Relevance of the nitrosative stress in medical practice © Dr. sc. med. Bodo Kuklinski Enquiries of the chronological medical history and of all organic symptoms from a pool of 1.900 patients of both sexes, when which complaints in which intensity with which accompanying symptoms in which duration and frequency took place, have shown, that
The accompanying multi organic symptoms included the following organic systems:
Independent from the fact, whether the patients visited us because of MCS, CFS, fibromyalgy, penetrating joint aches, headaches, spinal chord aches, migraine, psoriasis or neurodermatitis, syndrome of irritable intestine, colitis, Mb. Crohn, MS etc., the questioning always resulted in long-term symptoms as well as the paraplegic symptoms of several organs. The multi system illnesses were registered by the physicians involved with the therapy as comorbidities. By 65 % of the patients there were without provocation increased parameters of NO metabolism, detected in form of citrulline, citrullinated peptides or NO-concentration of the expired air. The aminoacid citrulline is formed as a by-product of the chemical reaction
The upper reference value for citrulline is given with 100 µmol/g creatinine. Healthy, efficient persons have values of < 10 µmol/g creatinine. In the expired air of healthy individuals, the level of NO is either non-existent or detectable up to approx. 10 µg. It is created by the alveolar cells and the cells of the nasal mucous membrane (14). In inflammations of the respiratory tract, bronchitis, chronic lung diseases and bronchial asthma, increased NO-concentrations are measured. Citrulline has a labile value. Measurements in our laboratories showed, that for individuals with a damaged CS, citrulline can be increased by mechanical CS-load such as car driving, motorbike-riding, vertical distress due to sprain caused by hard heels or jogging and can give rise to pathologically high levels, and these even from completely normal resting values of < 5 µmol/g creatinine. By MCS-Persons, unbearable odours also increase the synthesis of NO. The constant or intermittent high rates of NO synthesis are an indication of a chronical nitrosative stress by the above-mentioned multi system diseases. Its relevance in medical practice was until now underestimated. NO-Isomers: Nowadays there are four NO-Isomers known (Table 1). Table 1: Characteristics of the NO-Isoforms
According to the needs there are other cell types able to synthesize iNO, such as:
Strong stimulators of the iNO-synthesis by infectious processes are TNFα or IFγ. Interferon γ is increasingly released by MCS-patients intolerable to xenobiotics-expositions from Th1-Lymphocytes (26). Under the appropriate chronical duration of the nitrosative stress and specific growth-inducing conditions, cell systems can proliferate; e. g. mastocytosis, endometriosis, hypertrophal cardiomyopathia, arteriosklerosis, hypereosinophile syndrome, sklerodermitis, psoriasis, neurodermatitis etc can arise. Metabolic effects of the nitrosative stress: Function of mitochondria: NO shows a high affinity to iron and enzymes with a FeS-bond. It inhibits the FeS-bearing clusters in the mitochondrial respiratory complexes I and II, where FMN und FAD (= vitamin B2) and Coenzyme Q10 assure the electron transfer as redox substances between the respiratory complexes (13). As a physiological function, mt-NO inhibits the ATP-synthesis and the O2-consumption. Pathologically high NO concentrations, especially by a chronic Mn-deficit, inhibit initially, depending on the amounts of pO2, the mt-respiration. High oxygen particle pressure hinders the NO-coupling (with a resulting O2-deficit for the affected persons). The favourable effect of the oxygen shower by MCS-reactions should be derived thereof. In the long run however, the chronic energy deficit predominates. Especially organs with an intensive energy need such as the brain, the muscles, the retina, the mucous membranes of the intestines as well as the immune system indicate at clinical tests at first symptoms of energy deficit (neural or gastrointestinal encephalomyopathy). This chronic energy deficit forces the cell, to switch on the “emergency power supply aggregates“ in order to assure its survival, e.g. the membrane – bound NADH-Oxidoreductase or the aerobic glycolysis. With the aid of this enzyme, electrons can be transferred to oxygen molecules. The disadvantage lies in an amplified activation of proto-oncogenic substances and increased release of superoxide. By blocking the mt-electron transport, mitochondria become literally “free-radical-cannons“. They damage the mitochondrial genome, but also mitochondrial and cell-nucleus membranes, cell structural parts and the DNA of the cell nucleus. The consecutive oxidative stress oxidises polyene fatty acids of the inner mitochondrial membrane, SH-groups or enzymes, activates proteases and transcription factors. Structural damages set cardiolipids from the inner mitochondrial membrane free. As a result auto antibodies against this structure and other mitochondrial structures are formed. This chronic energy deficit sinks the activity of the energy dependent ATPases. Membranes become electrically unstable (myocardium-arrhythmia, brain-over irritability going as far as epilepsy); energy is lacking to pump out ions flowing into the cell. As a result of this effect, the glutamate receptor becomes overfed. Its Ca++-influx predominates and becomes a lethal risk. Besides, it sets increasingly superoxide (O2°−) free and activates the nNO- and eNO-syntheses. By reduced intracellular energy levels the ATP-dependent Mg++-block of the NMDA-receptor can not be maintained and thus its activation becomes neurotoxical (1). Apart from that, the NMDA-receptor activity is increased (15, 20, 29, 44, 49, 52)
In addition, reperfusion phenomena, as they appear in CS-instability cases, release high amounts of O2° . Sources of superoxides are the following:
Therefore in a nitrosative stress there is a development of an imbalance in favour of an increased O2°−-generation. NO has a three-fold higher affinity to O2°− than superoxide to superoxide dismutase. Accordingly, there is the creation of a lethal cocktail (40):
Peroxinitrite has an oxidative effect. It hinders irreversibly the mt-Mn-SOD, it oxidises vitamine C, uric acid, cholesterole, sulphuric hydryl-(SH-)-groups, polyene fatty acids of the inner mitochondrial membrane etc. According to the Petkau-effect, extremely high O2°−-concentrations are less toxic than lower concentrations. At an O2°−-concentration of 10-4 M (mol/l) the biological half-life (T½) lies at 0. 05 seconds, since there is a spontaneous dismutation of the superoxide molecules. Lower concentrations of 10-10 M on the contrary show a biological half-life of 14 hours (!). This long period of time facilitates the intrusion of the superoxide in the membranes. Through proton donation the peroxide radical HO2− is formed. The relatively inert superoxide thus forms the highly toxic HOO° radical with a redox potential of + 1.000 mV. This radical oxidises in the membranes polyene fatty acids, tocopherole, lycopine and coenzyme Q10. Citric acid metabolism: The aconitase as a FeS-bearing enzyme in the citrate metabolism is hindered by NO (7). The conversion of citric acid to isocitric acid is blocked. Thus the acetyl-CoA-expulsion through the citrate shuttle of the mitochondria and the pyruvate insertion in the citrate cycle are prevented. As a result of this, proteins, fatty acids and aminoacids can not be absorbed and used. The yield of NADH2 and FADH2 from the citrate cycle is reduced and there is a lack of them as a redox substance in the mitochondrial respiratory chain. Cholesterol metabolism: The FeS-bearing hepatic 7α-hydroxylase is blocked by NO. As a result, a cholesterol anaemia, which is resistant to diets, is developped, since the conversion of cholesterole to bile acids is no longer possible. If bile acids are lacking, then the digestion of fats and the frequency of stool release is disturbed (laxative effect of the bile acids). Patients suffering from such an illness tend to have an aversion against fatty nutrition. Since bile acids contain biliary cholesterol in a solution, during a bile acid deficit the lithogenous (stone-making) Index is increased and therefore with it the formation of gallstone. The 7α-hydroxylase together with the HMGCoA-reductase is the second limiting enzyme of the cholesterol homeostasis. The nitrosative stress induces a cholesterole anaemia. Adrenotoxine: The FeS-bearing adrenotoxine is involved, together with Cyt P450 at the hydroxylation of cholesterole in the inner mitochondrial membrane. It presents the first step for the synthesis of steroid hormones through pregnenolone. Consequently, disorders in the synthesis of steroid hormones (sexualhormones) can possibly arise by nitrosative stress. Further iron-bearing enzymes, which do not react with NO, are:
Since in the mitochondria all functions such as the cell respiration (Ox.Phos), the ATP-synthesis, the citric acid cycle, the oxidation of fatty acids, the synthesis of glutamine, partially the synthesis of steroid hormones and the beginning of the glucose-formation (gluconeogenesis) take place, massive metabolic deficits are developped, which in turn are manifested clinically in form of disorders during blood formation (e.g. porphyria), in lactose intolerances, above all however in a chronic energy deficit. The consequences are severe, since there is a rise of intolerances to fructose, glutene and lactose, of intracellular deficits in zinc-, ω3-PUFA and vitamine-B- complex and in the creation of toxic metabolites, such as homocysteine, oxy-LDL, and 4-hydroxyalkenes at increased rates of formation. Citrullination of peptides: Citrulline is stored on proteins. Thus citrullinated peptides are formed, which from the host organism are identified as foreign, i.e. as antigens and induce the formation of auto antibodies. They initiate aseptic inflammatory reactions and the CrP is increased. The citrullinated fibrinogen is detected as a first early sign in infected joints (21) by salt - effected arthralgias, palindromic rheumatism or unspecified joint effusions. Since the NO also activates the inflammation cascade through COX-enzymes, in several organs the readiness to be infected is increased altogether (43). If the corresponding chronic condition exists, an unspecified arthritis can lead to a seropositive rheumatoidal arthritis, to auto-immune responses, for persons being HLA-B27-positive it can turn to a Mb. Bechterew or a Reiter-syndrome. For every increase in CrP levels, a nitrosative stress with mitochondrial disease should be assumed. We count the unspecified pains in joints, back, the carpal tunnel syndrome, the shoulder-arm-syndrome, the Dupuytren-contractures, the arterial infections, the Heberden-, Bouchard arthroses of the fingers, the Cox arthrosis, the gonarthroses, colitis, Crohn, MS, many forms of auto-immune disease and the patellar femoral pain syndrome to the secondary diseases, because they always accompany nitrosative stress and react very well to NO-blocking therapies. Of course activated macrophages and infected cells contribute to the further formation of NO. Hypochloric acids, which are released by activated macrophages, form together with NO peroxynitrite. However, as long as the NO-synthesis and the formation of citrullinated peptides predominate against cleansing or infectious reactions, every therapy against inflammations will be a little late. On one hand it reduces the infectious activity, but on the other it does not decrease the progression of the illness, e.g. during the therapy of rheumatoidal arthritis. Nitration of aromatic aminoacids: Peroxinitrite shows a high affinity to aromatic aminoacids, which are the precursors of neurotransmitters or hormones. Nitration takes place, and as a result there is deposition of NO2. Such aminoacids are tryptophane, formed from serotonine, melatonine, NAD and NADP. The aromatic aminoacid tyrosine is a precursor for the formation of substances such as dopamine, noradrenaline, adrenaline, melanine and thyroxine. Nitrated aminoacids such as nitrotyrosine can be identified nowadays. High values of these aminoacids can be an indication towards an increased concentration of NO or citrulline and portray a stronger danger through nitrosative stress, since peroxynitrite is actively effecting the reactions. If during iodisation of tyrosine an iodine bond is blocked by NO2, no functional thyroid hormones can be formed. We notice here one of the triggers for a thyroid malfunction such as the Hashimoto-thyreoiditis. The serotonine-auto antibody formation in fibromyalgy is well known. If the nitration of tyrosine and serotonine are significant, this effect could be of importance for the rest neurotransmitters and hormones reaching up to melatonine and melanine. Melatonine, for example, is an effective NO-scavenger (collector). The β-cell of the pancreas reacts with a particular sensitivity to NO. It protects itself with the aid of melatonine and GABA-receptors from nitrosative stress. The fibromyalgy-syndrome is a typical mitochondrial disease (39) with nitrosative stress. Peroxynitrite oxidises SH-groups of cysteine, methionine, glutathion and other molecules (41). Peroxynitrite nitrates among others the nucleophile cysteine molecule in the active centre of the dimethylarginine-dimethylaminohydrolase (DDAH) as well (22). This oxidative activity is also shown among others by
With this oxidation of the DDAH molecule the asymmetric dimethylarginine (ADMA) can not be broken down. The ADMA molecule becomes an independent risk factor for cardiovascular diseases such as arteriosclerosis, hypertony, chronic kidney and myocardium insufficiency, since the endothelial synthesis of NO is inhibited and the arginine-influx in the thrombocytes is blocked, so that thrombocytes in turn form inadequate amounts of NO (5). The neuronal synthetic activity of NO is also blocked. Endothelial and neuronal NO are critical determinants in the fetal and neonatal organ maturation and growth. If they are lacking, the results can be among others symptoms of asymmetrical brain maturity and growth delay, hypertrophic pylorus stenoses, stomach overgrowth, disturbances in the maturity of the small intestine etc. The early loss of endothelial NO-synthesis during beginning arteriosclerosis is well known. Even by depressions and pre-eclampsia the eNOS is reduced and the ADMA is increased (38, 46), illnesses, which are accompanied by increased amounts of CrP. Nitrotyrosine is found to be increased in many chronic – infectious diseases, such as:
Nitrated tyrosine can at early stages be detected in the axes of neural pathways and induces there toxic neural diseases. NO has a toxic effect on the β-cells of the pankreas. Chronically relapsing stress in the sympathetic nervous system blocks its vagus nerve reactivity. By chronic deficits of zinc, magnesium and vitamine-B6, the synthesis of melatonine and γ-amino butyric acid is impaired. Melatonine and GABA-receptors of the pancreas are however important protective factors against nitrosative stress. Energy situation of the nitrosative stress: Mitochondria have an important key position in the aerobic metabolism. They are the place, in which the high energy substrates NADH2 and FADH2 from the citric acid cycle or the β-fatty acid oxidation are oxidised. Their electrons are gradually transferred to the oxygen molecule in 4 complexes. The released energy serves for the ATP- synthesis. Nitrosative stress inhibits the citrate cycle and the oxidative phosphorylation. The energy yield is adequate for basic functions. Despite that, symptoms of encephalopathy with weak brain activity, ataxias, myopathy, endocrinopathies particularly of the thyroid gland, vision disorders with retinal participation, exocrine and endocrine pancreatic functional malfunctions are developped. A tendency to diarrhoea arises at an early stage. NO-induced mitochondrial diseases include all organic systems. In the foreground appear the organs with the highest need for energy. Every additional mitochondrial metabolic acceleration by means of mental or physical workload, psychological stress, bacterial or viral infections (which increase the NO-synthesis up to 30-fold) can initiate dangerous decompensations. Sulphonamides or antibiotics can cause massive crises, since they impair either directly or indirectly the mitochondrial function. The increased energy requirement can only be covered by oxidation of fatty acids and anaerobic glycolysis, which are inhibited during nitrosative stress as well. As a final product there are lactate, ketone bodies and alanine formed. The inadequate formation of NADH2 from the citrate metabolism and fatty acid oxidation impairs the respiratory burst of macrophages and other infectious cells. Thus the way is prepared for the development of granulomatoses (sarcoidoses). By patients with such symptoms a high nitrosative stress was always detectable, which could not be eliminated by administering cortisone. The drop of activity of the infected cells must have played a role in the hyperactivity of Th1-lymphocytes (compensatory regulation). The mt-NO-concentration regulates the Ox-Phos-activity of mitochondria. In resting cells 85 % of the ATP is formed by the mitochondria and 15 % by aerobic glycolysis. During cell division the situation is reversed. The cell respiration sinks to 15 %, 85 % of the ATP is derived from anaerobic glycolysis. This low mitochondrial oxidative phosphorylation serves to protect the cell from radical strain in this particularly sensitive cell phase. The cell achieves this conversion through an increased mt-NO-concentration. In cases of chronically pathological high NO-strain the risk is very high, that cells have to remain at the glycolysis level. The “gates are opened” for the carcinogenesis (i.e. cancer formation). The chronic energy deficit during nitrosative stress is amplified by a rich intake of carbohydrates. Patients realise this effect at the extreme tiredness after rich meals. For some patients even the consumption of a single bread-roll leads to a “gorge-induced anaesthesia“ (according to a patient). Midday sleep after lunch is deep as in a state of coma, it can last only 10 minutes, but also several hours. The comatose sleep initiates a flow of saliva and at times strong snoring. People at work avoid for this reason a rich lunch. During the day most patients are chronically tired, have low energy reserves, are quickly exhausted by mental or physical workloads and need long relaxation breaks, which can last from hours to whole days. These patients show explicit muscle weakness and if they overcome their subjective estimated mental or physical workload limit most intensive muscle ache, muscle stiffing (lactacidosis) and very strong signs of exhaustion. Lactate and pyruvate measurements showed steadily a lactacidosis Type II. The lactate/pyruvate ratio was at a resting state in the area of 30 to 400: 1. The highest standard value at rest and after strain should never exceed the value 10: 1. The nutrition waiting period is shortened, i.e. in regular intervals of one to every four hours the patients must eat something, otherwise severe symptoms of mental or physical exhaustion, visual malfunctions or sympathetic nervous system reactions occur. Since in the evening no meals are taken, during the second half of the night energy deficits are developped, which either give rise to contrary sympathetic nervous system regulations such as tachycardias, sweating, phobia attacks or periods of long sleeping. In the next morning the brain, instead of having to deal with relaxation and energy storage, faces almost a hypoglycemic-schock, although there are no low concentrations of blood sugar. The run-up time is prolonged. It can range from half an hour but then reaches 4 to 6 hours. Thinking, speaking and parallel actions are almost impossible. Absurd actions are taken. Every step, every action demands slow procedures and concentration, parallel activities can not be executed. In light cases a breakfast with a sweet spreading (e.g. honey, jam) can be of help, in serious cases there is an absolute lack of appetite in the early morning hours. The chronic fatigue syndrome (CFS) is a result of the nitrosative stress. NO synthesis peaks can appear at night as well. This is typical by patients with previously damaged cervical spine. Along the trigeminus upper maxillary part the NO-synthesis is intensive, the vessels of the nasal mucous membrane are dilated, the blood circulation is increased, the nasal mucous is swollen and the nasal respiration is blocked. This situation is often also dependent on the laying position. If lying on the left side the left nasal opening is swollen, the right one is free and by lying on the right side the reverse situation occurs. In the next morning after the daily hygiene with sniffing or numerous sneezing the nose remains free during the day. The increased formation of NO in severe cases nevertheless blocks the nocturnal energy situation of the brain. Exactly as by rich consumption of carbohydrates symptoms such as snoring, apnoe and salivation followed by comatose or anaesthesia –like deep slumber accompanied by pulse irregularity occur. Own measurements of the NO-concentration among snorers with and without apnoe resulted in very high NO-concentrations in the early morning, which during the day become lower or even not detectable NO values of the expired air. The sleep apnoe syndrome is a nocturnal situation of cerebral energy deficit due to nitrosative stress. The therapy with respiratory devices does not change anything to the primary cause during apnoe. More succesful, because it acts upon the cause, is a pillow supporting the neck, before the night´s rest a strong late snack (eventually even at night by awakening) such as whole meal bread, thick layer of butter, cheese or rost meat and a NO-Blocker. Large amounts of consumed carbohydrates can often not be broken down by patients suffering from mitochondrial diseases. Carbohydrates are converted to fat and thus increase the CrP concentration. They even amplify the energy deficit. Putting on weight can therefore take place even with reduced meal intakes. Despite minimal energy intake the weight of the person increases year for year several kilogramms, especially the fat in the ventral region (spare tyre). Since cholesterole levels are increased as well, the thyroid is sensitive to NO and sympathetic nervous system attacks caused by sensitivity to stress and the presence of hypoglycemias give rise to an insuline resistance, the metabolic syndrome occurs. Initial hypotonic blood pressure cases become normotonic and later hypertonic (inhibition of the eNO by iNO). Oxidative stress blocks the esterification of cholesterole (LCAT-Blockade) (4), so that triglyceride levels can also be increased. Among normal-weight children of people with Type-II-Diabetes, with the aid of MRT of muscle tissue an increased new formation of fat due to intakes of carbohydrates could be proved. The reason is an inherited mitochondrial disease (37). The glucose absorption of the musculature was by 60 % lower and the accumulation of lipids by 80 % higher than that of healthy persons. The rate of mitochondrial ATP synthesis was by 30 % lower, the size of mitochondria was reduced by more than 50 % (9, 10, 18, 37). Lighter forms of mitochondrial diseases lead to obesity, to increases in blood lipids and blood pressure as well as to diabetes mellitus Type II. In severe cases the loss of weight is very rapid, although patients eat in intervals of one or two hours. The affected persons can emaciate to skeletons. We found severe forms of development among employees, which were exposed to nitrous gases, e.g. in the explosives industry or in branches, in which often fire of amines, nitrates and nitrites occured. Nitrous gases are insidious, since they are not irritants. The energy situation among mitochondrial diseases can be improved by taking simple measures. Chronically tired, fatigued persons however, blame their situation on the lack of physical condition. They begin to jog – which is the worst thing they could do. They thus increase the formation of NO and citrulline. This is especially recognisable among CS-affected persons. Subjectively, a feeling of well-being can be created due to the increased activity of the sympathetic nervous system and the release of endorphins, but in the long run these persons damage themselves (Diagram 1). Jogging while having nitrosative stress is like nailing on the own coffin. Diagram 1: Citrulline increase by four men after a 30-minute period of jogging
The best mitochondrial protection is the avoidance of activities with strong energy demands and a reduced intake of carbohydrates. Animal and plant fats as well as proteins can be used for the derivation of energy. In severe cases should 50 to 60 % of the total energy be consumed in form of fat (butter, cream, plant and animal fats). Carbohydrates are to be limited and fed only in small portions in a complex form. That is, whole-meal bread instead of white bread, more vegetables instead of noodles or potatoes etc. and shortly before the night´s sleep a strong snack. Sweets and sweet drinks are to be avoided. Obese persons have increased CrP values, which rise even more with a carbohydrate –rich nutrition. Dietetic restrictional nutrition is not succesful for persons with high levels of CrP. Low-fat nutrition, rich in fruit and vegetables even increases the concentration of oxy-LDL (47). Thus a weight reduction is possible only by reducing the carbohydrates and increasing the consumption of fat (36). With the support of a mitochondrial micro nutrient therapy the symptoms are improved. The metabolic syndrom and the Diabetes mellitus Type II are a form of mitochondrial disease. Mitochondrial diseasee and MCS: By all examined MCS patients an increased concentration of NO- and/or citrulline was detected. IFγ was also found in pathologically high levels in a basic state and after stimulation among MCS patients. IFγ is a strong stimulant of the enzyme iNO-synthase (43). The systematic examination of the phase -I- and -II- detoxifying enzymes gave hints towards increased risks for illness among expositions to xenobiotics (30, 48), but does not explain explicitly the formation of MCS (53). Other authors found a connection there (8, 28). Further pathogenic mechanisms are:
MCS accompanies nitrosative stress and is one of the consequences of mitochondrial disease. Mitochondrial genome damages: The circular mitochondrial genome contains 37 genes (= “47th chromosome“). The mt-genome codes 22 mt-transfer RNA, 13 structural proteins of the complexes I, III, IV and V as well as 2 ribosomal RNA. Over 300 further mt-enzymes are genetically encoded in the nucleus, in the endoplasmic reticulum of the cytosole synthetised and imported in die mitochondria. Since the mt-genome, as opposed to the nucleic DNA, contains only some histone - like proteins, it can not be repaired if damages occur. As a consequence of this fact, the mutation rate of the mt-DNA is 10- fold to 20-fold higher than that of the nucleic-DNA (17). It is not known, whether in the nucleic DNA there is a formation of NO, but it is conceivable. After all, the histone of the cell nucleus DNA is rich in arginine. Every fourth aminoacid position is occupied by basically charged arginine. In the presence of chronic nitrosative stress it is only a question of time, when irreversible mt-genome damages occur. They do not appear immediately at clinical tests, since the mt-genome portrays a special feature. As opposed to the cell nucleus -DNA every gene posseses from 2 to 15 copies. By amounts of 1.000 to 2.500 mitochondria per cell with 37 genes each, the number of copies is > 10.000. The ovocytes contain approx. 20.000 mitochondria and up to 100.000 mt-DNA-copies. During the ageing process, the number of damaged copies rises. Under conditions of nitrosative stress this process is accelerated. Mitochondrial DNA-damages include point mutations, depletions of structural proteins of the respiratory chain, transport- and assembly proteins. Nuclear DNA damages can also cause mitochondrial diseases. This refers to proteins of the pyruvate dehydrogenase-complex, enzymes of the citrate cycle, of the β-fatty acid oxidation, structural proteins of the respiratory complex, transport and assembly proteins etc. From the 13 mitochondrial coded proteins there are at least more than 100 point mutations known. The diabetes mellitus Type II can give rise to 30 diverse nuclear and mitochondrial DNA-damages. With an increasing part of the damaged copies there is a rise in heteroplasmy. If the threshold of defects is exceeded, multi organic symptoms are developped. If all DNA copies of a gene are defect, then a homeoplasmy exists. The majority of mt-genetic damages are acquired throughout life. They are inherited by the mother. In the hereditary process healthy or mutated DNA-copies can be transferred. Even in neighbouring ovocytes the degree of heteroplasmy can vary between 0 and 100 %. Consequently, mothers suffering from nitrosative stress can bear completely healthy children, but also children with homeoplastic mt-DNA-damages, e. g. with retinitis pigmentosa. This variability in the transfer of genetic copies according to the principle „Russian Roulette“explains why there is a lack in the correlation between genotype and phenotype. Different defects in the nucleic and mt-DNA can lead to similar disease schemes. In this way an encephalopathy is possible due to the disturbed β-oxidation of fatty acids, but also due to the damaged pyruvate dehydrogenase complex. On the other hand the same mt-DNA defect can lead to different disease schemesThe search for mutations in the mitochondria remains problematic, especially by cell nucleus coded DNA damages, which can also be inherited by the father. These genetic defects remain mostly undetectable; one can only notice them in a non specific reduction of the oxidative phosphorylation. In different tissues, even in neighbouring cells the degree of heteroplasmy remains significant, as e.g. in the musculature. The coupling of elektron transport and ATP-Synthesis requires intact mitochondrial membranes. If these membranes are defect, electrons can be transferred, but without the formation of ATP. In general, by multi organic or multi system illnesses a mitochondrial disease is to be assumed, especially when the brain, the musculature, the sense organs (ear, eyes) and the energy metabolism are affected. Gesicherte mitochondropathies are at present Mb. Parkinson, the ALS, migraine, Mb. Alzheimer, fibromyalgy, arthroses of the joints, the diabetes mellitus Type II and the hypertrophal cardiomyopathy. Morbus Parkinson and dilatative cardiomyopathy indicate the same mitochondrial genetic damage (33). These mitochondrial diseases explain also the what are known as Co-morbidities. A woman with migraine is suffering more often from bronchial asthma, depressions, irritated intestine syndrome, physical exhaustion, complaints in the joints. The same applies for patients suffering from FMS, MVS and CFS, since they always suffer from multi organic diseases. The door to the mitochondrial medicine has been opened a long time ago. Mothers suffering from FMS, CFS, MCS or chronically latent hypoglycemias have a higher risk to bear children with a mitochondrial damage. An indication is the threatening premature birth, which is initiated by a very high NO concentration. Nitrogen monoxide triggers the contraction of the uterine musculature and the opening of the cervix. Children born under such conditions suffer from disturbances in the brain maturity as well as from lacking dominance of the temporal lobe, inadequate interconnection of both brain hemispheres, a too small brain volume (e. g. ADHS), inadequate development of motoric ability and of the optical and acoustic centres. Their immune system is damaged and is shown in the form of allergies, neurormatitis, bronchial asthma or urticaria. The tendency to inflammations, nose polyps, nasal sinuses and inflammations of middle ear is very high. They complain of muscle weakness, light fatigue, back aches, joint aches and headaches. Not pollen, mites or the school satchet are the reason for their symptoms, but instead the nitrosative stress. The nitrosative stress can be detected already from the 1st year on the basis of pathologically high citrulline values. The uncritical use of antibiotics during childhood amplifies the mitochondrial disease. Risks of bronchial asthma and Mb.-Crohn rise. Children indicate most often disturbances in the carbohydrate metabolism. The chronically latent energy deficit forces these children to eat in short intervals. The greed for sweets is obvious. Due to the deficitary capacity of the pyruvate metabolism, fats are increasingly formed. The overweight of children and young generation, high blood lipids and the rising number of diabetes mellitus Type II has its cause in the hereditary mitochondrial disease. An increased synthesis of lipids at children of parents with diabetes mellitus Type II with normal weight has been confirmed. Modern life-style and nourishing habits uncover the mitochondrial disease of children at a very early age. High amounts of carbohydrates in the nutrition and too little physical activities of children or youths contribute to this effect. Iatrogenic mitochondrial diseases: Antibiotics and sulphonamides such as tetracycline, erythromycine, amoxicilline, trimetoprim, but also metotrexat, cyclosporine etc. are mitochondrial noxa. Their uncritical use takes place early in childhood against infections, inflammations of the middle ear, cystitis or auto- immune diseases. This trend is continued in adult life. For a period of months antibiotics are administered against unclear increases of CrP. The multi organic symptoms with chronic fatigue, unspecified neurological disturbances and joint aches tend to give an embarrassing diagnosis of borreliosis. Despite negative cerebrospinal findings, antibiotics are prescribed. The same applies for the therapy of titer reactivation as for example by chlamydia and viruses. These are typical symptoms of the nitrosative stress, although a real infection is not existent. Long lasting nitrates: NO has only a biological half-life (T½) of 1 to 5 seconds. Long lasting nitrates are converted to NO in the organism, in order to assure the vasodilation. They augment however the nitrosative stress. Statines: CSE-Inhibitors for the decrease of blood cholesteroles increase the NO synthesis. The rapidly effective clinical action for coronary heart diseases is based on this effect. We have already pointed out, that nitrosative stress leads to cholesterol increase. A further NO-burden is added to this. Clinical consequences can be among others impairment of the brain performance, polyneuropathies, increasing muscle and joint aches as well as myopathies (11, 35, 51), without a compulsory increase in enzyme levels. Since the persons affected suffer in any case from nitrosative stress with these symptoms, the danger is not recognised. For healthy persons, the treatment of the laboratory value of “cholesterol increase“over a period of years is extremely dubious. The sole treatment of the cholesterol anemia increases the lethality by senior patients (45). We could repeatedly detect increasing fatigue, muscular weakness, muscle aches and exercise –induced dyspnoe by elder patients under statine therapy. The lactate /pyruvate-quotient lay in pathologically high values, although the muscle enzymes such as CPK did not show particularly increased levels. In the Oxford Heart Protection Study, a 5-year intake of statine gave rise to an increased number of melanomas. This number on the one hand was still under the significance threshold, but in the sun-deprived England it was a serious hint. This trend indicates that the nitration of tyrosine can have an effect on the synthesis of melanine. Potency pills increase the NO-synthesis. Men perceive the side effects of these pills as headaches, heart burns and swelling of the nasal mucous membranes. Since the erectile malfunction in any case is a concomitant symptom of a multi system illness with nitrosative stress, the mitochondrial damage is amplified. Even Ginseng or arginine as potency remedies increase the NO-synthesis. The side effects are analogous. Cytostatics: After treatment of tumours with cytostatic agents, chronic syndroms of fatigue may remain, under which the affected persons suffer very heavily. Neurotoxic substances such as platinum preparations induce very rapid and at an early stage measurable S-100-increases and withthese also increased NO-formation rates. If the central brain barrier cells and peripheral barrier nerve cells are damaged, they release high amounts of NO. Tumour cells exhibit a mere mitochondrial activity of < 5 %. By metastases this is no longer detectable. The high NO-burden damages therefore only the healthy tissue in its defence, activates proto oncogenic substances and increases the endogenous formation of nitrosamine Arginine: In the therapy of chronic arterial disturbances in the blood circulation, the use of arginine in high daily dosages of > 9 g/die is considered. Cardioactive agents: Some antihypertonic agents increase the NO-synthesis and affect because of this negatively the myocardium inotropically. The ejection fraction and the blood pressure are lowered (e. g. Enalapril). Foodstuffs: Nitrogen containing fertilisers used in agriculture enrich the foodstuffs in the form of nitrate. Nitrate amounts of several hundred milligramm per 100 Gramm basic foodstuffs are not seldom. Especially leafy and root vegetables in the seasons of the year without sunlight are rich in nitrates. The threshold values in the food industry are set very high. They are directed towards the formation of Met-hemoglobin and do not take into consideration the NO-burden of the organism. The fresh crispy lettuce salad in winter may contain as much as 2.000 mg nitrate. According to a press report a piece of lamb-lettuce salad was at the top with 15 g nitrate in 100 g. As long as in agriculture what counts is the revenue per hectare (tonnage ideology), the often consumed articles of basic nutrition will still remain important sources of NO-formation in the organism. After consumption of certain drinks, ice-cream or fruits some patients react with swollen nasal sinuses and speak nasally. The reason fort his is the amplified NO-burden. Whether nitrates, other aromatic or foreign agents the initiators are,it is unclear for the time being. Patients should pay attention to their body signals and and avoid such products. As a summary we realised from the evaluation of more than 1.800 medical files, that
Therapy of the nitrosative stress and of the mitochondrial disease: Before every therapy there is always the diagnosis. Firstly, the possibility of mitochondrial disease must be seriously considered. Secondly the nitrosative stress must be measured. Here it is urgently necessary to demand the introduction to the nitrotyrosine-analytical method from laboratories. Thirdly, the fundamental reason for the increased formation of NO must be identified (whether it is hereditary, due to toxic substances or due to CS-instability). The elimination of the fundamental cause – if possible – has a priority. The Pediatric Society for Metabolism Diseases and the Deutsche Gesellschaft für Neurologie (German Society of Neurology) have worked out a consensus for the therapy of mitochondropathies. They refer to inherited mitochondropathies, but must be valid for the nitrosative stress as well. The therapy of the nitrosative stress is the domain of micronutrients. There are no standard therapies, since the forms of appearance of the disease are very heterogenous. The medical doctor must handle according to the principle „trial and error“. The diagnosis of acquired mitochondropathies is still in its infancy. The lacking standard therapy is not surprising. The importance of micronutrients and mitochondrial genome stability is still relatively unknown, as an international group of experts had to find out in 2001 (9). On the other hand, one third of gene mutations trigger enzyme disturbances with a reduced binding affinity. Of all 3.870 known enzymes, 22 % require a co-factor such as vitamines, trace elements and metabolites. By approx. 50 human genetic illnesses the symptoms can be improved by a high dosage of vitamine therapy, since these vitamines stimulate enzymatic activities (2). |
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