Use of Enzymes in Surgery

Use of Enzymes in Surgery

Use of Enzymes in Surgery EDWARD L. HOWES, M.D., F.A.C.S.* MANY of the physiological and pathological processes that the surgeon attempts to correct ...

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Use of Enzymes in Surgery EDWARD L. HOWES, M.D., F.A.C.S.*

MANY of the physiological and pathological processes that the surgeon attempts to correct by surgery are motivated by enzymes. Their role has become more apparent as the chemistry and physiology of the diseases he treats have been studied. To mention a few examples: the deposition of fibrin and its solvation, the liquefaction of necrotic tissue, digestion and its disturbance, and the growth of tissues-all are brought about by the help of enzymes. When, for example, the surgeon removes the pancreas or most, if not the entire, stomach or colon, the missing enzymes must be compensated for by some means. Furthermore, certain diseases like peptic ulcer, ulcerative colitis, regional enteritis, pancreatitis, and many other diseases must be related to, if not caused by, disturbances in enzymatic activity. These diseases could be more intelli gently dealt with if the distortion of the enzyme pattern were known. Parenthetically, the catgut the surgeon uses is "absorbed" through the action of enzymes. With this background in mind, the surgeon has added enzymes from an external source to pathophysiological processes in the hope that their evolution may be brought about more rapidly or completely. By this method he has attempted to aid inflammation, thrombosis, the resolution of infection and healing. The treatment of burns and empyemas has been attempted. Anyone who has had the experience of seeing large amounts of burn slough quickly, removed by the use of enzymes after two days, the bedsore converted to a clean granulating lesion in two or three days, necrotic slough removed from an x-ray burn, or the pus of the empyema cavity quickly liquefy and drain cannot help but be impressed that enzymes provide powerful adjunct help for the surgeon. On the other hand, when the lesion is not helped by the application of enzymes, the surgeon does not seem to understand that the naturally occurring enzymes have not worked, hence the lesion; that conditions were not or could not be established to allow the externally applied

* Associate Professor of Clinical Surgery, Columbia University; Attending Surgeon, Presbyterian Hospital, New York, N. Y. 497

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enzymes to work; or that the enzymes were asked to do an impossible task. For example, no enzyme has yet been discovered that will aid the separation of necrotic bone or to dissolve "leatherized" human skin. RULES FOR THE USE OF ENZYMES

When enzymes from an external source are to be used in therapy or when a disturbed enzymatic process is to be corrected, certain rules for their activity must be observed if they are to work. These rules have not always been followed, and failure to observe them accounts in part for some clinical failures in the use of enzymes. First, enzymes attack only specific substrates. For example, an enzyme that attacks fibrin will not attack collagen and it cannot be expected to dissolve burn slough that is composed largely of collagen. Neither streptokinase nor trypsin is a collagenase, yet both have been used and recommended to treat burns. Next, enzymes require a favorable chemical environment for their action. If they are used in an environment that is too acid or alkaline or if an unfavorable metallic ion or an inhibitor is present, the enzyme either will not work or its activity will be greatly reduced. Pepsin, for instance, requires a pH of about 4.5 for optimal activity and therefore it will not work in the presence of alkaline pus (pH 9) that may be found in a chronic infection. Litmus paper touched to the area where the enzyme is to be used will immediately disclose whether the enzyme will 'work in the pH of the environment. Many enzymes require a small amount of calcium or ferrous ions to work or the presence of a small amount of cysteine to attain peak activity. If these ions are not present, even the naturally occurring enzymes will not work. Some enzymes work only after other enzymes have completed their work. They work in a chain reaction as they do in the gastrointestinal tract, where tryptic digestion acts after peptic digestion, and then the peptidases act after trypsin. In each case the proper substrate is created by the action of the preceding enzyme. Enzymes are proteins; therefore characteristic difficulties found with the clinical use of proteins are encountered. First, they can be allergenic if the patient has a sensitivity. Next, antibodies can be elaborated against them-antibodies that neutralize their action. This neutralization would occur only if they are used for too long a period of time. The action of enzymes is so powerful that if the desired result is not obtained in the first four or five days, very little is going to be accomplished thereafter. Application of enzymes for four or five days is not long enough to cause the elaboration of interfering antibodies. However, a second course of, enzymes given later may be greatly influenced by the antibodies produced by a first application. As proteins enzymes are also coagulated by antiseptics. In fact, G-11 not only stops the action of enzymes but also remains so long adherent

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to the skin that an enzyme like proteinase-collagenase mixture, which will ordinarily attack burned skin, will not do so after the application of G-11. The use of antiseptics to clean skin or a lesion is contraindicated if burns or necrotic lesions are to be treated with enzymes. As proteins some of the enzymes are also attacked by bacteria, heat and by other enzymes. Some enzymes seem to digest themselves and are very short-lived. Trypsin is a very short-lived enzyme as compared to some other proteinases attacking the same group of substrates. For local application the enzyme employed ideally should be as long-lived as possible, relatively resistant to the action of bacteria, and continue to act in their presence. When used locally the enzymes should usually be protected by combining them with the use of antibiotics. Antibiotics do not interefere with the action of enzymes if the antibiotics are employed in bactericidal or bacteriostatic concentrations. Antibiotics such as streptomycin, penicillin and Sulfamylon that do not set up local tissue reactions should be used. Lastly, enzymes require a moist environment to act. When the substrate dries as in the dried eschar, the enzyme will not work on its surface unless the scab will remoisten and plumb. Even then the enzyme will work imperfectly. To act on the dried substrate the enzyme must be placed under the eschar. As our knowledge has increased about enzymes, the author has more frequently placed enzymes below the eschar by means of a hypodermic needle or by means of the hypo-spray. Favorable results are usually obtained in twenty-four hours and there have been fe,ver failures. We have increased the amount of enzymes injected very slowly to avoid toxic reactions, and in a case recently reported' we used on successive days as much as 30 cc. of a 1 mg. per 100 cc. solution of a powerful peptidase-type enzyme without any side reactions. Despite these many conditions that must be established for the enzymes to work, the enzymes are very potent agents when these conditions are correct. SOURCE OF ENZYMES

Enzymes cannot be used for therapy unless they are available in an abundant, not too expensive source. Enzymes from animal tissuespepsin and trypsin, obtained from the stomach and pancreas, were the first to be used clinically. Later enzymes recovered from plant juices were employed. They were extracted from fruits that were long known to have digestive properties. Among these were papain obtained from the papaya, ficin from figs, bromelin from pineapple. In nearly all instances these were .crude extracts and therefore contained considerable extraneous substances. Then two advances in enzyme chemistry considerably changed the outlook for the future use of enzymes for therapy. First, enzymes were

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crystallized by Northrop and Kunitz in the early 1930'S.2 Their true nature became better understood, and extraneous substances previously present in large amounts were eliminated. Next, enzymes were recovered in large amounts from growing organisms. Diastase was produced by the action of Aspergillus on rice. MacFarlane and his associates" showed that the alpha ·toxin produced by Clostridium perfringens was probably a lecithinase. Since this initial work, many of the bacterial toxins have been found to be enzymes and their substrates have been identified. In this way hyaluronidase, kinases, proteinases, collagenase and desoxyribonuclease have been obtained from growing bacteria. Growing yeast cells can be used to provide an abundant source of mixed enzymes. The methods used for the large scale production of penicillin and streptomycin have been adapted for the production of enzymes. The bacteria are grown in large vats on a suitable media that produces the most enzyme. The bacteria are then removed by filters, and the protein enzyme left in the filtrate is precipitated out by means of ammonium sulfate or methanol. This precipitate is then washed and lyophilized to a dry powder. In this form it can be stored indefinitely for future purification and use. When reconstituted in water, various methods can be used to purify and concentrate the particular fraction desired. With these advances not only have many new enzymes been obtained but adequate sources for clinical use are made available. A streptokinase-streptodornase mixture and taka-diastase have been produced and. purified for clinical use. Trypsin has been recrystallized and marketed in a crystalline form and has been used parenterally. The fruit enzymes are available now in more concentrated purified forms; but still consist of a mixture of enzymes. Hyaluronidase has been extracted from bovine testes. Lysozyme is obtained from egg white. Pancreatin is marketed as an aid to carbohydrate digestion, while lipases are sold to help the digestion of fat. Actually neither the uses of enzymes in the treatment of geriatric deficiencies nor after surgical removal of large portions of the digestive tract have been properly explored, and many reevaluations should be done with the newer methods that have been developed in enzyme chemistry. Commercially large quantities of enzymes are prepared from yeast and bacteria for the digestion of leather and for meat tenderizers, but these enzymes have not been purified for clinical use. Many enzymes, such as the collagenases and some of the proteinases, have been made by the investigators who tested their therapeutic properties; however, some of these enzymes have not been produced in quantity. MUCIN AS A PROTECTOR FROM ENZYMES

Tissues existing in a healthy state in the presence of an enzyme capable of digesting them either contain an inhibitor or:their surface is

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bathed in a secretion like mucin that the enzymes will not attack. When an organ such as the pancreas is suddenly attacked by its ever-present enzyme, as occurs in pancreatitis, either the inhibitor has been destroyed because the blood supply has been altered, a new enzyme is present, or the old enzyme has developed a new unusual activity. In general, most enzymes do not attack living tissues in the concentration of their secretions, but they attack this same tissue as soon as it loses its blood supply. Mucin that protects the entire inner surface of the gastrointestinal tract from its enzymes and the upper respiratory tract is composed of mucoproteins that are not attacked by the ordinary proteolytic enzyme. Neither pepsin, trypsin nor chymotrypsin attack mueoproteins.' Specific enzymes-the mucinases-are required to digest mucus. The presence of bacteria that secrete mucinases capable of digesting this protective layer renders the tissue beneath open to a digestive attack. Lysozyme that was first suggested as changing the viscosity of mucus does not affect mucoproteins but mucopolysaccharides. The mucin coming out everywhere on the surface forms a chemical "barrier" layer between the tissues and the enzymes. When mucus-secreting cells are destroyed and this layer is no longer present, the tissue becomes irritated, leukocytosis occurs and superficial ulceration develops. Esophagitis that occurs postoperatively as the end result of scarification and destruction of the mucus-secreting glands at the lower end of the esophagus may arise on this basis. The presence of an abundance of mucus in the lumen is no longer at the interface and does not serve as a protective layer. THE ENZYMES AND BACTERIAL INFECTIONS

It was originally hoped that the use of enzymes would starve out the bacteria present in an infection by depleting their food supplies. Our examination of local lesions treated with enzymes has shown that bacteria persist after enzymatic treatment. Apparently the bacteria are as capable of surviving on the products of dissolution of enzymatic digestion as on the original necrotic tissue. It is well known, of course, that bacteria continue to exist even when healthy granulations appear and continue to grow. Quantitative studies as to their numbers and types have not been done. They need to be, especially when enzymes are used in combination with antibiotics. On the other hand, the use of enzymes locally usually ends purulency by liquefying pus and ridding the area of necrotic tissue, and helps the granulations to appear sooner. Infections have not been made worse by the use of enzymes applied locally, when the enzymes were applied in ointments. Among 135 third degree burns treated with enzymes by various investigators, infection was usually reported as lessened. They have not been observed after the enzymes were injected.' However, Altemeier" and Connell" have each

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reported septicemia after the treatment of burns with continuous wet dressings of enzymes. Whether maceration was at fault or the particular enzyme (proteinase A) was not clear. In both cases the bacteria recovered were resistant to the antibiotics employed subsequent to the development of the infection. Thus the possibility of a spreading infection cannot be ignored. Any marked rise in temperature should lead to the discontinuation of the use of the enzyme and a blood culture should be taken. The other approach is to cover the patient with antibiotics when the enzymes are started. PARENTERAL USE OF TRYPSIN

The use of trypsin parenterally to treat inflammation has a long history. During the First World War, 1918-1919, Sir Almroth Wright and his followers? contended that it was impossible to sterilize a wound with an established infection by means of antiseptics and that the greatest benefit could be obtained by aiding the physiological processes that normally bring about recovery. Students of this problem will recognize that the situation has not changed with the 1!se of antibiotics, except that they will control the cellulitis spreading from the area of infection. Wright showed that the blood serum of soldiers suffering infection had greatly enhanced antitryptic action, and that further, this antitryptic action of the serum was related to the in vitro growth of bacteria in the serum. In serum with normal or increased antitryptic action, streptococci and some staphylococci were the only bacteria that would grow freely when transplanted in small numbers. Wright found that in a new wound where bacteria were growing, the discharges were tryptic and the flora of bacteria was mixed and abundant, while later only streptococci and staphylococci were found when the antitryptic action of the serum increased. Usually the last bacteria to disappear were the streptococci. Opie" showed that leukocytes liberate a trypsin-like enzyme when they disintegrate. According to this theory, then, a high antitryptic index of the blood serum was essential to combat an infection while the presence of a trypsin-like substance locally helped to solvate necrotic tissue. Kay and Lockwood 9 restudied the tryptic inhibitors of the serum in disease and found again that they were important; they and Britton-" ligated the appendix in the dog and studied the number of deaths from peritonitis in relation to tryptic inhibition. The number of deaths were reduced when the antitryptic factor was high. Certain strains of streptococci have been shown by Christensen 11 and Tillett" to elaborate a water-soluble substance that has the capacity to activate a proteolytic enzyme in human plasma. This enzyme "plasmin" comes from an inactive form plasminogen when "streptokinase," made from streptococci, is injected. This same series uf events occurs only in humans, not in animals. The enzyme plasmin digests fibrin only. Strepto-

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kinase has been made commercially, and its evaluation in the treatment of diseases was summarized by Tillett in the 1949-1950 Harvey lectures." Its chief use seems to be to dissolve fibrin and to promote better drainage in empyemas, thereby speeding up a process that occurs normally as the leukocytes disintegrate and liberate trypsin. Theoretically, a sufficient amount of streptokinase could be injected to keep the lumina of the vascular system free of clots of fibrin and therefore free of thrombosis, but this amount of streptokinase injected intravenously is pyrogenic. There is hope that this complication may be avoided. An important enzyme in the streptokinase-streptodornase mixture seems to be streptodornase that acts directly on desoxyribonucleic acid. This enzyme converts the thick pus of empyema into a thin milky fluid. It causes a striking fall in viscosity and the percentage of sediment in the empyema fluid, as has been shown by Sherry, Johnson and Tillett;" and a striking decrease in the number of degenerated leukocytes. ENZYMES AND THE CLOTTING OF BLOOD

Two problems are presented in the clinical treatment of thrombosis: first, to prevent clotting by the correction of the enzyme system involved, and second, to dissolve the formed clot. Only the second problem will be discussed here. Dissolution of the clot with enzymes seems to be an easy accomplishment because many of the enzymes dissolve fibrin in the test tube. Difficulties arise when the enzymes are given intravenously. Streptokinase, as has been pointed out, becomes pyrogenic. Tagnon'! feels that trypsin given intravenously is dangerous. Ferguson and Erickson" showed that crystallized trypsin coagulated blood in vitro. However, Innerfield'" gave this same amount slowly by means of an intravenous drip and could find no evidence of blood coagulation. Most of the proteolytic enzymes cause coagulation of heparinized blood in vitro either because they contain a thromboplastic substance or a specific type of prothrombin activator. Moreover in the presence of serum trypsin loses its capacity to dissolve fibrin, perhaps because of the presence of the inhibitor. In fact Innerfield, 16 who was most impressed by the lytic action of trypsin on chemically induced thrombi, did not assign its effect to direct action on fibrin. Instead he believed that either plasmin was activated or.that antitrypsin was changed in some way. Innerfield has made some unusual claims for the intravenous use of trypsin that will require substantiation. At least he has shown that parenterally this danger was not as great as was expected. STOPPING THE ACTION OF ENZYMES

It is as important to know how to stop the action of enzymes. as ·to how to make them work. The use of tannic acid in the treatment of

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burns and now the use of the dry state are two examples where enzymatic action is arrested to change the progress of a pathological process. With drying only the surface is affected, while with tannic acid the resistance extends deeper. Bacteria cannot grow on dried or tanned skin; neither can the enzymes liquefy dried slough. Chromatization of catgut is another example. Here collagen is treated to resist enzymatic digestion. The surgeon is sometimes faced directly by the problem of enzymatic digestion of tissue after an operation. An intestinal fistula or drainage of a certain type of pus rich in enzymes begins to erode the surrounding tissue. Fantastic treatments have been suggested to stop this erosion; most of these substances being in a class that provides a new cover for the area to keep it away from the enzyme. Some of the substances used may be mentioned-rubber sheeting, rubber cement, sprayed-on plastics, aluminum salts and greases of all types. The enzymes usually get under these covers. Actually the best solution is to establish the dry state; to get rid of maceration and stop enzymatic digestion. One hour of dryness in twenty-four will usually bring about healing. If nothing else, the enzymatic activity. can be kept to the small stream of the discharge if the dressings are removed. Most dressings just distribute the enzymes over a wide area. If these measures fail, the pH / of the location can be changed so that the enzyme cannot act. Dry powder neutralizers are best to again avoid maceration. As the surgeon obtains more potent enzymes and uses them more widely, knowledge as to how to stop their activation will become increasingly more necessary. In fact, one suspects that removal of enzyme inhibitors from certain growths and disease processes may allow the enzymes that naturally occur to act properly. Thus the study of enzyme inhibition may become as important as how to make enzymes act. SUMMARY

Enzymes take part in and are distorted in many diseases treated by the surgeon. Enzymes are active in blood clotting, inflammation, healing and tissue growth. A wide variety of crude enzyme preparations have been used by surgeons to help pathological processes and to treat diseases. At present, the clinical use of enzymes seems to be more promising than practical, but this state will not continue, for significant advances made in enzyme chemistry are so recent that they have not been transferred to practical use. Enzymes were only crystallized in the 1930's, and the methods for separating and producing them in bulk date from the late 1940's. Many more enzymes have been discovered and are recoverable from growing bacteria molds and yeasts. Their cultivation will provide an abundant source for clinical use. It is as important for the surgeon to know how to stop the action of enzymes as to know how to make them work. Rules for both are dis-

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cussed. Some excoriations, bacterial digestion of tissue and some inflammatory processes are caused by enzymatic digestion that could be cured by halting the enzyme action. Enzymes cannot be neglected in the future by those investigating surgical diseases and problems in geriatrics. REFERENCES 1. Howes, E. L., MacLennan, J. D.,~Mandl, I. and DeBellis, R.: Debridement of Burn Slough by Peptidases Recovered from Clostridium Histolyticum. Bull. New York Acad. Med. 31: 413 (May) 1955. 2. Northrop, J. H.: Crystalline Enzymes. Columbia University Press, 1939. 3. MacFarlane, R. L., et al.: Haemolysis and the Production of Opalescence in Serum and Lecitho-Vitellen by the Toxin of Clostridium Welchii. J. Path. & Bacteriol. 52: 99, 1941. 4. Howes, E. L. and Armitage, C.: Digestion of Mucoproteins by Enzymes. J. Expel'. Biol, & Med. In press. 5. Altemeier, W.: Personal communication. 6. Connell, J. F.: Personal communication. 7a. Wright, Almroth: Conditions Which Govern the Growth of the Bacillus of "Gas Gangrene" in Artificial Culture Media in the Blood Fluids in vitro and in the Dead and Living Organism. The Lancet 1: 1-9 (Jan. 6) 1917. 7b. Wright, A. and Flemming, A.: Further Observations on Acidaema in Gas Gangrene and on the Conditions Which Favour the Growth of Its Defective Agent in the Blood Fluids. The Lancet 1: 205-,-210 (Feb. 9) 1918. 8. Opie, Eugene: Intracellular Digestion. Physiol. Rev. 2: 552-585 (Oct.) 1922. 9. Kay, J. H. and Lockwood, J. S.: Experimental Appendiceal Peritonitis; Prognostic Significance of Certain Hematologic Factors, Especially Prothrombin Time. Surgery 20: 56-71 (July) 1946. Idem: Experimental Appendiceal Peritonitis; Significance of Imbalance of Circulating Fibrinolytic and Antifibrinolytic Factors in Course of Disease. Surgery 21: 155-167 (Feb.) 1947. 10. Britton, Richard: Unpublished data, personal communication. 11. Christensen, L. R.: Streptococcal Fibrinolysis; A Proteolytic Reaction Due to a Serum Enzyme Activated by Streptococcal Fibrinolysin. J. Gen. Physiol. 28: 363, 1944-1945. 12. Tillett, William: Studies on Enzymatic Lysis of Fibrin and Inflammatory Exudates by Products of Hemolytic Streptococci. The Harvey Lectures 1949-1950, p. 49. 13. Sherry, S., Johnson, A. and Tillett, W.: The Action of Strep. Desoxyribose Nuclease (Streptodornase) in vitro and on Purulent Pleural Exudations in Patients. J. Clin. Investigation 28: 1094, 1949. 14. Tagnon, Henry J.: Some Recent Developments in the Practical Application of Enzymes to Medicine and Surgery. Practitioner 174: 95-102 (Jan.) 1955. 15. Ferguson, J. H. and Erickson, B. N.: Coagulating Action of Crystalline Trypsin. Am. J. Physiology 126: 661, 1939. 16a. Innerfield, 1., et al.: Intravenous Trypsin, Its Anticoagulant Fibrinolytic and Thrombolytic Effects. J. Clin. Investigation 31: 1049 (July-Dec.) 1952. 16b. Innerfield, I.: Trypsin Given Intramuscularly in Chronic, Recurrent Thrombophlebitis. J.A.M.A. 156: 1056-1058 (Nov.-Dec.) 1954. 180 Fort Washington Avenue New York 32, N. Y.