In vitro evaluation of the cytotoxicity and genotoxicity of resorcylidene aminoguanidine in human diploid cells B-HNF-1.

RAG belongs to appropriate inhibitors of protein glycation, i.e. formation of advanced glycation end products, which are thought to be responsible for some complications of DM, including neuropathy, angiopathy, retinopathy and nephropathy. In the present study authors have evaluated the genotoxic effect of RAG on the cell culture of human neonatal fibroblasts (B-HNF-1) in regard to its potential clinical application as inhibitor of advanced glycation end products in relationships to the pathogenesis of chronic diabetic complications. The direct contact cytotoxicity assay and micronucleus test were performed. The results showed that RAG in the concentration range of 1 x 10-4 to 1 x 10-6 mol.l-1 did not induce any changes in the morphology of exposed B-HNF-1 cells. The frequency of micronuclei was not significantly increased as well. The inhibitive effect of resorcylidene aminoguanidine was directly proportional to its concentration. It can be concluded that RAG at the selected concentrations has an inhibitive effect on proliferation of the treated cells and, at the same time, does not display any genotoxic effects on B-HNF-1 cells.

Rooibos tea (Aspalathus linearis) partially prevents oxidative stress in streptozotocin-induced diabetic rats.

The aim of this study was to investigate the effects of rooibos tea as a natural source of a wide scale of antioxidants on the prevention and treatment of oxidative stress in streptozotocin-induced diabetic rats. Expected significant changes of biochemical parameters characteristic for experimental diabetic state were found in plasma and tissues eight weeks after single dose streptozotocin application. Administration of aqueous and alkaline extracts of rooibos tea (or N-acetyl-L-cysteine for comparison) to diabetic rats did not affect markers of the diabetic status (glucose, glycated hemoglobin and fructosamine). Besides the parameters characterizing hepatotoxic effect of streptozotocin, rooibos tea significantly lowered advanced glycation end-products (AGEs) and malondialdehyde (MDA) in the plasma and in different tissues of diabetic rats, particularly MDA concentration in the lens. From these results we can conclude that antioxidant compounds in rooibos tea partially prevent oxidative stress and they are effective in both hydrophobic and hydrophilic biological systems. Therefore, rooibos tea as a commonly used beverage can be recommended as an excellent adjuvant support for the prevention and therapy of diabetic vascular complications, particularly for protecting ocular membrane systems against their peroxidation by reactive oxygen species.

2,3-diphosphoglycerate and oxygen supply of tissues in cardiosurgical diabetics.

THE MAIN AIM OF THE STUDY: The oxygen supply of tissues was studied under haemodilution in cardiosurgical diabetic and non-diabetic patients. METHODS: There were 30 cardiosurgery patients examined, 9 were patients with diabetes mellitus.and 21 were non-diabetic patients. Venous blood samples were examined preoperatively, intraoperatively and for 10 days after operation. RESULTS: Haemodilution caused a decrease in haematocrit values in both groups, as well as in the erythrocyte count and haemoglobin concentration. Postoperatively, an increase was recorded in haematological values in both groups, the values had not reached the baseline even by 10th day. Increased values of blood oxygen saturation and partial oxygen pressure during the operation returned to baseline in both groups in the postoperative days. Values of p50 did not change in both groups for the period of observation. CONCLUSION: The obtained data suggest that sufficient oxygen supply to tissues was ensured under haemodilution in cardiosurgery patients in both groups. These results confirm multifactorial dependence of blood oxygen transport to tissues (Tab. 1, Fig. 3, Ref. 13).

Influence of 2,5-dihydroxybenzylidene aminoguanidine on lipid oxidative damage and on antioxidant levels in model diabetes mellitus.

2,5-Dihydroxybenzylidene aminoguanidine (BAG) is a structural analogue of the antidiabetic compound aminoguanidine, and is an example of a substance protecting diabetic rats from lipoprotein oxidation arising in oxidative stress conditions characteristic of diabetes mellitus. We found that administration of BAG to diabetic rats decreases their susceptibility to lipoprotein oxidation, decreases formation of conjugated dienes and 4-hydroxy-2-nonenal, and increases antioxidant potential of plasma. On the other hand, our results show that BAG has a negative influence on lipoprotein oxidation in control rats. Increased formation of 4-hydroxy-2-nonenal and conjugated dienes and a decrease in plasma antioxidant potential was observed when BAG was administered to control rats. It is therefore necessary to search for other structural modifications of this substance that would combine higher antidiabetic activity with less toxicity in healthy individuals.

The effect of selected membrane active substances on erythrocyte deformability.

Aminoguanidine improved the erythrocyte filterability by 4%, pyridoxyliden-aminoguanidine by 11% and pyridoxal by 13% in healthy subjects. In diabetic patients the aminoguanidine effect on erythrocyte filterability was improved by 7%, PAG effect by 9% and pyridoxal effect by 15% in comparison to the control group. The other investigated haematological variables in both groups were within the range of the physiological standard. All of the tested substances demonstrated a mild protective influence on erythrocyte elasticity both in healthy subjects and diabetic patients. Significant elasticity improvement was obtained only by pyridoxal (p<0.01) in patients with diabetes mellitus. (Fig. 4, Ref. 18.).

Cytotoxicity test and cytogenetic analysis of effects of aminoguanidine in vitro.

The potential genotoxic activity of chemical substances in vitro is usually assessed by the micronucleus test and by karyological analysis. Use of the fluorescent plus Giemsa (FPG) technique is also recommended in the event that positive results are found in the micronucleus test, or if there is an increased rate of structural and numerical chromosome aberrations compared with controls. The tested substance, aminoguanidine (AG), has a marked ability to inhibit the toxic effects of carbonyl products (carbonyl stress) that arise during the end-phases of non-enzymatic protein glycation both in vitro and in vivo. The importance of this ability follows the finding that the production of advanced glycation end-products (AGE) is a part of the molecular mechanism of the pathogenesis of chronic diabetic complications. The aim of this study was to test the cytotoxic and clastogenic effects of AG on cells of the diploid cell line B-HEF-2, derived from a three-month-old male fetus. The results of the test did not reveal any induction of micronucleus production in the analyzed cells at AG concentrations ranging between 1 x 10(-2) and 1 x 10(-4) mol.L-1. Karyological analysis showed no clastogenic effect of the tested substance nor any increased rate of structural chromosome aberrations. The positive properties of AG and to its potential use as a glycoxidation inhibitor and AGE production are somewhat dimmed by its ionic nature, which hampers hydrophobic interaction with the nonpolar components of biological membranes. For this reason, the authors will further study the cytotoxicity and cytogenetic analysis of Schiff bases of AG synthesis on the basis of natural aldehydes (resorcine aldehyde, pyridoxal, etc.) in which antiglycation activity has been detected.

Influence of beta-resorcylidene aminoguanidine on selected metabolic parameters and antioxidant status of rats with diabetes mellitus.

We studied the effects of administration of beta-resorcylidene aminoguanidine (RAG) to Wistar strain rats with experimental diabetes mellitus (DM) induced by streptozotocin. The effects studied included antioxidant levels in plasma and the liver, oxidative damage of lipids represented by the formation of substances reacting with thiobarbituric acid (TBARP) and selected biochemical indicators. The administration of RAG did not significantly affect antioxidant status of diabetic rats or hemoglobin glycation and plasma concentration of fructosamine. In diabetic rats, application of RAG decreased formation of TBARP in plasma but not in the liver. Moderate steatosis of liver and increased plasma levels of triacylglycerols in diabetic rats were significantly improved by application of RAG.

Energy transfer in acute diabetic rat hearts: adaptation to increased energy demands due to augmented calcium transients.

OBJECTIVES: Hearts of rats with diabetes mellitus (DM) are characterized by energy demands exceeding their energy production, but they might also exhibit decreased vulnerability to ischemia and calcium overload. This indicates adaptation in cardiac energetics (CE), where energy transport is not rate-limiting. Aim-This study was designed to elucidate the functional significance of the DM-induced adaptation in CE by investigating the formation of mitochondrial contact sites (MiCS), facilitating the Ca-dependent/high-capacity energy transfer from mitochondria, in conjunction with testing the ischemic tolerance (IT) of hearts. METHODS: After 1 week of streptozotocin-induced DM (45 mg/kg iv), the hearts of male diabetic and age-matched control rats (C) were isolated and Langendorff-perfused with either 1.6 or 2.2 mmol/L of CaCl(2). MiCS formation was assessed by cytochemical detection of mCPK octamers and was quantified stereologically as MiCS to mitochondrial surface ratio (S(S)). IT was evaluated in anesthetized open-chest animals subjected to 30-min occlusion of the LAD coronary artery followed by 4-h reperfusion, by monitoring ischemic arrhythmias and by measuring the size of infarction (tetrazolium double staining). RESULTS: In C hearts, increasing Ca2+ induced both positive inotropic response (dP/dt increase from 2270 +/- 220 to 2955 +/- 229, p < 0.01) and elevated MiCS formation (S(S) increase from 0.070 +/- 0.011 to 0.123 +/- 0.012, p < 0.01). In DM hearts, basic MiCS formation was already comparable with that induced by elevated Ca2+ in C hearts and could not be further stimulated by Ca2+. In C, ventricular tachycardia represented 55.4% of the total arrhythmias and occurred in 90% of the animals. In DM rats, the arrhythmia profile was similar to that in C, and the incidence of tachyarrhythmias and their severity were not enhanced (arrhythmia score: 3.18 +/- 0.4 vs. 3.30 +/- 0.3 in C). The infarct size normalized to the size of area at risk was smaller in the DM than in C hearts (52.3 +/- 5.8% vs. 69.2 +/- 2.2%, respectively; p < 0.05). CONCLUSIONS: Ca-signaling represents the link between energy delivery from mitochondria (via MiCS) and energy requirements of the heart. In DM hearts, energy transport via MiCS is elevated to the maximum value. This contributes to increased resistance of DM hearts to irreversible cell damage.

Effects of aminoguanidine Schiff's base on biomarkers of the oxidative stress, 4-hydroxy-2-nonenal and conjugated dienes, in the model diabetes mellitus.

Diabetes mellitus evoked by streptozotocine in rats is associated with the oxidative stress. We examined the effect of Schiff's base 2,5-dihydroxybenzaldehyde with a well-known antidiabetic drug aminoguanidine, 2,5-dihydroxybenzilideneaminoguanidine (BAG) on the production of markers of oxidative stress such as 4-hydroxy-2-nonenal (4HNE) and conjugated dienes in diabetic rats. BAG administration did not affect glucose level in diabetic rats but significantly decreased the production of 4HNE and conjugated dienes. On the other hand, BAG caused the elevation of conjugated dienes and an insignificant increase of 4HNE levels in the control animals.

Fluidising effect of resorcylidene aminoguanidine on sarcolemmal membranes in streptozotocin-diabetic rats: blunted adaptation of diabetic myocardium to Ca2+ overload.

The "remodelling" of cardiac sarcolemma in diabetes is believed to underlie the reduced sensitivity of diabetic hearts due to their overload with extracellular calcium. Along with a non-enzymatic glycosylation and the free radical-derived glycoxidation of sarcolemmal proteins there is ongoing reduction in cardiomyocyte membrane fluidity, the modulator of cardiac sarcolemmal functioning. Aminoguanidine derivatives, that inhibit glycation and glycoxidation, might suppress myocardium "remodelling" occurring in diabetic heart. To verify this hypothesis, we studied physical parameters of cardiac sarcolemma from the streptozotocin-induced diabetic rats (45 mg.kg(-1) i.m.) treated with resorcylidene aminoguanidine (RAG, 4 or 8 mg.kg(-1) i.m.). The treatment with RAG not only completely abolished protein glycation and a generation of free oxygen species (p < 0.001) in treated diabetic animals, but also considerably attenuated the decrease in sarcolemmal membrane fluidity (p < 0.001). In diabetic animals the "normalization" of the sarcolemmal membrane fluidity was accompanied by the vastly increased susceptibility of diabetic hearts to be overload with external calcium. We concluded that the decreased fluidity of the sarcolemmal membrane, apparently linked to the excessive glycation of sarcolemmal membrane proteins, might be intimately connected with the adaptation mechanism(s) that are likely to develop in diabetic heart to protect it against the overload with external calcium.

Serum levels of advanced glycation end products in poorly metabolically controlled children with diabetes mellitus: relation to HbA1c.

Serum levels of advanced glycation end products (s-AGEs) were investigated in children with poorly metabolically controlled diabetes to determine whether they may be correlated with HbA1c, fructosamine, glycaemia, albumin excretion rate, duration of diabetes and age. In this study, 17 children with Type 1 diabetes mellitus (age range 9 to 18 yr) and 8 healthy children (age range 7 to 17 yr) served as subjects. S-AGEs were found to be increased in poorly metabolically controlled children with diabetes (HbA1c>9%). A significant correlation (r=0.65;p<0.01) was found between s-AGEs and HbA1c in the group of diabetic children. In poorly metabolically controlled children with diabetes of pre-pubertal and pubertal age, the level of s-AGEs should be monitored as the risk of microvascular complications may be already present at this early stage of life.

Impaired erythrocyte transmembrane potential in diabetes mellitus and its possible improvement by resorcylidene aminoguanidine.

Erythrocytes of diabetic patients have abnormal membrane properties. We examined in vitro transmembrane potential and the possible effect of resorcylidene aminoguanidine (RAG) on its modulation in erythrocytes of diabetic subjects. The transmembrane potential was assessed in RAG-treated and untreated erythrocytes, respectively, using a fluorescent dye (3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)]). We confirmed earlier findings that the transmembrane potential of diabetic erythrocytes is significantly increased compared with control (P < 0.01). The membrane hyperpolarization found in diabetic cells seems to be a result of oxidative stress present in diabetes mellitus. On one hand, the RAG treatment induced decrease in abnormal transmembrane potential values in diabetic erythrocytes (P < 0.01), presumably via its antioxidant and antiglycation activity. On the other hand, RAG moderately hyperpolarized the control erythrocytes (P < 0.05). We suggest that the drug-induced transient membrane expansion leads to an intracellular potassium loss and a subsequent change of the transmembrane potential. However, if controlled by an appropriate dosage, RAG can eliminate certain types of erythrocyte membrane damage induced by diabetes mellitus.

[Oxygen transport values in patients with surgery performed under extracorporeal blood circulation]. / Hodnoty parametrov transportu kyslíka u pacientov operovaných v podmienkach mimotelového krvného obehu.

The aim of the work was to study the O2-transport changes to tissues in cardio-surgical patients suffering from CAD and operated during extra-corporeal blood circulation (ECC). The changes of selected haematologic variables, 2,3-diphosphoglycerate (2,3-DPG) and ATP concentration, acid-base balance parameters with p50 calculation were measured in the venous blood samples taken before the operation, during the operation and on the 1st, 2nd, 3d, 5th, 7th and 10th day after the operation. From the obtained results follows, that extreme haemodilution causes significant decrease of the haematocrit value (Htc) by 35%, the value of haemoglobin (Hb) by 37% and the count of erythrocytes (Er) by 37% from the initial values. The count of reticulocytes (Ret) was increased by 52%. In the days after operation the increase in Htc values, the values of Hb and count of erythrocytes was observed, whereby the initial values were not reach even on the 10th day after the operation. The increase of the reticulocytes count by 33% prevailed to the 10th day after the operation in comparison with the initial values. 2,3-DPG concentration was increased between 3d and 10th day after the operation by 30% and ATP concentration between 5th and 10th day was increased by 23% from the initial values. Hb-O2 saturation (SpO2) and pO2 were increased already during the operation, the increase prevailed until the 7th day by 27%, pO2 until the 3d day by 39% from the initial values. Calculated values p50 did not change in the course of this study--they fluctuated in range +/- 0.04 kPa from the initial value 3.55 kPa. Supposing multifactorial character of Hb oxygenation and deoxygenation process it is possible to conclude, that the determined changes of observed parameters did not significantly influence O2-transport to tissues during ECC. (Fig. 3, Ref. 12.)

[Effect of vitamin C and E on nonenzymatic glycation and physico- chemical properties of isolated erythrocyte membranes in diabetic patients]. / Vplyv vitamínu C a E na neenzýmovú glykáciu a fyzikálno-chemické vlastnosti izolovaných membrán erytrocytov u diabetických pacientov.

Non-enzymatic glycation, accompanied by the formation of free radicals, represents a serious problem in diabetes mellitus. It is supposed to be the cause of the development of long-term diabetic complications. The aim of this work was to estimate the effect of treatment with vitamin C (1 g per day) and E (600 mg per day) on selected biochemical parameters as well as to determine the physicochemical state of erythrocyte membranes in diabetics. The paper also compares the physicochemical state of diabetic and control erythrocyte membranes. The changes in the values of glycaemia, glycated haemoglobin, and fructosamine were insignificant after three months of treatment. This points out that the doses used could be low or that the patient compliance was poor. An anionic fluorescent probe merocyanine 540 (MC540) was used to monitor possible changes in the physicochemical properties of isolated diabetic erythrocyte membranes. Significantly higher affinity of MC540 monomers to the membrane in diabetics treated with vitamin E was observed, which can be the result of the antioxidative effect of the vitamin (p < 0.02). A comparison of absorption spectra of MC540 in diabetic and control membranes revealed significant changes in the position of the bands and in their absorbances (p < 0.01 and less). They result from substantial alterations in the structure, surface charge, and the fluidity of erythrocyte membranes in diabetes mellitus. (Tab. 2, Fig. 3, Ref. 22.)

Values of markers of early and advanced glycation and lipoxidation in serum proteins of children with diabetes mellitus.

BACKGROUND: Advanced glycation endproducts (AGEs) have been established as one of the major factors responsible for the multi-organ damage seen in diabetes. AGEs and lipoxidation products, as e.g. MDA, and their adducts with proteins appear to be formed together in serum and tissues. A link between AGEs formation and increased lipoxidation at tissue damage is under investigation. AIM: The aim of the present study was to determine fructosamine (FAM), glycated haemoglobin (HbA1c) AGEs-specific fluorescence and MDA-protein adducts specific fluorescence in diabetic and in healthy children, with statistical evaluation of the relationship between the parameters assessed. SUBJECTS AND METHODS: Values of FAM and HbA1c (spectrophotometry) and of AGEs-specific fluorescence and MDA-protein adducts specific fluorescence were investigated in serum proteins of 17 children with poorly controlled type 1 diabetes mellitus (age range 9 to 18 years). Eight healthy children (age range 7 to 17 years) served as controls. RESULTS: In the diabetic group, all the parameters evaluated were significantly higher than in the control group. Furthermore, MDA-linked specific fluorescence of MDA-protein adducts (a biomarker of oxidative stress) was correlated with AGEs-specific fluorescence. In patients this correlation was extremely significant (r = 0.8176, p < 0.0001). CONCLUSION: The increased oxidative stress in children with type 1 diabetes may not be attributed to complications, though it could contribute to the development of complications. (Tab. 2, Fig. 7, Ref. 30.)

Decreased fluidity of isolated erythrocyte membranes in type 1 and type 2 diabetes. The effect of resorcylidene aminoguanidine.

Changes in the physico-chemical properties of erythrocyte membranes induced by nonenzymatic glycation as well as the possible prevention of their rise were studied. Using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescence anisotropy values were determined in erythrocyte membranes isolated from type 1 and type 2 diabetic patients with and without complications. The mean anisotropy values for the groups of diabetic patients were significantly higher than those for the control group (p < 0.01). This indicated pathologically decreased fluidity in cell membranes in the diabetics regardless of the type of diabetes or the presence of complications. The fluorescence anisotropy positively correlated (p < 0.01) with clinical parameters, such as glycohaemoglobin and plasma cholesterol content, which are important for the monitoring of the compensation status of the diabetic patient. Our results support the suggestion that protein crosslinking and oxidative stress induced by nonenzymatic glycation contribute to changes in the physico-chemical properties of erythrocyte membranes. In vitro testing of a new potential drug resorcylidene aminoguanidine (RAG) showed its ability to increase significantly (p < 0.001), to various extent (p < 0.01), the fluidity of both diabetic and control erythrocyte membranes. Upon the administration of RAG, reduced fluorescence anisotropy values for the groups of diabetic patients approached the normal values obtained for the controls. This may play an important role in the improvement of impaired cell functions found in diabetes that are controlled by the cell membrane.

Inhibition of nonenzymatic protein glycation and lipid peroxidation by drugs with antioxidant activity.

We studied the effects of aminoguanidine (AG), beta-resorcylidene aminoguanidine (RAG), DL-penicillamine (PNCA) and captopril on early and advanced glycation of human serum albumin (HSA). We also assessed inhibition of lipid peroxidation by AG and RAG in erythrocytes. Incubation of HSA with D-glucose (20 mM, 37 degrees C for 21 days) led to the formation of Amadori products and fluorescent advanced glycation end-products (AGE). Only PNCA markedly reduced the formation of Amadori products, while all tested compounds markedly reduced the formation of AGE. AG and RAG also inhibited malondialdehyde formation in erythrocytes incubated with hydrogen peroxide. Addition of AG at concentrations from 1 microM to 1 mM caused a 10-80% inhibition of lipid peroxidation. Thus, AG and RAG inhibit toxic oxidative processes and may have therapeutic potential in a number of human diseases.

Prevention of processes coupled with free radical formation prevents also the development of calcium-resistance in the diabetic heart.

Recently it was shown that besides their negative role in pathogenesis of diabetes, reactive oxygen species (ROS) and particularly the products of non-enzymatic glycation of proteins (NEGP) may also participate in some processes of adaptation of the myocardium to diabetes, such as in the mechanism of development of calcium resistance of the heart. Our study revealed that the hearts of rats with experimentally induced diabetes (single dose of streptozotocin, 45 mg/kg i.v., 6 U/kg insulin daily) develop considerable resistance against calcium overload (induced by means of Ca-paradox). On the day 63 after the beginning of experiment, when the diabetic cardiomyopathy became fully developed but the hearts were still not failing, their calcium resistance was increased to 83.33%. Our results provide evidence that, when applied in a special regimen, resorcylidene aminoguanidine (RAG, 4 mg/kg) prevented both, the formation of fructosamine (a source of ROS generation), and also that of the advanced Maillard products, in the heart sarcolemma of diabetic rats. The effect of RAG was accompanied by a decrease in calcium resistance in the group of rats with chronic diabetes (63 days) from 83.3 to 46.7%. It is concluded that NEGP and ROS formation are inevitably needed for development of calcium resistance in the diabetic hearts.

[The effect of streptozotocin-induced diabetes in the neonatal period on hepatic mitochondrial bioenergetics in adult rats]. / Vplyv streptozotocínového diabetu vyvolaného v neonatálnom období na bioenergetiku mitochondrií pecene dospelých potkanov.

BACKGROUND: Previous experimental studies focused on the liver mitochondrial bioenergetic changes in diabetes mellitus type I induced in adult animals. Information about the effects of persisting neonataly induced diabetes mellitus type I on the mitochondrial bioenergetics are missing. AIM: The aim of the study was to assess the degree of diabetes mellitus compensation and parameters of oxidative phosphorylation in rats aged 3 months with DM persisting from neonatal period. METHODS: DM was induced in male Wistar rats by repeated intraperitoneal administration of streptozotocine 45 mg/kg on 2nd and 9th day after birth. The concentrations of glucose, glycosylated haemoglobin, fructosamine were detected in the blood and the concentration of cholesterol and triacylglycerols in the blood and liver tissue, respectively. After mitochondria isolation from the liver we measured parameters of oxidative phosphorylation by polarography using Clark oxygen electrode. RESULTS: In the group of neonataly induced DM the concentration of glucose (23.10 +/- 1.55 vs 8.3 +/- 0.56 mmol/l), glycosylated haemoglobin (6.04 +/- 1.17 vs 3.99 +/- 0.44%) and blood cholesterol concentration (2.15 +/- 0.11 vs 1.83 +/- 0.09 mmol/l) increased significantly (p < 0.001 and p < 0.005 for cholesterol) comparing to a group of healthy rats. No statistically significant differences were found in the remaining parameters when comparing these two groups. The parameters of oxidative phosphorylation were significantly (p < 0.001) decreased in the group with DM comparing to control group of healthy animals--the index of respiratory control (4.87 +/- 0.25 vs 9.57 +/- 0.34), the rate of oxygen consumption in the stage 3 in ADP presence (88.61 +/- 4.62 vs 165.08 +/- 4.5 natO.mg/protein/min) and phosphorylation rate (203.54 +/- 7.26 vs 332.87 +/- 7.39 nmolATP.mg/protein/min) with NAD substrate glutamate. Similar trend was also seen with FAD substrate succinate. The index of oxidative phosphorylation ADP:0 was not changed in both groups. CONCLUSIONS: In 3 months old rats with neonataly induced DM the development of steatosis was not observed and uncoupling of oxidative processes from phosphorylation did not appear. Energy production was sufficient enough for normal functions of the liver and to ensure all needs of the organism. (Tab. 4, Ref. 51.)