Action of gastric juice on microencapsulated invertase

Action of gastric juice on

microencapsulated invertase I. Garcia, R. B. Aisina, O. Ancheta and C. Pascual* *Department of Clinical Biochemistry, National Center for Scientific Research, P.O. Box 6990, Havana, Cuba Department of Chemistry, M. V. Lomonosov State University, Moscow, USSR (Received 30 November 1987; revised 24 April 1988) Invertase (fl-D-fructofuranoside-fructohydrolase) (E.C.3.2.1.2.6.) was microencapsulated in acetylphthalylcellulose membrane by the double emulsification method, which retains over 90% of the activity. The behavior of the microencapsulated enzyme was determined at p H values similar to those of the gastric juice and intestinal environment. While the free enzyme is extremely sensitive to low p H value, the microencapsulated enzyme maintained approximately 40% of the activity after 1 h incubation with gastric juice. Thus microcapsules could be potentially useful in the treatment of invertase deficiency.

Keywords: Gastric juice; microcapsules; invertase

Introduction It is known that patients with deficiency of the intestinal enzyme sucrase-isomaltase suffer sucrose intolerance and osmotic diarrhea upon ingestion of this sugar. 1 This is considered to be one of the most frequent diseases among hereditary disaccharidase deficiencies. 2 Attempts for ameliorative treatments of this disease have been made by adding large doses of yeast invertase to food. 3 A more logical approach to alleviate the symptoms in these patients would be to supplement the enzyme in the gastrointestinal tract, at least during the time of food ingestion. In principle, this could be achieved by oral administration if the enzyme was protected from the adverse conditions of the extreme acidic pH of the gastric juice. In this work we report the preparation of potentially therapeutically useful microcapsules made of acetylphtalycellulose polymer membrane, which contains a protective compound enclosed together with the enzyme. We have chosen acetylphthalylcellulose as a polymer to encapsulate invertase because the integrity of this type of microcapsules depends on the pH of the environment. At a pH value similar to the one of the gastric juice, the microcapsules are stable, and this could offer protection to the enzyme that is contained inside. At higher pH values in the range of the pH value of the small intestine, the microcapsules are

* To whom correspondence should be addressed.

© 1989 Butterworth Publishers

disintegrated and the enzyme is liberated to exert its catalytic action. Since it is of interest to have microcapsules that liberate as much of the enzyme as possible in the small intestine after its transit through the stomach, we describe the effect of conditions in vitro that stimulate the pH environment of the gastrointestinal tract on microencapsulated invertase. The microcapsules were treated first with acidic pH or gastric juice, since even integral and stable microcapsules are relatively permeable, especially to low-molecular-weight compounds, and this may cause inactivation. Afterwards, the preparation was incubated at higher pH values similar to that of the intestine, which causes the microcapsules to disintegrate and liberate the enzyme. Our results show that approximately 40% of the invertase activity used for microencapsulation was liberated at pH 8.0 after incubating the microcapsules for I h in gastric juice.

Materials and methods Invertase from Bakers' yeast grade VII (Sigma Chemical Co.) with specific activity of 400 U mg -j was used as the source of the enzyme. Bovine serum albumin fraction V was obtained from Sigma Chemical Co. Acetone and gelatin were from Merck. Chloroform and n-hexane were Analar from British Drug House Chemicals Co. Ltd. Carboxymethylcellulose sodium salt was from British Drug House Chemicals Co. Ltd. acetylphthalylcellulose was from Pharmaceutical Medical, Moscow. Stirring was performed with a homogenizer-Medengen GDR. The stirrer consisted of a 2.0-ram stain-

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Papers less steel hard wire having a triangular shape in its extreme with base and altitude of 3 cm.

Microencapsulation procedure In a typical experimental procedure, 10 mg of invertase was mixed with 1 g of protective filler (gelatin, carboxymethylcellulose, albumin, hemoglobin) and 1 or 2 ml of mineral oil. This mixture was added to 10-15 ml 15% (w/v) solution of acetylphtalylcellulose in acetone under stirring at 650 rev min -1 and afterwards an additional 50 ml of mineral oil was added. After 10 min, 25 ml of chloroform was added. The polymer membrane on the surface of the emulsion drop was formed by stirring for 90 min to the total evaporation of the organic solvent. Afterwards, the oil phase was washed off by filtration under vacuum with 20 ml of n-hexane. Residual levels of the organic solvents used for microencapsulation have not been measured and are presumed to be absent or to have very low levels due to evaporation which occurs during the process. These substances must be quantified before any attempt for use in humans. The microcapsules were kept in a desiccator at 4°C for 24 h. The microcapsules prepared by this procedure were spherical with sizes ranging from 250 to 300

tzm (Figure 1). Determination of microencapsulated invertase activity Determination of microencapsulated invertase activity was performed by disintegrating the microcapsules by means of incubation in a medium with a pH similar to that of the intestine and measuring the activity of the liberated enzyme. This was achieved by incubating the microcapsules in 0.1 M phosphate buffer pH 8.0 (or, depending on the experiment, at pH 6.0 or higher) at 37°C. An aliquot of the clear suspension was taken at 15 min or at another time for invertase determination.

100 8o

60 40

J

2 i

g 8 lb PH

Figure 2 Invertase activity of the free enzyme after 15 min incubation at different pH values

Invertase activity was performed according to Hestrin 4 by measuring the glucose liberated by the glucose oxidase method (GOD-PERID, Boehringer, Mannheim).

Effect of acid p H or gastric juice on microencapsulated invertase To test for the effect of acid pH or gastric juice on microcapsules containing invertase, the microcapsules were placed in a small stainless steel sieve having small porosity and a cover so that the microcapsules could not leak out. The sieve was immersed in HCI solution pH 1.5 or in natural gastric juice at 37°C. At different time intervals, the sieve containing the microcapsules was removed from the suspension medium and the microcapsules were thoroughly washed with distilled water and the excess of water was removed as much as possible. Immediately afterwards the determination of the microencapsulated invertase was performed as described above.

Results

Free invertase Effect of pH. It is important to know the behavior of

Figure I Scanning electron micrograph of invertase microcapsules containing carboxymethylcellulose (300x)

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the invertase preparation at different pH values, since one of the main goals that must be achieved by microencapsulation is to protect the enzyme against the acid medium of the gastric juice while still having a functioning enzyme in the intestine. The results in Figure 2 show that more than 80% of the activity was lost when the enzyme was incubated 15 min at a pH value below 2. This is in agreement with the literature data) Incubation of the enzyme above its pH optimum also provoked a loss of activity, although to a lower degree than at acidic pH, but it could have a considerable effect after 1 h at pH 8.0 (Table 1). Therefore

Action of gastric juice: L Garcia et al. Table 1 Effect of protective compounds on free invertase activity Protective compound

Concentration (mg ml -~)

None; free enzyme pH 8.0 (0.02 mg ml -~)

-

Hemoglobin

1.4

CMC

1.0

Albumin

1.0

Gelatin Gelatin and albumin None; free enzyme pH 6.5 (0.02 mg ml .~)

1.0 0.5 of each

0 min

Enzyme activity (%) after incubation at pH 8.0 during: 15 min 30 min 60 min

238,0 (100) 323.7 (136) 238.0 (100) 200.6 (84.3) 238.0 (100) 214.7 (90.2) 243.0 (100)

163.3 (68.6) 249.9 (105) 177.3 (74.5) 163.3 (68.6) 172.8 (72.6) 182.1 (76,5) 243.0 (100)

79.3 (33.3) 249.9 (105) 172.6 (72.5) 144.7 (60.8) 107.3 (45,1) 153.9 (64.7) 243.0 (100)

47.6 (20.0) 238.0 (100) 119.0 (50.0) 99.5 (41.8) 55.9 (23.5) 65.5 (27.5) 243.0 (100)

Enzyme activity is expressed as/zmol rain -1 mg enzyme-1. The effects of protective compounds were tested at pH 8.0 during the time indicated

it is also important to protect the enzyme from the pH environment of the intestine, because it is here that the enzyme should be liberated and would be exposed for a prolonged period of time to exert its catalytic action. For these reasons, compounds such as albumin, gelatin, carboxymethylcellulose (CMC), and hemoglobin, which are known to provide stability for some enzymes, were tested on invertase activity at alkaline pH (Table 1) in order to select the most appropriate. Several of these protective compounds also provided stability to the enzyme during microencapsulation, and it has been reported that they may even contribute to the shape of the microcapsules. 3

Microencapsulated invertase

found that hemoglobin precipitated inside the microcapsules, probably due to the action of the organic solvents employed (Figure3). This seemed to interfere considerably with the activity of the enzymes, and for this reason we preferred to use CMC as a protective agent which is dissolved at alkaline pH while it is kept insoluble at acid pH.

-Liberation of invertase from the microcapsules When the microcapsules containing CMC with invertase were incubated at pH 8.0 during the first 5 rain, most of the enzyme was liberated, and more than 90%

Hemoglobin offers the best protection against an alkaline medium and would be a good candidate to use in the microencapsulation procedure. However, we

c=60

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£o

6-o

INCUBATION TIME(rain) Figure 4 Remaining activity of invertase after incubation with

Figure 3 Microphotography of invertase microcapsules containing hemoglobin (164x)

0.1 M phosphate buffer, pH 8.0, for different times. IOl Free enzyme containing CMC (2 mg ml -~) and acetylnapnthylcellulose (6 mg ml-1). (0) Microencapsulated invertase. Invertase activity is expressed as percent related to the free enzyme in phosphate buffer at pH 8.0 at time 0 (100% = 332 U mg -1 enzyme)

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.

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.

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60

INCUBATION

TIME(min)

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60

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Figure 5 Remaining activity of invertase microcapsules after

Figure 7 Remaining activity of free enzyme and invertase

incubation with 0.1 M phosphate buffer pH 6.0 (O), pH 6.5 (&), and pH 7.0 (A) for different times. ( I ) Refers to a preparation of free enzyme at pH 6.5. Activity is expressed as percent related to the activity of the free enzyme at its corresponding pH

microcapsules incubated with gastric juice at different time. After incubation with gastric juice at the time indicated, the microcapsules were transferred to a solution of 0.1 a phosphate buffer, pH 8.0, for 15 min (©) o r t o a solution of 0.1 M phosphate buffer pH 6.5 for 15 min (A) for the liberation of the enzyme in order to make possible enzyme activity determination. (Q) Refers to free enzyme incubated in gastric juice. Activity is expressed as percent related to activity of microcapsules without incubation with gastric juice at time 0

of the original enzyme activity introduced in the microcapsules was recovered after I0 min. More than 60% of the activity still remained after 1 h (Figure 4). We also tested the enzyme activity of the microcapsules at pH 6.0, 6.5, and 7.0. It was observed that at these pH values practically all of the original enzyme activity was recovered even after I h incubation

(Figure 5).

2°k 30 60 90 120 incuboti0n time (rain} Figure 8 Remaining activity of free invertase in a medium with CMC (1 mg m1-1) and acetylphthalylcellulose (15%) (0) and invertase microcapsules (O) incubated at pH 1.5 for different times. After incubation at pH 1.5, the microcapsules were transferred to a solution of phosphate buffer pH 8.0 for 15 min for liberation of the enzyme in order to determine its activity. Activity is expressed as percent related to initial activity of microcapsules without incubation at pH 1.5 (time 0)

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Effect of acid pH and gastric juice on microencapsulated inuertase As expected, free invertase in the presence of CMC and acetylphthalylcellulose was completely inactivated after 2 h incubation at pH 1.5. However, the microcapsules prepared with CMC incubated at pH 1.5 during 1 h showed approximately 70% of the enzyme activity after its liberation from the microcapsules at pH 8.0, and after 2 h of incubation at pH 1.5 about 30% of the activity was recovered (Figure 6). When invertase was incubated with gastric juice, a more drastic inactivation was observed. After 20 min in the presence of gastric juice, the free enzyme was more than 90% inactivated. However, when the microcapsules were incubated for 1 h in gastric juice, 30 to 40% of the activity was recovered, depending on the liberating pH medium (Figure 7). The action of gastric juice on microencapsulated invertase may be due to the low pH as well as to the presence of other inactivating substances. It seems improbable that proteolytic enzymes of the gastric juice such as pepsin permeated the microcapsules. Discussion

Acetylphthalylcellulose seems to be suitable for preparation of invertase microencapsules. This compound is well tolerated by the gastrointestinal tract, and a previous report s indicates its use for the microencap-

Action of gastric juice: L Garcia et al. sulation of other digestive enzymes of medical pharmaceutical interest. In the microencapsulation of invertase, more than 90% of the enzyme originally introduced was recovered. This retention of activity can be considered highly satisfactory and could be due to the protective action of the microcapsule and of CMC. The other protective agents used for microencapsulation did not show better recovery of enzymatic activity (data not included). The most powerful action of the gastrointestinal tract upon invertase microcapsules is expected to be due to the low pH value of the gastric juice. The half-emptying time of the gastric content in humans after ingestion of a small meal is considered to be in the range of 30 min. 6 Microcapsules offer a good protection from the gastric juice, since approximately 40% of the activity still remains after 1 h incubation

In the extensive work of Rambourg et al.,3 these authors used various methods, protective proteins, and bifunctional acylating agents for the encapsulation of invertase different from the one we employed. The remaining activity of invertase microcapsules that they found after incubation with pepsin at pH 1.4 during 30 min in no case was higher than 30%, although a higher activity was reported for some of their microcapsules preparations when incubated at pH 1.5 alone. Enzyme microcapsules could be of use for restitutive treatments. Replacement therapy seems to be a promising therapeutic method which could be applied to a wide range of diseases, especially to genetic disorders where no other treatment could be used. Recently, with the development of enzyme biotechnology, this approach has been used to treat some severe inherited diseases. 10

(Figure 7). In normal humans, the invertase activity is high in the proximal and mid jejunum2; specifically, the highest activity is reported up to the anatomical region of the ligament of Treitz. The pH value of 6.5, into which the microcapsules were transferred for their disintegration and liberation of invertase after incubation with the gastric juice, is representative of the pH value of the jejunum. 6'7 Even when pH 8.0, as representative of pancreatic juice, was used for liberating the enzyme, more than 30% of active invertase activity was recovered from the microcapsules after incubating for 1 h in gastric juice. The Km value of the human intestinal invertase for sucrose is 1.8 x 10-2 M, while the Km value of the yeast Saccharomyces cerevisiae enzyme, which is the source of enzyme used, is approximately the same 8 or may even be lower, depending on the strain and preparation of the enzyme used. 9 This is a useful parameter to consider, which is related to the affinity of the enzyme for the substrate.

References 1 2

3 4

5 6 7 8

9 10

Lloyd, M. L. and Olsen, W. A. N. Engl. J. Med. 1987, 316, 438 Alpers, D. H. and Isselbacher, K. J. in Advances in Metabolic Disorders, Vol. 4 (Levine, R. and Luft, R., eds.) Academic Press, New York, 1970, pp 75-122 Rambourg, P., Levy, J. and Levy, M. C. J. Pharmaceutical Sci. 1982, 71, 753 Hestrin, S., Feingold, D. S. and Schramm, M. S. in Methods in Enzymology, Vol. 1 (Colowick, S. P. and Kaplan, N. O., eds.) Academic Press, New York, 1955, pp. 251-257 Aisina, R. B. et al. Chemical Pharmaceutical Journal (in Russian) 1984, 18, 1486 Dressman, J. B. Pharmaceutical Res. 1986, 3, 123 Wissenschaftliche Tabellen in Documenta Geigy, 1955, p. 269, Switzerland Ohlenbusch, H. D. and Vogele, P. Methods o f Enzymatic Analysis (Bergmeyer, H. U., ed.) Vol. 2 Academic Press, New York, 1974, p. 923 Garcfa, I. et al. lnterferrn y Biotechnologia 1986, 3, 39 Hirschhorm, R. N. Engl. J. Med. 1987, 316, 623

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