The utilization of Schardinger dextrins by the rat

TOXICOLOGY

The G.

H.

AND APPLIED

Utilization ANDERSEN,~

F.

of

Schardinger

M.

ROBBINS, AND

Research

5,257-266

PHARMACOLOGY

Center,

General

Dextrins

F. J. c. L.

Foods

Recrived

(1963)

DOMINGUES,~

the

R. G.

Rat MOORES,

LONG”

Corporation, Tarrytown, June

by

New York

29, 1962

Schardinger dextrins are a homologous series of cyclic dextrins composed of 6 or more -n-glucopyranose units linked by 13 4 bonds as in amylose. The 6- and 7-unit Schardinger dextrins are the most common and are called, respectively, a-dextrin and /3-dextrin. The Schardinger dextrins can be obtained from the breakdown of starch by a few known strains of bacteria and fungi-the most common being Bacillus macerans. These dextrins are resistant to hydrolysis by acid, c(- and fi-amylase, and yeast. They are of interest in food applications becauseof their ability to form complexes with many organic and inorganic substances. These observations were noted in the work of Schlenk et ,aZ. (1955). Although relatively insoluble, many of these complexes dissociate in aqueous solutions to release included compounds. This observation resulted in an increasedinterest in the cyclic dextrins for binding volatile or unstable substancesused in foods and attention was directed toward their potential toxicity. Very little is known about the metabolism of the Schardinger dextrins by animals. French (1957) has summarized the available data in his review of these compounds. He reported that a group of rats fed purified P-dextrin died after 1 week on the diet. Von Hoesslin and Pringsheim (1923) reported that no glycogen could be detected in the livers of fasted rabbits and guinea pigs 3 hours after they were fed the dextrins. The above references show that experimental animals have been fed diets containing Schardinger dextrins. However, the experiments were 1 Present address: State of New York Department of Labor, Division of Industrial Hygiene, Radiochemical Laboratory, 80 Centre Street, New York 13, New York. 2 General Measurements, Inc., Division of Precision Scientific Corporation, Garnerville, New York. 3 Present address: Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts. 257

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ANDERSEN

ET

AL.

too limited in the number of animals or duration of tests to yield meaningful data concerning utilization of the cyclic dextrins. The Schardinger dextrins are very resistant to attack by the common starch-hydrolyzing enzymes. It has been reported that the Schardinger dextrins are completely resistant to a- and fl-amylase action. However, French (1957) found that the a-dextrin is essentially completely resistant to salivary amylase, the fl-dextrin is very slowly utilized, and the y-dextrin (8 glucose units) is attacked about 1% as rapidly as starch. It has been reported by Ben-Gershom (1955) that certain fungal preparations contain enzymes that are capable of hydrolyzing Schardinger dextrins. Green and Stumpf (1942), in their work on purified potato phosphorylase, showed that this enzyme is competitively inhibited in its action on starch by a- or P-dextrins. They theorized that the Schardinger dextrins and starch were competing for the same active group in the enzyme. Cramer (1953) showed that l3-dextrin retards the cleavage of indican by emulsin or by acid hydrolysis. The formation of including compounds may have been involved in the above inhibition. Thoma and Koshland (1960) demonstrated a competitive inhibition of sweet potato l3-amylase with a- and fl-Schardinger dextrins. The results were interpreted as added support for the induced-fit theory of specificity and suggested that this new type of competitive inhibition by substrate exists in other enzyme systems. McCloskey and Porter (1945) reported that of 18 bacterial species and 4 yeasts, only Bacillus macerans and Bacillus polymyxa were able to utilize Schardinger dextrins. It is evident that uncertainty exists in the literature regarding the metabolism of Schardinger dextrins. The studies reported here were undertaken to clarify this situation by comparing the utilization by rats of radioactive o- and P-dextrins with radioactive potato starch. METHODS The Biosynthetic Preparation of Uniformly Labeled Carbon-14 Potato Starch Potted potato plants, in which the tubers were just beginning to form, were used to prepare the Cl’-labeled starch. The plants were maintained in the apparatus described by Weinstein et al. (1959) differing only in that the apparatus used in this work to enclose the plants was a 43-liter chamber. A bank of ten 40-watt fluorescent bulbs was arranged over the

SCHARDINGER

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UTILIZATION

259

chamber, and six incandescent spot lights were focused on it to supply light to the growing plants. A timing circuit was set up to turn the lights on and off so that the plants would have light for 11 hours and darkness for 13 hours each day. The plants were fed a total of 3 mc of C1-‘OZ in 3 doses of about 1 mc per dose. Two days after the initial millicurie was given, 91% of C’-‘O- given the plants was fixed by them. At that time, another portion of about 1 mc was given; a third 1 mc portion 3 days later. The second dose resulted in 89% fixation and the third 93%. These figures were obtained by sampling the atmosphere at the beginning and end of each fixation period as described by Weinstein et al. (1959). Two days after the last feeding period, the plants were opened to air for a week to allow the fixed C’“O:! to be converted into starch in the tubers. At the end of this period, the tubers were separated from the plant. The total net weight of the potatoes was 46 g. The potatoes were peeled and homogenizedin a Waring blendor three times with cold water. The filtrate was discarded and the cold water-insoluble crude starch was freeze-dried was washed with alcohol. The yield was 3.5 g of labeled potato starch of specific activity 214 PC/g. The Synthesis

of Radioactive

Schardinger

Dextrins

Radioactive a- and P-Schardinger dextrins were prepared by enzymatic digestion of a 2.5% dispersion of potato starch by B. macerans amylase at 40” C for 4 hours, using essentially the method described by Tilden and Hudson (1939). The digestion mixture contained 25 ml of enzyme solution equivalent to 288 units of enzyme (Tilden and Hudson, 1942). The starch consistedof 3.1 g of uniformly labeled C’” potato starch and 8.9 g of Maine potato starch giving a specific activity of 55.3 nc/g. The resulting a- and P-dextrins were isolated as trichloroethylene and bromobenzene complexes as described by French (1957). The yield of a-dextrin was 9.4% and P-dextrin 14.77r, based on the weight of starch used. The cyclic dextrins had the same specific activity; 56 I&g. The specific rotations of the a- and P-dextrins in water were 150.6 t 1.0’ and 161.O F 1.6‘, respectively. The specific rotations reported by French (1957) are 150.5 -+ 0.5” and 162.5 rt 0.5”. Metabolism

Studies

Charles River rats, Wistar strain, weighing 185 % 50 g were starved 18 hours prior to use. Six rats were used in the metabolic study; of these rats, two were fed a-dextrin, two /3-dextrin, and two potato starch.

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ET AL.

The Schardinger dextrin solutions used for these studies were prepared by dissolving the solid materials in warm water. The starch was suspended in boiling water and homogenized in a Virtis homogenizer. Approximately 2.5 ml of material (2.5%) were used for intubation. The quantitative amount delivered to each rat was determined by weight difference of the syringe before and after intubation. Using the samematerial, weighed amounts were immediately placed in weighed platinum boats for assay. After intubation, the rats were placed in a metabolic chamber for 16 to 23 hours and respiratory CO2 was drawn through a continuous radioactive gas analyzer, as described by Long et al. (1958), which automatically recorded the rate and total Cr40z respired. The radioactive carbon dioxide was collected in 2 N NaOH traps, which were assayed for total carbon-14. At the end of the experiment, the animals were sacrificed by a blow on the head and the organs were removed for assay. The urine, feces, and carcasseswere also assayedfor radioactivity as described below and a carbon-14 material balance obtained. Radioactive Determination Measurement of carbon dioxide excretion. The continuous measurement of total C1402 respired was accomplished by a method similar to that reported by Long et al. (1958). Combustion. All samples for radioactive assay were handled in the manner described by Domingues et al. (1960). Correction for sample absorption was that describedby Calvin et al. ( 1949). RESULTS

The utilization of Schardinger dextrins by the rat has been evaluated using radioactive C’ 1 substrates. The amount of tY40z respired by the rats after oral administration of a-dextrin was B-62% of the dose in 17-23 hours. The fl-dextrin was utilized to a similar extent as shown by Cl402 excretions of 49-69s in a similar time interval (Table 1) . The Cl402 respiratory patterns from the a- and fl-dextrins were similar, but they differed greatly from the pattern obtained from radioactive starch (Figs. 1 and 2). The breath pattern from the animal that received the a- or P-dextrin indicated a very slow utilization of the cyclic compound. Since each spike on the recorder chart indicates a s-minute time interval, it is evident from Fig. 1 that very small amounts of C140Z were initially respired, using 10 units on the chart as background level. The slope of the respiratory curve showeda definite change after the third

SCHARDINGER

DEXTRIN

hour postintubation and reached a broad 9 hours. Small amounts of radioactive hours postintubation. Figure 2 depicts a respiratory pattern Maximum output of C1402 was observed reached background level approximately DISTRIBUTION

Tissue

a-Dextrin (% of dose)

Breath Cecum content Cecum GI tract Urine Feces Liver Heart Lungs Kidneys Sex organs Spleen Carcass Recover) Time on test (hours)

Q The radioactivity and its contents.

maximum output between 8 and C1403 persisted even after 20 obtained from radioactive starch. within an hour of intubation and 8 hours later. The rat has shown

TABLE 1 OF RADIOACTIVITY

a-Dextrin

261

UTILIZATION

fl-Dextrin

IN

ANIMALS Starch control

fi-Dextrin

Starch control (% of dose)

(%

(% of dose)

(%

(70 of dose)

58.2 1.7 0.t 7.0 3.6 14.4 1.3 0.1 0.3 0.3 0.3 0.1 16.7 104.6

62.4 3.8 0.4 8.0 8.0 Sone 3.2 0.1 Lost 0.4 0.3 0.3 16.2 103.2

48.6 4.0 1.6 4.0 5.1 5.4 3.0 0.1 0.1 0.4 0.2 0.1 22.5 95.1

66.8 4.9 1.1 8.8 3.6 None 0.2 0.1 0.1 0.3 0.2 0.1 14.2 lOC.2

64.3 1.2 02 2.6 1.8 None 0.4 0.1 0.1 0.3 0.4 0.1 26.0 97.4

58.6

17

22

17

23

22

22

Male

Male

Male

of dose)

in the gastrointestinal

tract

of dose)

Male of this

Male animal

includes

-

6.la 3.0 Lost 3.1 0.04 0.04 0.1 0.02 0.1 15.6 86.7

Female the cecum

the ability to utilize starch quite rapidly since significant amounts of C140Z were evident in the breath within 15 minutes. Although a difference in the rate of Cl402 production was noted, the total amount respired was essentially the same as the Schardinger dextrins. The distribution of radioactivity in the various tissues and organs of the rats subjected to the above-mentioned compounds is given in Table 1. It should be noted that the cecum and its contents were analyzed separately from the rest of the gastrointestinal tract. The gastrointestinal tract, as analyzed, contains any contents present at the time of sacrifice of the

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ANDERSEN

ET AL.

71

0 2 V

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DEXTRIN

UTILIZATION

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ANDERSEN

ET AL.

animal. The total radioactivity in the cecum, cecum contents, and the gastrointestinal tract range from 9 to 15% from the a- and fi-dextrins and from 4 to 65% from starch. In general, there are no marked differences in the distribution of radioactivity in the other tissues between the rats fed dextrins and those fed starch. The distribution of radioactivity in the urine and feces is also given in Table 1. No comparison was made of the feces as one of the animals given radioactive starch was void of feces during the experiment and the other animal’s feces was lost. Nevertheless, there was a significant amount of radioactive material detected in the feces of the animals fed a- and P-dextrins. This would imply to a certain degree that these compounds were digested slowly. No attempt was made to identify the radioactive components present in any of the urine or feces samples. DISCUSSION

As one criterion generally used to determine the utilization of a radioactive substrate by the mammalian system is the production of carbon dioxide, it may be stated that the Schardinger dextrins are metabolized by the rat. One may also conjecture that the utilization or partial degradation of the substrate occurred by intestinal flora; followed by absorption of the microbial products and finally metabolic conversion of these absorbed products into a wide variety of compounds. The data presented in this study do not allow for a differentiation between the animal’s ability to metabolize the compounds or their prior conversion to digestible products by the intestinal flora preceding absorption. In animal experiments, von Hoesslin and Pringsheim (1923) reported that they were unable to detect any synthesis of liver glycogen after 3 hours when Schardinger dextrins were fed to fasted rabbits and guinea pigs. Using a 4%hour starved rabbit, they noted 0.14 g of glycogen was recovered from 1.5g of the p-dextrin. It is quite possible that the reserves of the animal were depleted to such an extent that glycogen synthesis was not possible, A 24-hour fasted animal given 40 g of glucose resulted in the isolation of 7 g of liver glycogen. It one assumes that the starvation regime above did not completely deplete the reserves of the animal, the data reported here would suggest that von Hoesslin and Pringsheim did not wait a sufficient length of time for absorption of the dextrins or microbial products to take place. The effect of a- and fi-dextrins in the animal diet for long periods of time has been evaluated in this laboratory. These data, to be published

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at a later date, indicate that rats maintained for 3 months on a diet containing 1% of the a- or l3-dextrins showed growth curves and food efficiencies equal to the control diet containing no a- or l3-dextrins. In unpublished data referred to by French (1957), rats maintained on a diet in which part of the carbohydrate was supplied by highly purified /3-dextrin refused to eat the test diet except in small quantities. The rats maintained on this diet died within 1 week. It is possible that the rats died because of insufficient food intake rather than from toxic effects of the dextrin. Considering the stability of these cyclic glucose polymers toward fracture by enzymes and the general limited utilization by a few bacterial species, it is quite significant that the rat, by some metabolic route, is capable of oxidizing these compounds to COIL. It is evident that the cyclic ring must be opened prior to any oxidation. The splitting of the ring should result in the formation of an oligosaccharide containing 6 or more glucose molecules. This being the case? it would appear that the utilization of these cyclic compounds are not different from simple oligosaccharides which are easily metabolizable. SUMMARY Comparative metabolism studies were made with radioactive starch and cyclic Schardinger dextrins. This paper describes the growth of radioactive potato tubers, the isolation of radioactive starch from the tubers, and the preparation of radioactive CI- and fi-cyclic dextrins. The radioactive starch and the two cyclic dextrins were administered to rats and the expired ClaO., was measured continuously for 17-23 hours, The distribution of residual radioactivity in the individual organs and carcasses of animals fed starch and Schardinger dextrins was determined by standard combustion techniques. The results show that the cyclic dextrins are metabolized, but at a slower initial rate than starch. However. after 24 hours, the total amounts metabolized are about the same. The distribution of radioactivity within the animals is similar to that of starch. ACKNOWLEDGMENTS Grateful acknowledgment is made to Dr. John Campbell, New Jersey Agricultural Experimental Station, for supplying potato plants, and to Dr. Leonard H. Weinstein, Boyce Thompson Institute for Plant Research, Inc., for advice on growing the plants. The authors also wish to acknowledge the technical assistance of Messrs. Kenneth J. Gildner and Donald J. Rizzo. REFERENCES BEN-GERSHOM. E. (1955). M.. HEIDELBERGER, (1919). Isotopic Carbon,

CALVIN,

Anomeric inversions by glycosidases. C., REID, J. C., TOLBERT, B. M., and p. 30. Wiley, New York.

Nature YANKWICH,

1’75, 593. B. F.

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ET AL.

CRAMER, F. (1953). iiber einschlusse Verbindungen. IV. Die Hemmung drr Glykosidspaltung durch Cyclodextrin. Amz. Chew Liebig’s 579, 17-22. DOMINGUES, F. J., LONG, C. L., ANDERSEN, G. H.? BALDWIN, R. R., THIESSEN, R. JR., and ZEITLIN, B. R. (1960). The suitability of B-n-glucose pentaacetate for food use. II. Studies on the absorption and metabolism. Toxicol. Appl. Pkarmac-ol. 2, 281-294. FRENCH, D. (1957). The Schardinger dextrins. .4dvances in Carbohydrate Chew 12, 189-260. GREEN, D. E., and STUMPF, P. K. (1942). Starch phosphorylase of potato. .I. Biol. Chem. 142, 355-366. HOESSLIN, H. VON, and PRIN~SHEIM, H. (1923). Zur Physiologie der Polyamylosen. II. Glykogenbildung und Tierische Verbrennung. 2. Physiol. Chem. Hoppe-SfFk’r’s 131, 168-176. LONG, C. L., DOMINGUES, F. J., STUDER, V., LOWRY, J. R., ZEITLIN, B. R., BALDWIN, R. R., and THIESSEN, R., JR. (1958). Studies on absorption and metabolism of propylene glycol distearate. Arch. Biochem. Biophys. 77, 428-439. MCCLOSKEY, C. M., and PORTER, J. R. (1945). Utilization of certain rare sugars by microorganisms. Proc. Sot. Exptl. Biol. Med. 60, 269-271. SCHLENK, H., SAND, D. M., and TILLOTSON, J, A. (1955). Stabilization of autoxidizable material by means of inclusion. J. Am. Chem. Sot. 77, 3587-3590. TIIOMA, J. A., and KOSHLAND, D. E., JR. (1960). Competitive inhibition by substrate during enzyme action. Evidence for the induced-fit theory. J. .4m. Chem. Sot. 82, 3329-3333. TILDEN, E. B., and HUDSON, C. S. (1939). The conversion of starch to crystalline dextrins by the action of a new type of amylase separated from cultures of Aerobacillus macerans. J. Am. Chem. Sot. 61, 2900-2902. TILDEN, E. B., and HUDSON, C. S. (1942). Preparation and properties of the amylases produced by Bacillus macerans and Bacillus polymyxa. J. Bacterial. 43, 527-544. WEINSTEIN, L. H., PORTER, C. A., and LAURENCOB, H. J., JR. (1959). Quinic acid as a precursor in aromatic biosynthesis in the rose. Contribs. Bo?lCe Thompsola Inst. 20, 121-134.