Gut, 1976, 17, 450-455

Factors affecting the absorption of vitamin K-Iin vitro

D. HOLLANDER' AND ELENA RIM

From the Division of Gastroenterology, Wayne State University and Harper Hospital, Detroit,Michigan, U.S.A.

SUMMARY Factors which might affect the absorption of vitamin K of dietary origin were investigatedusing everted small bowel sacs. Increasing the bile salt concentration to 20 mM or the addition oflong chain fatty acids, monoolein, or lecithin all resulted in significant (p < 0.05) decrease in theabsorption rate of the vitamin. The addition of 2-5 mM short and medium chain fatty acids didnot change the absorption rate of vitamin K-1 (p > 0-05). The absorption rate of vitamin K-1appears to be modified by the presence of compounds in the incubation medium which either alterthe partition of the vitamin between the micelle and the cell membrane or which change the per-meation characteristics of the compound through the unstirred water layer or modify the physicalcharacteristics of the cell membrane itself. It is possible that some of the above factors modify theabsorption of lipid soluble compounds in general.

Vitamin K-1 (phylloquinone) is the major dietaryform of the vitamin. It is absorbed in vitro by anenergy requiring absorption process which displayssaturation kinetics and is inhibited by nitrogenatmosphere, the addition of 2,4-dinitrophenol, orlow incubation temperatures (Hollander, 1973).In normal circumstances, absorption of the vitaminwould be expected to take place in the presence offatty acids of varying chain lengths and degree ofsaturation as well as differing bile acid concentra-tions. Therefore, the influence of the presence orabsence of the above compounds on the intestinalabsorption of vitamin K-1 was investigated.

Methods

INCUBATION MATERIALSRadioactively labelled vitamin K-1 (phylloquinone)had a specific activity of 41-4 /iCi/,umol and waslabelled with tritium at the 2-methyl position.Unlabelled vitamin K-1 was purchased fromNutritional Biochemicals Co., Cleveland, Ohio.The purity of the radioactive as well as non-radio-active vitamin preparations was ascertained bythin layer chromatography on silica gel-G developedin 10% methanol in benzene (Doisy and Matschiner,

"Address for correspondence: Daniel Hollander, Department ofMedicine, Scott Hall, Wayne State University, Detroit, Michigan48201, U.S.A.

Received for publication 11 February 1976

1970). The compounds were purified by silicic acidcolumn chromatography using increasing concen-trations of benezene in hexane if more than 5%impurities were found (Matschiner et al., 1967).Sodium taurocholate (K & K Laboratories, PlainView, N.Y.) was found to have less than 5%impurities by thin layer chromatography (Gregg,1966). Oleic acid, monoolein, octanoic acid,lecithin, linoleic acid, and butyric acid were pur-chased from Signa Chemical Co., St. Louis, Mo.The micellar incubation solution was prepared in

Krebs phosphate buffer, pH 7-4, with dextroseconcentration of 200 mg/100 ml (Umbreit et al.,1964). The final micellar solution was 10 mM withrespect to sodium taurocholate and containedvarious concentrations of monoglycerides andfatty acids as indicated. The solution was preparedby ultrasound irradiation for five minutes with asonicator (Artek Corporation, Farmingdale, N.Y.).Varying concentrations of radioactive as well asnon-radioactive compounds were added to themicellar solution after sonication by homogenisa-tion of the compounds with the micellar solution in atissue homogeniser. The pH of the incubation me-dium was measured after incubations and was foundto vary between 7 30 and 7 39.

INCUBATION METHODSMale Sprague-Dawley rats weighing between 100and 180 g were fed regular chow (Check-R-Board,

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Factors affecting vitamin K-4 absorption

Novis, Michigan) ad libitum and were not fastedbefore experimentation. The rats were killed bycervical dislocation and the bowel was rinsed insitu with iced 0 9% sodium chloride solution. Theentire small bowel was removed and everted. A10 cm segment immediately distal to the insertionof the common bile duct was designated as theproximal small bowel segment. A second portiondesignated as the distal segment was the distal 10 cmof the small bowel immediately proximal to theileocecal valve. Each segment was subdivided withligatures into five 1 cm everted sacs which wereidentified with tags indicating their exact origin.Peyer's patches were avoided The sacs were placedin a Plexiglass incubation chamber with internaldimensions of 2 x 6 x 30 cm containing 50 mlmicellar incubation medium which had beenaerated with 100% 02 gas and equilibrated to 37°Cbefore experimentation. The oxygen stream waspresaturated with water in order to minimise evap-oration during the procedure. Continuous bubblingof oxygen was maintained throughout the experi-ment. The chamber was positioned in a metabolicwater bath (at 37°C) which was agitated at 40 oscil-lations per minute. The length of all incubationexperiments was five minutes. Immediately afterincubations, the sacs were rinsed for 60 seconds ina beaker containing 300 mll mM sodium taurocho-late solution not containing the vitamin in orderto remove some of the incubation solution whichhad remained adherent to the sac walls. Less than1% of the total radioactivity present on and withinthe sac walls was washed by the taurocholatesolution within the 60 seconds' rinse. The sacs werethen dried at 50°C under 51 cm (20 in.) of mercuryvacuum for 24 hours. All calculations pertaining totissue weight refer to the constant dry weight ofthe tissue.

RADIOACTIVITY DETERMINATIONSTissue radioactivity was determined by total combus-tion into tritiated water or 14C labelled C02 gasusing a sample oxidiser (306 Tri-Carb sample oxi-dizer, Packard Instruments, Downers Grove, Illi-nois). Measurements of radioactivity were carriedto a counting error of 1% using a Beckman LS 250liquid scintillation counter with automatic quenchcalibration at ambient temperature.

SUBSTANTIATION OF RADIOACTIVE PURITYOF ABSORBED VITAMINIntestinal tissue was extracted in a 1:1 mixture ofacetone ethanol. The extract was spotted on silicagel G thin layer plates developed in chloroform(Doisy and Matschiner, 1970). More than 90% ofthe radioactivity from the absorbed phylloquinone

migrated with the same Rf values as that of purephylloquinone standards, indicating that the radio-active label did represent unaltered vitamin K-I.

Results

MORPHOLOGICAL INTACTNESS OF INTESTINALTISSUEAfter incubation, intestinal sacs were placed in 10%formaldehyde solution and processed for lightmicroscopy. Intestinal tissue slides were examinedby two individuals who were not aware of the timesource of each specimen. No morphological changeswere detected during the five minutes of incubatiortime indicating the lack of visible tissue damageduring the experimental procedures.

DETERMINATION OF ADHERENT MUCOSALFLUID COMPARTMENTDespite the taurocholate rinse, a certain volume ofincubation fluid remained adherent to the sacwalls adding radioactive counts to those emittedby the absorbed vitamin. In order to subtract thesecounts from those emitted by the absorbed vitamin,a non-absorbable marker 14C-inulin was addedto the incubation medium of multiple experiments(Esposito and Czaky, 1974). Assuming an equaldistribution of inulin between the bulk aqueousphase and the adherent fluid compartment, thevolume of the adherent layer was determinedspecifically for each segment at each specific timeinterval, and the amount of vitamin K-1 present inthat volume was then subtracted from the apparenttotal absorption value of the vitamin. The mean± SE steady state volumes of proximal and distalintestinal adherent fluid compartments were 49 4 ±0-8 and 46-4 ± 2-3 ,ul/100 mg of tissue respectively.All experimental results reported in this com-munication were corrected by subtraction of theadsorbed amount of vitamin K-1 from the totalapparent amount of absorption.

REQUIREMENT FOR BILE SALTS IN VITAMINK-1 ABSORPTIONClinical observations have suggested that theabsence of bile could result in malabsorption ofvitamin K (Butt et al., 1938). The influence of bilesalt concentration on vitamin K-1 absorption wastherefore investigated. Absorption of the vitaminwhen solubilised in a 10 mM sodium taurocholatemicellar solution was examined in six animals.The mean ± SE absorption rates of the vitamin were26-1 ± 1i4 and 12-7 ± 1 1 nmol/min/100 mg oftissue by the proximal and distal small bowelrespectively (Fig. 1). When the bile salt concentrationwas increased to 20 mM the absorption rate of

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D. Hollander and Elena Rim

30'

SMALL INTESTINE[VIT K-3 . 300,UM

O PROXIMAL= DISTAL* p<0.05

00

z

z

NA PLURONIC NATAUROCHOLATE TAUROCHOLATE

10 mM I93zAM 20mM

Fig. 1 The effect on the absorption rate ofvitamin K-iby theproximal and distal smal bowel ofchanges in theconcentration ofbile salts or their complete absence.The number ofanimals used is indicated by the numeralat the base ofthe bars. Each bar represents the mean ± SEcalculatedfrom absorption values of10 different sacsperanimal. The statistical significance ofthe differencesbetween experiments was calculated using Student's t testforpairedobservations.

vitamin K-1 was decreased significantly (p <0-02)to 13-7 ± 1.9 and 6-9 ± 0-8 nmol/min/100 mg oftissue respectively (Fig. 1). The effect of completeremoval of sodium taurocholate from the incubationsolution was tested by the use of non-ionic solubiliser-pluronic F-68 (polyoxypropylene-polyoxyethyleneMW 8350) at 19-3 ,tM concentration. The rate ofabsorption of the vitamin by the proximal and distalsmall bowel segments solubilised in pluronic solu-tion was 20-3 ± 1-7 and 14 3 + 2-3 nmol/min/100mgof tissue respectively (Fig. 1). The above rates were

significantly lower (p = *03) only in the proximalsmall bowel when compared with baseline absorp-tion rates with 10mM sodium taurocholate solution.

INFLUENCE OF FATTY ACID CHAIN LENGTH

AND SATURATION ON VITAMIN K-I ABSORPTION

RATEThe influence of chain length and degree of satura-

tion of fatty acids on the absorption rate of vitaminK-1 by the proximal and distal small bowel wasinvestigated. The absorption rate of the vitaminwhen solubilised in sodium taurocholate micellarsolution not containing fatty acids was 26-11 +1-4 and 12-7 ± 1 1 nmol/min/100 mg of proximaland distal small bowel respectively (Fig. 2). Whenbutyric acid (C4:0) and octanoic acid (C8:0) wereadded in a 2-5 mM concentration to the 10 mMsodium taurocholate solution no significant changein the absorption rate of the vitamin was observed(p > 0-05) (Fig. 2). The separate additions ofthe long chain unsaturated fatty acids oleic (C18:1)and linoleic (C18:2) resulted in a significant (p <0 05) decrease in the absorption rate of vitamin K-1both by the proximal and distal small bowel seg-ments (Fig. 2). The decrease in the absorption rateof the vitamin was greater with the addition ofthe polyunsaturated linoleic acid (C18:2) than withthe monounsaturated oleic acid (C18:1).

INFLUENCE OF MONOGLYCERIDES ANDLECITHIN ON VITAMIN K-1 ABSORPTIONAs the intestinal milieu contains monoglyceridesand lecithin, their effect on the absorption rate ofvitamin K-1 was compared with absorption of thevitamin out of pure sodium taurocholate micellarsolution. Monoolein and 3-phosphatidyl choline(lecithin) were added to the 10 mM taurocholatemicellar solution in a 2-5 mM concentration inseparate groups of experiments. A significant(P < 0 05) decrease in the absorption rate of vitaminK-1 both by the proximal and distal small bowel wasseen with the addition ofeither monooleate or lecithinto the incubation medium (Fig. 3).

Discussion

Previous studies of vitamin K-1 absorption (Hol-lander, 1973) did not define the influence of thevarious constituents of the intestinal lumen on theabsorption of the vitamin. Elucidation of the possibleinfluence of fatty acids, bile salts, monoglycerides,and lecithin on vitamin K-1 absorption is importantnot only because of the physiological role of thevitamin but also because of the applicability ofthe concepts derived from such investigation tothe general field of absorption of lipid solublecompounds.The major determinants which could modify the

absorption rate of lipid soluble compounds arethe chemical preparation of the compound forabsorption-that is, pancreatic factors (Simmonds,1974)-the composition and characteristics ofthe micelle (Simmonds, 1974), partition of thecompound between the micelle and the mono-

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Factors affecting vitamin K-i absorption

SMALL IIjTESTINEVIT K-IJ * 300u

TF1i.

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NOWTY ACID BUT'(C4l

I

P0)

±

S.

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rCf PROXIMAL- INSTAL* P<O.O5

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FIg. 2 The effect on the absorption ofvitamin K-1 oftheaddiionof2*5 mMfatty acidsofvayin chain kngthandsaturation. The results represent the mean + SEforabsoption ofthe vitamin by 10 different sacsper animal.The nwnber ofanima per experiment is indicated at thebas ofthe bar. The statistical significance of thedifferences between experinents was calculated usitnStudent's t testforpairedobservations. The incubationsolution was conmosedof10mMsodium taurocholateinphosphate buffer.

IL3lCIG(CI:l)

LINUUK.(Cl6:2)

I PROXIMAL- DISTAL* p<0.O5

Fig. 3 The effect on the absorption rate ofvitamin K-iofthe addition of2.5 mMmonoolein and lecithin to thesodium taurocholate incubation solution. The valuesplotted are mean ± SEof10 sacsper animal. The numberofanimalsper experiment is indicated by the numeral atthe base ofthe bargraph. The statistical significance ofthedifferences between experiments was calculated usingStudent's t testforpaired observations. The incubationsolution was composedof10mMsodium taurocholateinphosphate buffer.

20 -

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SMALL INTESTINECVIT K-13 . 300,uM

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11SEUNE MONOOLEATE LECITHIN2t5 ml 2S5 mM

p- o ip-

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19% M. m-r%9kf :p"

(CO :M

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D. Hollander and Elena Rim

molecular aqueous solution (Diamond and Katz,1974), the permeation coefficient of the compoundthrough the unstirred water layer (Westergaardand Dietschy, 1974) the characteristics of the cellmembrane itself (Vandenheuvel, 1971), and, finally,factors within the cell which affect the rate of transferof the compound from the cell into the circulation(Thompson et al., 1969).

In this investigation, pancreatic factors have beencircumvented by the use of chemically definedcomponents of triglyceride hydrolysis. The completeremoval of bile salts from the incubation mediumand the substitution of a non-ionic detergent,pluronic F-68, resulted in only a minimal decrease inthe absorption rate of vitamin K-1 by the proximalsmall bowel and no decrease in the absorptionrate by the distal small bowel (Fig. 1). Thus, bilesalts appear to have no direct role in the absorptiveprocess of vitamin K-1 except for their micellarsolubilising properties. Increasing the concentrationof sodium taurocholate up to 20 mM resulted in asignificant (p < 0-05) decrease in the absorptionrate of vitamin K-1 both by the proximal and distalsmall bowel segments (Fig. 1). The above may bedue to changes in the partition of vitamin K-1between the micelle and the cell membrane or theaqueous monomolecular phase (Simmonds, 1974)in favour of the micelle. A shift of vitamin K-1 intothe micelle would be expected to result in diminishedabsorption of the vitamin due to decreased releaseofthe vitamin from the micelle (Diamond and Wright,1969). It is also possible that a high concentration ofbile salts may in some way modify the character-istics of the cell membrane itself, making it lesspermeable to lipid soluble compounds such asvitamin K-1 (Harries and Sladen, 1972).The effect of expansion of the micelle by the

addition of fatty acids of varying chain lengths anddegree of saturation on the absorption of vitaminK-1 was examined in the next series of experiments.When compared with the absorption rate of thevitamin out of a pure bile salt micelle, the additionof a short chain fatty acid-butyric acid (C4:0) andthe medium chain fatty acid-octanoic acid (C8:0)caused an increase in the absorption rate of thevitamin primarily by the distal small bowel(Fig. 2) which was not statistically significant (p >005). The addition of the long chain fatty acids-oleic (C18 :1) and linoleic (C18 :2) significantly(p < 0-05) decreased the absorption rate of thevitamin when compared with its rate of absorptionout of pure bile salt micelles (Fig. 2). The decreasein the absorption rate observed in the presence oflong chain fatty acids could be due to interactionsat two major points in the absorptive pathway ofvitamin K-1. The long chain fatty acids are known

to change the physical characteristics of the micelle(Simmonds, 1974) which could result in a greateraffinity of the micelle for vitamin K-1, hence dim-inishing its transfer to the absorptive cell membrane(Lee et al., 1971). An alternative explanation is thepossibility that the expanded micelle containinglong chain fatty acids may diffuse through theunstirred water layer (Wilson and Dietschy, 1974)at a slower rate because of its large size, resulting ina decrease in the absorption rate of the vitamin.Because the incubation period in our experimentalprocedure is short. it is unlikely that the decreasein the absorption rate ofvitamin K-1 that is seen withthe addition of the long chain fatty acids would bedue to competition between the compounds fortransport out of the cell intothelymphaticcirculation.The inhibition of the rate of absorption of vitaminK-1 noted with the addition of 2-5 mM monooleinor lecithin (Fig. 3) is likely to be due to mechanismsand considerations as described above for the effectof the long chain fatty acids. It should also be con-sidered that the decrease in vitamin K-1 absorptionseen after the addition of lecithin (Fig. 3) may bedue to changes induced by lecithin in the physicalcharacteristics of the cell membrane itself (Vanden-heuvel, 1971). The observed increased absorption ofthe vitamin with the additon of short and mediumchain fatty acids even though statistically insig-nificant is still noteworthy. The effect of these fattyacids is most noticeable in the distal bowel segments.The reason for these observations is unknown. Theshort chain fatty acids are less likely to affect themicellar characteristics than are the long chain fattyacids because of their greater aqueous solubility andindependence from micellar solubilisation as arequirement for absorption (Jackson, 1974).Whether the short and medium chain fatty acidschange the characteristics of the lipid membraneitself, thereby affecting vitamin K-1 absorption rate,is unknown.The above experiments demonstrate that the

composition of the micelle and the presence orabsence of other fat soluble compounds, can haveprofound effects on the absorption rates of a lipidsoluble compound-vitamin K-1. These factors couldmodify the absorption of other lipid soluble com-pounds as well. Indeed, polyunsaturated fatty acidshave been shown to interfere with vitamin E-1absorption (Horwitt, 1962). Further investigationinto the factors which may modify the absorptionof other lipid soluble compounds such as digitaliscompounds, steroids, and thiazide diuretics shouldenlarge our understanding of this important facet ofabsorption.

This study was supported by grants AM17607 and

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Factors affecting vitamin K-i absorption 455

RR05384 from the National Institute of Health ofthe United States Public Health Service. Researchsupport was also generously supplied by the Skill-man Foundation of Detroit, Michigan, and theHarper Hospital research and education fund. Theauthors are grateful to Miss Barbara Glispin forsecretarial assistance.

References

Butt, H. R., Snell, A. M., and Osterberg, A. E. (1938). Theuse of vitamin K and bile in treatment of the hemorrhagicdiathesis in cases of jaundice. Proceedings of the StaffMeetings of the Mayo Clinic, 13, 74-80.

Diamond, J. M., and Katz, Y. (1974). Interpretation of non-electrolyte partition coefficients between dimyristoyllecithin and water. Journal of Membrane Biology, 17,121-154.

Diamond, J. M., and Wright, E. M. (1969). Biologicalmembranes: the physical basis of ion and nonelectrolyteselectivity. Annual Review of Physiology, 31, 581-646.

Doisy, E. A. Jr, and Matschiner, J. T. (1970). Biochemistryof vitamin K. In Fat-Soluble Vitamins, pp. 293-299.Edited by R. A. Morton. Pergamon: Oxford.

Esposito, G., and Csaky, T. Z. (1974). Extracellular space inthe epithelium of rats' small intestine. American Journal ofPhysiology, 226, 50-55.

Gregg, J. A. (1966). New solvent systems for thin-layerchromatography of bile acids. Journal ofLipid Research, 7,579-581.

Harries, J. T., and Sladen, G. E. (1972). The effects of differentbile salts on the absorption of fluid, electrolytes, and mono-saccharides in the small intestine of the rat in vivo. Gut,13, 596-603.

Hollander, D. (1973). Vitamin K1 absorption by evertedintestinal sacs of the rat. American Journal of Physiology,225, 360-364.

Horwitt, M. K. (1962). Interrelations between vitamin E andpoly-unsaturated fatty acids in adult men. Vitamins andHormones, 20, 541-558.

Jackson, M. J. (1974). Transport of short chain fatty acids.In Intestinal Absorption, pp. 673-709. Edited by D. H.Smyth. Plenum Press: New York.

Lee, K. Y., Simmonds, W. J., and Hoffman, N. E. (1971).The effect of partition of fatty acid between oil andmicelles on its uptake by everted intestinal sacs. Bio-chimica et Biophysica Acta, 249, 548-555.

Matschiner, J. T., Taggart, W. V., and Amelotti, J. M. (1967).The vitamin K content of beef liver. Detection of a newform of vitamin K. Biochemistry, 6, 1243-1248.

Simmonds, W. J. (1974). Absorption of lipids. In Gastro-intestinal Physiology, pp. 343-376. Edited by E. D. Jacobsonand L. L. Shanbour. University Park Press: Baltimore.

Thompson, G. R., Ockner, R. K., and Isselbacher, K. J.(1969). Effect of mixed micellar lipid on the absorption ofcholesterol and vitamin D3 into lymph. Journal of ClinicalInvestigation, 48, 87-95.

Umbreit, W. W., Burris, R. H., and Stauffer, J. F. (1964).Manometric Techniques, 4th edn, p. 132. Burgess: Min-neapolis.

Vandenheuvel, F. A. (1971). Structure of membranes androle of lipids therein. Advances in Lipid Research, 9,161-248.

Westergaard, H., and Dietschy, J. M. (1974). Delineation ofthe dimensions and permeability characteristics of thetwo major diffusion barriers to passive mucosal uptake inthe rabbit intestine. Journal of Clinical Investigation, 54,718-732.

Wilson, F. A., and Dietschy, J. M. (1974). The intestinalunstirred layer; its surface area and effect on active trans-port kinetics. Biochimica et Biophysica Acta, 363, 112-126.

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