In Viv Absrptin and Phsphrylatin f Pyridxine Hel in Rat Jejunum HNRY M. MIDDLTON III, M.D. Assciate Prfessr f Medicine, Medical Cllege f Gergia, and Principal Investigatr and Assistant Chief, Gastrenterlgy Service, Veterans Administratin Hspital (FHD), Augusta, Gergia The absrptin and phsphrylatin f 3H-pyridxine HCI were studied in viv in islated lps f rat jejunum. Uptake was rapid and linear ver the cncentratin range f 0.2-1 mm; tissue cntent f absrbed vitamin was saturable and cnsisted primarily f phsphrylated frms f vitamin B 6 Phsphrylatin was saturable with a Km f 11.6 p.m and a V max f 1.65 nmlj5 min/g wet tissue. Inhibitin f phsphrylatin changed neither the t}f 3H_ pyridxine HCI disappearance frm the lumen nr the calculated uptake after either 5-min r 3D-min absrptin perids. Inhibitin f phsphrylatin, hwever, significantly decreased the t} fr transmural absrptin and significantly increased the calculated transmural absrptin after a 5-min absrptin perid. These studies indicate that (a) pyridxine absrptin, ver the cncentratin range tested, is nnsaturable and cnsistent with passive diffusin; (b) jejunal phsphrylatin f pyridxine ccurs in viv, is saturable, and results in saturatin f tissue cntent f the absrbed vitamin; and (c) phsphrylatin has n significant effect n the uptake f pyridxine but significantly delays the transmural absrptin f a finite amunt f the vitamin. The in vitr absrptin f pyridxine. Hel by the rat jejunum appears t ccur by a mechanism mst cnsistent with passive diffusin. Such a cnclusin Received April 4, 1978. Accepted August 16, 1978. This study was published in part in abstract frm (Gastrenterlgy 72:1102, 1977). Address requests fr reprints t: Dr. Henry M. Middletn III, Gastrenterlgy Research Labratries, Veterans Administratin Hspital (FHD), Augusta, Gergia 30904. This study was supprted by the Veterans Administratin (MRIS n. 5702). The authr wishes t express his appreciatin t Mrs. Anne Brsius and Mr. Carey Hall fr technical assistance and t Mrs. Bena Clary fr clerical assistance. is derived in large measure frm studies that have dcumented either (a) nnsaturable, nnenergy-dependent uptake, 2 r (b) failure f pyridxine t achieve a significant sersal-t-mucsal fluid cncentratin gradient in everted sacs. 3-5 Cnfirmatin f these studies in an in viv mdel, hwever, has been very limited. Brain and Bth evaluated the absrptin f pyridxine HCI in viv in bth rat 6 and human 7 and cncluded that it ccurred by passive diffusin. Hwever, the quantities f pyridxine administered in these studies were high in relatin t dietary needs in bth rat and human, and absrptin was tested nly indirectly by measuring the urinary excretin f the vitamin. In vitr studies have clearly demnstrated that phsphrylatin f absrbed pyridxine ccurs within the jejunal wall f the rat during uptake f the vitamin.' z Pyridxal kinase, which is present in the small intestine, presumably mediates this prcess. R The characteristics and effects f this phsphrylatin during in viv pyridxine absrptin, hwever, have nt been well defined. The purpse f the present studies was t extend bservatins n the intestinal uptake and phsphrylatin f pyridxine in the rat by evaluating these prcesses in the in viv islated lp mdel. The specific aims were (a) t define the mechanism f intestinal absrptin f pyridxine in viv; (b) t study the characteristics f jejunal phsphrylatin f pyridxine in viv; and (c) t determine the effects f that phsphrylatin n pyridxine absrptin. Materials and Methds Basic xperimental Mdel Male Sprague Dawley rats weighing 75-150 g were maintained n cmmercial stck diet ad libitum. They were fasted vernight befre use but were permitted free access t water. The animals were anesthetized with 55
44 MIDDLTON GASTRONTROLOGY Vl. 76, N. ] mg/kg f pentbarbital sdium by intraperitneal injectin. The prximal jejunum was identified thrugh a midline abdminal incisin, and a lp, which was 4-6 em in length, was selected. A test slutin, in a ttal vlume f 0.25 ml, was injected int the lumen between silk ligatures psitined s as nt t cmprmise the mesenteric circulatin. The slutins cnsisted f 3H-pyridxine HCI eh PN) (Amersham/Searle Crp., Arlingtn Hts., Ill., 5.5-7.3 mcijmg; 94-96% radi purity) with r withut unlabeled pyridxine. HCI (Sigma Chemical C., St. Luis, M.) in Krebs bicarbnate buffer, gassed with 95% O 2-5% CO 2 (ph 7.40; 37 C). The lp f intestine was returned t the abdminal cavity and the abdmen was clsed with surgical clamps. The animal was cvered; bdy temperature was maintained with an incandescent lamp placed a shrt distance abve the animal and was mnitred by rectal thermmeter. At the end f the desired absrptin perid, the abdmen was repened; the islated lp, alng with sutures, was dissected free f attached mesentery and was cut free frm remaining bwel. The lp was rinsed in 0.9% saline. One end f the lp was cut and the residual luminal fluid was permitted t drain int a vial. The lumen was flushed with 10 ml f 0.9% saline; the flushing slutin was added t the vial. The resulting slutin and tissue were then prcessed as described belw fr each type f study. Measurement f Jejunal Uptake, Tissue Cntent, and Transmural Absrptin The flushed lp f jejunum was pened lngitudinally and washed vigrusly in 10 ml f 0.9% saline. Further washing did nt significantly increase the recvery f radiactivity. The tissue was placed in a preweighed vial and dried fr 18 hr at 80 C fr determinatin f dry weight. Validatin studies demnstrated n significant further weight change with lnger drying perids. The tissue was then sapnified in 5 ml f 0.75 N NaOH. Aliquts (100,u1) f the flushed luminal slutin, the tissue washing slutin, and the sapnified tissue were cunted in a scintillatin spectrmeter (Packard Tricarb mdel 3320, Packard Instrument C., Inc., Dwners Grve, Ill.) using 15 ml f a scintillatin fluid described by Sallee et a1." fficiency f cunting was similar fr all samples. Uptake was defined as luminal disappearance and was calculated as the difference between injected and residual pyridxine in the lumen. Residual luminal pyridxine was calculated as the sum f that in the flushed slutin and that in the wash slutin. Tissue cntent f 3H-vit Ba (ttal 3H-Iabeled vitamin Ba withut reference t frm(s) f the vitamin present; it may cnsist f bth free and phsphrylated frms) was determined frm the sapnified sample. Transmural absrptin was defined and calculated as ttal injected pyridxine less bth residual luminal pyridxine and tissue 3H-vit Ba. Measurement f Tissue 3H-B6PO. Rats t be used fr the measurement f tissue 3H_ BaPO. e H-Iabeled, phsphrylated frms f vitamin Ba islated by chrmatgraphy as a single grup) were han- died similarly t thse used t measure uptake, tissue cntent, and transmural absrptin. xperimental cnditins during absrptin were the same. After the islated lp had been stripped f mesentery and cut free frm the rest f the bwel, it was likewise rinsed and then flushed with 0.9% saline. The sutures were cut away; the tissue was bltted gently; and the wet weight was determined. The tissue was then placed in 5 ml f 5% TCA and hmgenized. The precipitate was washed nce with 5% TCA, and the supernatants were cmbined. With this prcedure, nly negligible amunts f radiactivity remained in the precipitated fractin. The resulting supernatant was then prcessed and the cntent f 3H-BaPO. was determined by paper chrmatgraphy as previusly described. ' 2 With this technique 3H-BaPO. was islated as a single grup and was presumed t include pyridxine phsphate, pyridxal phsphate, and pyridxamine phsphate. ' N attempt was made t separate the varius cmpnents f 3H-B6PO. Such a prcedure was beynd the scpe f this study and wuld have affected neither the infrmatin sught nr the cnclusins reached. valuatin f Data All data were expressed as mean ± SM. Statistical significance between means f cntrl and experimental data was determined by unpaired, tw-tailed, Student t-test.lo Regressin analyses were by least-square methd.lo Statistical significance fr the differences between regressins was determined by cmparisn f regressin slpes.lo V max and Km fr phsphrylatin were calculated by least-square analysis f Lineweaver-Burke plts. Results Absrptin as a Functin f Time T define the characteristics f pyridxine HCl absrptin frm the islated lp, absrptin f finite quantities was studied after varying time intervals. Figure 1 demnstrates the results f such studies. Pyridxine (0.05 nml in 0.25 ml; 0.2 JLM) disappeared rapidly frm the lumen fllwing first-rder kinetics (Figure 6A). Tissue cntent f 3H_ vit Ba reached a maximum after 30-45 min and then decreased thereafter. Between 5 and 30 min the tissue cntent was 36-40% f uptake; thereafter, it represented prgressively smaller fractins f ttal uptake, e.g., nly 7.2% at 240 min. Ttal transmural absrptin increased thrughut the perid f absrptin; its rate f increase, hwever, was less than the rate f disappearance f pyridxine frm the lumen. The difference was a functin f the tissue cntent f 3H-vit B 6 Similar curves were btained when 2 JLM "H-PN (0.5 nml in 0.25 ml) were used. After 5 min, apprximately 27% f luminal "H-PN had been absrbed; after 30 min, apprximately 80% had been absrbed. Figure 2 demnstrates that pyridxine uptake, tissue cntent f 3H-vit Ba, and transmural absrptin were all linear with respect t time fr the 1st 5 min f absrptin.
January 1979 PYRIDOXIN ABSORPTION IN VIVO 45 0.06 ~ 0.04 ~8 I-...J 5> :a 0.03 I J: c: rtl - 0.02 005 ---------------------------------- Lumen Transmural absrptin 0.0 I Q +--..,.----r-=:...:~==;==*=;==~_ii_4l. 0 20 40 60 80 100 120 240 TIM (MIN) 0.03 01 0.02 100 1112 I-~ 0.01 5> ~ I c: J:- rtl! ;:: >... Figure 1. Absrptin f 0.2 JIM pyridxine. HCl (0.05 nml in 0.25 ml Krebs bicarbnate buffer) in islated in viv lps as a functin f time. The luminal cntent f residual pyridxine (e--e) is expressed as nml/lp. Tissue cntent f 3H-vit Ba is expressed as nml/lp (A-A) and as nml/1oo mg dry tissue (L>-L». Transmural absrptin (0--0) is expressed as nml/lp. Pints n the graph represent the means f three t fur determinatins. S averaged 17% f the means. Chrmatgraphic studies were perfrmed t determine the nature f the 3H-labeled vitamin Ba in tissue during absrptin. Tissue 3H-vit Ba cnsisted primarily f 3H-BaP04' which was 62%,72%, and 82% f ttal 3H-vit B6 after 5-min, 3D-min, and 120-min absrptin perids, respectively. Values fr 3H-PN in tissue were 33%, 22%, and 8% after the same time intervals. The remainder f 3H-vit B6 activity crrespnded t 3H-pyridxal-pyridxamine CH-PL-PM), which had previusly been nted t be present during in vitr studies. 2 16 This prtin f 3H-vit Ba is nt the cncern f this study and is nt discussed further. Figure 3 depicts the calculated quantities f 3H_ 0.30 ~ 0.25 Q) :l III III i= 020 &-c!::: DI 0.15 > 10 :1:0,., ::::: 0.10 5005 Uptake 2 3 4 5 TIM (MIN) Figure 2. Absrptin f 2 JIM pyridxine. HCl (0.5 nml in 0.25 ml Krebs bicarbnate buffer) in islated in viv lps as a functin f time. ach pint represents the mean ±SM fr three t seven determinatins.... 0.20.,-------------, <II :J CI) CI) i= 0.15 >. W' enc t: e. >0 l I_ 10 I'/') '005 c: TIM 2 HR Figure 3. Tissue cntent f 3H-vit B6 after absrptin fr 5 min, 30 min, and 2 hr. The pen bars represent 3H-BaP04; the shaded bars, 3H_PN. Results are expressed as mean ± S f fur t seven determinatins. B a P04 and 3H-PN in tissue during absrptin f 2,aM 3H-PN. Absrptin as a Functin f Pyridxine Cncentratin T determine if the uptake f pyridxine r the tissue cntent f 3H-vit Ba was saturable, the initialluminal cncentratin f pyridxine was varied ver a wide range. Because uptake, tissue cntent, and transmural absrptin were linear with respect t time fr 5 min, 5-min studies were used t define the kinetics f absrptin. Figure 4 demnstrates the effect f luminal cncentratins f pyridxine ranging frm 0.2,aM t 75,aM. Uptake was linear with respect t cncentratin ver the entire range, demnstrating n evidence f saturatin kinetics. Further studies (Figure 7), which revealed n difference in uptakel,am 3H-PN at pyridxine cncentratins f 2,aM and 1 mm, in effect extended this demnstrated lack f saturatin kinetics thrugh 1 mm. Tissue cntent demnstrated saturatin ver the same cncentratin range. 3H-B6P04 in Tissue as a Functin f Pyridxine Cncentratin Because a large prtin f tissue 3H-vit B6 was fund t be phsphrylated and because tissue cntent demnstrated saturatin, the kinetics f phsphrylatin were als studied. Figure 5 illustrates the effect f the luminal cncentratin f pyridxine n tissue 3H-BaP04 after absrptin fr either 5 min r 30 min. It is readily apparent that saturatin ccurs at bth time intervals with qualitatively similar results. Values fr Km and V max fr 5-min absrptin were 11.6,aM and 1.65 nm1!5 minlg wet tissue. Since uptake and tissue cntent were nt linear with respect t time at 30 min, Km and V max values were
46 MIDDLTON GASTRONTROLOGY Vl. 76, N. 1 8.0 7.0 w ::::> ~6.0 I- 10 '- >- W t: 5.0 ~O ~Ol a.. ::::>0 4.0 zo 0..' I Z 30 I. f()~ 10, 02.0 c: (5 ~~~~----~----~----~ ~. 0 0.5 1.0 1.5 2.0 PYRIDOXIN HCI CONCNTRATION (,um w ~ (I) (I) i= ID> (Xl Ir.t CI > 0> I IO rt) 0 w_ ~'- 0.1 ~ ~ -~ 1-10 '- I \upt... ~Ol m 1- _0 0 2.0 >,_ I zw rl ~~ 1.0 1.0 W 10 (f) ::::>~I (f) 0 > (f) t: O-jlll'---..,----r---.------,----,.----r---.---;-O 1-..5.0 10 20 30 40 50 60 70 80 PYRIDOXIN HCI CONCNTRATION (,um) Figure 4. Uptake f 3H_PN and tissue cntent f 3H-vit Ba as functins f pyridxine. HCl cncentratins during 5-min absrptin perids. Units fr uptake (O-D) are indicated n the left side f the graph. Units fr tissue cntent (~~) are indicated n the scale t right f the graph. The inset illustrates mre detailed infrmatin at lw cncentratins (0.2 /-IM-2.O /-1M). Values represent mean ± SM fr six determinatins. nt calculated fr 3D-min studies. Tissue cntent f 3H-B6P04 was essentially maximal at pyridxine cncentratins abve 50 JLM. ffect f Phsphrylatin n Pyridxine Absrptin The effect f tissue phsphrylatin n the uptake f pyridxine frm the lumen was evaluated in tw different ways. First, the effect f 100 JLM 4-dexypyridxine HCI n the time-curse f 0.2 JLM 3H-PN absrptin was studied (Figure 6). 4-Dexypyridxine HCI is a structural analgue f pyridxine and an inhibitr f pyridxine phsphrylatin. ll As illustrated in the upper panel f Figure 6, the presence f 4-dexypyridxine HCI had n significant effect n the disappearance f pyridxine frm the jejunal lumen. Calculated t}values fr the disappearance f 3H-PN frm the IUffi,m were 12.5 min and 11.4 min withut and with 4-dexypyridxine, respectively. The effect n transmural absrptin was evaluated by determining the disappearance f label frm lumen and tissue cmbined (Panel B, Figure 6). Since the ttal 3H-vit Ba in the system remained cnstant at all time perids (equivalent t amunt injected at 3.0 zer time), the study f disappearance f 3H frm lumen plus tissue permitted the applicatin f first r. der kinetics t transmural absrptin. Thus, in Figure 6B, ttal 3H-vit B6 (dtted line) minus 3H-vit Ba in lumen plus tissue (slid line) defined transmural absrptin. The rate f disappearance f 3H frm lumen plus tissue was significantly greater in the pres- ~ 2.5 W v~ 0(1) CL ~ 20 ID I-. ~I- I W rt) 3: 1.5 W '" ~ "- (f) (5 (f) 1.0 i=..s 0.5 10 20 30 40 bo 60 70 80 PYRIDOXIN HCI CONCNTRATION (flm) Figure 5. Tissue cntent f 3H-BaP04 as a functin f pyridxine HCl cncentratin after either 5-min (li-b.) r 3D-min (0--0) absrptin. Results are expressed as mean ± SM f three determinatins.
January 1979 PYRIDOXIN ABSORPTION IN VIVO 47 0.05 0.025 Z~ a.. a.. 0.015.0 IO I\')~ ZO IJ.J :::!: c: :::>.~...J 0.005 0.0025 0.0015 0.05 0.025 r P>0.05 10 20 30 40 TIM (MIN) 50 60 as a percent f cntrl values (Figure 7). Phsphrylatin was prfundly depressed by 4-dexypyridxine (Figure 7). Thugh the ttal quantity f :ih_ B 6 P04 was greater in tissue during 1 mm absrptin than during 2 JLM absrptin, phsphrylatin relative t 3H-PN cncentratin ('H-BaP04/JLM 3H-PN) was markedly decreased with the 1 mm 3H_PN. Under nne f the experimental cnditins was uptake significantly altered (Figure 7). Tissue 3H-vit Ba was significantly decreased by 38-77%. Transmural absrptin was increased by 13-65% ver cntrls; nly the 13% increase was nt statistically significant. 0.015 0.005 O~r--~~I--~I--~~--~ 10 20 30 40 50 60 TIM (MIN) Figure 6. ffect f 100 I'M 4-dexypyridxine n the time-curse f absrptin f 0.2 I'M 3H_PN. The upper panel (A) illustrates the disappearance f 3H_PN frm the lumen either in the absence f (0-0) r in the presence f (--.) 4-dexypyridxine. The lwer panel (B) illustrates the effect f 4-dexypyridxine n transmural absrptin f 0.2 I'M 3H_PN. Transmural absrptin is defined as ttal 3H_PN injected (0.05 nml/lp-dtted line) minus lumen-plus-tissue 3H-vit Ba (slid lines). Pints represent the mean f three t fur determinatins. S averaged 17% f the means. ence f 4-dexypyridxine than under cntrl cnditins (n 4-dexypyridxine). Thus, the presence f nrmal phsphrylatin was assciated with a significant slwing f transmural absrptin. Calculated t}values fr transmural absrptin were 31.5 min and 18.3 min withut and with 4-dexypyridxine, respectively. Secndly, the effects f phsphrylatin were evaluated after specific perids f absrptin (5 min and 30 min). Five-minute studies were used t reflect early, linear, absrptin kinetics; 3D-min studies, t demnstrate net changes ccurring late in the absrptin sequence. Absrptin f 2 JLM 3H_PN was selected as a cntrl fr each study (cntrl rats). Fr experimental rats, the quantitative significance f phsphrylatin relative t the cncentratin f vitamin injected was minimized at bth time intervals either by using high cncentratins f pyridxine (1 mm) that vastly exceeded the phsphrylating capacity f the tissue r by using high cncentratins f 4-dexypyridxine (1 mm). Data fr bth cntrl and experimental rats were expressed as uptake/jlm 3H-PN, tissue cntent/jlm 3H_PN, etc. Cmparisns between cntrl and experimental cnditins were made by expressing experimental results Discussin Pyridxine is rapidly absrbed by the rat jejunum. a The islated lp mdel as used here permits an evaluatin f the absrptin f small, finite quantities f the vitamin under nn-steady-state cnditins. Unlike earlier studies f in viv pyridxine absrptin, the quantities used in these studies are well belw the usual dietary requirements fr the...j 200,------------------------------------. 175 a: I- 150 Z u Z 125 a.. ::c '1: ~ 100 ::t~ N LL 75 I- Z UJ U a: w a.. 50 25 ~--~~~--~~~~--~~~~~~L-~ UPTAK TISSU CONTNT + TRANSMURAL 3H-BsP04 ABSORPTION IN TISSU Figure 7. ffect f high cncentratins f either 3H_PN r 4-dexypyridxine Hel n intestinal absrptin f pyridxine in islated in viv lps. All cntrls cnsisted f 2 I'M 3H_PN. xperimental cnditins included: 1 mm 3H-PN during 5-min studies (pen bars), 1 mm 4-dexypyridxine during 5-min studies (bars with diagnal shading), 1 mm 3H_PN during 30- min studies (stippled bars), and 1 mm 4-dexypyridxine during 30-min studies (bars with hrizntal shading). Data fr bth cntrl and experimental rats were calculated as uptake/i'm 3H-PN, tissue cntent/i'm 3H_ PN, transmural absrptin/i'm 3H-PN, and 3H-BaP04/ I'M 3H-PN. With all data n a per I'M 3H_PN basis, experimental results were expressed as a percent f cntrl (vertical axis). The numbers f rats used are indicated in the bars. Statistical significance: *, P < 0.001; +, P < 0.005; and t, P < 0.05.
48 MIDDLTON GASTRONTROLOGY Vl. 76, N. 1 rat. 6 Whereas a rat requires apprximately 45 pg f vitamin B6 per day, 0.25 ml f a 2-pM slutin f pyridxine cntains nly 0.1 pg.'2 With these small amunts f vitamin, n evidence can be fund t indicate the presence f a carrier mechanism fr the uptake f pyridxine. First, 5-min absrptin studies demnstrate a linear relatinship between uptake and luminal cncentratins f pyridxine (Figures 4 and 7). They are cmparable with in vitr experiments that have demnstrated lack f saturatin f uptake with pyridxine cncentratins as high as 10 mm.2 Secndly, uptake is nt affected by 4-dexypyridxine, a structural analgue f pyridxine and an inhibitr f active pyridxine transprt in yeast (Figures 6 and 7).'3 The pssibility f a carrier with a Km that is much higher than the cncentratins used cannt ttally be ruled OUt. '4 Hwever, the results btained here are in agreement with in vitr studies and are mst cmpatible with passive diffusin as the mechanism fr jejunal uptake in viv als. In vitr studies have demnstrated that phsphrylatin f pyridxine ccurs in rat jejunum during uptake f the vitamin.,,2 Presumably, it is mediated by pyridxal kinase, which is knwn t be present in small intestine and t phsphrylate free frms f vitamin B6. 8 The present experiments indicate that such phsphrylatin als ccurs during in viv absrptin, is saturable, and is a quantitatively significant event, especially at lw 3H-PN cncentratins. Fr the 1st 30 min, greater than ne-third f 2 pm 3H_ PN absrbed frm the lumen f the islated lp is present in jejunal wall. This seemingly high percentage is due t the metablism f the absrbed vitamin t phsphrylated cmpunds, the principal frms f the vitamin in tissue (Figure 3). When phsphrylatin is inhibited, the tissue cntent f 3H-vit B6, expressed as a percent f ttal absrbed vitamin, is significantly reduced. One questin that has nt been well answered by previus in vitr studies relates t the effect that phsphrylatin has n the rate f pyridxine absrptin. Tsuji et al. have demnstrated increased accumulatin f vitamin in intestinal rings during in vitr incubatin in media cntaining pyridxine.' As a result, they have suggested that phsphrylatin may enhance uptake f the vitamin by the bwel wall. Other studies by these same investigatrs, utilizing in vitr everted sacs f rat intestine, als appear t supprt such a cnclusin.15.16 Presumably, enhancement f uptake in vitr results frm the maintenance f a high lumen-t-tissue gradient thrugh intracellular cnversin f pyridxine t phsphrylated frms f the vitamin. Such an effect can be expected t be mst bvius after relatively lng incubatins when metablism has its greatest effect n cellular levels f pyridxine. The in vitr studies just cited, in fact, were carred ut ver prlnged incubatin perids.','5,'6 Unfrtunately, in vitr studies d nt reprduce accurately all the dynamic fluxes that are present in viv.17 The remval f absrbed vitamin by the mesenteric circulatin in viv is nt present in the in vitr mdel. As a result, lng-term in vitr studies may exaggerate the apparent effect f cellular metablism n intestinal uptake studies.17 The questin cncerning the effect f phsphrylatin n intestinal absrptin f pyridxine can best be answered in an in viv mdel. The present studies indicate clearly that phsphrylatin has n discernable effect n the uptake f finite quantities f pyridxine in the in viv is~ lated jejunal lp. This is true whether the effect is measured after 5-min absrptin, after 3D-min absrptin, r cntinuusly ver an absrptin perid f 1 hr (Figures 6 and 7). This failure t affect uptake exists in spite f the fact that phsphrylatin/pm 3H_PN is inhibited by 83-87%. Any effect f phsphrylatin n cncentratin gradients acrss the mucsal membrane, therefre, must be f little quantitative significance in viv. The effect f phsphrylatin n the net absrptin f pyridxine acrss the jejunal wall (transmural absrptin), hwever, is quantitatively quite different. The presence f nrmal phsphrylatin results in a decreased rate f transmural absrptin as manifested by a significantly prlnged t}. This effect is als demnstrable by the enhanced transmural absrptin after 5 r 30 min that ccurs when phsphrylatin is inhibited. Because uptake is unchanged by phsphrylatin, the effect f phsphrylatin n transmural absrptin must be ne f hindering efflux f absrbed vitamin frm the mucsal cell. Such a trapping f vitamin within the bwel wall may be cmparable with the intracellular accumulatin f pyridxal phsphate that ccurs in erythrcytes during incubatin with pyridxine.'s In summary, in viv studies that utilize the islated jejunal lp have demnstrated (a) that pyridxine is rapidly absrbed frm the islated lp by first-rder kinetics, (b) that the in viv intestinal absrptin f pyridxine Hel is cmpatible with passive diffusin, (c) that phsphrylatin f the absrbed pyridxine ccurs in the jejunal wall and is saturable, and (d) that the presence f phsphrylatin has the net effect in the islated lp f delaying exit f the vitamin frm the tissue withut affecting the rate f uptake f pyridxine frm the lumen. The bilgic significance f this delaying effect f phsphrylatin is nt presently knwn; a mre cmplete understanding must await further investigatin.
January 1979 PYRIDOXIN ABSORPTION IN VIVO 49 References 1. Tsuji T, Yamada R, Nse Y: Intestinal absrptin f vitamin B6. 1. Pyridxl uptake by rat intestinal tissue. J Nutr Sci Vitaminl 19:401-417, 1973 2. Middletn HM: Uptake f pyridxine hydrchlride by the rat jejunal mucsa in vitr. J Nutr 107:126-131, 1977 3. Spencer RP, Bw TM: In vitr transprt f radilabeled vitamins by the small intestine. J Nucl Med 5:251-258, 1964 4. Serebr HA, Slmn HM, Jhnsn JH, et al: The intestinal absrptin f vitamin B6 cmpunds by the rat and hamster. Jhns Hpkins Med J 119:166-171, 1966 5. Tsuji T, Yamada R: Mechanism f intestinal absrptin f vitamin B6. II. Studies with the everted sacs f rat intestine. Vitamins (in Iapanese) 48:471-476, 1974 6. Bth CC, Brain MC: The absrptin f tritium-labelled pyridxine hydrchlride in the rat. J Physil (Lnd) 164:282-294, 1962 7. Brain MC, Bth CC: The absrptin f tritium-labelled pyridxine HCl in cntrl subjects and in patients with intestinal malabsrptin. Gut 5:241-247, 1964 8. McCrmick DB, Gregry M, Snell : Pyridxal Phsphkinases. 1. Assay, distributin, purificatin, and prperties. J Bii Chern 236:2076-2084, 1961 9. Sallee VL, Wilsn FA, Dietschy JM: Determinatin f unidirectinal uptake rates fr lipids acrss the intestinal brush brder. J Lipid Res 13:184-192, 1972 10. Snedecr GW, Cchran WG: Statistical Methds. Sixth editin. Ames, Iwa, Iwa State University Press, 1967 11. Chern q, Beutler : Purificatin and characterizatin f human erythrcyte pyridxine kinase. Clin Chim Acta 61:353-365, 1975 12. Driskell JA, Strickland LA, Pn CH, et al: The vitamin B6 requirement f the male rat as determined by behaviral patterns brain pyridxal phsphate and nucleic acid cmpsitin and erythrcyte alanine amintransferase activity. J Nutr 103:670-680, 1973. 13. Shane B, Snell : Transprt and metablism f vitamin B6 in the yeast sacchamyces carlsbergensis 4228. J Bii Chern 251:1042-1051, 1976 14. Wilbrandt W, Rsenberg T: The cncept f carrier transprt and its crllaries in pharmaclgy. Pharmacl Rev 13:109-183,1961 15. Tsuji T, Yamada R: Studies f intestinal absrptin f pyridxal with everted sacs f rat intestine. Vitamins (in Japanese) 50:97-101, 1976 16. Tsuji T, Yamada R: Studies f intestinal absrptin f pyridxamine with everted sacs f rat intestine. Vitamins (in Japanese) 50:103-108, 1976 17. Crane RK: Na+-dependent transprt in the intestine and ther animal tissues. Gastrenterlgy 24:1000-1006, 1965 18. Andersn BB, Fulfrd-Jnes C, Child JA, et al: Cnversin f vitamin B6 cmpunds t active frms in the red bld cell. J Clin Invest 50:1901-1909, 1971