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In Vitro Conversion of Carotene to Vitamin A by Chick Liver and Intestine1
In Vitro Conversion of Carotene to Vitamin A by Chick Liver and Intestine 1 E. M. OLSEN, J. D. HARVEY, 2 D. C. H I L L AND H. D. BRANION
Department of Nutrition, Ontario Agricultural College, Guelph, Ontario, Canada (Received for publication February 10, 1959)
I
REVIEW OF LITERATURE
There is a large volume of literature dealing with problems of conversion of carotene to vitamin A and there is considerable diversity in conclusions and procedures, to say nothing of the possibility of species variation. A review of research on the site of conversion of carotene to vitamin A has been published by Kon and Thompson (1951) and by Lowe and Morton (1956); the latter also reviewed 1 This work formed a part of a thesis submitted by the senior author to the School of Graduate Studies, University of Toronto, in partial fulfilment of the requirements for the degree of Master of Science in Agriculture. 2 Present address: Topnotch Feeds, Ltd., Seaforth, Ontario.
work on the mechanisms involved in the conversion. Hence, the review of previous work in this field will be limited largely to in vitro conversion. In vitro conversion of carotene to vitamin was first claimed by Olcott and McCann (1931), who incubated liver from vitamin A-deficient rats with carotene and claimed the formation of vitamin A by a liver carotenase. However, Ahmad (1931) and Rhea and Drummond (1932) were unable to confirm this finding. Negative results were reported from in vitro experiments using shark liver by von Euler and von Euler (1931), and using cat liver by Drummond and MacWalter (1933). Parienti and Ralli (1931) obtained one positive result in four trials using dog livers, while von Euler and Klausman (1932) reported conversion of carotene to vitamin A when beef liver was incubated with a carotene solution. Wilson et al. (1937) reported positive results with rabbit liver. Sibbald and Olsen (1958) and Sibbald and Hutcheson (1959) obtained conversion of carotene to vitamin A in a ligatured loop of the duodenum of the living chick. The latter workers showed that the conversion took place in the wall, but that, if the blood supply to the loop is ligated, conversion is prevented, and suggested that some factor or factors present in the loop, but absent in an excised loop, is essential to the absorption of carotene by the duodenal tissue. Wiese et al. (1947) and Glover et al. (1948) obtained conversion with carotene
950
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T IS generally accepted that the small intestine is a major site for conversion of ingested carotene to vitamin A. Originally, a specific enzyme, carotenase, was assumed to be involved in the process. Recently, however, the hypothesis has been advanced that conversion may take place by /3-oxidation of carotene to retinene, followed by reduction of retinene to vitamin A alcohol. With such an hypothesis, a special enzyme, carotenase, would not be required and only normal /3-oxidation processes need be postulated. This hypothesis is supported by recent evidence that conversion of carotene to vitamin A can take place in several tissues in addition to the intestine. However, much of the work in this field is inconclusive and contradictory and further information on many aspects of the problem is required.
IN VITRO CONVERSION OF CAROTENE
951
ride reagent was added from a rapiddelivery pipette. To avoid error, due to differences in time consumed in the manipulation of the samples between the addition of the reagent and reading, all readings were taken 15 seconds after the addition of the reagent. Owing to the delay, some fading of the blue colour occurred before the reading was taken. However, compensation for the effect of this fading was made by referring the density value to a calibration curve made by the same procedure. Calculations were made in the same way as in the macro-determinations, with the correction for carotenoids. The liver and intestinal tissues, used in these experiments, were obtained from Columbian Rock chicks fed a vitamin A and carotenoid free diet, and hatched from eggs laid by hens fed a carotenoidfree and vitamin A-low diet. Details of these diets were given by Olsen et al. (1959b).
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Assays for vitamin A and carotene were made by the procedure of Olsen et al. (1959a), except that a semi-micro procedure was used for the determination of vitamin A in some of the samples with low concentrations of the vitamin. After chromatography, the eluate, containing vitamin A, was evaporated under vacuum in a hot water bath at 50-60°C. The cooled residue was dissolved in 2 ml. of hexane. One ml. of the hexane solution was transferred to an Evelyn microcolorimeter cuvette and the percent transmittance at 400 m/j. was determined. The hexane solution was evaporated to dryness, using a vacuum hot-plate, care being taken to avoid loss by splattering. The residue was dissolved in 0.1 ml. of chloroform and one drop of acetic anhydride was added before the sample was placed in the colorimeter. One ml. of antimony trichlo-
1. Comparison of liver homogenate and whole intestine as sources of enzymes for conversion of carotene to vitamin A. A suspension of carotene3 was prepared by adding slowly a solution of carotene in acetone to a 5 percent solution of Tween 60 in distilled water, while heating on a steam bath under nitrogen. Heating and agitation was continued until all the acetone had evaporated. The resulting mixture was an opaque suspension with no visible crystals of carotene. The concentration was adjusted to the desired level of carotene with 5 percent Tween solution. 3
Unless otherwise stated, carotene suspensions used in other experiments were prepared in the same way. For this experiment, 10%a-90% |3-carotene was used, but, in succeeding experiments, pure 0-carotene, purchased from General Biochemicals Inc., Chagrin Falls, Ohio, was used.
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incubated with rat small intestine, but Kon and Thompson (1951), using a perfusion technique, failed to do so. Stallcup and Herman (1950), observed conversion when carotene in Tween 80 was incubated with either calf liver or intestine. McGillivray (1951), using colloidal carotene, incubated anaerobically in Ringer-Locke solution, with sheep jejunum, found significant conversion into vitamin A. Rosenberg and Sobel (1953) also obtained conversion of j3-carotene, dispersed in Tween 80 and with a-tocopherol, in rat intestine incubated in Ringer solution. However, De and Sundarajan (1951) could not demonstrate in vitro conversion of carotene by rat intestine, and Bieri and Pollard (1953), using rat, calf, and rabbit intestine, under a variety of conditions, concluded that the conversion of carotene into vitamin A proceeded at a greatly reduced rate, if at all, in the isolated intestine.
952
E. M. OLSEN, J. D. HARVEY, D. C. H I L L AND H. D. BRANION
4
This solution contairs0.8% NaCl, 0.02% KC1, 0.01% CaCl2, 0 . 1 % NaHC0 3 , 0.01% MgCl 2 , 0.005% NaH 2 P0 4 , and 0 . 1 % dexi.cse.
TABLE 1.—A comparison of intestine and a liver homogenate as enzyme sources for the conversion of carotene to vitamin A Analysis following incubation1 Tissue Carotene incubated f^ded (mc«-> Intestine Intestine Liver Liver
2,000 — 3,000 —
Carotene (meg.)
Vitamin A Non-carotene* Ca (I.U.) (mcg)
1,516+65.8 20.8+1.5 43 1.2 2,278+61.2 29.3+4.6 — 3,3
13.1 — 16.2 —
1 Fifteen test samples and two control samples were analyzed for each tissue type. Variation, where given, is fiducial limits for p=0.05. 2 Non-carotene carotenoids eluted from the chromatographic column together with vitamin A.
Attention should be drawn to the fairly high contamination of vitamin A with carotene pigments, with the attendant uncertainty of the correction for these impurities. It is possible that this may have contributed to the vitamin A levels found. In any event, the data indicate that liver preparations are likely to be as satisfactory as the intestine for in vitro experiments on conversion, in so far as amount of conversion obtained, is concerned. Furthermore, the preparation of the liver samples was less time consuming, a factor which might be of importance in investigations of enzyme activity. In view of these considerations, subsequent in vitro experiments were conducted with liver. 2. Influence of concentration of carotene on the conversion of carotene to vitamin A by liver homogenate. Although only about 1 percent of the carotene, which was added to the liver and duodenal preparations in the preceding experiment, was converted to vitamin A, it was considered possible that a similar absolute amount of vitamin A might be found, following incubation, even if a much lower concentration of the provitamin was added to the tissue. A lower concentration would be desirable, since difficulty was encountered in separating the different components when large amounts of carotene were added to
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One week old chicks, depleted in vitamin A, were killed and their livers and duodena immediately removed. The duodenum was rinsed with 0.9 percent saline and tied off at one end. Two ml. of the carotene suspension was placed in the lumen, using a syringe, and the other end tied. The intestinal section was then placed in an Erlenmeyer flask, containing 20 ml. of Tyrode's solution.4 All livers were pooled and homogenized in a Waring blendor with 1 ml. of Tyrode's solution for each gram of liver. Aliquots of the homogenate, each weighing 3 grams, were placed in 100 ml. beakers and 3 ml. of carotene suspension were added to each and mixed with the liver homogenate. The samples were placed in an air incubator at 37°C, immediately after preparation, and incubated for 6 hours. Control samples, containing only the tissue and Tyrode's solution, were incubated at the same time. The results are shown in Table 1. There was more conversion of carotene in the liver preparations, 26 I.U. per sample compared to 19.6 I.U. per duodenal sample. These results are in agreement with a report by Harvey (1954) that a liver homogenate was more active than the duodenum in converting carotene to vitamin A, and confirm the reports of previous investigators that the liver, as well as the intestine, is capable of the conversion. The superior conversion in the liver preparation could be due either to the fact that a greater amount of carotene was added to the liver tissue, or it could be attributed to a greater activity in this tissue of the enzymes required for the conversion.
IN VITRO CONVERSION OF CAROTENE
T A B L E 2.—Recovery oj carotene and vitamin A liver homogenate samples incubated with different levels of carotene
from
Average recovery per sample Level of carotene (mcg.) 0 100 200 600
N
t
™°-?'e s sam P'
Carotene (meg.)
Vitamin A (I.U.)
0 54 82 317
1.8 2.9 3.5 3.8
NonIncrease in carotene vit. A durcarotenoids ing incuba(mcg.) tion (I.U.) 0.2 0.8 1.0 1.5
1.1 1.7 2.0
However, since the small amount of vitamin A, obtained by adding 100 micrograms of carotene, could be measured satisfactorily, using the semi-micro technique, and the advantages of working with a low level of carotene were considerable, this level of carotene was used in the experiments to follow 3. Influence of incubation time on the conversion of carotene to vitamin A by liver homogenate. A much higher proportion of carotene, than could be accounted for as vitamin A, disappeared from the incubation mixtures in both of the preceding experiments. This suggested that considable destruction of carotene took place during incubation. Some destruction of the vitamin A, which had been formed, could also be expected, and the amount of vitamin A found, following incubation, would, therefore, be the difference between that formed and that destroyed. An experiment, not reported in detail herein, in which vitamin A was incubated with liver homogenate for 1 hour, indicated that at least one-third of the vitamin was destroyed. The length of time that conversion will proceed during incubation, under the conditions employed, is not known, but it might be assumed that, as incubation time is prolonged, the rate of formation of vitamin A decreases, while the rate of destruction remains unchanged. Hence, there should be an incubation time which is optimum for the maximum recovery of vitamin A. In view of these considerations it was thought worthwhile to investigate the effect of different incubation times on the conversion of carotene into vitamin A. Two experiments were conducted. In the first experiment, liver homogenates containing 5 grams of fresh liver were incubated with 100 micrograms of carotene in Tween 60, for periods varying from 1 to 6 hours. In the second experiment the
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the tissue. The major part of the colour present in the eluate, containing vitamin A, was carotenoids which could not be separated from vitamin A on the chromatographic column. In addition, some contamination with carotene from the receiving cylinder was difficult to avoid, in spite of several rinses, following the removal of the carotene and before vitamin A was eluted. Non-carotene carotenoids, present as impurities in the sample of carotene used, were found to be contributing significantly to the contamination of vitamin A on the chromatographic column. Accordingly, in this experiment and all subsequent experiments, the carotene was recrystallized before dissolving it in acetone. In the present experiment, designed to study the effect of different levels of carotene addition, 3 gram samples of liver homogenate were incubated for 6 hours with carotene in Tween 60 suspension, at levels of 100, 200 and 600 micrograms of carotene per sample. The results are given in Table 2. The recovery of vitamin A was low at all levels of added carotene, being about 1 percent in the case of the 100 and 200 microgram levels, of about the same order as those found in the preceding experiment, but only 0.3 percent for the 600 microgram level. It appeared, therefore, that under the conditions of this experiment, the amount of carotene converted was :nfluenced by the amount added.
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E. M. OLSEN, J. D. HARVEY, D. C. HILL AND H. D. BRANION
TABLE 3.—Influence of incubation time on the conversion of carotene to vitamin A by liver homogenate1 Carotene and vitamin A found Incubation time (hours)
Unheated liver homogenates
Heated liver homogenates
Carotene Vitamin (meg.) A (I.U.)
Carotene Vitamin (meg.) A (I.U.)
81 61 63 75 72 78 76
0 1 2 3 4 5 6
80 48 46 50 46 46 46
2.8 5.0 5.4 5.8 5.6 5.4 5.1
2.6 3.0 2.8 2.9 2.9 2.7 2.3
length of the incubation time was varied from 15 to 60 minutes and three levels of carotene were used, 20, 60 and 100 micrograms. In both experiments the control samples were liver homogenates in which the enzymes were inactivated by heating on a steam bath for 2 minutes. Previous tests indicated that this treatment destroyed only negligible quantities of the vitamin A contained in the liver. The results of the two experiments are given in Tables 3 and 4. The evidence indicated that, after only 15 minutes of incubation of the homogenate, a small, but significant, amount of conversion of carotene into vitamin A,
CONCLUSIONS
Although conversion of carotene to vitamin A was accomplished, the amount of conversion obtained was too small to
TABLE 4.—Influence of incubation time and level of added carotene on the conversion of carotene to vitamin A by liver homogenate Level of carotene added (meg.) Incubation time
60 (control1)
100
60
20 Carotene
Vit. A
Carotene
Vit. A
Carotene
Vit. A
Carotene
Vit. A
(minutes)
(meg.)
(I.U.)
(meg.)
(I.U.)
(meg.)
(I.U.)
(meg.)
(LIT.)
0 15 30 45 60
4.7 9.2 11.5 10.9 11.5
1.8 2.1 2.3 2.1 2.5
37 29 31 32 33
1.8 2.3 2.9 2.9 3.0
85 76 69 73 77
1.8 2.7 3.2 3.5 3.9
35 33 36 33 35
1.9 1.8 1.9 1.8 1.6
1
Liver homogenate heated to render enzymes inactive.
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1 100 meg. of carotene added to 5 gm. liver homogenate.
had occurred. In the first experiment (Table 3) vitamin A content reached a maximum in 2 to 3 hours; in the second experiment, the maximum was reached in 30 minutes. However, the differences would seem to be too small to justify definite conclusions as to optimal incubation times. In all comparisons between unheated and heated, prior to incubation, homogenates, the former yielded the higher level of vitamin A. The amount found in the heated preparations was relatively constant, regardless of the time of incubation, and it seems likely that the quantity found was that present in the liver prior to incubation, i.e., the liver stores of the chicks, in spite of their previous dietary regime and origin, were not exhausted. Further, since there was little difference between the amounts of vitamin A found in the unheated and heated homogenates not subjected to incubation, it seems clear that destruction of vitamin A, during heat treatment of the liver, could not account for the differences observed in the incubated preparations.
IN VITRO CONVERSION or CAROTENE
SUMMARY
A series of experiments was conducted to study the in vitro conversion of carotene to vitamin A. A small, but definite, amount of conversion of carotene to vitamin A was obtained when preparations of chick duodenum or liver homogenate were incubated with an aqueous suspension of carotene in Tween 60. The two tissues would appear to be about equally active as sources of the enzymes required for the conversion, although the amount of vitamin A found in the liver preparations was actually greater. Under the conditions of these experiments, the amount of carotene which was converted to vitamin A, appeared to be related to the amount of provitamin added to the incubated liver preparations. In general, about 1 percent of the carotene could be accounted for as vitamin A in the samples following incubation. The conversion process was a rapid reaction, since a measurable amount took place within the first 15 minutes of incubation. Increasing the incubation time, beyond 30 minutes, did not have any great effect on the amount of conversion.
REFERENCES Ahmad, B., 1931. The fate of carotene after absorption in the animal organism. Biochem. J. 25: 1195-1204. Bieri, J. G., and C. J. Pollard, 1953. The in vitro conversion of carotene to vitamin A. Texas Repts. Biol. Med. 11:402-411. De, N. K., and A. R. Sundarajan, 1951. The site of in vivo conversion of carotene to vitamin A. Indian J. Med. Res. 39: 479-489. Drummond, J. C , and R. MacWalter, 1933. About the biological relation between carotene and vitamin A. Biochem. J. 27: 1342-1347. Glover, J., T. W. Goodwin and R. A. Morton, 1948. Studies in vitamin A. 8. Conversion of |3-carotene into vitamin A in the intestine of the rat. Biochem. J. 43: 512-518. Harvey, J. D., 1954. Studies on the utilization of vitamin A and carotene by chickens during embryonic development and early life. Ph.D. thesis, Kansas State College, Manhattan. Kon, S. K., and S. Y. Thompson, 1951. Site of conversion of carotene to vitamin A. British J. Nutrition, 5: 114-119. Lowe, J. S., and R. A. Morton, 1956. Some aspects of vitamin metabolism. Vitamins and Hormones, 14: 97-137. McGillivray, W. A., 1951. The conversion of carotene to vitamin A in sheep and cattle. Australian J. Sci. Res. (B) 4: 370-376. Olcott, H. S., and D. C. McCann, 1931. Carotenase. The transformation of carotene to vitamin A in vitro. J. Biol. Chem. 94: 185-193. Olsen, E. M., J. D . Harvey, B . C. Hill and H. D. Branion, 1959a. Distribution of carotene and vitamin A in the tissues of chicks following administration of carotene. Poultry Sci. 38: 688693. Olsen, E. M., J. D. Harvey, D. C. Hill and H. D. Branion, 1959b. Utilization and stability of commercial vitamin A supplements. Poultry Sci. 38: 929-942. Parienti, A. C , and E. P. Ralli, 1931. Presence of carotenase in the liver of the dog. Proc. Soc. Expt. Biol. Med. 29: 1209-1210. Rhea, J. L., and J. C. Drummond, 1932. Zur die Bildung von vitamin A aus Carotene in tierischen Organismus. Zeit. Vitaminforsch. 1: 177183. Rosenberg, A., and A. E. Sobel, 1953. In vitro conversion of carotene to vitamin A in the isolated small intestine of the rat. Arch. Biochem. 44: 320-325. Sibbald, I. R., and L. M. Hutcheson, 1959. The conversion of /3-carotene to vitamin A in the liga-
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provide for further study of the possible reactions involved in the process, and of the factors which may influence it. One possibility, worthy of future study, is evident. In view of the finding of Sibbald and Hutcheson (1959), that a blood supply to the ligatured duodenum was necessary for conversion, additions of various blood fractions to in vitro preparations would seem obvious. In fact, the possibility of actual conversion by blood constituents should not be overlooked. Further, it is not beyond the realm of speculation, that destruction of the vitamin A formed might be alleviated, to some extent, by blood constituents.
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tured duodenum of the chick. Poultry Sci. 38: 701-706. Sibbald, I. R., and E. M. Olsen, 1958. The conversion of /3-carotene to vitamin A in the duodenum of the chick. Poultry Sci. 37: 1465-1467. Stallcup, O. T., and H. A. Herman, 1950. In vitro studies on the conversion of carotene to vitamin A in dairy calves. J. Dairy Sci. 33: 237-242. von Euler, H., and E. Klausman, 1932. Carotene und Vitamin A. Svensk. Kem. Tidschr. 44: 223. von Euler, H., and B. von Euler, 1931. Zur Kennt-
nis der Leberole von Fischen and Vogeln. Svensk. Kem. Tidschr. 43: 174-178. Weise, C. E., J. W. Mehl and H. J. Deuel, Jr., 1947. Studies on carotenoid metabolism. 8. The in vitro conversion of carotene to vitamin A in the intestine of the rat. Arch. Biochem. 15: 75-79. Wilson, H. E., B. Ahmad and B. N. Mazumdar, 1937. Transformation of carotene into vitamin A in liver autolysates. Indian. J. Med. Res. 25: 85-88.
R. C. PATHAK AND C. M. SINGH Department of Pathology and Bacteriology, U.P. Veterinary College, Mathura, U.P., India (Received for publication February 12, 1959)
T
HE pleuropneumonia-like organisms (PPLO) are a group of those fastidious microorganisms which are filtrable, soft, fragile, and highly pleomorphic. Nocard and his associates in 1898 studied these microorganisms for the first time, and they considered these as the causative agents of bovine pleuropneumonia. The first isolation of these organisms from humans was reported in 1937 by Dienes and Edsall who recovered them from a suppurating Bartholin's gland. Since then a number of reports have appeared regarding the isolation of the PPLO either parasitic or saprophytic, the majority of the instances being from humans. Markham and Wong (1952) isolated the PPLO from exudates of chickens and turkeys with the chronic respiratory disease (CRD) and indicated that these could be cultivated in cell free medium. The CRD has been recognized as the
* This report is a portion of research work being carried out by R. C. Pathak in partial fulfillment of the requirements for the degree of Master of Veterinary Science in Advanced Bacteriology of the Agra University.
disease of great economic importance to the poultry industry in the U.S.A., Canada, and other countries. In recent years the knowledge regarding the etiology, epizootiology, immunity and control has expanded to a considerable extent, and much literature has been put forth by the American workers. In India also there have been indications of the presence of the CRD for some time past. However, Rao (1958) could not establish the presence of the PPLO in his studies on infectious coryza in chickens by inoculation of chick embryo technique, which was the only method employed by him for the isolation of these organisms. During the routine post-mortem examinations of poultry submitted to this department for the last few years it has been observed that quite a few birds show lesions similar to that of the so called CRD. However, the isolation and identification of the PPLO could not be attempted earlier. The present report deals with the isolation and identification of PPLO from poultry maintained on the poultry farm attached to the U. P. Veterinary College, Mathura. This poultry
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A Preliminary Report on the Isolation and Identification of Pleuropneumonia-like Organisms from Poultry*
Department of Nutrition, Ontario Agricultural College, Guelph, Ontario, Canada (Received for publication February 10, 1959)
I
REVIEW OF LITERATURE
There is a large volume of literature dealing with problems of conversion of carotene to vitamin A and there is considerable diversity in conclusions and procedures, to say nothing of the possibility of species variation. A review of research on the site of conversion of carotene to vitamin A has been published by Kon and Thompson (1951) and by Lowe and Morton (1956); the latter also reviewed 1 This work formed a part of a thesis submitted by the senior author to the School of Graduate Studies, University of Toronto, in partial fulfilment of the requirements for the degree of Master of Science in Agriculture. 2 Present address: Topnotch Feeds, Ltd., Seaforth, Ontario.
work on the mechanisms involved in the conversion. Hence, the review of previous work in this field will be limited largely to in vitro conversion. In vitro conversion of carotene to vitamin was first claimed by Olcott and McCann (1931), who incubated liver from vitamin A-deficient rats with carotene and claimed the formation of vitamin A by a liver carotenase. However, Ahmad (1931) and Rhea and Drummond (1932) were unable to confirm this finding. Negative results were reported from in vitro experiments using shark liver by von Euler and von Euler (1931), and using cat liver by Drummond and MacWalter (1933). Parienti and Ralli (1931) obtained one positive result in four trials using dog livers, while von Euler and Klausman (1932) reported conversion of carotene to vitamin A when beef liver was incubated with a carotene solution. Wilson et al. (1937) reported positive results with rabbit liver. Sibbald and Olsen (1958) and Sibbald and Hutcheson (1959) obtained conversion of carotene to vitamin A in a ligatured loop of the duodenum of the living chick. The latter workers showed that the conversion took place in the wall, but that, if the blood supply to the loop is ligated, conversion is prevented, and suggested that some factor or factors present in the loop, but absent in an excised loop, is essential to the absorption of carotene by the duodenal tissue. Wiese et al. (1947) and Glover et al. (1948) obtained conversion with carotene
950
Downloaded from http://ps.oxfordjournals.org/ at Periodicals Dept on June 30, 2015
T IS generally accepted that the small intestine is a major site for conversion of ingested carotene to vitamin A. Originally, a specific enzyme, carotenase, was assumed to be involved in the process. Recently, however, the hypothesis has been advanced that conversion may take place by /3-oxidation of carotene to retinene, followed by reduction of retinene to vitamin A alcohol. With such an hypothesis, a special enzyme, carotenase, would not be required and only normal /3-oxidation processes need be postulated. This hypothesis is supported by recent evidence that conversion of carotene to vitamin A can take place in several tissues in addition to the intestine. However, much of the work in this field is inconclusive and contradictory and further information on many aspects of the problem is required.
IN VITRO CONVERSION OF CAROTENE
951
ride reagent was added from a rapiddelivery pipette. To avoid error, due to differences in time consumed in the manipulation of the samples between the addition of the reagent and reading, all readings were taken 15 seconds after the addition of the reagent. Owing to the delay, some fading of the blue colour occurred before the reading was taken. However, compensation for the effect of this fading was made by referring the density value to a calibration curve made by the same procedure. Calculations were made in the same way as in the macro-determinations, with the correction for carotenoids. The liver and intestinal tissues, used in these experiments, were obtained from Columbian Rock chicks fed a vitamin A and carotenoid free diet, and hatched from eggs laid by hens fed a carotenoidfree and vitamin A-low diet. Details of these diets were given by Olsen et al. (1959b).
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Assays for vitamin A and carotene were made by the procedure of Olsen et al. (1959a), except that a semi-micro procedure was used for the determination of vitamin A in some of the samples with low concentrations of the vitamin. After chromatography, the eluate, containing vitamin A, was evaporated under vacuum in a hot water bath at 50-60°C. The cooled residue was dissolved in 2 ml. of hexane. One ml. of the hexane solution was transferred to an Evelyn microcolorimeter cuvette and the percent transmittance at 400 m/j. was determined. The hexane solution was evaporated to dryness, using a vacuum hot-plate, care being taken to avoid loss by splattering. The residue was dissolved in 0.1 ml. of chloroform and one drop of acetic anhydride was added before the sample was placed in the colorimeter. One ml. of antimony trichlo-
1. Comparison of liver homogenate and whole intestine as sources of enzymes for conversion of carotene to vitamin A. A suspension of carotene3 was prepared by adding slowly a solution of carotene in acetone to a 5 percent solution of Tween 60 in distilled water, while heating on a steam bath under nitrogen. Heating and agitation was continued until all the acetone had evaporated. The resulting mixture was an opaque suspension with no visible crystals of carotene. The concentration was adjusted to the desired level of carotene with 5 percent Tween solution. 3
Unless otherwise stated, carotene suspensions used in other experiments were prepared in the same way. For this experiment, 10%a-90% |3-carotene was used, but, in succeeding experiments, pure 0-carotene, purchased from General Biochemicals Inc., Chagrin Falls, Ohio, was used.
Downloaded from http://ps.oxfordjournals.org/ at Periodicals Dept on June 30, 2015
incubated with rat small intestine, but Kon and Thompson (1951), using a perfusion technique, failed to do so. Stallcup and Herman (1950), observed conversion when carotene in Tween 80 was incubated with either calf liver or intestine. McGillivray (1951), using colloidal carotene, incubated anaerobically in Ringer-Locke solution, with sheep jejunum, found significant conversion into vitamin A. Rosenberg and Sobel (1953) also obtained conversion of j3-carotene, dispersed in Tween 80 and with a-tocopherol, in rat intestine incubated in Ringer solution. However, De and Sundarajan (1951) could not demonstrate in vitro conversion of carotene by rat intestine, and Bieri and Pollard (1953), using rat, calf, and rabbit intestine, under a variety of conditions, concluded that the conversion of carotene into vitamin A proceeded at a greatly reduced rate, if at all, in the isolated intestine.
952
E. M. OLSEN, J. D. HARVEY, D. C. H I L L AND H. D. BRANION
4
This solution contairs0.8% NaCl, 0.02% KC1, 0.01% CaCl2, 0 . 1 % NaHC0 3 , 0.01% MgCl 2 , 0.005% NaH 2 P0 4 , and 0 . 1 % dexi.cse.
TABLE 1.—A comparison of intestine and a liver homogenate as enzyme sources for the conversion of carotene to vitamin A Analysis following incubation1 Tissue Carotene incubated f^ded (mc«-> Intestine Intestine Liver Liver
2,000 — 3,000 —
Carotene (meg.)
Vitamin A Non-carotene* Ca (I.U.) (mcg)
1,516+65.8 20.8+1.5 43 1.2 2,278+61.2 29.3+4.6 — 3,3
13.1 — 16.2 —
1 Fifteen test samples and two control samples were analyzed for each tissue type. Variation, where given, is fiducial limits for p=0.05. 2 Non-carotene carotenoids eluted from the chromatographic column together with vitamin A.
Attention should be drawn to the fairly high contamination of vitamin A with carotene pigments, with the attendant uncertainty of the correction for these impurities. It is possible that this may have contributed to the vitamin A levels found. In any event, the data indicate that liver preparations are likely to be as satisfactory as the intestine for in vitro experiments on conversion, in so far as amount of conversion obtained, is concerned. Furthermore, the preparation of the liver samples was less time consuming, a factor which might be of importance in investigations of enzyme activity. In view of these considerations, subsequent in vitro experiments were conducted with liver. 2. Influence of concentration of carotene on the conversion of carotene to vitamin A by liver homogenate. Although only about 1 percent of the carotene, which was added to the liver and duodenal preparations in the preceding experiment, was converted to vitamin A, it was considered possible that a similar absolute amount of vitamin A might be found, following incubation, even if a much lower concentration of the provitamin was added to the tissue. A lower concentration would be desirable, since difficulty was encountered in separating the different components when large amounts of carotene were added to
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One week old chicks, depleted in vitamin A, were killed and their livers and duodena immediately removed. The duodenum was rinsed with 0.9 percent saline and tied off at one end. Two ml. of the carotene suspension was placed in the lumen, using a syringe, and the other end tied. The intestinal section was then placed in an Erlenmeyer flask, containing 20 ml. of Tyrode's solution.4 All livers were pooled and homogenized in a Waring blendor with 1 ml. of Tyrode's solution for each gram of liver. Aliquots of the homogenate, each weighing 3 grams, were placed in 100 ml. beakers and 3 ml. of carotene suspension were added to each and mixed with the liver homogenate. The samples were placed in an air incubator at 37°C, immediately after preparation, and incubated for 6 hours. Control samples, containing only the tissue and Tyrode's solution, were incubated at the same time. The results are shown in Table 1. There was more conversion of carotene in the liver preparations, 26 I.U. per sample compared to 19.6 I.U. per duodenal sample. These results are in agreement with a report by Harvey (1954) that a liver homogenate was more active than the duodenum in converting carotene to vitamin A, and confirm the reports of previous investigators that the liver, as well as the intestine, is capable of the conversion. The superior conversion in the liver preparation could be due either to the fact that a greater amount of carotene was added to the liver tissue, or it could be attributed to a greater activity in this tissue of the enzymes required for the conversion.
IN VITRO CONVERSION OF CAROTENE
T A B L E 2.—Recovery oj carotene and vitamin A liver homogenate samples incubated with different levels of carotene
from
Average recovery per sample Level of carotene (mcg.) 0 100 200 600
N
t
™°-?'e s sam P'
Carotene (meg.)
Vitamin A (I.U.)
0 54 82 317
1.8 2.9 3.5 3.8
NonIncrease in carotene vit. A durcarotenoids ing incuba(mcg.) tion (I.U.) 0.2 0.8 1.0 1.5
1.1 1.7 2.0
However, since the small amount of vitamin A, obtained by adding 100 micrograms of carotene, could be measured satisfactorily, using the semi-micro technique, and the advantages of working with a low level of carotene were considerable, this level of carotene was used in the experiments to follow 3. Influence of incubation time on the conversion of carotene to vitamin A by liver homogenate. A much higher proportion of carotene, than could be accounted for as vitamin A, disappeared from the incubation mixtures in both of the preceding experiments. This suggested that considable destruction of carotene took place during incubation. Some destruction of the vitamin A, which had been formed, could also be expected, and the amount of vitamin A found, following incubation, would, therefore, be the difference between that formed and that destroyed. An experiment, not reported in detail herein, in which vitamin A was incubated with liver homogenate for 1 hour, indicated that at least one-third of the vitamin was destroyed. The length of time that conversion will proceed during incubation, under the conditions employed, is not known, but it might be assumed that, as incubation time is prolonged, the rate of formation of vitamin A decreases, while the rate of destruction remains unchanged. Hence, there should be an incubation time which is optimum for the maximum recovery of vitamin A. In view of these considerations it was thought worthwhile to investigate the effect of different incubation times on the conversion of carotene into vitamin A. Two experiments were conducted. In the first experiment, liver homogenates containing 5 grams of fresh liver were incubated with 100 micrograms of carotene in Tween 60, for periods varying from 1 to 6 hours. In the second experiment the
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the tissue. The major part of the colour present in the eluate, containing vitamin A, was carotenoids which could not be separated from vitamin A on the chromatographic column. In addition, some contamination with carotene from the receiving cylinder was difficult to avoid, in spite of several rinses, following the removal of the carotene and before vitamin A was eluted. Non-carotene carotenoids, present as impurities in the sample of carotene used, were found to be contributing significantly to the contamination of vitamin A on the chromatographic column. Accordingly, in this experiment and all subsequent experiments, the carotene was recrystallized before dissolving it in acetone. In the present experiment, designed to study the effect of different levels of carotene addition, 3 gram samples of liver homogenate were incubated for 6 hours with carotene in Tween 60 suspension, at levels of 100, 200 and 600 micrograms of carotene per sample. The results are given in Table 2. The recovery of vitamin A was low at all levels of added carotene, being about 1 percent in the case of the 100 and 200 microgram levels, of about the same order as those found in the preceding experiment, but only 0.3 percent for the 600 microgram level. It appeared, therefore, that under the conditions of this experiment, the amount of carotene converted was :nfluenced by the amount added.
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E. M. OLSEN, J. D. HARVEY, D. C. HILL AND H. D. BRANION
TABLE 3.—Influence of incubation time on the conversion of carotene to vitamin A by liver homogenate1 Carotene and vitamin A found Incubation time (hours)
Unheated liver homogenates
Heated liver homogenates
Carotene Vitamin (meg.) A (I.U.)
Carotene Vitamin (meg.) A (I.U.)
81 61 63 75 72 78 76
0 1 2 3 4 5 6
80 48 46 50 46 46 46
2.8 5.0 5.4 5.8 5.6 5.4 5.1
2.6 3.0 2.8 2.9 2.9 2.7 2.3
length of the incubation time was varied from 15 to 60 minutes and three levels of carotene were used, 20, 60 and 100 micrograms. In both experiments the control samples were liver homogenates in which the enzymes were inactivated by heating on a steam bath for 2 minutes. Previous tests indicated that this treatment destroyed only negligible quantities of the vitamin A contained in the liver. The results of the two experiments are given in Tables 3 and 4. The evidence indicated that, after only 15 minutes of incubation of the homogenate, a small, but significant, amount of conversion of carotene into vitamin A,
CONCLUSIONS
Although conversion of carotene to vitamin A was accomplished, the amount of conversion obtained was too small to
TABLE 4.—Influence of incubation time and level of added carotene on the conversion of carotene to vitamin A by liver homogenate Level of carotene added (meg.) Incubation time
60 (control1)
100
60
20 Carotene
Vit. A
Carotene
Vit. A
Carotene
Vit. A
Carotene
Vit. A
(minutes)
(meg.)
(I.U.)
(meg.)
(I.U.)
(meg.)
(I.U.)
(meg.)
(LIT.)
0 15 30 45 60
4.7 9.2 11.5 10.9 11.5
1.8 2.1 2.3 2.1 2.5
37 29 31 32 33
1.8 2.3 2.9 2.9 3.0
85 76 69 73 77
1.8 2.7 3.2 3.5 3.9
35 33 36 33 35
1.9 1.8 1.9 1.8 1.6
1
Liver homogenate heated to render enzymes inactive.
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1 100 meg. of carotene added to 5 gm. liver homogenate.
had occurred. In the first experiment (Table 3) vitamin A content reached a maximum in 2 to 3 hours; in the second experiment, the maximum was reached in 30 minutes. However, the differences would seem to be too small to justify definite conclusions as to optimal incubation times. In all comparisons between unheated and heated, prior to incubation, homogenates, the former yielded the higher level of vitamin A. The amount found in the heated preparations was relatively constant, regardless of the time of incubation, and it seems likely that the quantity found was that present in the liver prior to incubation, i.e., the liver stores of the chicks, in spite of their previous dietary regime and origin, were not exhausted. Further, since there was little difference between the amounts of vitamin A found in the unheated and heated homogenates not subjected to incubation, it seems clear that destruction of vitamin A, during heat treatment of the liver, could not account for the differences observed in the incubated preparations.
IN VITRO CONVERSION or CAROTENE
SUMMARY
A series of experiments was conducted to study the in vitro conversion of carotene to vitamin A. A small, but definite, amount of conversion of carotene to vitamin A was obtained when preparations of chick duodenum or liver homogenate were incubated with an aqueous suspension of carotene in Tween 60. The two tissues would appear to be about equally active as sources of the enzymes required for the conversion, although the amount of vitamin A found in the liver preparations was actually greater. Under the conditions of these experiments, the amount of carotene which was converted to vitamin A, appeared to be related to the amount of provitamin added to the incubated liver preparations. In general, about 1 percent of the carotene could be accounted for as vitamin A in the samples following incubation. The conversion process was a rapid reaction, since a measurable amount took place within the first 15 minutes of incubation. Increasing the incubation time, beyond 30 minutes, did not have any great effect on the amount of conversion.
REFERENCES Ahmad, B., 1931. The fate of carotene after absorption in the animal organism. Biochem. J. 25: 1195-1204. Bieri, J. G., and C. J. Pollard, 1953. The in vitro conversion of carotene to vitamin A. Texas Repts. Biol. Med. 11:402-411. De, N. K., and A. R. Sundarajan, 1951. The site of in vivo conversion of carotene to vitamin A. Indian J. Med. Res. 39: 479-489. Drummond, J. C , and R. MacWalter, 1933. About the biological relation between carotene and vitamin A. Biochem. J. 27: 1342-1347. Glover, J., T. W. Goodwin and R. A. Morton, 1948. Studies in vitamin A. 8. Conversion of |3-carotene into vitamin A in the intestine of the rat. Biochem. J. 43: 512-518. Harvey, J. D., 1954. Studies on the utilization of vitamin A and carotene by chickens during embryonic development and early life. Ph.D. thesis, Kansas State College, Manhattan. Kon, S. K., and S. Y. Thompson, 1951. Site of conversion of carotene to vitamin A. British J. Nutrition, 5: 114-119. Lowe, J. S., and R. A. Morton, 1956. Some aspects of vitamin metabolism. Vitamins and Hormones, 14: 97-137. McGillivray, W. A., 1951. The conversion of carotene to vitamin A in sheep and cattle. Australian J. Sci. Res. (B) 4: 370-376. Olcott, H. S., and D. C. McCann, 1931. Carotenase. The transformation of carotene to vitamin A in vitro. J. Biol. Chem. 94: 185-193. Olsen, E. M., J. D . Harvey, B . C. Hill and H. D. Branion, 1959a. Distribution of carotene and vitamin A in the tissues of chicks following administration of carotene. Poultry Sci. 38: 688693. Olsen, E. M., J. D. Harvey, D. C. Hill and H. D. Branion, 1959b. Utilization and stability of commercial vitamin A supplements. Poultry Sci. 38: 929-942. Parienti, A. C , and E. P. Ralli, 1931. Presence of carotenase in the liver of the dog. Proc. Soc. Expt. Biol. Med. 29: 1209-1210. Rhea, J. L., and J. C. Drummond, 1932. Zur die Bildung von vitamin A aus Carotene in tierischen Organismus. Zeit. Vitaminforsch. 1: 177183. Rosenberg, A., and A. E. Sobel, 1953. In vitro conversion of carotene to vitamin A in the isolated small intestine of the rat. Arch. Biochem. 44: 320-325. Sibbald, I. R., and L. M. Hutcheson, 1959. The conversion of /3-carotene to vitamin A in the liga-
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provide for further study of the possible reactions involved in the process, and of the factors which may influence it. One possibility, worthy of future study, is evident. In view of the finding of Sibbald and Hutcheson (1959), that a blood supply to the ligatured duodenum was necessary for conversion, additions of various blood fractions to in vitro preparations would seem obvious. In fact, the possibility of actual conversion by blood constituents should not be overlooked. Further, it is not beyond the realm of speculation, that destruction of the vitamin A formed might be alleviated, to some extent, by blood constituents.
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tured duodenum of the chick. Poultry Sci. 38: 701-706. Sibbald, I. R., and E. M. Olsen, 1958. The conversion of /3-carotene to vitamin A in the duodenum of the chick. Poultry Sci. 37: 1465-1467. Stallcup, O. T., and H. A. Herman, 1950. In vitro studies on the conversion of carotene to vitamin A in dairy calves. J. Dairy Sci. 33: 237-242. von Euler, H., and E. Klausman, 1932. Carotene und Vitamin A. Svensk. Kem. Tidschr. 44: 223. von Euler, H., and B. von Euler, 1931. Zur Kennt-
nis der Leberole von Fischen and Vogeln. Svensk. Kem. Tidschr. 43: 174-178. Weise, C. E., J. W. Mehl and H. J. Deuel, Jr., 1947. Studies on carotenoid metabolism. 8. The in vitro conversion of carotene to vitamin A in the intestine of the rat. Arch. Biochem. 15: 75-79. Wilson, H. E., B. Ahmad and B. N. Mazumdar, 1937. Transformation of carotene into vitamin A in liver autolysates. Indian. J. Med. Res. 25: 85-88.
R. C. PATHAK AND C. M. SINGH Department of Pathology and Bacteriology, U.P. Veterinary College, Mathura, U.P., India (Received for publication February 12, 1959)
T
HE pleuropneumonia-like organisms (PPLO) are a group of those fastidious microorganisms which are filtrable, soft, fragile, and highly pleomorphic. Nocard and his associates in 1898 studied these microorganisms for the first time, and they considered these as the causative agents of bovine pleuropneumonia. The first isolation of these organisms from humans was reported in 1937 by Dienes and Edsall who recovered them from a suppurating Bartholin's gland. Since then a number of reports have appeared regarding the isolation of the PPLO either parasitic or saprophytic, the majority of the instances being from humans. Markham and Wong (1952) isolated the PPLO from exudates of chickens and turkeys with the chronic respiratory disease (CRD) and indicated that these could be cultivated in cell free medium. The CRD has been recognized as the
* This report is a portion of research work being carried out by R. C. Pathak in partial fulfillment of the requirements for the degree of Master of Veterinary Science in Advanced Bacteriology of the Agra University.
disease of great economic importance to the poultry industry in the U.S.A., Canada, and other countries. In recent years the knowledge regarding the etiology, epizootiology, immunity and control has expanded to a considerable extent, and much literature has been put forth by the American workers. In India also there have been indications of the presence of the CRD for some time past. However, Rao (1958) could not establish the presence of the PPLO in his studies on infectious coryza in chickens by inoculation of chick embryo technique, which was the only method employed by him for the isolation of these organisms. During the routine post-mortem examinations of poultry submitted to this department for the last few years it has been observed that quite a few birds show lesions similar to that of the so called CRD. However, the isolation and identification of the PPLO could not be attempted earlier. The present report deals with the isolation and identification of PPLO from poultry maintained on the poultry farm attached to the U. P. Veterinary College, Mathura. This poultry
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A Preliminary Report on the Isolation and Identification of Pleuropneumonia-like Organisms from Poultry*