eluted by the stepwise procedure. Sample 3 (untreated) was eluted by combined gradient and stepwise pro cedure and the salt (3-1) and acetone fraction (3-2) [untreated and acid treated (A)] were rechromatographed by the stepwise procedure.
1273
RIBOFLAVIN METABOLITES 25,
20
40
60
100 120 -H20-
-0.05N(NH4)2$O4—
FRACTIONS
140
160 -*-10-6055 180 200ACETONE 220 240 GRADIENT-» 260 280
( tub« number )
Fig. 2 "C elution profile (cpm) of urine (sample 3) from an R-15 column by stepwise and gradient elution ( 150 ml 0.05 M ammonium sulfate, 250 ml water and a linear gradient formed with 250 ml each of 10 and 60% acetone in water).
tions. The total 14C recovery from the columns was somewhat disappointing and less than 1% of the 14C was eluted with 100% acetone. Ninety-eight percent of the [2-14C]riboflavin added to rat urine was eluted from an R-15 column in the 50% acetone fraction and identified as riboflavin by TLC. Figure 2 shows the R-15 14Celution pro file of urine sample 3 chromatographed by
WMBC
the combined stepwise and gradient elu tion procedure. The majority of the radio activity was eluted either in the salt frac tion or the early part of the acetone gra dient. When the combined salt and acetone fractions (untreated and acid treated) were chromatographed on R-15 resin, 96% of the 14C in the initial salt fraction (un treated and acid treated) was again eluted in the salt fraction. The acid treatment of the acetone fraction caused a marked shift of 14C from the 10% to the 50% acetone eluant (table 2). Figures 3 and 4 illustrate the TLC sepa ration of the "C and fluorescent com pounds in the 3 R-15 fractions of sample 2 (untreated and acid treated) in three solvent systems. The 14C TLC profiles of the four urine samples were similar. Fol-
BAW TLC SAMPLE NUMBER
Fig. 3 The "C
and fluorescent (A)
profiléof TLC separations of individual R-15 fractions [Sample ¿—untreated and acid treated (A)]; 3—water fraction; 4—10% acetone frac tion, and 5—50% acetone fraction. Solvents: WMBC—water: Methanol: n butyl alcohol: chloroform (2:2:8:1.5 (v/v); BAW—n butyl alcohol: acetic acid: water [5:1:2.2 (v/v)]. (a) Flavin mononucleotide; (b) Flavin dinucleotide; (c) riboflavin.
WMBC TLC SAMPLE NUMBER
Fig. i-is. i4 The *..- "C ~ v() and fluorescent (A) TLC profile of R-15 urinary fractions [S-4—10% acetone fraction (untreated)]; [S-4A—50% ace tone fraction (acid treated)]. Solvents: WMBC and water. Standards—see figure 3.
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0
1274
J. A. TILLOTSON
AND M. S. KARCZ, JR.
part the large amounts of 14C excreted in the feces during the repletion phase. The urinary and fecal 14C excretion suggests that the mature rat will absorb more ribo f118272090263020181423Sample32195014711755229141641867171006711150592429 flavin during riboflavin repletion and me SpecificUrineUrine tabolize the tissue riboflavin faster during depletion than the growing rat. The uri nary 14C excretion during depletion be tween the two groups suggest parallel re sponse of tissue metabolism differing only (A)R-15(NH4), in magnitude. No differences were seen between the fecal 14C of the two groups SO«(NH«)2 during the 2nd depletion period. The fecal (A)H.OH,OSO« 14C in both groups decreased significantly (A)10% with time as would be expected. The Acetone10% fecal 14C during depletion most likely (A)50% Acetone represent mucosal sloughing and bile se Acetone50% cretion (17). Tailcups prevented coproAcetone (A)Activity1 Ehagy, however, reabsorption of the la1 (A) = acid treated. 2Specific activity eled compounds from the intestines can = DPM/ng riboflavin. 3Samples as listed in not be ignored. Fecal 14Ccompounds were table 1. not investigated as intestinal microflora would alter all or part of the [2-l4C]ribolowing the periodic acid treatment, a com pound^) in the acetone fractions had an flavin or its metabolites. Degradation of the isoalloxazine ring was Rf similar to formyl methyl flavin. Other ascertained by the isolation of 14C-urea solvent systems (12) were also used to from urine. Our values were higher than further demonstrate qualitatively the dif ferences between the 14C compounds in Christensen reported (3); however, they the individual R-15 fractions. The 14C represent a percentage of the urinary 14C in the absence of excess di areas on the TLC plates were shown to be metabolites etary 14C-riboflavin. distinct bands by autoradiography. In previous studies, riboflavin was gen Riboflavin was measured microbiologically by measuring growth of L. casei in erally fed in excess of the daily mainte urine samples 1, 2, and 4 (untreated and nance requirement of the rat and any ex treated) and in the R-15 fractions. The cess riboflavin was excreted in the urine. This riboflavin would mask the small biological activity of the unidentified compounds was compared with that of amounts of flavin metabolites, thus prevent riboflavin. The biological activity of the ing detection and identification. Only min l4C-metabolites was increased by acid imal increases in the tissue flavin concen tration were measured in normal rats fed treatment, table 3. An increase in I/specific activity X IO3 indicates increased L. casei high levels of riboflavin (18), therefore, adequate time should be allowed durifig growth. Preliminary extraction of 14Ccompounds labeling experiments for the turnover of the 14C-riboflavin with tissue flavins. In in selected tissues has demonstrated re tention of 14C riboflavin 10 days after re the present study, the tissue flavins were partially depleted of riboflavin by dietary moval of riboflavin from the diet. These restriction. Thus, the uptake of l4C-riboresults will be reported in a separate flavin was enhanced. During the 2nd ribo paper. flavin depletion period, the tissue 14CDISCUSSION flavins were utilized by the rat to satisfy its riboflavin requirements and the 14C Optimal riboflavin absorption is ob urinary compounds reflected only ribo tained only when dietary riboflavin is in flavin metabolism. The urinary 14C-comcorporated into the food ( 16). Our facili ties prevented incorporation of the 14C- pounds were true flavin metabolites or riboflavin in the diet, thus explaining in catabolites since 14C-riboflavin added to TABLE 3 Microbiological assays of urine and R-16 resin fractions
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RIBOFLAVIN
1275
We have shown that the tissue of rats, partially depleted of riboflavin, will ab sorb and retain [2-"C]riboflavin or com pounds containing the riboflavin. After removal of dietary riboflavin, the tissue 14C-flavin compounds were utilized to sat isfy the rats riboflavin requirements. These compounds were metabolized and/or chemically degraded and at least 10 14C flavin related compounds were isolated from urine by the R-15 and thin layer chromatography. This metabolism or degradation of riboflavin explain in part the requirement of the rat and probably other mammals for a constant dietary source of riboflavin. LITERATURE
CITED
1. Yagi, K., Nagatsu, T., Nagatsu-Ishibashi I. & Ohashi, A. (1966) Migration of injected "C labeled riboflavin into rat tissues. J. Biochem. 59, 313-315. 2. Yang, C. & McCormick, D. B. (1967) Degradation and excretion of riboflavin in the rat. J. Nutr. 93, 445-453. 3. Christensen, S. (1971) Studies of ribo flavin metabolism in the rat. No. 6 Properties of radioactive metabolites excreted after ad ministration of 1*C-2-riboflavin. Acta Pharmacol. Toxicol. 30, 177-184. 4. Whitby, L. G. (1952) Riboflavinyl glucoside: a new derivative of riboflavin. Biochem. J. 50, 433-438. 5. Kasai, S., Isemura, S. & Masuoka, Matsui, K. (1972) Identification of riboflavinyl o D glucósido in cat liver. J. Vitaminology IS, 17-23. 6. Faulker, R. D. & Lambooy, J. P. (1961) Intestinal synthesis of riboflavin in the rat. J. Nutr. 75, 373-378. 7. Fazekas, A. G., Mendendez, G. E. & Rivlin, R. S. (1974) A competitive protein bind ing assay for urinary riboflavin. Biochem. Med. 9, 167-176. 8. Bessey, O. A., Lowry, O. H., Davis, E. B. & Dorn, J. L. (1958) The riboflavin economy of the rat. J. Nutr. 64, 185-202. 9. Owen, E. C., Montgomery, J. P. & Proudfoot, R. (1962) Properties of a metabolite of riboflavin. Biochem. J. 82, 8 p. 10. Tillotson, J. A. & Sauberlich, H. E. (1971) Effect of riboflavin depletion and repletion on the erythrocyte glutathione reducÃ-ase in the rat. J. Nutr. 101, 1459-1466. 11. Koziolowa, A. & Koziol, J. (1968) A new method of isolation of natural flavins using phenol type resins. J. Chromatog. 34, 216221. 12. Treadwell, G. E., Jr. & Metzler, D. E. ( 1972) Development and application of methods for the study of free flavins in plant tissues. Anal. Biochem. 46, 261-275.
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rat urine was recovered and unchanged following R-15 and thin-layer chromatography. This study has demonstrated that prior to fluorometric assays or TLC, riboflavin and its metabolites should be isolated from nonflavin compounds in urine or biologi cal extracts. The R-15 resin quantitatively absorbs flavins (11), and it has been as sumed that the ammonium sulfate and water washes remove only proteins and nonflavin compounds from resin. How ever, 14C was measured in all R-15 frac tions and the 14C compounds in three fractions were shown to be different by TLC. Several of the unidentified "C com pounds had similar Rt values in one or more solvent systems. This similarity would prevent the identification and sepa ration of some compounds when urine is chromatographed directly on PC or TLC. Conjugation of one or more 14C com pounds derived from [2-14C]riboflavin was shown by the shift of a percentage of the 14C in the 10% acetone fraction to the 50% acetone fraction following acid hy drolysis. Further, conjugation of one or more flavin compounds was shown by in creased growth of L. casei in the 50% acetone fraction as well as the original urine samples following acid hydrolysis. Measurement of microbiological growth in all R-15 fractions suggest that other com pounds as well as riboflavin and the flavin nucleotides are biologically active. Also, the conjugated forms have less biological activity. Tentative identification of formylmethyl flavin in the acetone fractions after the periodic acid treatment imply that the ribityl group remains intact (12). Figures 3 and 4 show that measurement of fluorescence fail to detect all 14C com pounds containing or derived from [2-1JC]riboflavin. Also, not all fluorescent areas contain 14C. Treadwell and Metzler (12) obtained similar findings with plant flavins. Therefore, until the riboflavin me tabolites are identified and new assays de veloped, labeling experiments with 14C compounds is the only reliable method of detection of the flavin metabolites. Excess 14C-riboflavin when present should be sep arated from the metabolites by R-15 resin chromatography.
METABOLITES
1276
J. A. TILLOTSON
AND M. S. KARCZ, JR. 16. Levy, G. & Jusko, W. J. (1966) Factors affeeling the absorption of riboflavin in man. J. Pharma. Sci. 55, 285-289. 17. Christensen, S. (1969) Studies of riboflavin metabolism in the rat; #1—urinary and fecal excretion after oral administration of FMN. Acta. Pharmacol. Toxicol. 27, 27-33. 18. Rivlin, R. S. (1970) Riboflavin metabolism. New Eng. J. Med. 283, 463-472.
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13. Pearson, W. N. (1967) Riboflavin in the Vitamins edited by P. Gyorgy and W. N. Pearson, Academic Press, New York, Vol. 7, 99-136. 14. Winer, B. J. (1971) Statistical principle in experimental design. McGraw-Hill, New York. 15. Dixon, W. J. (1973) Biomedicai Computer Programs. University of California Press, Los Angeles.
