THE INFLUENCE'OF ENTEROSORPTION ON ENDOTOXAEIVIIA IN BURN ILLNESS

Nedeliaeva A., OvinG.Y.

Research Institute of Traurnatology and Orthopaedics, Nizhniy Novgorod, Russia

SUMMARY. In this paper we report our experience in mice and rats with the use of enterosorbents such as vaulen (a carbonfibrillar adsorbent), Dneeper ACFM (activated carbonfibrillar material), enterodes (low-molecular polyvinylpyrrolidone), enterosgel (polymethylsiloxan) and polyphepan (a lignin derivative). It was established that enterosorption greatly reduced GI bacteria endotoxin concentration in the blood and the mass molecular peptide level, and suppressed their cytolytic action. Enterosorption-induced endogenic intoxication decrease improved hepatic glycogen- and protei n -synthesizing functions.

Introduction
Toxaemia develops in the first hours after thermal injury, determining not only the gravity and irreversibility of the shock but also the course of the burn illness. The-nature of burn toxaemia has been insufficiently investIgated in the past.
Taking intec0As,10eration Babb and Scribner's (2) concept of "middle moleculars", the level of which is significantly ele '. vai0ed,' due to uraemia and some other pathological states, the assumpton of the pathogenetic role of middle:. mass, molecular peptides (MMP) as burn toxaemia factors is of great importance (6, 14).
Until now there e,ase, been no single point of view regarding the nature,eof bum toxaemia. The gravity of the burn disease and- its possible sequelae are thought to be related to peQteolytic activity (7) and lipid peroxidation (18).
The role of e G1 endotoxins in endogenous intoxication has b,dee.,,1i,,ttle investigated. In 1964 Fine (9) showed that graffl-pegative intestinal flora was the source of endotoe,ic",faetors in different types of shock. There are few data about the role of endotoxins in bums, and the opinions of different authors do not coincide.
It was shown In the experiments of Little and Stoner (13) that end6toxins do not play a decisive role in the pathogenesise of burn disease. However, according to other.'e researchers, a large number of intestinal bacteria:',e'e(. ' hdde'tieoxins reach the bloodstream owing to the .esi!4den increase of intestinal permeability, whie, leads to the endogenous intoxication conditi6n (4, 15, 16).
The nature of.the endotoxic factor in the acute period of bum,",edisease and the dynamics of endotoxaemia in the_early period after thermal injury need further investigation. This question is of great importance nowadays owing to the appearance and application of various enterosorbents decreasing endotoxaemia of practically any aetiology in various fields of medicine.

Material and methods
1860 adult animals (360 white rats and 1500 mice) of both sexes kept on standard diet were used in the experiment. The burns were inflicted for 10 sec on two areas of the back with a hot (650 'C) plate under pentobarbital sodium (Nembutal) anaesthesia. The rats received 3rd-4th degree burns on 20-25% TI3SA. The investigations were performed on the same day, over 3 days and over a week. Blood was taken by decapitation. The NIMP level was determined for the assessment of the common toxicity of the blood (10). The method was that of trichloracetic acid which induces protein precipitation (in our case these were proteins with molecular mass over 5000 Dalton). MNIP concentration was determined in the proteinfree supematant by waves of two lengths (E2,4 and E2W). For NIMP secretion the method of blood serum ultrafiltration through the YAM-100 acetatecellulose membrane was used. The immune cellular reactions were assessed with the specific cellular leukocytolysis reaction. This method is based on the phenomenon described by Favour (8). In sensitized organisms the "white" blood elements are hypersensitive to the allergen that results in leukocyte destruction.
We also used a method of our own that makes it possible to increase the accuracy of autosensitization measurements in leukoeytolysis and to determine blood serum toxicity (1). The essence of the method is that both autologic blood serum (in leukocytolysis) and from the experimental probe -u ltraf 1 ltrate comprising only NIMP (in the blood serum toxicity test) are added to leukocytes.
Biotesting in mice (12) with dactinomycin administration for sensitization (cf. Selye's method (19)) was used to determine the GI endotoxin level in blood. Mice were intraperitoneally infilsed with 0.5 ml of rat blood serum mixed with NaCl solution (1:5) and 0.5 ml of dactinomycin solution with 12.5 mcg of active agent. The percentage of dead mice was calculated on the 4th day of observation. On the calibrated curve we found the bacterial endotoxin dose (BET) corresponding to the lethal percentage. The calibrated curve reflected a logarithmic dependence beetwen Escherichia coli endotoxin (E. Coli) and the lethal percentage. It was constructed according to the data of the experiment on mice intraperitoneally injected with dactinomycin and 7 doses of endotoxin (0.001; 0.002; 0.01; 0.02; 0. 1; 0.2; 0.4 mcg). We also carried out histological investigations on the burned rats' liver. The glycogen content (Best's staining) and adipose degeneration were determined. We investigated the total protein content (refrac tometry) and protein fractions (electrophoresis orithe paper carrier) in the blood of burned animals.
By enterosorption the sorbent was perorally infused through the probe once a day for 1, 3 and, 5 days, depending on the aim of the experiment. Five types of enterosorbents were used:

• carbonfibrillar adsorbent (vaulen) - 40 mg/kg;
• Dneeper activated carbonfibrillar material (ACFM) - 40 mg/kg;
• low-molecular polyvinylpyrrolidone (enter- odes) - 200 mg/kg;
• polymethylsiloxan (enterosgel) - 1000 mg/kg;
• lignin derivative - polyphepan - 250 mg/kg.

Optimal enterosorbent concentration was determined in burned mice (3rd-4th degree bums in about 40% TBSA). The percentage of lethality in mice was calculated 2 days post-burn.

Results
Endotoxaemia is significantly expressed in the first 3 days post-burn: administration of blood serum drawn from rats 24 h after thermal injury to the sensitized mice resulted in the death of 72.7t4.2% of the animals (BET dose - 0.4 mcg); after 3 days post-burn - 61.2-+3.3% (BET dose - 0.0158 mcg); in 7 days - 46.3.t5.2% (BET dose - 0.0398 mcg).
MMP content (both aromatic and non-aromatic) in the rat blood increased greatly and the largest concentration was observed immediately after the bum trauma. In 24 h MMP level increased 2 times. After 3 and 7 days this value remained high (Fig. 1).

We also studied MMP cytolytic action after the burn trauma., The serum toxicity; value (STV) increased 3.5 times in a day after the bum trauma and remained high enough during the investigation (Fig. 2). It was shown that autolsensitivization processes evaluated by leukocyte autolysis level become active in the bum shock period (Fig. 2).

Severe endogenic burn intoxication (especially connected with GI endotoxins) is associated with organ pathology, above all hepatic dysfunction. According to the data of histological investigations 3 days post-burn hepatic adipose degeneration developed in all the burned rats and glycogen reduction affecting hepatic protein production was observed. Together with vascular permeability disturbance it resulted in essential hypo- and dysproteinaemia.
The largest decrease of total protein was observed one day after the burn injury: 56.61+3.3 g/1, in 3 days; up to 60.3±1.8 g/1, in one week; up to 69.3±1.4 g/1 (in intact rats the total protein content was 78.1±4,5 g/1). Hypoproteinaemia was mostly connected with albumin dynamics. The albumin level one day after the burn trauma was 15.4±0.9 g/1 (in intact animals 26.8_+3.2 g/1), in 3 days 15.7_+0.8 g/1, and in one week 16.0±2.1 g/1.
Further investigations considered the possibility of endotoxaemia correction with enterosorption. Enterosorbent administration caused a significant decrease in endotoxaemia, according to biotesting. Lethality in mice injected with serum of burned treated rats decreased by 26-48% in the first day after the burn trauma, by 12-26% after 3 days, and by 3-18% after a week, depending on the type of sorbent. The enterosorption efficacy was shown to be different. The greatest effect was observed with enterosgel and Dneeper ACM Vaulen had the maximum detoxication effect on the first day post-burn.
Enterosorbents greatly reduced not only the GI endotoxin level but also NIMP concentration (Fig. 1). The maximum NINIP decrease was observed after polyphepan application (one day and one week after the burn trauma). Three days after the thermal injury the NINIP level was normal after enterosgel and ACFM application.
Enterosorption also caused a large decrease of the NIMP cytolytic effect. The serum toxication value (STV) fell to 4.6% (Dneeper MFM suspension) and to 11.7% (enterosgel).
Autoaggression manifestations decreased after enterosorption. The general rate of leucocyte lysis (GRLL) was practically normal after enterosorbent application. After vaulen administration GRLL decreased from 28.6_+1.5% to 12.6_+1.8% 3 days after the burn trauma. After the application of the Dneeper ACFM suspension, GRLL decreased to 19.3±2.6% and enterodes to 15.0±0.8%. It was shown that the GRLL increase in the early period after thermal injury was connected mainly to the increase of blood serum toxicity, which decreased significantly after enterosorption. After the application of enterosorbents the difference between GRLL and STV decreased significantly, which determined the immediate role of autoinimune reactions in leukocytolysis, After the bum the difference was 6.8%; in, treated rats this reduced to 0.6% after application of vaulen dnd,-to 3.2% after the application of enterodes (Fig. 2).
The reduction of endogenic intoxication due to the influence of enterosorption improved morphological and functional hepatic values. Enterosgel had a clear hepatoprotective effect (glycogen content in liver was normal, and adipose degeneration manifestations were insignificant or practically absent).
In our study we investigated the influence of enterosorption on the mixture of protein fractions of blood serum in burned rats. In seven days every sorbent reduced dysproteinaemia and hypoalbuminaemia, and one day after the burn trauma prevented acute total protein fall. Polyphepan was the most effective; it prevented the reduction of total protein concentration (65.6 g/1) one day after the burn trauma (control: 56.6 g/I).
The significant reduction of endogenic intoxication under the influence of enterosorption is thus the main reason for the reduction of degenerative changes in the hepatic parenchyma, and results in the improvement of its most important function: protein production. Polyphepan and enterosgel seem to be the most efTective in this respect.

Discussion
Enterosorbents are widely used in the treatment of severe intoxication and metabolic disturbances (7), and as an antidote (3). In Ditter's experimental model (5) the most effective material in the prevention of endotoxaemia proved to be bentonite, followed by kaopectate and charcoal particles.
Few data testify to the fact that the application of enterosorbents is positive in the burn disease (11, 20, 21). Enterode administration positively influences the burn disease: intoxication manifestations are eliminated, and clinical and immunological values become normal (20, 21). Enterosorption with enterodes and enterosgel is one of the methods for prevention of complications in the burn disease.
There is no other evidence for the application of other enterosorbents. The optimal enterosorbent concentration and the time for providing enterosorption are also unknown.
We established that a twofold increase of the usual dose of enterodes and vaulen reduces mice lethality twofold. A further increase of the dose proved to be unreasonable owing to the absence of the effect.
Our investigations thus showed that toxaemia _developing in the first hours after thermal injury played a decisive role in the pathogenesis of the acute period of the burn disease.
Enterosorption greatly reduces GI endotoxin concentration and the NINIP level. Enterosgel and the Dneeper ACFM show the greatest advantages, although their effect is different at different times of observation.
It must also be mentioned that vaulen entero.: sorption suppresses autoimmune and cytotoxic reactions, which constitutes the advantage of this enterosorbent. The essential decrease of enterosorption-induced endogenic intoxication is the most significant reason for the reduction of degenerative changes in the hepatic parenchyma and the improvement of its most important function: protein production. In this respect polyphepan and enterosgel seem to be the most effective enterosorbents.
Our investigations thus show that enterosorption must be controlled in the first hours after thermal injury. It has been established that enterosorption makes it possible not only to reduce endogenic intoxication caused by GI bacterial endotoxins but also to decrease NINIP concentration and autoaggression manifestations and to improve hepatic protein- and glycogen-synthesizing functions.

RÉSUME. Les auteurs décrivent leurs résultats chez les souris et les rats avee l'emploi des entérosorbants comme le vaulen (adsorbant carbonfibrillaire), le Dneeper ACFM (matériau carbonfibrillaire activé), les entérodes (polyvinylpyrrolidone de masse moléculare basse), l'entérosgel (polymethylsiloxane) et le polypliépan (dérivé de la ligninc). lls ont trouvé que l'entérosorption réduit fortement la concentration hématique des endotoxines des bactéries gastro-intestinales el le niveau de la masse moléculaire des peptides, en supprimant leur action cytolytique. La réduction de I'intoxication endog¿ne provoquée par l'entérosorption améliore les fonctions hépatiques de synthétisation des glycogénes et des protéines.

1. A.c. 1691747 SU, G 01 N33/53: Method of endotoxaemia determination in burn disease. Levin G.Y., Korabliov S.B., Nedeliaeva A.V., Kushner R.M., Nov. 1991, Discoveries, Inventions, 42, Nov. 1991 (Russian).
2. Babb L., Popovich R.P., Christopher T.G., Scribner B.H.: The genesis of the square meterhour hypothesis. ASAIO, 17: 81-95, 1971.
3. Cooney D.O.: "Superactive" charcoal for antidotal use in poisonings. Clin. Toxicol., 2: 387-390, 1977.
4. Deitch E.A., Maejima K., Berg R.: Effect of oral antibiotics on the translocation of the GI tract microflora in burned rats. J. Trauma, 25: 385-392, 1985.
5. Ditter B., Urbaschek R., Urbaschek B.: Ability of various adsorbents to bind endotoxins in vitro and to prevent orally induced endotoxernia in mice. Gastroenterology, 84: 1547-1552, 1983.
6. Dolgushin 1. 1., Ebert L.Y., Lifshits R. I.: "Trauma
7. Immunology", Sverdlovsk, 1989 (Russian).
8. "Enterosorption", Beliakov N.A. (Ed.), Centre of the Sorption Technologies, Leningrad, 1991 (Russian).
9. Favour C.B.: Lytic effect of bacterial products on lymphocytes of tuberculous animals. Proc. Soc. Exp. Biol. Med., 65: 269-272, 1947.
10. Fine J., Boston M.: Septic shock. JAMA, 188: 427-432, 1964.
11. Gabrielian N.I., Lipatova V.I.: The experience of the MMP index usage in diagnostics of nephrologic diseases in children. Lab. Work, 3: 7-12, 1984 (Russian).
12. Gur-Arjie B.N., Levshunov V.S., Beliakov N.A. et al.: Hemosorption and enterosorption influence on burn disease.
13. Third All-Union Conference on the problem: "Modern first aid means and treatment methods of the burn disease", 163-164, Moscow, 1986 (Russian).
14. Kiseliev P.N., Shu1jie G.S.: Bacterial endotoxin determination in blood of irradiated animals and humans. Lab. Work, 9: 561-563, 1981 (Russian).
15. Little R.A, Stoner H.B.: Studies of the mechanism of shock. Br. J. Exp. Path., 53: 381-389, 1972.
16. Lifshits R.I.: Metabolic principles of burn intoxication and methods of detoxication therapy. Kazan Med. J., 6: 405-407, 1987 (Russian).
17. Ma Li F.: Endogenous microbial dissemination following severe burns in rats. Burns, 12: 325-329, 1986.
18. Manson W.L., Westerveld A.W " Klasen H.J., Sauer E.W.: Selective intestinal decontamination of the digestive tract for infection prophylaxis in severely burned patients. Scand. J. Plast. Reconstr. Surg., 21: 269-272, 1987.
19. Minstry S.: Cellular immune response following thermal injury. Burns, 12: 318-324, 1986.
20. Nishigaki I., Hagihara M., Hiramatsu M. et al.: Effect of thermal injury on lipid peroxide levels of rat. Biochem. Med., 24: 185-189, 1980.
21. Selye H., Tuchweber B., Bertok L.: Effect of lead acetate on the susceptibility of rats to bacterial endotoxin. J. Bacteriol., 91: 884-890, 1966.
22. Suzdaleva V.V.: Modern concept of detoxication problem. Hematology & Transfusiology, 7: 3-5, 1987 (Russian).
23. Suzdaleva V.V., Makarova N.Y., Kiselieva A.A. et al.: Enterodes: a new detoxication peroral agent. Exp. Inform. New Medicinal Agents, 1: 1-8, 1988 (Russian).