In vitro conversion of carotene to vitamin A in the isolated small intestine of the rat

In Vitro Conversion of Carotene to Vitamin A in the Iso-

lated Small Intestine of the RatIs Abraham Rosenberg3 and Albert Edward Sobel From the Department of Biochemistry, Jewish Hospital Department of Chemistry, Polytechnic Institute Brooklyn, New York

of Brooklyn;

and the

of Brooklyn,

Received January 12, 1953

A number of reports have appeared in recent years which indicate that, at least in experimental animals, the small intestine, rather than the liver, is a major site of conversion of carotene to vitamin A. Most of the evidence for this contention is indirect, as given below: 1. Following administration of carotene to a wide variety of animals, vitamin A appears first in the intestinal wall and contents, and much later in the liver (l-6). 2. Vitamin A fails to appear in either the blood or the liver when intestinal lymph is diverted following a carotene meal (7). 3. Vitamin A deficiency in rats is not cured following parenteral injection of carotene, while unchanged carotene accumulates in the liver (8): Direct evidence for the conversion of carotene to vitamin A in the intestinal loop of rats has been reported by Wiese, Mehl, and Deuel (9), 1 Presented in part before the Division of Biological Chemistry, American Chemical Society, 122nd National Meeting, Atlantic City, Sept. 15, 1952. 2 These studies were supported by Endo Products, Inc., and by the New York Diabetes Association. a Taken in part from the thesis submitted to the Polytechnic Institute of Brooklyn in partial fulfilment of the requirements for the degree of Master of Science in Chemistry, June, 1952. 4 Since the completion of this manuscript a preliminary paper has appeared [Bieri, J. G., and Pollard, C. J., Federation Proc. 12,409 (195311,in which the statement is made that some conversion of carotene to vitamin A can take place even following the removal of the small intestine after the injection of carotene dispersed in Tween 49. 320

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while under somewhat different conditions, no conversion was observed by Thompson, Ganguly, and Kon (4). Data obtained by Wiese et al. are based on the appearance of a fleeting blue color on addition of antimony trichloride to nonsaponifiable lipide extracted from the gut wall following incubation with an aqueous carotene and tocopherol dispersion. Instrument readings were regarded as invalid when the reaction mixture failed to display the blue color of the vitamin ASbC13 reaction. On this basis, they found 2.7 pg. of vitamin A in the isolated intestinal wall of “A’‘-depleted rats after incubation with carotene and tjocopherol. The findings of Wiese et al. are of such a fuudamental nature that an attempt to confirm their results was undertaken. RESULTS On repeating the experiments of these workers, we observed only a brownish color with antimony trichloride, and a greenish-yellow rather than a magenta color with activated glycerol dichlorohydrin (10). In view of the dubious nature of these calorimetric reactions in providing evidence for the conversion of carotene to vitamin A, we developed a more satisfactory method of measuring vitamin A by the difference in absorption spectrum of the gut nonsaponifiable lipide before and after irradiation with ultraviolet light. Vitamin A estimation in this manner was proposed by Chevalier and Chabre (ll), developed further by others (12-14), and applied to the analysis of liver (15) and serum (16, 17). Bessey et al. indicated the need for saponification as a preliminary step and demonstrated that the difference in absorption spectrum between 310 and 390 rnp before and after irradiation closely resembles the spectrum of pure vitamin A. After the isolated gut loop from vitamin A-depleted rats had been incubated with carotene by a procedure similar to that of Wiese et al., a mean spectrum was obtained from ten experiments by subtracting post- from preirradiation spectra of the nonsaponifiable gut lipide (Fig. 1) and compared with that of crystalline vitamin A acetate. It appears from these results that the extinction of the “difference spectrum” at 328 rnp may be used to calculate vitamin A content of the gut lipide in a manner analogous to that of Bessey et al. for serum vitamin A. It should be pointed out that deviations of the individual spectra from the mean spectrum were within the estimated experimental error. Errors due to carotene corrections, necessary in calorimetric “A” estima-

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tion, do not arise (10). Such corrections, made on the basis of B-carotene, may not be valid in the presence of partially reacted carotene. Using the destructive irradiation procedure for vitamin A estimation, we reinvestigated the conversion of carotene to vitamin A in the isolated small intestine. In this, except for the method of analysis, the procedure of Wiese et al. was followed with a few modifications (see Experimental). 14 xylem - k.?rcKene -

WAVE

FIG.

LENGTH,

Ddference between we- 0r-d post-wrodtotion spectrum d gut nonsap.miftoMe lipid tn I I xylem -kerosene

mp

1. Light-absorption spectrum difference between pre- and postirradiated Iipide compared with the spectrum of crystalline vitamin A.

gut

Results obtained with ten successive animals from four separate litters are given in Table I. The mean value was 4.24 pg. of vitamin A produced with a standard deviation of f1.53 (f36 % of mean), and a standard error of the mean of 0.39. This may be compared to the mean value found by Wiese et al. (2.6 pg.), with a standard deviation of f1.7 (65% of mean) based on 18 animals. The extreme variation from the mean observed by Wiese et al. did not occur in this series. The results of this study corroborate the findings of Wiese et al. The inability of Thompson et al. (4) to observe a conversion may be related to the poor absorption of oily solutions (18), particularly after death,

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0

when an emulsifying system is no longer provided by the flow of pancreatic and bile juices. These experiments may be regarded as direct evidence that, at least in the rat,, the small intestine contains a major system for t,he conversion of carotene to vitamin A. Further study of this system, particularly of enzymes t)hat may be involved, should provide a fruitful subject for future work. TABLE

I

Light Absorption of Gut Nonsaponijiable Lipide Before and ilfter Irradiation, Calculated Vitamin A Content of Gut Incubated with an iiqueorrs Dispersion Carotene and Tocopherol

OpticalDensityat 328rnp

Animal Preirradiation

-

Postirradiation

and

of

Vitar$ Agntent

-,I Pg.

la lb IC

2a 2b

3a 3b la 4b

4c Mean....

0.448 0.285 0.421 0.311 0.830 0.377 0.430 0.311 0.808 0.427 0.465

0.235 0.125 0.242 0.206 0.493 0.107 0.194 0.206 0.494 0.222 0.252

0.213 0.158 0.179 0.105 0.337 0.270 0.236 0.105 0.314 0.205 0.212

4.21 3.60 3.54 2.08 6.65 5.34 4.65 2.08 6.20 4.05 4.24

EXPERIMENTAL

Vitamin A Depletion albino rats of an inbred Wistar strain were maintained on the stock diet of Bills et a/. (19). The young were removed at 3 weeks of age and placed on the IT. S. P. XIV vitamin A test diet for a period of 3-4 weeks. The breeding and experimental rooms were kept at 25-26°C. by thermostatic control. Vitamin A depletion was evidenced by failure to gain weight for several days and by xerophthalmia.

Carotene Dose An aqueous carotene dispersion was prepared by mixing 3.0 mg. carotene (90% S-10% a) with 5.0 mg ol-tocopherol, dissolving in 500 mg. of sorethytan oleate (Tween 80, Atlas Powder Co.), and adding distilled water very slowly with thorough mixing. A water-clear, nonviscous aqueous dispersion of the lipides was produced. This was diluted to 10.0 ml. with distilled water. The animals were fasted overnight and 0.5 ml. of this dispersion (150 pg. carotene) was placed directly int,o the stomach of each animal with a 1.0.ml. syringe and a stomach tube made from

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a 16-gage needle, the end of which was bent at a 30” angle, the point ground flat, and fitted with a spherical tip.

In Vitro Conversion of Carotene to Vitamin A The animal was sacrificed by a sharp blow on the head immediately after dosing. The stomach contents were squeezed into the small intestine which was then tied off with surgical silk thread. The tied off intestine was removed intact and transferred to a 125-ml. Erlenmeyer flask filled to the brim with sterile Ringer’s solution and capped with an inverted IO-ml. beaker. The flask was incubated at 45°C. for 2 hr. Peristaltic movement of the intestine continued for about 1 hr. At the end of the incubation period, the intestinal contents were flushed out with 20 ml. of 0.9% NaCl solution using a 20-ml. syringe and a 20-gage needle.

Ancdysis of Gut Tissue for Vitumin A The intestinal wall tissue was placed in a small flask containing 10 ml. of 95% ethanol, redistilled over KOH, and 1.0 ml. of 60% aqueous KOH solution. The tissue was refluxed with the alcoholic base until a homogeneous solution appeared, usually within 5-10 min., and not more than 15 min. This was transferred to a 59ml. glass-stoppered centrifuge bottle with an equal volume of distilled water. The nonsaponifiable lipide was extracted by shaking in a shaking machine with 2-lo-ml. portions of petroleum ether (A. R. grade, b.p. 30&6O”C.), the phases separated by centrifugation, and the supernatant petroleum ether removed and collected. The extract was evaporated in a 40” water bath with a stream of nitrogen and redissolved in a 1:l mixture of xylene (A. R. grade) and kerosene (odorless, Fisher Scientific Co.). The optical density of the solution was read in a Beckman model DU spectrophotometer from 310 to 390 mr, using a hydrogen-discharge lamp as a light source. The solution was then irradiated for 1 hr. in several quartz tubes 2-3 mm. in diameter and 2.5-3.5 cm. in length, sealed at one end. A Coleman spectrophotometer fluorescence accessory was used as a source of irradiating energy in front of which was placed a Farrand Optical Co. filter transmitting light only from 300 to 490 mp. After irradiation, the spectrum was read again. Subtraction of the postirradiation spectrum from the preirradiation spectrum gave a curve which closely resembles that of pure vitamin A. (Fig. 1). The optical density at 328 rnMwas used to calculate the vitamin A content of the gut lipide in accordance with Beer’s law which holds for light absorption by vitamin A at 328 me, where EiFrn is 1570 in xylene-kerosene. No carotene correction is required in this scheme of analysis. However, care must be exerted in handling both the carotene dispersion and the gut nonsaponifiable lipide, which contains some carotene. Prolonged exposure to strong light or prolonged heating in saponification may result in some cis inversion of the all-trans-p-carotene which is accompanied by the appearance of a cis peak (20) with an absorption maximum near 340 rnp. Part of this absorption is destroyed by irradiation and may therefore occasion a false estimation of vitamin A content (17) by interfering with the measurement of vitamin A absorption at 328 rnp.

SUMMARY Estimation of vitamin A in gut tissue after incubation with a carotene and tocopherol dispersion appears to be nonspecific with the Carr-Price

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reagent previously employed and with activated glycerol dichlorohydrin. In contrast to this, measurement of light absorption of t,he nonsaponifiable gut lipide at 328 rnp before and after destructive irradiation of vitamin A can be employed to estimate vitamin A accurately since the difference in spectrum, between 310 and 390 rnp, before and after destructive irradiation closely resembles the absorption curve of pure vitamin A. Employing this method of estimation, conversion of carotene to vitamin A in the isolated intestinal loop of the rat was demonstrated. From this evidence, in conjunction with the prior findings of other workers, it may be concluded that the small intestine is an important site of conversion of carotene to vitamin A in the rat’. REFERENCES 1. GOODWIN, T. W., DEWAR, ,4. D., AND GREGORY, R. A., Biochem. J. (London) 40, lx (1946). 2. GLOVER, J., GOODWIN, T. W., AND MORTON, R. A., Biochem. J. (London) 43, lxv (1947). 3. MATTSON, F. H., MEHL, J. W., AND DEUEI,, J. H., JR., Arch. Biochem. 15 65 (1947). 4. THOMPSOX, S. Y., GANGULY, J., AND KON, S. K., Brit. J. Nutrition, 3,50 (1949). 5. GANGULY, J., MAWSON, E. H., AND KON, S. K., Proc. 12th Intern. Dairy Gong. (Stockholm) 2, 238 (1949). 41,619 (1950). 6. CHENG, A. L. S., AND DEUEL, H. J., JR., J. Nutrition 7. THOMPSON, S. Y., BRANDE, R., COATES, M. E., CORVIES, A. T., GANGULY, J., AND KON, S. K., Brit. J. Nutrition 4, 398 (1950). S. SEXTON, E. L., MEHL, J. W., AND DEUEL, H. J., JR., J. Nutrition 31,299 (1946). 9. WIESE, C. E., MEHL, J. W., AND DEUEL, H. J., JR., Arch. Biochem. 16, 75 (1947). 10. SOBEL, A. E., AND WERBIN, H., Ind. Eng. Chem., Anal. Ed. 18, 570 (1946). Il. CHEVALIER, A., AND CHABRE, I?., Bull. sot. chim. biol. 16, 1451 (1934). 12. NEAL, R. H., HACRAXD, C. H., AND LUCKMAN, F. H., Znd. Eng. Chew, Anal. Ed. 13, 150 (1941). 13. NEAL, R. H., AND LUCKMAN, F. H., Ind. Eng. Chem., flnnl. Ed. 16,358 (1944). 14. LITTLE, R. W., Ind. Eng. Chem., Anal. Ed. 16,288 (1944). 15. LITTLE, R. W., THOS~AS, A. W., AND SHERMAN, H. C., J. Biol. Chem. 143, 441 (1943). 16. BESSEY, 0. A., LOWRY, 0. H., BROCK, M. J., AND LOPEZ, J. &4., b. Biol. Chem. 166, 177 (1946). li. BIERI, J. G., AND SCHULTZE, M. O., Arch. Biochem. and Biophys. 34,273 (1951). 18. SOBSL, A. E., SHERMAN, M., LICHTBLAU, J., SNO’IV, S., .IND KRAI\IER, B., J. Nutrition 36, 225 (1948). 19. BILLS, C. E., HONEYWELL, E. M., WIRICK, A. M., AND KUSSYEIER, .J., J. Riol. Chem. 90, 619 (1931). 20. ZECHXEISTER, L., AND POLK&R, -4., J. ilm. Chem. Sot. 66, 1522 (1943).