Studies on the Transport and Cellular Distribution of Vitamin A in Normal and Vitamin A-deficient Rats with Special Reference to the Vitamin A-binding Plasma Protein*

Abstract The retinol-binding protein (RBP) and thyroxine-binding prealbumin were isolated from rat serum. Under physiological conditions RBP and prealbumin form a protein complex. Rat RBP exhibited characteristics very similar to those previously encountered for the human and monkey counterparts, including the association constant to prealbumin (8 x 106 m-1). Thyroxine-binding prealbumin in rat is in contrast to its human counterpart the major thyroxine carrying plasma protein. Its molecular weight differs from that of the human protein (51,000 and 62,000, respectively) in spite of its similar binding constant for thyroxine. The role of the vitamin A-transporting protein complex was investigated with respect to the tissue distribution of [3H-]-vitamin A in normal and vitamin A-deficient rats. It was demonstrated that the hepatocytes which stored virtually all liver vitamin A could rapidly mobilize newly administered vitamin, and the retinol occurred exclusively bound to RBP. The mobilization of liver vitamin A was unaffected by prior actinomycin D treatment of the rats, which suggests that newly synthesized RBP requires retinol for its release from the hepatocytes. An investigation was also undertaken of the role of the kidney as a storage organ for vitamin A. Kinetic measurements showed that the kidney received most of its vitamin A from RBP in contrast to the liver which gets its main supply from the chylomicrons. Fractionation of kidney cortex cells on a discontinuous silica gel gradient indicated that most vitamin A resided in tubular cells. The vitamin-containing cell fractions were shown to possess the property to take up RBP in vivo. These results suggest that some vitamin A may reach the kidney by a process of glomerular filtration followed by tubular reabsorption.


SUMMARY
The retinol-binding protein (RBP) and thyroxine-binding prealbumin were isolated from rat serum. Under physiological conditions RBP and prealbumin form a protein complex. Rat RBP exhibited characteristics very similar to those previously encountered for the human and monkey counterparts, including the association constant to prealbumin (8 x lo6 M-l).
Thyroxine-binding prealbumin in rat is in contrast to its human counterpart the major thyroxine carrying plasma protein.
Its molecular weight differs from that of the human protein (51,000 and 62,000, respectively) in spite of its similar binding constant for thyroxine.
The role of the vitamin A-transporting protein complex was investigated with respect to the tissue distribution of [3H]vitamin A in normal and vitamin A-deficient rats. It was demonstrated that the hepatocytes which stored virtually all liver vitamin A could rapidly mobilize newly administered vitamin, and the retinol occurred exclusively bound to RBP. The mobilization of liver vitamin A was unaffected by prior actinomycin D treatment of the rats, which suggests that newly synthesized RBP requires retinol for its release from the hepatocytes.
An investigation was also undertaken of the role of the kidney as a storage organ for vitamin A. Kinetic m.easurements showed that the kidney received most of its vitamin A from RBP in contrast to the liver which gets its main supply from the chylomicrons.
Fractionation of kidney cortex cells on a discontinuous silica gel gradient indicated that most vitamin A resided in tubular cells.
The vitamin-containing cell fractions were shown to possess the property to take up RBP in vivo. These results suggest that some vitamin A may reach the kidney by a process of glomerular filtration followed by tubular reabsorption.
The basic features for the intestinal uptake and transport of vitamin A are now well understood.
From the intestines, retinylesters are transported together with the chylomicrons to the liver, the main storage site for vitamin A (cf. Reference 1). Detailed studies on the enzymat.ic processing of vitamin A in the intestinal cells and in the liver have been published (2)(3)(4)(5)(6). Results on the distribution of vitamin A among hepatocytes and Kupfer cells within the liver are, however, conflicting (cf. Rcference 7). Goodman et al. (8) have in an elegant study shown that newly absorbed vitamin A is present in second greatest abundance in the kidneys.
No detailed information is, however, available concerning the uptake or the cellular distribution of the vitamin in this organ.
Recent studies by Goodman and co-workers (9) and in this laboratory (10,11) have shown that vitamin A in human blood is transported by a small plasma protein, the retinol-binding protein.
Previous studies have shown that in plasma of humans suffering from vitamin A deficiency the levels of serum RHP, vitamin A, and prealbumin are substantially lowered and a high degree of correlation exists for the amounts of RBP and prealbumin over a IO-fold concentration range (17). Similar findings have also been reported in liver disease (16). These results warrant a detailed study of the in viva regulation of the synthesis and degradation of the individual components constituting the vitamin A-transporting protein complex. Since the two-protein transporting system for vitamin A occurs in humans as well as in monkeys (18), it was reasonable to assume that a similar system is present also in other vertebrates.
To obtain a suitable animal model for future studies on the biosynthesis of RBP and prealbumin, now well under way in this laboratory, as well as on the metabolism of vitamin A in target cells, we describe in the present communication basic characteristics for the transporting system of vitamin A in normal and vitamin A-deficient rats. During the course of this work the prealbumin-RBP complex of the rat was isolated and charalterized.
The time course of the distribution of radioactively labeled vitamin A in liver, kidney, and blood following intravenous administration has been studied. In addition, data are given for the relative distribution of vitamin A among fract,ionated liver and kidney cells.

Materials
Serum for the isolation of RBP and prealbumin was obtained from blood collected from the abdominal aortas of male Sprague-Dawley rats. The serum was processed immediately or stored frozen until further processing.
Retinol and retinylacetate were purchased from Sigma Chemical Co. Radioactive [II, (specific activity 0.25 mCi per mg) was a kind gift by Dr. 0. Wiss, Hoffmann-La Roche Inc., Basle. Carrierfree i*sI and [1251]thyroxine were obtained from Amersham (Buckinghamshire, England). Sephadex G-75, G-100, and G-200, Sepharose 6B, and DEAE-Sephadex, products of Pharmacia Fine Chemicals (Uppsala, Sweden), were prepared according to the instructions supplied by the manufacturer. Highly purified guanidine HCl was obtained from Heico (Delaware Water Gap, Pennsylvania).
All other reagents used were of the highest quality available.

Methods
Preparation of Animals-Weanling (46 to 54 g) male Sprague-Dawley rats were made vitamin A-deficient by use of the USP 11 rat test diet (Nutritional Biochemicals, Cleveland, Ohio). The animals were housed in individual hanging wire cages in a room with automatic lighting from 8 a.m. to 8 p.m. Normal control animals were fed a commercial laboratory chow (Anticimes, Stockholm, Sweden).
Both groups of animals had free access to food and drinking water.
The normal and vitamin A-depleted animals did not differ in growth rate until after 6 weeks. The vitamin A-depleted rats continued to grow during 10 weeks on the diet, then the body weights were constant for approximately 1 week to subsequently declitic. All animals in this study were used for experiments after 8 to 10 weeks on the deficient diet except when specifically stated otherwise.
The depleted rats were in apparently good health up to the 10th week, although overt signs of vitamin A deficiency were apparent at the end of this time period.
Intravenous injections into the rats were accomplished either through the jugular vein or through the tail vein. Blood was usually obtained from the abdominal aorta from animals to be exsanguinated.
Serial blood samples were collected from the tail vein.
Polyacrylamide disc gel electrophoresis was carried out with 4% polyacrylamide (Eastman-Kodak) in the spacer gel and 8% in the running gel. A discontinuous buffer system with 0.4 M Tris-HCl buffer, pH 8.9, in the running gel, 0.1 M Tris-HCl buffer, pH 6.7, in the spacer gel, and 0.04 M Tris-glycine buffer, pH 8.3, in the electrode compartment was used (21).
Agarose gel electrophoresis was performed according to the procedure outlined in detail by Johansson (22). weights were estimated at 20" in a Spinco model E analytical ultracentrifuge, equipped with an RTIC temperature control unit and an electronic speed control de,vice. All experiments were conducted in 0.02 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl.
The samples were diluted to the appropriate concentrations and dialyzed in the cold against two changes of of the solvent. Standard 12.mm double sector cells with sapphire windows were used throughout. The centrifuge was operated at 60,000 rpm for the sedimentation velocity determination.
The sedimenting boundary was recorded every 4 min with either the phase plate schlieren optics or with the photoelectric scanning system set at 280 or 330 nm. Calculations were carried out according to Schachman (23).
Sedimentation equilibrium experiments were performed with the meniscus depletion technique of Yphantis with speed settings according to his suggestions (24). Recordings were made with the Rayleigh interference optics. Equilibrium times were estimated according to the procedure of Teller et al. (25). In practice, equilibrium was assessed by comparing recordings taken several hours apart.
The experiments were discontinued when no significant redistribution of material could be observed over a period of several hours.
Partial specific volumes for the calculations were estimated from the amino acid analyses reported below (26). Densities of the solutions were determined by pycnometry.
Analytical Gel Chromatography and Calculations of Difusion Constants, Molecular Weights, and Frictional Ratios-Stokes molecular radii were determined by analytical gel chromatography on a Sephadex G-200 column (140 x 2 cm) equilibrated with 0.02 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl. All experiments were performed at +4" and analyses were carried out in duplicate.
The experimental details were identical with those previously published (27).
Stokes molecular radii were computed from the equation given by Laurent and Killander (28) as described elsewhere (29). Apparent diffusion constants (D20,w) were calculated from Stokes radii by use of the Stokes-Einstein equation (30). Molecular weights were determined by sedimentation equilibrium ultracentrifugation as described above or calculated from sedimentation constants, diffusion constants, and partial specific volumes by the equation of Svedberg (31). Frictional ratios (f/fo) were calculated from Stokes radii, sedimentation constants, molecular weights, and partial specific volumes (31).
Measurements of Circular Dichroism-The circular dichroism spectra of rat prealbumin and RBP were measured with a Jasco model J-20 spectropolarimeter as outlined previously for their human counterparts (32). The reduced mean residue ellipticities [0] were calculated using a mean residue weight of 115 for RBP and 110 for prealbumin.
Amino Acid Analyses-Quantitative amino acid analyses were carried out as described by Spackman et al. (33). The protein samples (0.5 to 1.0 mg) were hydrolyzed in 6 N HCl at 110" for 24 hours. Chromatography was carried out on a Jeol-6AM fully automatic amino acid analyzer (Jeol Co., Tokyo, Japan). Tryptophan was measured spectrophotometrically (34). Preparation of '*51-labeled Protein-Iodination of highly purified RBP and prealbumin was accomplished with 125I using the iodine monochloride technique of McFarlane (35). The labeled proteins were freed from excess iodine by passage over a Sephadex G-25 column (14 x 0.6 cm) equilibrated with 0.02 M 'Iris-HCl buffer, pH 7.4, containing 0.15 M NaCl.
There was always less than 0.7 atom of iodine per protein molecule in the Ultracentrifugation-Sedimentation coefficients and molecular final products. The labeling efficiency was routinely on the order of 70% and the obtained specific activity of the labeled protein was 10 to 100 PCi per mg. Over 98% of the radioactivity of all preparations was precipitable with 10% trichloroacetic acid.
Gel Chromatography in 6 Jw Guanidine WC&Reduced and alkylated proteins usually behave as linear random coils in 6 M guanidine HCl (36). Accordingly, their elution from a gel chromatography column equilibrated in this medium is according to size, i.e. the length of the polypeptide chain. The subunit structure of reduced and alkylated prealbumin and RBP labeled with lzeI was explored by chromatography on a column (120 X 1.5 cm) of Sepharose 6B equilibrated with 6 M guanidine HCl. The same marker proteins as previously employed were used to calibrate the column (cf. Reference 37). All proteins were reduced with 0.1 M dithiothreitol (Mann) for 1 hour and alkylated with 0.3 111 iodoacetic acid (Schuchardt, Munich, Germany) for !s hour prior to application.
The elution volumes of the proteins were determined by weighing the effluent.
Fractions of about 0.9 g were collected at a flow rate of 5 g per hour.
Fluorescence Measurements-All fluorescence measurements were carried out with an Aminco-Bowman spectrofluorometer. lYnless otherwise stated, the fluorescence measurements were performed at ambient temperature (22 f 2") in a buffer composed of Tris-HCl (0.02 M) and NaCl (0.15 M) adjusted to pH 7.4.
Association constants for the binding of RBP and thyroxine, respectively, to prealbumin were measured by estimation of the prealbumin protein fluorescence on complex formation. The optical densities were never allowed to exceed 0.1 at the escitation wave length in the two sets of experiments, rendering inner filter effects negligible.
In both experiments, a constant concentration of a prealbumin solution was used to which various amounts of either RBP or thyroxine were added to obtain the desired molar ratios. The contribution of RBP to the emission at 335 nm was subtracted from the measured values by using blanks of RBP with appropriate concentrations. Details of the experimental procedure have been published (13,14). The quenching data were treated according to the equation of Scatchard (38).
In addition, the association between thyroxine and prealbumin was studied using the fluorescent probe 1,8-anilinonaphthalene sulfonic acid. Titrations of 1 ,8-anilinonaphthalene sulfonic acid and thyroxine were followed spectrofluorometrically.
Relative fluorescence values were uniformly corrected for dilution and for attenuation of the exciting and emitted energy (13). Complete emission spectra were recorded at each observation.
Vitamin A Assay--Vitamin A was extracted from liver samples according to the method of Bligh and Dyer (39). The cont,ent of vitamin A in the chloroform extract was estimated by the trifluoroacetic acid procedure of Dugan et al. (40). The recovery of vitamin A from the extracted liver samples was better than 957, as indicated by the amount of radioactivity obtained in the chloroform layer after extraction of liver [3H]vitamin A.
Quantitative determinations of vitamin A in serum were accomplished by a fluorometric procedure (41).
Determinations of Serum RBP-Serum concentrations of RBP were determined employing a single radial immunodiffusion technique (42). Due to the low amounts of RBP present in serum of vitamin A-deficient rats, the immunodiffusion plates had to be dried and stained with Coomassie blue (Mann) prior to recording.
The antiserum used was raised in a rabbit by footpad injec-tions of highly purified rat RBP. The immunization schedule was the same as earlier (43). Highly purified rat RBP was used as the standard in the immunological quantitations. Liver Cell Fractionation-Liver cells were prepared and separated by centrifugation in colloidal silica gel essentially as described earlier (44). The livers were perfused in situ through vena cava with 100 ml of 0.1 M NaCl followed by 100 ml of 0.15 M NaCl containing 27 mM sodium citrate.
The liver was subsequently removed, blotted on filter paper, and a portion (usually 1 to 3 g) was minced.
Dispersion of the liver cells was performed by subjecting the minced liver to a solution of 40 ml of 16% polyethylene glycol (PEG 4000, Union Carbide Corp., Chicago, Ill.), adjusted to pH 7.4, in a modified Potter-Elvehjem homogenizer furnished with a loose fitting, cone-shaped rubber pestle. The dispersed liver cell solution, obtained by seven strokes with the pestle was subsequently filtered through a nylon sieve to remove cell aggregates and connective tissue.
Centrifugation was performed for 40 min at 800 X g. The content of the centrifuge tube was emptied by pumping 52.4% silica sol into the bottom of the tube and 3-ml fractions were collected from the top (45). Each one of the fractions was washed twice with 16% polyethylene glycol to remove the silica and the cells were recovered by centrifugation for 10 min at 800 X g. Finally, the cells in each fraction were suspended in 0.15 M NaCl.
The entire procedure was carried out at room temperature.
Prior to w-ashing, densities of the fractions were measured in a density gradient column prepared from kerosine and carbon tetrachloride (46). Fraclionation of Kidney Cells-Kidney cell suspensions were prepared with collagenase and hyaluronidase (47) and the cells were fractionated by centrifugation on gradients of colloidal silica.3 The kidneys were perfused in situ by injections into the abdominal aorta (approximately 1 cm below the orifices of the renal arteries) of 100 ml of ice-cold calcium-free Hanks' balanced salt solution containing 0.5 mg per ml of collagenase (Type I from Clostridium histolyticum, Sigma) and 1.0 mg per ml of hyaluronidase (type I from bovine testes, Sigma). The kidneys were removed and freed from their capsule, and the kidney cortex was dissected out. The cortex was minced and incubated in 10 ml of the perfusion solution for 60 min at 37" in a rocking water bath (100 oscillations per min).
The incubation solution was then passed through a nylon sieve and the liberated cells were recovered by centrifugation at 150 X g for 2 min after washing in calcium-and glucose-free Hanks' balanced salt solution.
The cells from two kidneys were suspended in 10 ml of a solution of 1.2% silica sol containing 15% polyethylene glycol and the resulting suspension was layered on top of a discontinuous density gradient consisting of: (a) 25 ml of 4% silica sol and 15% polyethylene glycol (d = 1.04) ; (b) 25 ml of 9% silica sol and 15% polyethylene glycol (d = 1.07) ; (c) 25 ml of 11% silica sol and 147, polyethylene glycol (d = 1.08) ; and (d) 10 ml of 1470 silica sol and 14% polyethylene glycol (d = 1.10). All solutions had an osmotic pressure of 280 to 310 mosm as determined by freeze point depression (Advanced Instrument, New Highland, Mass.). The tubes were centrifuged at 800 x g for 60 min at room temperature.
The separated cells were recovered and washed, and the densities of the fractions were measured as described above for the liver cells.
Radioactivity Measurements-Tissue samples containing [%H]vitamin A were extracted according to the procedure of Bligh and Dyer (39). The chloroform phase, containing the radioactivity, was taken to dryness in a scintillation vial by flushing with nitrogen.
The residue was taken up in the scintillation solution containing 5 g of 2,5-diphenyloxazole (PPO) and 100 g of naphthalene per liter of dioxane.
Liver and kidney cell fractions obtained after density gradient centrifugation and containing [3H]vitamin A were treated as described above prior to radioactivity measurements. Serum samples and gel chromatography fractions containing [3H]vitamin A were directly subjected to radioactivity measurements in the scintillation solution. Tritium-containing samples were counted in a Beckman LS-250 liquid scintillation spectrometer equipped with an automatic external standardization system. When necessary, appropriate quench corrections were performed with the channel ratio method.
Samples containing iodine isotopes were counted in a gamma well type scintillation counter (Tri-Carb 3003, Packard). Other Methods-Protein fractions obtained during the isolation of prealbumin and RBP were concentrated by ultrafiltration with use of 2342 inch Visking dialysis tubing (Union Carbide Corp., Chicago, Ill.) as the ultrafiltration membrane (48). Protein in unpurified materials was estimated according to the method of Lowry et al. (49) with human serum albumin as the standard.
In preparations of greater purity, protein was usually measured by reading the absorbance at 280 nm. DNA was quantitatively estimated with use of the diphenylamine method (50). Lipoproteins were separated from nonlipoproteins in rat serum by centrifugation for 24 hours at 55,000 rpm in rotor 50.1 in a Spinco preparative ultracentrifuge (51). Prior to centrifugation the serum samples were adjusted with KBr to a density of 1.21.

Identification of Vitamin A-transporting
Protein in Rat Serum-Serum, which had been obtained from 15 rats 2 hours after intravenous administration of lipoprotein-bound [aH]retinylacetate, was subjected to gel chromatography on a column of Sephadex G-200.
The result is depicted in Fig. 1. It is evident from the figure that the protein-bound radioactivity was mainly eluted in a single peak appearing somewhat later than rat albumin. This elution position corresponds to that of human prealbumin, indicating a molecular weight for the rat vitamin Abinding protein of approximately 60,000. It will, however, be shown below that the appearance of the vitamin A-binding protein at this elution position is due to its forming a complex with rat prealbumin. tein was detected by means of the specific retinol fluorescence and thyroxine-binding components were traced by measuring the thyroxine radioactivity.
Prealbumin and RBP were isolated from a total of 1500 ml of rat serum. Table I gives a summary of the purification procedure. The yield of highly purified prealbumin and RBP, with the procedure employed, amounted to approximately lo%, based on the assumption that the starting material contained 200 pg per ml of prealbumin and 50 pg per ml of RBP. The material containing radioactivity was in both cases quanti-First Gel Chromatography Step on Sephadex G-gOO-Six 250-m] portions of [1251]thyroxine-containing rat serum were serially chromatographed on a column (110 X 7 cm) of Sephadex G-ZOO equilibrated with 0.02 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl. The column was eluted with a flow rate of 120 ml per hour and fractions of 30 ml were collected. The eluted protein showed a similar distribution as that shown in Fig. 1. Viitually all vitamin A fluorescence appeared in a single peak eluted somewhat later than albumin (cf. Fig. 1.). The distribution of [1251]thyroxine was superimposed on that containing the retinol fluorescence. Accordingly, fractions containing radioactivity and exhibiting retinol fluoresence were pooled and concentrated by ultrafiltration.
The retinol fluorescence also appeared in two positions. About 30% of the fluorescence was eluted with its maximum in the tail part of the first protein peak, corresponding to the elution position for RBP-I, whereas the remaining fluorescence coincided with the second protein peak. Fractions comprising the last eluted material containing retinol were pooled and concentrated. This material constituted highly purified free RBP, as will be shown below.
First DEAE-Sephadex Chromatography Step--The concentrated protein constituting the RBP fraction obtained from the Sephadex G-200 chromatography step was subjected to chromatography, after exhaustive dialysis against the starting buffer, on a DEAE-Sephadex column (40 X 7 cm) equilibrated with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.05 M NaCl. The applied material, containing 25,000 mg of total protein, was eluted with a 5,000-ml linear gradient from 0.05 M to 0.20 M NaCl in 0.05 M Tris-HCl buffer, pH 7.4. The resulting chromatogram exhibited one dominating protein peak, eluted at 0.15 M NaCI. Retinol fluorescence and [iZ51]thyroxine radioactivity appeared together on the frontal part of the main protein peak. Fractions containing radioactivity were combined and concentrated.

Zone Electrophoresis in Barbital
Buffer-The concentrated RBP-I fraction obtained after the second gel chromatography on Sephadex G-200 was subjected to zone electrophoresis in barbital buffer, pH 8.6, ionic strength 0.05. This low ionic strength was chosen due to the fact that interaction of the human prealbumin and RBP is very sensitive to low ionic strength.
After completed electrophoretic separation, three well resolved protein peaks were apparent. The peak with the highest anodal mobility was the only one containing appreciable amounts of [1261]thyroxine as revealed by radioactivity measurements. The peak with the lowest anodal mobility comprised all of the retinol fluorescence. Accordingly, fractions constituting the first and the third peaks were separately pooled and concentrated. It will be demonstrated below that the two fractions contained highly purified thyroxine-binding prealbumin and free RBP, respectively.

Second DEAE-Sephadex Chromatography
Step-Preliminary electrophoretic experiments in agarose gels showed that a further resolution of RBP and protein-bound [1251]thyroxine from the major contaminants could be achieved at pH 9.0. Accordingly, the concentrated protein fraction containing RBP and [12SI]thyroxine (2250 mg of total protein) was subjected to chromatography on a column (60 x 3 cm) of DEAE-Sephadex equilibrated with 0.05 M Tris-HCl buffer, pH 9.0, containing 0.1 M NaCl. Prior to application the material was dialyzed against three changes of the starting buffer. Elution was performed with a 2000-ml linear NaCl gradient from 0.1 to 0.2 M in the pH 9.0 buffer. The retinol fluorescence, which appeared in two equally large, distinct but partly overlapping peaks, was well separated from the bulk of the eluted protein. Most of the unrelated protein appeared later than RBP. The [1251]thyroxine radioactivity emerged from the column in a single peak coinciding with the earliest eluted retinol-containing material. It was thus obvious that RBP had resolved into two components, and fractions corresponding to each of the two peaks were separately pooled and concentrated. The two fractions will henceforth be termed RBP-I and RBP-II according to the order of their elution positions.
Purity and Characteristics of Prealbumin and RBP-The purity of prealbumin and RBP was assessed by polyacrylamide gel electrophoresis in Tris-glycine buffer, pH 8.9. It can be seen in Fig. 2 that prealbumin exhibited a single protein zone with high anodal mobility, whereas RBP-II displayed a total of five bands. The same electrophoretic distribution was also encountered for RBP-I. Only two of the five RBP bands exhib-Second Gel Chromatography on Sephadex G-,%)-Each of the two fractions (RBP-I and RBP-II) obtained from the second DEAE-Sephadex chromatography step was separately subjected to gel chromatography on a column of Sephadex G-200 (130 x 2 cm) equilibrated with 0.02 M Tris-HCI buffer, pH 7.4, containing 0.15 M NaCl. The RBP-I fraction gave rise to a single protein peak but the retinol fluorescence and the [1251]thyroxine, again coinciding, were eluted in the latter part of this protein peak, clearly indicating that the RBP in this fraction was still contaminated with unrelated protein. Accordingly, fractions containing retinol fluorescence and [1251]thyroxine were pooled and concentrated.
The RBP-II fraction was resolved into two protein peaks on Sephadex G-200. The earliest eluted material appeared in the same position as the protein peak obtained for the RBP-I fraction, whereas the second peak was eluted considerably later. ited retinol fluorescence (demonstrated prior to protein staining). Although this result may indicate that the RBP fractions were contaminated with unrelated protein, a more likely explanation is that rat RBP exhibits micro-heterogeneity on polyacrylamide gel electrophoresis analogous to that found for its human counterpart (11).
Additional support for the purity of RBP was obtained when a mixture of prealbumin and RBP was chromatographed on a column of Sephadex G-75 equilibrated with 0.02 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl.
It can be seen in Fig. 3 that RBP was quantitatively eluted together with prealbumin, clearly demonstrating that prealbumin and RBP under physiological conditions appear as a protein complex.
Furthermore, the figure demonstrates that RBP is highly purified, since no protein could be detected in the elution position of free RBP.
The generated prealbumin-RBP complex displayed on polyacrylamide gel electrophoresis the sum of the patterns obtained for the individual proteins establishing that all five RBP protein zones were the result of micro-heterogeneity.
Some physical properties for prealbumin and RBP were determined and are summarized in Table II. Sedimentation velocity analyses were carried out at protein concentrations of 0.02% to 0.08%.
Both prealbumin and RBP behaved as single components within this concentration range. The sedimentation constant (s&,~~) for prealbumin was 3.0 S and for RUP 2.1 S.
Stokes molecular radius was determined by gel chromatography on a calibrated column of Sephadex G-100.
The values obtained for the prealbumin-RBP complex, prealbumin, and RBP were 32 A, 28 A, and 19 A, respectively.
This demonstrates that the rat prealbumin-RBP complex is smaller than the human prealbumin-REP complex (38 A). The frictional ratios were calculated from the sedimentation coefficients and the molecular weights obtained from equilibrium a globular structure, provided the proteins have "normal" hydration.
The molecular weights of prealbumin and RBP were estimated by sedimentation equilibrium ultracentrifugation. Fig. 4 shows the resulting graphs for prealbumin and RBP when In c was plotted versus r2. The measured molecular weights were 51,200 for prealbumin and 20,500 for RBP, and the linear relationships obtained in the plots indicated homogeneity for the two examined preparations.
The molecular weights measured by sedimentation equilibrium ultracentrifugation and those computed from the sedimentation constants and the Stokes' radii were in good agreement (cf . Table II).
In order to investigate the possibility of a subunit structure for prealbumin and RBP, the two proteins were separately subjected to gel chromatography on a column of Sepharose 6B equilibrated with 6 M guanidine hydrochloride.
The column was calibrated with reference proteins as described under "Experimental Procedure." The reduced and alkylated RBP appeared slightly after human RBP, indicating a molecular weight of approximately 19,000. Prealbumin appeared somewhat earlier than the human @-microglobulin, which has a molecular weight of 11,800. From the elution position of prealbumin a molecular weight of 12,700 could be calculated.
Without reduction and alkylation, prealbumin appeared exactly at the same elution position. It thus appears that prealbumin is composed of 4 subunits.
It is also evident that the subunits are not held together by covalent interactions since the subunit structure is apparent without reduction and alkylation.
In analogy with findings for the human and the monkey prealbumins, it may be inferred that the rat prealbumin is composed of four apparently identical polypeptide chains.
Rat prealbumin and RBP were separately subjected to measurements of their circular dichroism. Fig. 5 depicts the circular dichroism spectra for prealbumin and RBP in the ultraviolet region.
It is evident from the figure that prealbumin exhibited a strong ellipticity at about 213 nm. This trough is quite similar to that found for human and monkey prealbumins although the magnitude is somewhat smaller.
Calculations indicate that the prealbumin structure contained at the most 10 to 20% /3 structure. No c11 helix content could be compatible with the observed ellipticity distribution, and it is probable that 80 to 90% of the structure is composed of some unordered yet rigid structure. RBP exhibited a more complex circular dichroism.
It is evident from Fig. 5 that there is a positive ellipticity around 320 nm, followed by a negative trough with minimum at 275 nm. A new band exhibiting positive ellipticity is apparent at 240 nm which may be due to disulfrde bridges, and this band is followed by the main negative ellipticity.
The complex over-all spectrum for RBP is strikingly similar to that recorded for the human counterpart, although the magnitudes of the individual bands differ.
The fluorescent properties of prealbumin and RBP were investigated.
RBP had in addition to its absorption band at 280 nm a band with maximum at 330 nm due to the presence of retinol.
On excitation of the protein at these two wave lengths, two types of fluorescence were obtained: one with maximum at 335 nm caused mainly by the protein tryptophyl residues, and the other at 470 nm caused by the vitamin.
By excitation at 280 nm, prealbumin exhibited a single fluorescence with maximum at 335 nm.
The prealbumin fluorescence at 335 nm undergoes dramatic changes with addition of RBP containing retinol. Fig. 6 shows the progressive quenching of the prealbumin fluorescence on increments of the molar ratio of RBP to prealbumin.
It is evident that interpolation of the linear regions of the quenching curve intersects at a 1: 1 molar ratio of the two proteins.
In this region the tryptophan fluorescence of prealbumin is considerably quenched.
The finding that the prealbumin fluorescence is partially quenched on complex formation with retinol-containing RBP made investigations on the affinity. between the two proteins possible.
Furthermore, the binding of thyroxine to prealbumin is also accompanied by a quenching of the tryptophan fluorescence suitable for use in evaluation of the binding constant. Fig.  7 shows two Scatchard plots resulting from the binding of RBP and thyroxine, respectively, to prealbumin.
The apparent asso- 6. Effects on the rat prealbumin fluorescence emission at 335 nm (excitation at 280 nm) at various molar ratios of RBP to prealbumin.
To a constant concentration of a prealbumin solution, 7 X 10e7 M, small amounts of RBP were added to obtain the desired molar ratios of the two proteins.
The contribution of RBP to the emission at 335 nm was subtracted from the measured values by using blanks of RBP with appropriate concentrations. 7. Binding of rat RBP (0) and thyroxine (0) to rat prealbumin determined by fluorescence quenching in 0.02 M Tris-HCI buffer, pH 7.4, containing 0.15 M NaCl. The data were plotted according to Scatchard (38). v is the molar ratio of bound RBP or thyroxine to prealbumin, and c the concentration of free RBP or thyroxine, respectively. ciation constants were 8 x lo6 M-' for the RBP binding and 4 x 10' M-' for the thyroxine binding.
It has earlier been noted that the fluorescent probe 1 ,%anilinonaphthalene sulfonic acid binds competitively to the human prealbumin thyroxine-binding site.2 Therefore, a study was undertaken to investigate whether rat prealbumin also bound 1,8-anilinonaphthalene sulfonic acid. On addition of 1,8-anilinonaphthalene sulfonic acid to a solution of rat prealbumin the fluorescence of 1,8-anilinonaphthalene sulfonic acid was increased approximately loo-fold compared to that of 1,8-anilinonaphthalene sulfonic acid alone in aqueous solution at the same concentration.
The emission maximum of 1,8-anilinonaphthalene sulfonic acid, 510 nm, underwent a marked hypsochromic shift to 460 nm in the presence of prealbumin.
Furthermore, in the presence of 1,8-anilinonaphthalene sulfonic acid, the prealbumin emission spectrum (excitation at 280 nm) exhibited a quenched tryptophyl fluorescence but, in addition, the characteristic 1,8anilinonaphthalene sulfonic acid fluorescence at 460 nm was evident. Thus, 1,8-anilinonaphthalene sulfonic acid was shown to bind to prealbumin by the criteria of fluorescence yield enhancement, shift of maximum emission, and energy transfer.
The change in relative fluorescence at 460 nm of a solution of prealbumin when 1,8-anilinonaphthalene sulfonic acid was added is shown in Fig. 8  The solid line in the figure is the theoretical binding curve for this K,,,,, value.
When thyroxine was added in small increments to a solution of prealbumin containing saturating amounts of 1,8-anilinonaphthalene sulfonic acid, the 460 nm fluorescence decreased progressively.
The quenching was almost complete when 1 mole of thyroxine was added per mole of protein, confirming the existence of one high affinity thyroxine-binding site per molecule (cf. Fig. 8B). Assuming that 1,8-anilinonaphthalene sulfonic acid competes with thyroxine for the binding to prealbumin, the association constant for the thyroxine-prealbumin interaction could be calculated.
The value obtained (2.7 x 10' M-') was in good agreement with the data obtained by measuring the thyroxine quenching of the prealbumin protein fluorescence (see above).

Amino
Acid Composition of Prealbumin and RBP-Due to scarcity of material only single analyses of prealbumin and RBP were accomplished.
The results, shown in Table III, suggest the presence of 113 amino acid residues in prealbumin, assuming that it is composed of 4 identical subunits, and 174 residues in RBP. These values for RBP are, of course, somewhat inexact since they were calculated on the basis of a single 24-hour hydrolysis value only. Thus, leucine, isoleucine, and valine are most probably too low, especially if they occur as nearest neighbors in the primary structure.
The values for cysteine and methionine may also ave yielded too low values, since they were not determined after prior performic acid oxidation. In spite of these uncertainties, it appears that there exists a reasonable agreement in amino acid composition between rat prealbumin and human prealbumin as well as between RBP from the two species.
It may thus be concluded that most of the physicochemical and chemical parameters examined for the rat proteins appear to be similar to those of the human counterparts.

Kinetics and Tissue Distribution of Vitamin A in Normal and Vitamin A-deficient Rats
Following the isolation of the specific plasma carrier protein for vitamin A in the rat (see above) a study was undertaken to investigate the tissue distribution of vitamin A in normal and vitamin A-deficient rats. Since it is understood that RBP plays a key role in the distribution of the vitamin, quantitative immunological estimations of RBP were nerformed in vitamin A depletion and deficiency.

Tissue Distribution of [3H]Vitamin A-Lipoprotein-bound [3H]retinylacetate
was injected intravenously into normal and vitamin A-deficient rats. The animals were killed at different time intervals following the administration of vitamin A. The amounts of radioactivity present in liver, kidney, and serum were measured and expressed as percentages of the injected radioactive dose. The data obtained are summarized in Fig. 9. It is evident from the figure that the liver contained its maximal amount of radioactive vitamin A already at the earliest time studied (15 min after administration).
In normal rats the liver was only slowly depleted of the radioactive vitamin; at 16 hours it still retained two-thirds of its maximal content.
In vitamin deficiency, the initial liver content of 3H was similar to that found for the normal rats, but the radioactivity declined rapidly and only about one-third of its initial content was retained at 16 hours.
In blood the [3H]vitamin A radioactivity declined in an exponential fashion for normal rats, whereas in vitamin A-deficient animals a rebound of the radioactive vitamin was apparent. Following a rapid decay during the first hour after administration, the levels increased to a peak value around 3 hours to subsequently decline again. This rebound effect was due t.o elimination from serum of 13H]retinylacetate, bound to the lipoproteins, during the first phase, and reappearance of [3H]retinol, bound to RBP, in the second phase. This was demonstrated by subjecting serum, obtained at the indicated times, to gel chromatography on Sephadex G-200. During the early time points a considerable amount of 3H radioactivity appeared close to the void volume, whereas at 2 hours after the administration and subsequently virtually all radioactivity appeared in the elution position of the prealbumin-RBP complex (cf. Fig. 1). The deposition of [3H]vitamin A in the kidney increased steadily with time both in normal and deficient rats. The relative magnitude of the content of 3H radioactivity in kidneys of the two groups of animals was, however, different.
More than twice as much [3H]vitamin A was recovered in the kidneys of the deficient animals compared to the normal ones. It may thus be concluded that newly administered vitamin A is more easily mobilized from the vitamin A-deficient liver than from the normal liver.
It also seems likely that the increased content of RBP-bound [3H]retinol in plasma of vitamin A-deficient rats compared to normals reflects the differences encountered in content of 3H radioactivity in the kidneys of the two groups of animals. Cellular Distribution of [3H] Vitamin A in Liver-Dispersed liver cells were separated by centrifugation in colloidal silica gel. Cells were obtained from normal and vitamin A-deficient rats at different time intervals after the intravenous administration of [3H]retinylacetate (cf. Fig. 9). The relative distribution of the liver cells following centrifugation was very similar for normal and vitamin il-deficient cells. It was noted, however, that in all fractions the normal cells contained approximately 20% more protein per mg of DNA than those of vitamin A-deficient animals.
A typical separation of normal liver cells by centrifugation in colloidal silica gel is depicted in Fig. 10. Four fractions of cells, banding at different densities, were obtained. Fig. 11 shows typical cells obtained in the various fractions. Approximately 70% of the total liver cells banded at densities of 1.08 to 1.10. These cells had a larger particle diameter than the cells present at higher densities.
The figure shows that there seems to be an enrichment of binuclear cells at the lowest density. It has earlier been shown that the three top fractions mainly contain hepatocytes (44). At the highest density (1.12 < d < 1.20) the Kupffer cells were recovered, occasionally contaminated with minor amounts of free nuclei.
The relative distribution of [3H]vitamin A in the four fractions of cells was identical within experimental error regardless of whether the cells were obtained from normal or vitamin A-deficient rats, although, of course, the total radioactivity differed significantly (cf. Fig. 9). It was shown above that the total 401s amount of [sH]vitamin A in livers of vitamin A-deficient rats Kupffer cell fraction only contained about one-tenth of the total declined considerably with time. The relative distribution of DNA recovered in spite of the fact that the Kupffer cells are the radioactivity in the four cell fractions obtained was, however, known to constitute approximately one-third of the total liver unchanged during the time period studied (15 min to 16 hours).
DNA (52-54). Making allowance for this discrepancy the The [aH]vitamin A radioactivity was present in greatest abun-Kupffer cells would still contain less than 3% of the total liver dance in the two cell fractions of lowest density ( Fig. 9 and Table  vitamin A (the corrected figure, based on the value 0.8% in IV).
About equal amounts of radioactivity were recovered in Table IV, is accordingly 2.4%). Furthermore, when doses of these two fractions despite approximately twice the content of vitamin A 100 times exceeding those commonly employed in DNA in Fraction II (cf. Table IV).
It is evident from the table the present study were administered intravenously to normal that Fractions III and IV contained only minor amounts of both and deficient rats, no more than 3% of the total liver content of the total DNA and the total radioactivity.
The specific radio-vitamin A could be recovered in the Kupffer cell fraction. Thus, activity (per mg of DNA) was 5-to 25-fold higher in the hepato-it may be concluded that virtually all vitamin A in the liver is cyte fractions (I to III) than in the Kupffer cell fraction.
The present in the hepatocytes. from Liver-It is well known that on vitamin A depletion the liver and serum levels of the vitamin decrease rapidly.
Determinations of vitamin A in liver and serum and of RBP in serum were performed in rats reared on a vitamin A-free diet and in normal control animals.
The result is shown in Fig. 12. It is evident from the figure that the content of liver vitamin A decreased rapidly in the rats on the deficient omop FIG. 11. Phase contrast micrograph of typical liver cells recovered in Fractions I to IV.  Fig. 10). b Expressed as percentage recovered in the fraction of the total amount recovered in all fractions. c Average of three normal rats injected with 1.7 FCi of [aH]retinylacetate.
The livers were excised 16 hours after administration. Serum vitamin A also decreased in concentration in the depleted animals, whereas a slight increase in the serum level of the normal rats was noted.
The serum concentration of RBP diminished also in deficiency in spite of a slight elevation with increasing age in the serum of the normal rats. That the effect of vitamin A on the serum levels of RBP is specific was shown by determinations of total plasma protein concentrations in normal and depleted animals at the times indicated in Fig. 12.
No differences were noted between the two groups of animals during the time period studied.
The mean levels in both normal and depleted animals increased from 58 mg per ml on the first day to 69 mg per ml on day 48. The data clearly show that the effects of vitamin A depletion are appearing in the order: liver vitamin A, serum vitamin A, and serum RBP, respectively. The steepest part of the RBP decline curve did not appear until virtually all vitamin A in the liver had been exhausted.
It thus seems likely from the above findings that the liver content of vitamin A may regulate the serum level of RBP.
Further investigations were initiated to study the effects of vitamin A repletion on the serum levels of RBP.
Groups of vitamin A-deficient rats were injected with three separate doses of retinol and blood was obtained at different times during the following 6 hours. The result is shown in Fig. 13. The concentration of serum RBP increased considerably after repletion with the vitamin.
In all three groups of rats, varying in amounts of vitamin A obtained, the maximum RBP value was recorded within 2 hours. The data in the figure indicate that administration of 5 pg of retinol was not sufficient to restore the RBP concentration to normal values, whereas 125 pg, as well as 25 pg, of retinol gave normal and sustained serum levels of RBP during the following 6 hours.
This experiment indicated that vitamin A might induce synthesis of RBP in the liver.
To test this idea, actinomycin D was injected into vitamin A-deficient animals 256 hours prior to the intravenous administration of 125 pg of retinol. Blood was obtained at the same time intervals as previously. The values are the mean of three rats.
to current concepts, most organs, except for the liver, obtain vitamin A from serum RBP. An experiment was therefore devised to study the cellular distribution of vitamin A in the kidney since this tissue is the second most important storage site for the vitamin.
Dispersed kidney cortex cells, obtained from rats previously injected with lipoprotein-bound [3H]retinylacetate, were separated by centrifugation in colloidal silica gel. Four fractions of cells, banding at different densities, were obtained.
The kidney cells exhibited a tendency to aggregate under the conditions employed, and the recovery of cells in the four fractions varied by as much as 20'% in different experiments.
In spite of this difficulty, the morphological appearance of the cells in the four fractions was quite reproducible.
The top fraction always contained the smallest cells, with a diameter of 10 to 15 EL, sometim.es contaminated with small amounts of cell debris. In the density range 1.07 to 1.08, approximately 60 to 80% of the cells were banded.
The two well resolved fractions at these densities contained considerable amounts of cells with brush borders. The bottom fraction contained large cells with a diameter of 15 to 20 p. The total recovery of cells from 1 g of kidney amounted to 1 to 2 X lo* cells.
[3H]Vitamin A was exclusively present in the two main, middle fractions, as shown in Fig. 14, regardless of whether the kidney cells were obtained from normal or deficient rats. Cells in these two fractions contained about 80% of the total [3H]vitamin A of the kidney since the amounts in the marrow represented less than one-fifth of the total.
The same experiment was repeated after lz51-labeled RBP had been administered intravenously into vitamin A-deficient rats. It can be seen in Fig. 14  sively confined to the same two kidney cell fractions which contained all [3H]vitamin A.
The trichloroacetic acid-precipitable 1251 radioactivity recovered in the kidney amounted to approximately 2% of the injected dose.
In conclusion, this experiment shows that virtually all [3H]vitamin A is present in kidney cells of two distinct densities. Furthermore, the same two fractions contained cells with the ability to concentrate 1251-labeled RBP.

DISCUSSION
The results of the present report establish that vitamin A in rat plasma is transported by a protein complex composed of thyroxine-binding prealbumin and the retinol-binding protein. The properties of rat prealbumin and RBP are similar to those of the human and monkey counterparts (18). RBP of the rat exhibits the same molecular weight and size as that of the primates. It also carries a single molecule of retinol, the fluorescence properties of which are indistinguishable from that of human retinol-RBP. It thus appears likely that the retinol molecule occupies a site of similar configuration and hydrophobicity in the two proteins.
The binding of rat prealbumin to RBP increased the quantum yield of retinol, indicating that the specific protein-protein interaction also may be similar to that of the human proteins (13). However, some differences were noted. On measurements of the circular dichroism, the RBP from the rat differed from the human.
However, the over-all spectra were similar, which signifies that probably only minor differences in structure are present between the two proteins. Differences in negative net charge were apparent on polyacrylamide gel electrophoresis; rat RBP exhibited, on the average, a somewhat slower electrophoretic mobility. Both the human and the rat RBP are however micro-heterogeneous and display multiple electrophoretic bands. This heterogeneity most probably reflects a tendency for the protein to lose amide groups, and it is interesting that the protein from both species exhibits this property (55).
Prealbumin differs somewhat more from the primate counterparts. The most significant difference found is the molecular weight: 51,000 for the rat protein compared to 62,000 for the human prealbumin (56). In spite of this difference, it is highly likely that the rat protein is composed of four apparently identical polypeptide chains in agreement with findings earlier reported for the human protein (56-59).
Prealbumin isolated from the rat exhibited on polyacrylamide gel electrophoresis considerably slower mobility than the human protein.
This diminished negative charge of the rat prealbumin made the isolation of the rat prealbumin-RBP complex much more difficult than was previously encountered for human or monkey prealbumin-RBP complexes.
The most interesting observation with regard to the vitamin A-transporting protein complex in the rat is its ability to bind thyroxine.
This property is also shared by the human and the monkey prealbumins, but in the latter species it is probably only of minor physiological importance since their plasma contains a more efficient thyroxine-binding globulin. It appears, however, that rat prealbumin is the only protein with any significant thyroxine-binding capacity in rat plasma. Thus, vitamin A and thyroxine are transported by the same vehicle in the rat, raising the important possibility that the hormone and the vitamin may exhibit cooperative metabolic interactions (11). There are no reports presently available establishing such an interaction, but it is noteworthy that on vitamin A deficiency the level of RBP in plasma decreases almost lo-fold (see below) concomitant with a similar decrease in the plasma prealbumin concentration. 2 Vitamin A deficiency in the rat would accordingly cause not only a decreased vitamin A transport capacity but also thyroxine "buffering" ability. It may thus be suggested that some of the effects of vitamin A deficiency are indeed the result of altered thyroxine metabolism. However, further studies are needed to clarify this point.
The major aim of this study was to investigate the transport of vitamin A to the liver and subsequently to target cells. An apparent difficulty in performing such studies is the number of conflicting reports on the type of liver cells storing the vitamin (cf. Reference 7). An attempt was made, therefore, to establish which cell type in the liver took up the vitamin from the chylomicrons (simulating the postabsorptive state) and which cell type stored the vitamin.
Employing liver cell separation on colloidal silica gel gradients, it was unambiguously shown that the hepatocytes took up and stored virtually all vitamin A. This finding is in contrast to that of Popper and Greenberg (60), who concluded that most of the vitamin was stored in the Kupffer cells. We did not observe more than 3% of the administered amount of vitamin A in the Kupffer cells even at early times. The discrepancy between our result and that of Popper and Greenberg is probably explained by the differences in techniques employed.
Popper and Greenberg examined liver cell slices by fluorescence microscopy and they noted serious problems of autofluorescence.
Recently, the distribution of vitamin A among hepatocytes and Kupffer cells was studied by Linder et al. (61). They found that virtually all vitamin A was present in the hepatocytes, in agreement with the present data.
The hepatocytes could be fractionated into cells of three different densities.
The cells with the lowest density contained most of the vitamin. This is interesting since it was noted that cells in this fraction were to a considerable extent binucleated. We would suggest that these cells are the major cell type responsible for RBP and prealbumin synthesis.
The relative distribution of vitamin A among the hepatocytes was invariant with regard to time and total content of vitamin A as demonstrated in normal and vitamin A-deficient livers. The only difference encountered between normal and vitamin A-deficient livers was the retention time of the administered radioactive vitamin, the radioactivity declining much faster in vitamin A deficiency than in normal conditions. This rapid turnover of vitamin A in the deficient liver was reflected in the plasma level of RBP-bound radioactivity.
It was thus noted that considerably higher levels of radioactively labeled vitamin A were noted in the deficient animals than in the control rats. This, of course, does not reflect the total content of plasma vitamin A but indicates that the vitamin is rapidly mobilized from the liver in the deficient state.
In vitamin A deficiency it is well known that the liver becomes exhausted of its vitamin A content, and subsequently diminished plasma levels of retinol are encountered (cf. Reference 1). As expected, the rat plasma RBP concentration decreased considerably on vitamin A deficiency approaching levels IO-fold less than those of normal animals.
This decrease of RBP is probably a primary effect of vitamin A depletion since the total plasma protein concentration appeared normal even in severe deficiency.
The low plasma levels of RBP would be easily explained assuming that vitamin A affects the synthesis of RBP in the liver. This assumption was, however, most probably disproven since actinomycin D did not inhibit the appearance of RBP in plasma of replenished animals.
A number of possible interpretations have bearing on this result. It could conceivably be inferred that vitamin A affects the synthesis of RBP on the translational level and that the messenger RNA for RBP is extraordinary long lived since actinomycin D affects the transcription rather than the translation of the messenger.
A more plausible explanation may be afforded by the recent observation that vitamin A is present in greatest abundance in the Golgi apparatus (62). It is well established that most plasma proteins leave the liver cells through the membranous network constituting the Golgi apparatus.
Many plasma proteins get their carbohydrate moieties within this structure and it may well be that RBP attaches its retinol at the same place. In vitamin A deficiency no retinol is available for the newly synthesized RBP, and this could result in the protein not being released into the blood. Administration of the vitamin to the liver cells would accordingly give rise to a rapid increase in the plasma RBP concentration due to release of preformed RBP. to a considerable extent by the kidney (64). Furthermore, preliminary experiments suggest that retinol, attached to RBP, is released from the protein on its degradation in the tubular cells. The released vitamin seems subsequently to be transported to the liver by lipoproteins.
It may thus be concluded that vitamin A recirculates in contrast to its carrier protein.
In a study parallel to this, Goodman and coworkers have isolated prealbumin and RBP from rat plasma and investigated the levels of RBP in liver and plasma in vitamin A deficiency (65,66). RBP was characterized in some detail and their data are in excellent agreement with ours. They have also found that the plasma levels of RBP decrease on induction of vitamin A deficiency and that the levels can be restored on replenishment of the vitamin, in accord with the present results.
In addition, they have made the important discovery that the liver cell content of RBP is approximately 4-fold higher in vitamin A deficiency compared to normal conditions. This observation, together with the present results on actinomycin D-treated rats, clearly indicates that vitamin A deficiency affects the release of RBP from the liver cells.
One of the most intriguing facts about the metabolism of vitamin A is the high concentration of the vitamin in the kidney (8). This observation, together with the well known fact that the kidney is the main catabolic site for low molecular weight plasma proteins like RBP, warranted a detailed study of the cellular distribution of the vitamin in the kidney. The present results indicated that more than 80 y0 of the total kidney content of radioactive vitamin A was confined to the cortex.
Furthermore, only two out of the four cell fractions recovered after colloidal silica gel gradient centrifugation contained appreciable amounts of the vitamin.
On morphological examination of the vitamin-containing cells, it was apparent that a majority of these cells exhibited brush borders.