In vivo evidence that intrinsic factor-cobalamin complex traverses the human intestine intact

328

Biochimica et Biophysica Acta, 675 (1981) 328 333

Elsevier/North-Holland Biomedical Press BBA 29646 IN VIVO EVIDENCE THAT INTRINSIC FACTOR-COBALAMIN COMPLEX TRAVERSES THE HUMAN INTESTINE INTACT JEAN-PIERRE NICOLAS, MARLENE JIMENEZ, GEORGE MARCOULLIS * and YVES PARMENTIER Laboratoire de Biochimie Medicale, Universitd de Nancy I, U.E.R. Alimentation et Nutrition, B.P. 1080-54019, Nancy Cedex (France)

(Received October 14th, 1980)

Key words: Vitamin B l 2 transport; Cobalamin; Intrinsic factor," R type protein; (Human intestine)

Ingested cyano[ s 7Co]cobalamin was recovered as coupled to intrinsic factor from the jejunum of healthy humans. This vitamin-protein complex and the analogous complex from patients having exocrine pancreatic insufficiency were indistinguishable from each other in terms of molecular radius (3.30 nm), ionic nature (eight well-defined isoproteins isoelectric at pH 4.71, 4.84, 4.90, 5.13, 5.23, 5.31, 5.40 and 5.69), and antigenic structure. These findings indicate that the pancreatic proteases do not alter the intrinsic factor cobalamin complex in vivo. Purified R type protein-cyano[S~Co]cobalamin complex recovered from patients with exocrine pancreatic insufficiency was similar to the analogous gastric protein in terms of molecular radius (a = 4.78 nm) and types of isoproteins (seven well-defined isoproteins isoelectric at pH 2.70, 3.03, 3.38, 3.74, 3.87, 4.05 and 4.20). However, this R protein complex from patients and the intrinsic factor complex from both control subjects and from patients was comprised of more of the acidic type of isoproteins, thereby supporting the notion that carbohydrate-rich isoproteins are more resistant to digestion in the intestine.

Introduction Recent in vivo studies demonstrated that ingested cyano [s 7Co ]cobalamin traverses the upper intestinal lumen coupled to R proteins** in patients with exocrine pancreatic insufficiency as opposed to healthy subjects where the vitamin is transported coupled to intrinsic factor [1]. These observations provided substantial evidence for the validity of the 'inhibited cobalamin absorption theory' [1,2], which includes that diminished pancreatic secretion leads to impaired * To whom reprint requests should be sent. Permanent address: Section of Hematology/Oncology, Brooklyn Veterans Administration Medical Center, Brooklyn, NY 11209, U.S.A. ** The term 'R protein' was originally devised to denote a cobalamin-binding protein in human gastric juice that was devoid of intrinsic factor activity, but the same term is currently used for other immunologic,ally cross-reacting proteins present in most biologic fluids and body tissues.

cobalamin absorption due to non-degradation of the unabsorbable vitamin complexes with R proteins. Another current theory (henceforth referred to as 'the defective intrinsic factor-activation theory') suggests that pancreatic proteases exert a direct effect on the 'precursor' gastric intrinsic factor molecule thereby converting it to a functionally active substance [3]. In this context intrinsic factor in the jejunum ought to differ more or less from the analogous protein in patients with exocrine pancreatic insufficiency. Consequently, systematic comparative physicochemical analysis of cobalamin coupled to intrinsic factor before as well as after in vivo interaction of that complex with pancreatic proteases would help clarify the question about the fate of intrinsic factor in the intestine. The present report addresses itself to this experimental approach and demonstrates that no detectable alteration of intrinsic factor occurs during intraluminal transport in man. In analogy to the terminology used for enzymes

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329 and their complexes with cofactors, the term 'holo' form will henceforth be referred to for transport proteins carrying cobalamin, and the term 'apo' form for the carrier proteins free of cobalamin [4]. Subjects, Materials and Methods All radioactive compounds were purchased from the Radiochemical Centre, Amersham, Buckinghamshire, U.K. All other chemicals, proteins and enzyme inhibitors were obtained as described elsewhere [ 1]. Sub/ects. Eight volunteer control subjects, and eight patients with proven exocrine pancreatic insufficiency (as indicated by positive medical history including pancreatic calcifications, steatorrhea, findings obtained using ultrasonography or computerized tomography and absence of pancreatic enzyme activity in the jejunal fluid or a combination thereof) [ 1] were studied. Collection of intestinal fluid. A triple lumened polyvinyl tube which was opaque to radioactivity [ 1] was used for intubation. One tube (naso-gastric tube opened into the gastric succus and the other two into the jejunum (130-140 cm from the incisor teeth). Following intubation all subjects fasted and did not receive pancreatic supplement. The subsequent morning, 6 ~g pentagastrin/kg body weight was injected subcutaneously and this was followed 20 min later by administration through the nasogastric tube, first of a test meal [1] lacking cobalamin, and second, of 250 ng cyano[STCo]cobalamin (2 pCi), coupled 53% to intrinsic factor and 47% to R proteins in gastric juice. This was followed by continuous aspiration of jejunal fluid containing cyano [s 7Co ] cobalamin. The pooled fluid from each subject was depepsinized by pH adjustments and mixed with ~ volume of 0.1 M phosphate buffer, pH 7.4, containing 0.02% (w/v) NAN3, 0.02 mM phenylmethylsulfonyl fluoride, 5 0 0 0 U aprotinin per 1 and 0.154 M NaC1. Chromatographic procedures. Sephadex G-200 columns were packed and calibrated as described recently [1 ]. Isoelectrofocusing of intrinsic factor and R proteins was carried out at 4°C in l l0-ml standard LKB columns packed with 1% (w/v) Ampholines of pH range 4.0-6.0 and 2.5-5.0, respectively. Purification procedures. Intestinal fluid from control subjects contained cyano[STCo]cobalamin cou-

pled to intrinsic factor (vide infra). The aspirates from exocrine pancreatic-insufficiency patients contained in addition cyano [s 7Co ] cobalamin coupled to R proteins. Aliquots containing 0.2-0.3 btCi cyanois 7Co ] cobalamin from each individual intestinal fluid were concentrated to 1.0-2.0 ml final volume by ultrafiltration, clarified by centrifugation at 27 000 × g for 30 min and chromatographed through Sephadex G-200 as such and also after 1 h incubation with excess pernicious anaemia serum type II [5]. The fractions that composed the intrinsic factor-cyano[STCo]cobalamin complex and the R protein-cyano[STCo]cobalamin complex peaks in the included volume of the respective columns were pooled, concentrated by ultrafiltration, and designated 'purified intestinal intrinsic factor and R protein', respectively. Results

Only one STCo-radioactive protein complex, identified as intrinsic factor-cyano[ s 7Co]cobalamin, was found (data not shown) to be present in the intestinal juice of healthy subjects. The intestinal juice from patients having exocrine pancreatic insufficiency contained in addition R protein-cyano [s 7Co ] cobalamin complex. Thus, intrinsic factor-cyano[S 7Co ]cobala. min complex was prepared from patients and control subjects whereas, the R protein-cyano [s 7Co ] cobalamin complex was derived from patients only. Molecular radii. The molecular radii were estimated by the formula of Ackers [6] illustrated below:

Kd =

(1 - (or[r)) 2 (1 - 2 . 1 0 4 ( a / r )

+ 2.09(c~/r) 3

- 0.95(a/r) s) where Kd, distribution coefficient; a, Stokes radius and r, column effective pore radius. Estimations for the roots of the polinomial were carried out by a programmed computer (Mitra, France) using the method of Newton [7] after substitution of x for a/r. The numerical solution of the equation of Ackers for x enabled the estimation of the column constant rl (21.2 -+0.8 nm) and r2 (20.3 -+0.9 nm) when human serum albumin (Stokes radius, 3.61 nm) and immunoglobulin-G (Stokes radius, 5.22 nm) were used as the reference proteins, respectively. The Stokes

330 radii were computed using the column constants r 1 and r2 and the experimental Kd values obtained with the two proteins under study. When immunoglobulin-G was used as marker, the Stokes radius for the holo intrinsic factor and holo Rprotein from exocrine pancreatic insufficiency patients were 3.17-+ 0.18 nm and 4.59 -+0.10 nm, respectively. The corresponding figures estimated when se.rum albumin was used were 3.30 -+0.08 nm and 4.78 -+0.10 nm. Similar estimations made with holo intrinsic factor purified from the controls agreed within 2.6% with the values calculated for the analogous protein from exocrine pancreatic insufficiency patients. Isoprotein patterns. Preliminary isoelectrofocusing analysis indicated that the purified holo intrinsic factor from control subjects did not differ from that obtained with samples from exocrine pancreatic-insufficiency patients. The holo intrinsic factor resolved into eight isoproteins (Fig. 1A)isoelectric at pH4.71 (-+0.03), 4.84 (-+0.01), 4.90 (-+0.03) 5.13 (-+0.02), 5.23 (-+0.02), 5.31 (-+0.02), 5.40 (-+0.02) and 5.69 (-+0.01). The corresponding figures for the purified hole R protein were (Fig. 1B): pI values 2.70 (-+0.02), 3.03 (-+0.04), 3.38 (-+0.04), 3.74 (-+0.03), 3.87 (_+0.03), 4.05 (-+0.02) and 4.20 (-+0.03). The main isoproteins for the hole R protein were isoelectric at pH 3.03 and 3.38 and those of hole intrinsic factor had pl values of 4.90 and 5.13. The estimated [I] mean pI for hole intrinsic factor was 5.09 (-+0.10) and for the hole R protein was 3.51 (-+0.10).

Integrity of the antigenic structure of the intn'nsic factor-cyanocobalamin complex in intestinal juice. Several type II anti-intrinsic factor sera wereincubated separately with cyano [s 7Co ] cobalamin coupled to human gastric juice and then filtered through Sephadex G-200. The STCo-radioactivity precoupled to intrinsic factor in the gastric juice shifted from the region after serum albumin (Fig. 2A) to the region before immunoglobulin-G (Fig. 2B). Four sera formed STCo-radioactive immunocomplexes with an estimated [I] molecular mass and a Stokes radius of 265 000 and 6.95 -+0.07 rim, respectively. As the molecular mass of IgG and hole intrinsic factor estimated by gel filtration are 150000 [8] and 56000 [9], respectively, the inference was drawn that the observed 265000 dalton immunocomplexes contained a single (divalent) anti-intrinsic factor IgG-type antibody, each one reacting with two molecules of

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Fig. 2. A. Cyano[ s 7Co]cobalamin coupled to normal h u m a n gastric juice eluted from Sephadex G-200 (solid line) 47% bound to R protein and 53% bound to'intrinsic factor. B. Preincubation of a similar sample with excess type II antiintrinsic factor serum prior to chromatography shifted (closed circles) the cobalamin-intdnsic factor complex (CblIF) peak into the region before liSI-labeled IgG because of the formation of immunoeomplexes ([CbI-IF]-IgG). Four sera which formed immunocomplexes duting slightly b e f o r e 12s I-labeled IgG (e.g. see immunocomplexes in Fig. 2B) w e r e pooled together. C. Preincubation of this pooled serum with a sample of s 7Co.radioactive gastric juice shifted (opened circles) the cobalamin-intrinsic factor complex peak into

greater than 950000 daltons [10]. The four divalent, but yet monodeterminant, antibody sera were pooled together and 0.1 ml aliquots were incubated with 3 ng cyano[STCo]cobalamin coupled to gastric juice and then filtered through Sephadex G-200. Now the STCo-radioactive immunocomplexes of intrinsic factor shifted into the region of the totally excluded column volume (Vo), corresponding to a molecular mass of 553 000 (Fig. 2C). In subsequent experiments 1 ml aliquots of the pooled anti-intrinsic factor serum were incubated with concentrated intestinal samples collected from each patient and also from each control subject and then filtered through Sephadex G-200. As observed in the experiments with gastric juice, the STCo-radioactive peaks which previously eluted after serum albumin now eluted near the Vo, with an average estimated molecular mass and Stokes radius of 550000 and 14.16 +-0.15 nm, respectively (e.g. see Fig. 2C). The molecular composition of the 550000 dalton immunocomplex in terms of number of IgG molecules per intrinsic factor-cyano[STCo]cobalamin complex(es) can be deduced by taking into account the fact that only divalent monodeterminant antibodies have been used in the reactions described above (see Fig. 2). Thus, the increase of molecular mass from 265 000 to 550 000 indicated that the immunocomplexes contained three divalent antiintrinsic factor antibodies since the expected molecular mass of an immunocomplex containing two divalent IgG molecules would not exceed a molecular mass value of 420000. This conclusion shows that each intrinsic factor molecule contained in the 550 000 dalton immunocomplex, whether from intestinal or from gastric fluid, comprises three different antigenic determinants. Therefore, the 'intestinal' intrinsic factor comprises three distinct antigenic determinants which crossreact with three analogous configurations present on the polypeptide core of the known gastric intrinsic factor. the region of V0 corresponding to a molecular mass o f 553 000. Repetition of the experiments illustrated in Figs. A, B, C, using s 7Co.radioactive intestinal fluids instead of gastric juice resulted in similar observations, indicating that three antigenic determinants present in the 'intestinal' intrinsic factor molecule crossreaet with respective eonfigttrations present on the analogous gastric protein. BD, Blue Dextran; HSA, human senlrn albumin; 12sI-, liSI-labeled; CbI-R, eobalamin-R protein complex.

332 Discussion

Previous studies in the pig [11] and more recent studies in man [12] have indicated that intrinsic factor free of cobalamin in basal jejunal fluid is similar to the known gastric intrinsic factor but the fate of the holo form of that protein under stimulation of intestinal and pancreatic secretion has not been determined. Regardless of whether pancreatic proteolysis is or is not directly involved in the intrinsic factor function, clarification of the exact physicochemical and immunochemical nature of intrinsic factor carrying cobalamin in the intestine is needed for complete understanding of normal intrinsic factor-mediated absorption of cobalamin. All recognized activation mechanisms include a chemical modification which directly or indirectly alters the physicochemical or conformational or antigenic properties of the proteins involved. In many instances these alterations can be readily demonstrated because the activation mechanisms require the removal of long polypeptide fragments from the intact precursor molecules [10,13]. In some rare cases the modifications may involve merely the removal of a single type of carbohydrate residues or loss of an amide or a simple cleavage of disulfide bridges with or without fragmentation [ 13,14]. These changes are relatively 'mild' but they may as well be determined if the modified proteins were analyzed by high-resolution power procedures, e.g. isoelectrofocusing. The considerations outlined above suggested to us that if intrinsic factor were actually activated then changes either in molecular dimensions, in isoprotein composition or in antigenic structure ought to accompany this hypothetical phenomenon during intraluminal transport. The molecular radius of 3.30 nm estimated for the 'intestinal' holo intrinsic factor does not significantly differ from the value of 3.28 nm previously estimated [15] for the human gastric protein, and it nonetheless precludes with certainty the possibility that a polypeptide fragment of considerable size splits off from intrinsic factor during transport. Furthermore, the results illustrated in Fig. 2 demonstrate that the 'intestinal' intrinsic factor reacts with three monodeterminant anti-intrinsic factor antibodies, each one directed against a distinct antigenic determinant on the polypeptide core of the known gastric intrinsic

factor. This failure to demonstrate any configurational changes or alterations in the reactivity of any one out of the three antigenic determinants identified on the gastric factor further diminishes the possibility that the intrinsic factor molecule is subject to alteration during transport. Lastly, isoelectrofocusing fractionated the 'intestinal' intrinsic factor isoprotein array into eight distinct components [cf. Refs. 1 6 18]. As shown in Fig. 1, the 'intestinal' intrinsic factor molecule is comprised ofisoproteins typical for the known gastric intrinsic factor. No additional more acidic or more neutral components were observed. Thus, any chemical modification of the type of selective removal of terminal carbohydrate residues or loss of an amide also appears to be an unlikely possibility. These similarities of antigenic structure, isoprotein composition, and molecular dimensions of the gastric and 'intestinal' intrinsic factors demonstrate that there occur no structural changes of the molecule during intraluminal transport of cobalamin and that, therefore, the interaction of intrinsic factor with pancreatic proteases does not bear on the physicochemical integrity and, presumably, on the biologic activity of the intrinsic factor-cobalamin complex. This inference is consonant with other in vitro findings where: I, intrinsic factor purified from intestinal juice and also from gastric juice exhibited similar avidity for binding to solubilized intrinsic factor receptors [11, 12] ; 2, intrinsic factor-cyano [S7Co]cobalamin complex incubated in isolated ileal loops of guinea pigs was fully absorbed and transposed intracellularly [ 19]; and 3, biosynthetically prepared intrinsic factor (that is, the protein which has never interacted with pancreatic proteases) [20] enhanced by 40-fold the uptake of cyano[SVCo]cobalamin by isolated guinea pig ileal cells. The isoprotein phenomena observed with the 'intestinal' intrinsic factor support previous suggestions [21-23] about the possible protective role of the carbohydrate residues of intrinsic factor and R proteins. In brief, the 'intestinal' intrinsic factor cobalamin complex, as well as the R protein complex, compared to the previously studied gastric analogous proteins [9] were now found here to contain higher relative concentrations of the acidic type of isoproteins. This is clearly shown in Fig. 1 and it is reflected by the shift of the mean pI of intrinsic factor and R protein from 5.20 (gastric intrinsic factor) [9] and

333

395 (R protein in gastric juice) [9] to 5.09 ('intestinal' intrinsic factor) and 3.51 ('intestinal' R protein), respectively. Furthermore, the intrinsic factorcobalamin and R protein-cobalamin complexes in the intestinal juice had a mean Stokes radius of 3.30 nm and 4.78 nm and an estimated molecular mass [1] of 5 7 0 0 0 and 125000, respectively. Both values are slightly higher than the corresponding figures estimated by the same procedures for the gastric analogous proteins [9,15]. These differences reflect variations in carbohydrate content (see Refs. 21 and 23) and, along with the isoelectrofocusing profdes, indicate that the intrinsic factor-cobalamin and R protein-cobalamin complexes in the intestinal lumen consist of isoproteins highly enriched in carbohydrate content. Presumably, the appearance of carbohydrate highly enriched isoproteins in the jejunum cannot be attributed to any synthetic biological process taking place in the intestinal lumen but simply to a selective in rive survival of glycoisoproteins which are generally considered to be more resistant to digestion [ 2 1 23]. It is, therefore, concluded from the present findings and from other recently reported observations from this laboratory that the single detectable chemical modification that the intrinsic factor-cobalamin complex undergoes during intraluminal transport concerns a selective diminution o f its more neutral type isoproteins [1,9,1 2]. Acknowledgements G.M. is a visiting Professor of Medicine under the auspices o f I.N.S.E.R.M. in the University of Nancy (Decision No. 79-659-DPC-E-GW-AP). Institutional grants were received from I.N.S.E.R.M. (No. 78.1.249.7). We thank Mr. Philippe Gerard for excellent technical assistance.

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