Insoluble glycogen, a metabolizable internal adsorbent, decreases the lethality of endotoxin shock in rats.

Insoluble glycogen is an enzymatically modified form of naturally occurring soluble glycogen with a great adsorbing capacity. It can be metabolized by phagocytes to glucose. In this study we used insoluble glycogen intravenously in the experimental endotoxin shock of rats. Wistar male rats were sensitized to endotoxin by Pb acetate. The survival of rats were compared in groups of animals endotoxin shock treated and non-treated with insoluble glycogen. Furthermore, we have determined in vitro the binding capacity of insoluble glycogen for endotoxin, tumour necrosis factor alpha, interleukin-1 and secretable phospholipase A2. Use of 10 mg/kg dose of insoluble glycogen could completely prevent the lethality of shock induced by LD50 quantity of endotoxin in rats. All animals treated survived. Insoluble glycogen is a form of 'metabolizable internal adsorbents'. It can potentially be used for treatment of septic shock.


Introduction
In the pathogenesis of endotoxin or septic shocks the main roles are played by secondary mediators, e.g. tumour necrosis factor alpha (TNF-a), interleukin-1 (IL-1), phospholipase A 2 (PLA 2 ) and platelet activating factor (PAF). 1 -5 These substances cause generalized lesions of capillaries resulting in hypotonia with the symptoms of concomitant multiple organ dysfunction syndrome. 1 Numerous biologically potent materials, toxins, cytokines, enzymes, lipid and peptide mediators can be simultaneously present in the circulation during septic shock. Blocking only one of these mediators cannot be enough. 6,7 Besides extracorporeal adsorbents, 8,9 intravascular adsorbents like kaolin, aluminium and magnesium silicate were found to have bene cial, simultaneously detoxi cating effects in animal experiments. 10 However, at a certain level, they were also toxic. Therefore, there is a need to develop drugs regarded as 'metabolizable internal adsorbents'.
Insoluble glycogen (ISG) is an enzymatically modi ed new form of naturally occurring glycogen. In consequence of its enlarged surface, it has an increased adsorbent capacity compared with natural glycogen or amylum. Phagocytes can engulf ISG and degrade it to glucose in the lysosomes, helping the metabolism of phagocytosing cells. Insoluble glycogen represents a potent adsorbing capacity in blood. In phagocytes it helps the metabolism during the biological neutralization of toxic materials. It is a 'metabolizable internal adsorbent' able to decrease the lethality in endotoxin (Escherichi a coli lipopolysaccharide) induced shock of rats.

Preparation of insoluble glycogen
Insoluble glycogen was prepared and made by our method in our laboratory. 11 Brie y, the enzymatic elongation of natural, soluble glycogen took place by phosphorylase-a and glucose 1 phosphate, resulting in a new, arti cial form, called insoluble glycogen (ISG) forming insoluble particles with 1 mm average diameter in water or buffer milieu. Sterile and endotoxinfree dishes and devices were used throughout the preparation. ISG represents an enlarged surface for aspeci c and simultaneous adsorption of various molecules in a solution. The suspensions of insoluble glycogen were prepared for intravenous treatment: 20 mg/ ml of ISG was used in sterile and endotoxin-free phosphated buffer in saline (PBS).
Rat model of endotoxin shock 12 Wistar male rats (200 g) were presensitized to endotoxin (E. coli lipopolysaccharide) by intravenous (i.v.) injection of 5 mg, endotoxin free Pb (lead) acetate (Reanal, Hungary). The LD 50 dose of i.v. E. coli lipopolysaccharide (LPS, endotoxin, ET) was found to be 2 mg, whereas the LD 100 dose of LPS was 5 mg. Ten mg/ kg of i.v. insoluble glycogen was applied just before LPS. ISG, Pb acetate, LPS were injected i.v. through the same needle but from various syringes in the sequence: ISG, Pb acetate, LPS. The lethality and survival were evaluated after 12 h. The results were calculated on the basis of three separate experiments.
The experimental protocol was approved by the Local Animal Experimental Ethical Committee in 1996.
Binding assays 99m Tc labelling of E. coli LPS (endotoxin), 125 I labelling of human phopholipase A 2 (14 kD puri ed in our laboratory), rabbit phosphorylase-a (puri ed in our laboratory), human recombinant TNF-a (prepared by Duda et al.), human IgG (Human Serobacteriological Institute, Hungary) were carried out in our laboratories. The C3b activating and binding capabilities of ISG was measured in citrated rabbit blood by determination of the amount of C3b consumed during the activation of alternative way of complement system. The in vitro binding capacity of 1 mg of ISG was calculated for various substances and the masses of bound materials were expressed in mg of bound molecules/1 mg of ISG. We have preferred to use this form of calculation instead of counting in mols, because this formula re ects better the relations of quantities to each other. For example: 0.1 mg of phosphorylase-a represents 10 -9 M, likewise 0.015 mg of phospholipase A 2 , showing that the expression of the masses of bound molecules in mg and not in M can describe better the real binding situations on the surfaces of ISG particles. The results represent the average values of two separate binding measurements with duplicated samples (n = 4).

Results
The LD 50 dose of E. coli endotoxin (ET, lipopolysaccharide, LPS) was found to be 2 mg in rats sensitized to endotoxin by Pb (lead) acetate. We have found that 10 mg/ kg dose of ISG given intravenously, simultaneously with Pb acetate and ET (by the same needle but from different syringes in the series: ISG, Pb acetate and LPS) could completely prevent the lethal effect of LD 50 dose of ET. In this group, all 12 animals survived (Table 1).
In the pathomechanism of endotoxin shock, the effects of circulating ET, TNF, IL-1, PLA 2 , PAF are regarded to be the main causative factors. For the explanation of the in vivo protective effect of ISG in the rat model of endotoxin shock, we have measured its binding capacity of these materials in vitro. The aim of the binding assays was to determine the amount of these biologically important molecules bound by ISG aspeci cally, by adsorption, in order also to prove the possibility of the occurrence of the phenomenon in vivo. Table 2 shows that ISG could bind ET labelled with 99m Tc dose dependently, in vitro.
Insoluble glycogen was found to bind 125 Ilabelled human recombinant TNF-a, IL-1, human secretable PLA 2 and human IgG ( Table 3). The greatest binding capacity of ISG was found on phosphorylase-a, it was still rather high on IgG, C3b and phospholipase A 2 , whereas on the cytokines, TNF-a and IL-1, it was rather low (Table 3). Table 4 shows that the luminol ampli ed chemiluminescence of phagocytes. ISG, like zymosan, increased the chemiluminescence in the samples of both human and rat bloods. However, when phosphorylase-a and the ISG complex was added to the cells together, chemiluminescence elevated signi cantly (P , 0·001) suggesting that the level of intracellular glucose had been elevated on the in uence of phosphorylase-a, metabolizing glycogen. These observations proved functionally (we have also unpublished morphological evidences) that the particles of ISG could be internalized by phagocytes, and the intracellular glucose produced could increase the free radical production, 6 and it could serve also as a source of energy for the phagocytosing cells. These data show that cells can tolerate insoluble glycogen having dual effects: transporting toxic materials to the phagocytes, 1 and producing intracellular glucose for the cells. 2

Discussion
Insoluble glycogen represents a new molecular and theoretical approach for the solution of the therapeutic problems of endotoxin or septic shock. In these pathologic states, like in other forms of systemic in ammatory response syndrome (SIRS), the simultaneous presence of numerous toxic substances is the main reason for the dif culties. 1 Insoluble glycogen partly follows the principle published previously concerning the bene cial effects of adsorbents, kaolin or silicates, used endogenously in experimental intestinal infections. These materials, however, are themselves toxic. 4 Insoluble glycogen is also a potent adsorbent with the capability to adsorb, to bind simultaneously many kinds of molecules in plasma, toxic or nontoxic, as well. The opsonization of ISG by C3b and IgG has still an important advantage in plasma. These molecules enable insoluble glycogen to penetrate into the phagocytes via CR1 and Fc receptors transporting some quantities of TNF-a, IL-1, PLA 2 , PAF, LPS, etc. into the lysosomes of phagocytes simultaneously. The special advantage of insoluble glycogen is manifested in its capability to improve the metabolism of phagocytes in toxic states. The effects of triglycerid-rich lipoproteins in preventing septic death, their in uences on the transport and metabolism of endotoxin or cytokines suggest similar mechanisms as in the case of ISG. 15 We have to mention that the protecting effect of ISG on the lethality of endotoxin shock of rats could be observed only when we injected it just before plumbum acetate and LPS. There was no bene cial effect of ISG applied in the 4th hour of shock. This nding suggests that the ef cacy of an adsorbent therapy can be strongly related to the time of application and  to the relative amount of toxic agents adsorbed in the acute forms of toxic shock. 16 In the in vitro binding experiments for the various substances the sedimentation of ISG particles caused a technical problem during incubation. This was why we measured the binding constants using the same amount of ISG and we tested the gradually elevating concentrations of the materials. From these studies it has been apparent that ISG, as a natural substrate of phosphorylase-a, can bind this enzyme to the greatest extent. Besides, there are great differences in the binding capacities of ISG to the various substances. From the aspect of therapeutical effects, we emphasize that ISG can bind C3b, IgG, endotoxin, PLA 2 , TNF-a and IL-1 simultaneously.
According to our unpublished data, ISG applied i.v. in a concentration of 10 mg/ kg did not result in a signi cant change in the level of blood glucose. Thus, no blood sugar elevating component can be expected to take part in its protecting effect in endotoxin shock, where the fall of blood sugar is regarded as a factor of lethality. 17 The bene cial in uence of ISG on the glucose metabolism can be basically related to the peripheral blood cells, mainly to phagocytes. ISG was able to increase the free radical production in the peripheral granulocytes suggesting that its particles could be readily internalized and they could be metabolized to glucose intracellularly, elevating the capability of cells for increased production of free radicals. 6 Recently we have found that ISG has a protective effect in the experimental necrotizing pancreatitis of dogs. 18 Insoluble glycogen is representative of a family of potentially new drugs with multiple functions, serving special pharmacological purposes like detoxi cation, transportation, etc., besides directly helping the metabolism of some types of cells. For such an aim, the chemical modi cation of various natural, not toxic materials, mainly of carbohydrates or polynucleotides, can give further examples in the future, effecting as 'metabolizable internal adsorbents'.