A long-term study of the excretion of folate
`and pterins in a human subject after ingestion
`of 14C folic acid, with observations on the
`effect of diphenylhydantoin administration 1• 2
`
`Carlos L. Krumdieck, 3 Kazuo Fukushima,4 Takeshi Fukushima, S Tetsuo Shiota, 6
`and C. E. Butterworth, Jr. 7
`
`After the administration of 2-14C folic acid to a human volunteer, urinary and
`ABSTRACf
`fecal radioactivity, as well as urinary excretion of folate (Lactobacillus casei assay) and
`biopterin-like material (Crithidia fasciculata assay) were determined at intervals over a 129 day
`period of observation. From two 24 hr urine samples erythroneopterin, bioterin, threoneopterin,
`pterin, isoxanthopterin, and xanthopterin were isolated by chromatographic procedures, quan(cid:173)
`titated, and their specific activities were determined. The effect on the pattern of elimination of
`urinary radioactivity and biological activity resulting from the administration of diphenylhydan(cid:173)
`toin was studied on two occasions. Urinary radioactivity plots suggest the decay of two forms of
`folates with markedly different biological half lives. One short-lived (tIll "" 31.5 hr), correspond(cid:173)
`ing to newly absorbed folate, and one long-lived (tl/z "" 100 day) thought to represent the
`decay of body pools. Diphenylhydantoin does not alter the rate of elimination of the long-lived
`component but may accelerate losses of newly absorbed folate. The analysis of pterins does not
`support the hypothesis that diphenylhydantoin increases the breakdown of folates to pterins.
`Am. J. Clin. Nutr. 31: 88-93, 1978.
`
`In recent years there has been considera(cid:173)
`ble interest in folate metabolism and turn(cid:173)
`over in man both as a means to determine
`nutritional requirements and to study the
`effect of interfering drugs.
`It is well recognized that the amount of
`folate in the diet (1, 2) is at least ten times
`as great as the quantity found in the urine
`by microbiological assay (3). This might
`suggest very incomplete intestinal absorp(cid:173)
`tion were it not for the fact that 50 to 75 %
`of tracer doses are not recoverable in 72-hr
`stool collections (4). An alternative expla(cid:173)
`nation would be that folate is converted
`into metabolites that do not support growth
`of assay organisms such as Lactobacillus
`casei. Such metabolites could include, for
`example, various pterins (5) or fragments
`thereof, as well as p-aminobenzoylgluta(cid:173)
`mate derivatives such as the acetamidoben(cid:173)
`zoylglutamate recently demonstrated in rat
`urine (6).
`It has been known since 1952 that di(cid:173)
`phenylhydantoin is capable of producing a
`folate-responsive megaloblastic anemia un-
`
`der certain conditions. Since the report of
`Mannheimer et al. (7) in that year, there
`have been many articles indicating that the
`prolonged use of anticonvulsants leads to a
`reduction in plasma folate and occasionally
`to overt deficiency symptoms (8-10). Nei(cid:173)
`ther the mechanism of this depletion nor of
`the anticonvulsant effect of these drugs is
`known. Worsening of seizure control after
`administration of folate to folate-depleted
`epileptics (11) suggests that an alteration of
`folate metabolism may play a role in the
`
`I From the Department of Nutrition Sciences and
`the Department of Microbiology, University of Ala(cid:173)
`bama in Birmingham, School of Medicine, Birming(cid:173)
`ham, Alabama 35294.
`• Supported by National Science Foundation Grant
`BMS-74-17348 and by National Foundation March of
`Dimes Grant 6-94.
`3 Professor of Biochemistry; Department of Nutri(cid:173)
`tion Sciences.
`• Faculty of Horticulture, Chiba Uni(cid:173)
`versity, 648 Tojo Matsudo City, Chiba-Ken,
`5 Department of Biology, Tokyo Metropoli(cid:173)
`Japan.
`tan University, Setagaya-Ieu Tokyo 158, Japan.
`S Professor of Microbiology.
`7 Professor of Medi(cid:173)
`cine; Chairman, Department of Nutrition Sciences.
`
`88
`
`The American Journal of Clinical Nutrition 31: JANUARY 1978, pp. 88-93. Printed in U.S.A.
`
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`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1114-0001
`
`

`
`EXCRETION OF FOLATE AND PTERINS IN HUMANS
`
`89
`
`pathogenesis of some forms of this disease
`or be essential to the mechanism of action
`of anticonvulsant drugs. The hypothesis has
`been advanced that diphenylhydantoin in(cid:173)
`terferes with folate absorption by inhibiting
`intestinal conversion of dietary pteroylpoly(cid:173)
`glutamates to monoglutamyl forms but it
`was not possible to confirm such an effect
`in vitro (12). Evidence that diphenylhydan(cid:173)
`toin impedes the normal intestinal absorp(cid:173)
`tion of free folic acid in the rat has been
`provided by Hepner (13), and human stud(cid:173)
`ies indicating inhibition of free folic acid
`absorption by therapeutic doses of diphen(cid:173)
`ylhydantoin have been reported by Gerson
`et al. (14). The possibility remains however
`that diphenylhydantoin enhances the excre(cid:173)
`tion or degradation of folates. In this report
`we present the results of a study designed
`to test the hypothesis that diphenylhydan(cid:173)
`toin administration might increase the elim(cid:173)
`ination of folates or the degradation of
`folate coenzymes to pterins or other break(cid:173)
`down products leading eventually to a defi(cid:173)
`ciency state. Data on the biological half-life
`of folate and an assessment of the contribu(cid:173)
`tions of urinary and fecal losses have also
`been obtained.
`
`Experimental design
`Purified 2_14C folic acid was administered
`orally in four equal doses of 10 /LCi given
`every 12 hr to a fully informed human
`volunteer. A total of 40 /LCi (0.725 /Lmoles,
`320 /Lg of folic acid) was given. The subject,
`a highly intelligent and cooperative 36-year(cid:173)
`old woman, was diagnosed as having Hodg(cid:173)
`kin's disease at age 25 and received inter(cid:173)
`mittent chemotherapy for 8 years thereaf(cid:173)
`ter. She had been in a state of complete
`remission for 3 years at the time of this
`study and remains symptom free. She was
`hematologically normal, weighed 75 kg, and
`was receiving no medication other than es(cid:173)
`trogen replacement (Evex 1.25 mg/day,
`Syntex Laboratories, Inc. brand of esteri(cid:173)
`fied estrogens, expressed as estrone sulfate)
`to compensate an ovarian insufficiency sec(cid:173)
`ondary to antitumor therapy. Studies in the
`hospital were conducted under metabolic
`ward conditions. Complete daily collection
`of urine (under toluene) and stools were
`made for 9 consecutive days. A 300 mg
`
`dose of diphenylhydantoin was adminis(cid:173)
`tered orally (100 mg three times daily) on
`the sixth day after the first dose of radioac(cid:173)
`tive folic acid, at which time the amount of
`radioactivity in the urine had begun to level
`off. She was discharged 4 days later. At
`home she collected 24-hr specimens of urine
`and feces for several periods of observation.
`The last collection period was ended 129
`days after ingestion of the isotope. A second
`period of diphenylhydantoin administration
`(300 mg/day) was started on day 20 and
`continued for 4 days. The urine samples
`were assayed for radioactivity, and bioas(cid:173)
`sayed with L. casei and Crithidia Jasciculata.
`Total radioactivity was determined in the
`fecal samples. If the administration of di(cid:173)
`phenylhydantoin were to cause increased
`loss of folate in the urine, an elevation of
`both radioactivity and L. casei active mate(cid:173)
`rial would be anticipated; if, on the other
`hand, the drug should cause increased
`breakdown of folate to pterins or other
`degradation products bearing the 2-C14 la(cid:173)
`bel, the urinary radioactivity would increase
`with no concomitant elevation of L. casei
`activity. The isolation and characterization
`of urinary pterins was carried out to search
`for possible conversion of radioactive folate
`into this class of compounds.
`
`Materials and methods
`
`2_'4C folic acid (specific activity 55.3 mCi/mmole)
`was purchased from Amersham Corporation. It was
`purified by DEAE cellulose chromatography as previ(cid:173)
`ously described (15). Ecteola-Sephadex and phospho(cid:173)
`Sephadex were prepared from Sephadex G-25 (fine)
`by the method of Peterson and Sober (16) for the
`corresponding celluloses. The pH 6 or pH 7 Ecteola(cid:173)
`Sephadex's were prepared as described before (5).
`
`Microbiological assays
`Urinary folates were determined by bioassay with
`L. casei as described by Baker and Frank (17). Urinary
`levels of Crithidia active substances (as biopterin equiv(cid:173)
`alents) were determined by C. Jasciculata by a modifi(cid:173)
`cation of the procedure described by Dewey and Kid(cid:173)
`der (18). The medium was prepared to give a 5-fold
`increase in the concentration of all the ingredients and
`Kanamycin, 500 ",glml was added. The concentrated
`medium was stored at -10 C and, for use, it was
`appropriately diluted and sterilized by use of a 0.45
`",m Millipore filter. The filtered-sterilized medium can
`be stored at 5 C for several weeks. Urinary radioactiv(cid:173)
`ity was determined by counting 0.5 ml of untreated
`urine using Aquasol as counting fluid. All counts were
`obtained with the automatic quench correction mode
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1114-0002
`
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`
`90
`
`KRUMDIECK ET AL.
`
`in a carbon" channel that excluded counts due to
`chemiluminescence. The efficiency of counting was
`51 %. Fecal radioactivity was obtained after homoge(cid:173)
`nizing the weighed samples in 1.0 liter of 0.05 N NaOH
`and oxidizing an aliquot with an excess of potassium
`permanganate as previously described (4). Packard
`Tricarb (model 3375) and Beckman (LS-250) liquid
`scintillation spectrometers were used to measure radio(cid:173)
`activity.
`
`Analysis ofpterins in urine
`The pterins in urine were purified by the column
`chromatography procedures previously reported (5,
`19). These compounds were identified by thin layer
`chromatography (MN-Polygram Cell 300 sheets,
`Brinkman, Inc.) employing five solvent systems (5).
`The R, values of biopterin, xanthopterin, pterin, ery(cid:173)
`throneopterin, threoneopterin, and isoxanthopterin
`were the same as those reported earlier (5).
`Spectrophotometric quantitations of pterins were
`performed whenever possible using the appropriate
`extinction coefficients (20-23). A fluorescent method
`was applied when the amount of pterin was small (5).
`
`Results
`
`Table 1 summarizes the results of the
`radioactivity measurements in urine and
`
`feces as well as the urinary levels of folates
`and biopterin-Iike compounds.
`It may be seen that maximal urinary ex(cid:173)
`cretion of 14C occurs on the second day of
`labeled folate ingestion, and declines rap(cid:173)
`idly for several days thereafter. A very
`efficient absorption is indicated by the small
`residual radioactivity, amounting to 3.16
`ILCi of the 40 ILCi dose (7.8 % of the
`administered dose), recovered in the feces
`collected during the first 6 days of the
`experiment. Urinary losses during the same
`period amounted to approximately 1.44
`ILCi, or 3.9% of the absorbed material.
`Figure lA shows the semilogarithmic plot
`of urinary radioactivity (cpm/24 hr) versus
`days after administration of the labeled folic
`acid. The graph suggests the decay of two
`forms of folates with markedly different
`biological half-lives. By averaging the re(cid:173)
`sults obtained during the five periods of
`urine collection indicated in Figure IB a
`half-life of approximately 100 days is ob(cid:173)
`tained for the long-lived component. Sub-
`
`TABLE 1
`
`Days from stan
`
`Urine
`
`Volume
`
`cpm (total)
`
`L. casei activity
`
`C. fusciculiJuJ activ-
`ity, biopterin equiva-
`lents
`
`Feces
`
`cpm (total)
`
`mJ
`o
`1340
`940
`1·
`za
`895
`1450
`3
`1500
`4
`5
`1150
`fI
`1980
`925
`7
`8
`1290
`970
`16
`1540
`2()1>
`21b
`1260
`22"
`1080
`920
`23b
`24
`1300
`1240
`25
`1160
`26
`1200
`27
`1690
`54
`55
`1410
`1260
`56
`74
`2290
`75
`1800
`76
`1450
`790
`77
`127
`1360
`128
`825
`129
`1250
`• 2-I4C folic acid administered.
`
`~/24hr
`
`7.4
`4.4
`9.0
`6.6
`7.4
`6.4
`9.9
`6.2
`8.4
`7.7
`8.8
`6.3
`6.4
`7.4
`8.1
`11.5
`10.5
`11.9
`10.3
`11.2
`7.3
`11.7
`8.8
`7.8
`9.2
`
`o
`216,200
`365,160
`324,800
`246,600
`230,000
`249,480
`223,850
`216,720
`184,300
`184,800
`168,840
`145,150
`168,540
`161,720
`157,232
`169,128
`180,720
`144,320
`148,890
`135,070
`135,540
`109,440
`95,120
`104,280
`92,480
`77,550
`72,500
`b Diphenylhydantoin administration.
`
`630
`620
`322
`435
`480
`675
`620
`481
`722
`621
`755
`605
`476
`619
`536
`462
`777
`754
`670
`536
`642
`
`565,000
`1,210,000
`1,120,000
`688,000
`
`224,000
`132,000
`
`200,600
`81,900
`
`84,000
`70,000
`57,900
`52,200
`57,120
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1114-0003
`
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`

`
`EXCRETION OF FOLATE AND PTERINS IN HUMANS
`
`91
`
`to estimate the rate of decay of the short(cid:173)
`lived component. This plot is shown in Fig(cid:173)
`ure 1 C and indicates a half-life of about
`31.5 hr.
`
`400 A
`
`Urine
`cpm x 10-5
`
`200
`
`cpm
`x 10-5
`100
`
`50
`
`Days
`
`100
`
`130
`
`B
`~~~'I,~ ..
`
`~~
`
`50~------~----____ ~ ____ __
`50
`100
`Days
`XI .. ·23-170.010 _'Z.'
`10 , .. , •• ..,..-
`10 "-""41.110
`.. 1'4·11-111,100
`10'_10."0
`
`c
`
`200
`
`100
`
`50
`
`20
`
`\
`\
`\
`'.
`
`DPH
`10~UU~---7-L ______ ~_
`10
`
`FIG. lAo Semilogarithmic plot of urinary radioactiv(cid:173)
`ity. B, Semilogarithmic plot of average (x) urinary
`radioactivities obtained during five periods of collec(cid:173)
`tion. Half-life of long-lived component. C, Half-life of
`short lived component. See text for explanation.
`
`tracting the counts corresponding to the
`long-lived component (obtained by extrap(cid:173)
`olation to zero time) from the actual counts
`obtained in specimens collected during the
`first 6-days of the experiment, it is possible
`
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`Effect of diphenylhydantoin
`Following the administration of diphenyl(cid:173)
`hydantoin on day 6 of the experiment, the
`L. casei activity in the urine increased and
`the urinary radioactivity also increased fail(cid:173)
`ing to continue its downward course (as
`predicted by the dotted line in Fig. lC).
`The second period of diphenylhydantoin
`administration of 4 days duration (rather
`than only 24 hr) was started on the 20th
`day of the experiment in the hope of show(cid:173)
`ing a more pronounced increase in folate
`and/or radioactivity losses. Contrary to ex(cid:173)
`pectations no discernible change in the pat(cid:173)
`tern of elimination was observed.
`The results of C. fasciculata assays which
`are presented in Table 1 indicate that there
`was no discernible effect on the pattern of
`elimination of Crithidia active pterins fol(cid:173)
`lowing administration of the drug. The
`urines from days 23 (the last of a 4-day
`period on DPH administration on 300 mgt
`day) and of day 25 were analyzed quantita(cid:173)
`tively for certain pterins. In Table 2 are
`shown the amounts and specific radioactivi(cid:173)
`ties found. Neither seems to be affected by
`diphenylhydantoin. Pterin and isoxanthop(cid:173)
`terin were radioactive whereas erythroneop(cid:173)
`terin, biopterin, and threoneopterin con(cid:173)
`tained little or no radioactivity.
`
`Discussion
`
`The high efficiency of absorption and
`retention of the radioactive folic acid admin(cid:173)
`istered (92.2 % of the label absorbed and
`only 3.9% of the absorbed radioactivity
`voided during the first 6 days) is in keeping
`with previously reported observations indi(cid:173)
`cating that small amounts of folic acid of
`the order of 1 #Lg/kg of body weight, even
`when administered intravenously, are al(cid:173)
`most quantitatively retained in the body
`(24).
`To the best of our knowledge, the biolog(cid:173)
`ical half-life of folate has not been precisely
`determined in man because the size of the
`total body folate pool has not been estab-
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1114-0004
`
`

`
`92
`
`KRUMDIECK ET AL.
`
`TABLE 2
`The amount and specific activity of certain pterins excreted in 24-
`Hr urine on day 23 and 25 of experimental period
`
`PI.rins
`
`Day 23 urine
`
`Day 25 urine
`
`/JII/24 h,
`14430
`Erythroneopterin
`439"
`Biopterin
`5(fl
`Threoneopterin
`5f!'
`Pterin
`21'
`lsoxanthopterin
`Xanthopterin
`8.5 b
`b These values
`o These values were determined by use of the extinction coefficient for each pterin (19).
`C Compound was identified but
`were determined by a fluorescence assay described in a previous paper (5).
`not quantitated (+ ).
`
`dpm/,.",ok
`<50
`<50
`<50
`3300
`1800
`
`/JII/24 h,
`1277"
`4840
`5~
`6f!'
`If!'
`+c
`
`dpm/,.,mol.
`<50
`<50
`<50
`3100
`2100
`
`Iished and because the degree of its deple(cid:173)
`tion needed to produce clinical manifesta(cid:173)
`tions is unknown. From our data, the bio(cid:173)
`logical half-life of folic acid in this subject
`was approximately 100 days. It is of interest
`that in the elegant study of Herbert (25),
`intermediate megaloblasts were observed in
`the bone marrow on the 99th day of re(cid:173)
`stricted folate intake, and frank megaloblas(cid:173)
`tosis was observed on the 134th day. Thus,
`it would seem that clear manifestations of
`deficiency may apear at a time when only
`about one-half of the body stores have been
`exhausted.
`A second aspect of this study to be em(cid:173)
`phasized is the recognition of a short-lived
`pool of newly absorbed folate that is clearly
`not in equilibrium with other folate forms.
`In humans, newly absorbed folate may,
`according to Eichner and Hillman (26),
`represent as much as 50% of serum folate
`and constitute a labile pool which is rapidly
`depleted on a folate-deficient diet or follow(cid:173)
`ing hemodialysis. The possibility that di(cid:173)
`phenylhydantoin may interfere with tissue
`uptake of newly absorbed folate is suggested
`by the increased biological activity and ra(cid:173)
`dioactivity found in the urine in day six.
`Further investigations are required to con(cid:173)
`firm or refute this possibility. Conventional
`doses of diphenylhydantoin (100 mg three
`times daily) given on days 20, 21,22,23 of
`the present study failed to produce any
`noticeable perturbation of the folate excre(cid:173)
`tion pattern. There is no discernible effect
`on the pattern of elimination of C. fascicu(cid:173)
`lata active pteridines following administra(cid:173)
`tion of diphenylhydantoin. Pterin and isox(cid:173)
`anthopterin were radioactive whereas ery(cid:173)
`throneopterin, biopterin, and threoneop-
`
`terin contained little or no radioactivity.
`The results are consistent with the observa(cid:173)
`tions of Fukushima and Shiota (27) con(cid:173)
`cerning pterin biosynthesis in growing cul(cid:173)
`tures of Chinese hamster ovary cells. Based
`on other studies, erythroneopterin, threo(cid:173)
`neopterin, and biopterin would not be ex(cid:173)
`pected to incorporate label from folic acid
`since these pterins are synthesized de novo
`from guanosine 5'triphophate (28-31). On
`the other hand, pterin and isoxanthopterin,
`which are formed as breakdown products
`of folic acid (and from other pterin deriva(cid:173)
`tives), would be expected to be radioactive.
`It is interesting to compare the levels of
`Crithidia active substances and the levels of
`biopterin and erythroneopterin reported
`here with those reported earlier (5). The
`present results give an average excretion of
`biopterin equivalents of 0.7 mg/24 hr by
`Crithidia assay, with the urines of days 23
`and 25 containing respectively 2.6 and 3.3
`times more erythroneopterin than biop(cid:173)
`terin. In the earlier report (5), the Crithidia
`activity/24 hr assayed 1.5 mg of biopterin
`equivalents with the daily outputs of biop(cid:173)
`terin exceeding those of erythroneopterin
`(1.0 and 0.4 mg/24 hr, respectively). The
`reason for the inverted ratio of biopterin/
`erythroneopterin found in this study is not
`known but could conceivably be attributed
`to the estrogen supplements that the patient
`was receiving. The possibility that the estro(cid:173)
`genic therapy may in some way influence
`our results on the half-life of folates, the
`bimodal curve of elimination of the tracer
`dose, and the e(.fects of diphenylhydantoin
`administration is thought to be rather re(cid:173)
`mote.
`Our results do not support the hypothesis
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1114-0005
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`
`EXCRETION OF FOLATE AND PTERINS IN HUMANS
`
`93
`
`that diphenylhydantoin increases the degra(cid:173)
`dation of folates to urinary pteridines (or
`derivatives thereof still bearing the 2_14C
`label) and thus hastens the onset of folate
`deficiency.
`The data on fecal radioactivity clearly
`indicate that this is an important avenue of
`elimination of folate or derivatives thereof.
`Late in the experiment there were days
`when the radioactivity in feces exceeded
`n
`that in the urine.
`The technical assistance of Mrs. Barbara Hudson is
`gratefully acknowledged.
`
`References
`
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`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1114-0006
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