CH704480A2 - Canceling or reducing negative effects of oxidation of cell membranes, preferably mitochondria and other organelles of human organs comprises providing molecules of biochemical energy during or shortly after occurrence of triggering event - Google Patents

Abstract

Canceling or reducing the negative effects of oxidation of cell membranes, preferably the mitochondria and other organelles of human organs, comprises providing molecules of the biochemical energy to the affected person during or shortly after the occurrence of the triggering event. The molecules exhibit reducing- and antioxidant effect. The molecules act as methyl group supplier and are administered with complexing effect for free metal ions through respiratory air. ACTIVITY : None given. MECHANISM OF ACTION : None given.

Description

Organs with a high metabolism such as brain, heart and liver consume a lot of energy and oxygen. Although the brain accounts for only about 2% of the total body mass, it requires 20% of the converted oxygen and 60% of the glucose to meet its energy needs. The brain and the heart are dependent on the constant supply of glucose and oxygen through the blood for their energy production, since they have hardly any reserves themselves. This chemical energy is obtained in the form of ATP (Adenosine Tri Phosphate) mainly by aerobic glycolysis and subsequent oxidation to carbon dioxide and water.

The human body consists of a plurality of different cells. Each one fulfills specific tasks. The cell and also the cell organelles contained inside the cell are surrounded by a membrane. The most important cell membranes are the outer, surrounding plasma membrane, the inner and outer cell membrane as well as the membranes of the endoplasmic reticulum, the Golgi apparatus, the inner and outer membrane of the mitochondria and the membranes of lysosomes, peroxisomes and other vesicles of the cytoplasm.

Most of these membranes consist of a continuous bilayer of amphiphatic lipids (each of these molecules having a lipophilic fat-soluble portion and a hydrophilic, water-soluble portion) in which proteins and other molecules are embedded. The best known membrane lipids are the phospholipids such as lecithin (phosphatidylcholine) and cholesterol.

Brain and nerve cells contain a very high proportion of about 70% of lipids. [For more details see: Jan Koolmann, Klaus-Heinrich Röhm, Pocket Atlas of Biochemistry, Georg Thieme Verlag, Stuttgart].

The lipophilic part of the phospholipids is formed by fatty acids. These are carboxylic acids with a more or less long unbranched carbon chain. This ranges from C4 with butyric acid to C24 with nervonic acid. Some of these fatty acids have a saturated hydrocarbon chain (e.g., palmitic and stearic acids), some are unsaturated and have 1-2-3-4 and even 6 double bonds. These are: oil - linoleolols - arachidone and docosahexaenoic acid (DHA). Especially brain and nerve cells are rich in DHA. The unsaturated fatty acids are fluid at body temperature and important for maintaining the fluidity of the cell membrane. Their double bonds are particularly susceptible to oxidation and therefore constitute a sensitive part of the cell membrane which is the main subject of the present patent application.

Functions of cell membranes

Demarcation and isolation of cells and organelles Control of mass transport Structure of proton gradients and energy conservation in it Reception of extracellular signals Enzymatic catalysis of biochemical reactions Interaction with other cells Anchoring of the cytoskeleton

This very sketchy list makes it clear that the cell membrane is the place for numerous biochemical reactions with intensive energy exchange.

Oxygen and energy production of the cell

The fire in the fireplace that gives us warmth and the energy in our cells are based on the same chemical reaction - the oxidation. This is due to the transfer of electrons from the electron donor (which is therefore itself oxidized) to the electron acceptor (which therefore is itself reduced). These two substances form a redox system. In biological redox systems for energy production, oxygen is the most important electron acceptor (= oxidizing agent) and is itself reduced to water and CO2. The energy gained is stored in the form of ATP (adenosine triphosphate). Cleavage of a phosphate group from ATP releases energy which is used by energetic coupling for movement, biosynthesis and transport processes in the so-called ion pumps of the membranes.

Important ways of energy (= ATP) generation

1st citrate - cycle

The citrate cycle takes place in the mitochondria (= power plant of the cell). It receives the acetyl residues (CH3-CO ») from the fatty acid oxidation. In this reaction sequence, one molecule of GTP (guanosine triphosphate), three molecules of NADH (reduced nicotinamide adenine dinucleotide) and one QH2 (reduced ubiquinone) are formed from one acetyl residue. Subsequent oxidative phosphorylation gives the cell a total of 10 ATP per acetyl group.

2. Oxidative phosphorylation

It forms most of the cellular ATP and also takes place in the mitochondria. As the name implies, it can only occur in the presence of oxygen. A subprocess of oxidative phosphorylation is the respiratory chain. It uses the enzyme cytochrome c oxidase to catalyze the stepwise transport of electrons from the reduced molecules NADFI + and QH2 (coming from the citrate cycle) to molecular oxygen. Since all these reactions take place on the membranes of the mitochondria, one can imagine that an intact membrane structure is of great importance for the conservation of energy.

3. Aerobic glycolysis

This takes place in the cytoplasm of the cell. It provides two molecules of pyruvate per molecule of glucose (CH3-CO-COO <->), ATP and NADH. Pyruvate and NADH enter the mitochondria where they are metabolized with oxygen for further ATP production.

4. Anaerobic glycolysis

For animal cells, it is the only way to gain ATP in an oxygen deficiency and represents an important possibility of survival in oxygen deficiency. Anaerobic glycolysis takes place via pyruvate for ATP production and is energetically far less favorable because of a molecule of glucose only 2 ATP arise. This forms lactate which is released into the blood. An accumulation of lactate can be considered as a biomarker of oxygen deficiency.

5. Hexose monophosphate way

This oxidative mechanism, like glycolysis, takes place in the cytoplasm of the cell and provides important intermediates from glucose. The cell may utilize a portion of it via the glycolysis-citrate cycle and respiratory chain for ATP recovery.

Oxygen deficiency (hypoxia, ischemia

Hypoxia is a lack of oxygen at the cellular level. There are 4 types: <tb> 1. <sep> Ischemic or stagnant hypoxia - the blood flow is interrupted or too weak to ensure adequate oxygenation. <tb> 2. <sep> Histotoxic hypoxia - a toxin prevents cells from using the oxygen they already have. <tb> <sep> Anemic hypoxia - the hemoglobin content of erythrocytes is too low to provide enough oxygen. <tb> 4. <sep> Hypoxic hypoxia - the oxygen content in the air is too low. Therefore, its content in the arterial blood is too low.

When the oxygen supply to the cell is interrupted, O2 lacks an electron acceptor for the respiratory chain. As a result, ATP synthesis and the entire metabolism of the mitochondria come to a standstill. NADH + H + and QH2 can no longer be oxidized to NAD <+> <>. This prevents the citrate cycle and other important reactions (pyruvate dehydrogenase, beta oxidation, malate shuttle, degradation of amino acids). The only way to ATP recovery remains anaerobic glycolysis. This ends with the consumption of the stock of glucose. ATP also provides the energy for the function of ion pumps in the cell membrane which maintain membrane potential and proton gradient. Lack of oxygen also brings the ion pumps to a halt. In ischemia, an accumulation of the excitatory amino acid glutamate is observed. This also indicates a disruption of the membrane in the mitochondria. The enzyme glutamate dehydrogenase converts glutamate into ketoglutarate and ultimately into urea by NAD + or NADP +. Like other urea-producing enzymes, it is localized in the mitochondria. This compartmentalization in the mitochondria also separates the free, toxic NH3.

Formation and reaction of free radicals

Free radicals (R °) are very reactive molecules with one or more unpaired electrons. O2 itself has 2 unpaired electrons, so it is a diradical. By the stepwise absorption of the oxygen molecule of one electron each, the superoxide radical (° 02 ~), the peroxide anion (O2 »), and by protonation hydrogen peroxide (H2O2) and the hydroxyl radical (° OH) are formed. Generally these molecules are called ROS (Reactive Oxygen Species). Many biochemical reactions proceed via free radicals as short-lived intermediates. Free radicals and peroxides (ROS) are caused by the following factors: Ionizing radiation (for example in sunburn) Biochemical reactions, especially in the production of energy in the mitochondria, heat (burns), cold (frostbite), action of leukocytes (inflammation), catalytic action of free metal ions, especially iron and copper (free hemoglobin in case of injury).

The healthy cell has sufficient protection mechanisms to protect against the destructive effect of the ROS. These are: Enzymes: catalase (decomposes hydrogen peroxide), superoxide dismutase (against superoxide anion) and glutathione peroxidase (detoxifies peroxides).

Antioxidants: also called "free radical scavengers". They delocalise unpaired electrons so that they can no longer react with other molecules. The most important are: tocopherols, carotenes, coenzyme Q (ubiquinone), bioflavonoids and others, mainly phenolic compounds (polyphenols).

Reducing agents: vitamin C, vitamins of the B group, glutathione and others. However, if the cell is weakened or if the action of the ROS exceeds these protective mechanisms, then an oxidative damage of the cell membrane takes place, which can lead to inflammation through to cell death. The most sensitive molecules against oxidation by ROS are the unsaturated fatty acids in the membranes of cell and cell organelles, especially the mitochondria.

Practical occurrence of oxidation of cell membranes by uncontrolled ROS

Many acute and chronic diseases are due to the action of ROS. Of particular importance are: Release of iron and copper ions through injury or uncontrolled decomposition of the erythrocytes. The natural content of free iron ions in the cytoplasm, blood and extracellular fluid is extremely low (<10 <-> <10> <tb> 1st <sep> mol per liter). The iron is firmly bound in hemoglobin, myoglobin and other molecules. This blocks its strong, catalytic effect on the oxidation of cell membranes. <tb> 2. <sep> Oxygen deficiency (hypoxia, ischemia) and reoxygenation of the cells This occurs during stroke and heart attack by clogging or bursting of arteries, as well as drowning or suffocation. A particularly important event in a person's life is the birth. The bloodstream of the baby has to decouple within a short time from the blood of the mother and switch to its own pulmonary respiration. This can occur due to unfavorable circumstances, a lack of oxygen. Prolonged oxygen deficiency with subsequent reoxygenation has fatal consequences, first at the cellular level, a little later at the whole organism. The brain, an organ which consumes the most oxygen, is particularly sensitive to hypoxia and is thus more or less irreversibly damaged, depending on its duration.

Hypoxia and reoxygenation

The occlusion of the blood flow (often called ischemia) to the brain after the short time of about 10 seconds unconsciousness result. The vegetative structures in the brain stem are more resistant than the cells of the cerebrum. Patients who survive a more or less long period of ischemia, such as a heart attack or stroke, retain their autonomic functions but often suffer intellectual damage. Hypoxia and subsequent reoxygenation can cause severe brain damage [Lipton, P., Ischemic Cell Death in Brain Neurons, Physiological Reviews 79 (4): 1431-1568 (1999)].

If the affected person survives the lack of oxygen, the breathing air via the blood again leads to fresh oxygen (reoxygenation). In many critical clinical situations, concentrated oxygen is given as breathing air. This facilitates the resumption of natural cell metabolism but significantly increases the possibility of uncontrolled oxidation of cell membranes. The membranes of brain cells and mitochondria contain a particularly high proportion of polyunsaturated fatty acids and are therefore sensitive to oxidation.

It is known that most lasting damage does not arise in the phase of hypoxia but in the subsequent reoxygenation [A. Watsuki et al., Effect of ischemia - reperfusion on xanthine oxidase activity in fetal rat brain capillaries, Am. J. Obstet. Gynecol. 1999 Sep 181 (3): 731-5]. In babies who have had hypoxia during birth, partial success has been used to cool the baby's head with ice water to alleviate the effects of reoxygenation [N. Jayne Robertson, Autour de l'enfant à naitre, presentation by CHUV Lausanne, June 2006], [T. Horiguchi, Postischemic hypothermia inhibits the generation of hydroxyl radical following transient forebrain ischemia in rats, Neural Star Cells Compendium, May 2003, Vol. 20, No 5: 511-520]. Cooling slows down the biochemical reactions - including the formation of harmful ROS caused by reoxygenation.

The administration of allopurinol also showed similar positive results as it inhibits the enzyme xanthine oxidase. During the initial hypoxia, some ATP is decomposed to xanthine. In the subsequent reoxygenation, the endothelial enzyme xanthine dehydrogenase is converted into xanthine oxidase. This xanthine oxidase catalyzes the formation of superoxide (= ROS) and uric acid from xanthine and oxygen. The byproducts of this reaction include hydrogen peroxide and ammonia, both of which have a toxic effect in the brain. Allopurinol inhibits the enzyme xanthine oxidase and thus the formation of ROS. The ratio of uric acid to creatine in neonatal urine can be used as a criterion for a possible lack of oxygen at birth [G. Ciler Erdag, A. Vitrinel, CanUric Acid / Creatine Ratio be Used as an Additional Marker for Neonatal Asphyxial. Int. Pediatr. 2004, 19 (4): 2.17 - 219]. Both treatments are only effective if they are performed immediately after hypoxia, ie before or at the beginning of reoxygenation. Practically, a window of 1-3 hours was found after a treatment is unsuccessful because the chemical processes of cell damage are irreversible. A large part of the energy of the neurons stored in the form of ATP is used to operate the Na - K pump. This maintains the concentration of extracellular K low and extracellular Na high. Another ATP driven ion pump keeps the concentration of extracellular calcium 10 000 times higher than in the cytoplasm of the cell. After only a few minutes without blood supply, the neurons lack the energy (ATP) to operate these ion pumps. Potassium leaves the cells and sodium and chloride ions flow into it as the ion gradient collapses at the cell membrane (depolarization). The neurons try to gain some ATP by anaerobic glycolysis, resulting in the formation of lactic acid. Therefore, within a few minutes hypoxia, the extracellular pH may drop from 7.3 to 6.7 [Best, B., Ischemia and Reperfusion Injury in Cryonics, HYPERLINK "http://www.benbest.com/cryonics/ischemia.html» http : //wvvw.benbest.com/cryonics/ischemia.html].

The accumulation of glutamate in ischemia indicates a glial cell disorder which normally converts glutamate to glutamine with the aid of ATP and ammonium ions. Glutamate is an excitatory neurotransmitter and opens the NMD receptor (N-methyl-D-aspartate), an ion channel that is particularly common in neurons. At normal membrane potential this channel is blocked by magnesium ions. Because of the partial or total depolarization of the membrane, the blockade is abolished and calcium ions flow into the cell [William F. Ganong; Review of Medical Physiology, 22nd ed., 2005, Mc Graw-Hill Comp., ISBN 0-07-144040-2].

A high concentration of calcium in the cytoplasm also indicates damage to the membrane of the mitochondria, which in addition to the generation of energy also serve as an intracellular calcium reservoir. Calcium excess in the cell's interior activates the enzymes phospholipase and lipoxigenase which cause further damage and swelling of the cells and their death. See Best B., cited above and [Kriegelstein, J. et al., The Neuroprotective Effect of Dihydrolipoic Acid, Thioctic Acid, 2nd Int. Thioctic Acid Workshop 1991, ISBN 3-89143-018-3].

Summary ischemia

Hypoxia brings the metabolism of the cell largely to a standstill. As a result, neither energy nor important molecules (antioxidants) are produced for cell protection against oxidation. Brain and heart have hardly any reserves. In reoxygenation, the unsaturated fatty acids of the defenseless cells are oxidized. Depending on the duration of hypoxia, this can result in lasting damage to the affected organs, especially the brain.

Purpose of the present invention is to bring the natural mechanism of energy production (ATP generation) as quickly as possible to get started again while avoiding or preventing the adverse consequences of Reoxigenation that is the oxidation of lipids in the cell membranes by ROS.

Description of the invention

The fastest way to bring a substance into the body is by inhalation through the lungs if it is distributed in gaseous form or as a fine aerosol in the breathing air. After an ischemic period, the first attack of oxygen on the membranes of the pulmonary alveoli takes place during reoxygenation. For the purposes of the present invention, the vapors of antioxidants and reducing agents are added directly to the respiratory air. Therefore, the oxidation-sensitive fatty acids and thiols of cell membranes are protected against oxidation. By suitable selection and combination of radical scavengers, peroxide inactivators and reducing agents even already oxidized biomolecules, especially -SH groups of proteins can be reactivated.

Method and device for admixing and testing the antioxidant protective effect of substances in the respiratory air

As experimental animals for this test, preferably young rats or rat embryos are used. One group of experimental animals is exposed to an ischemia period of 10-30 minutes. This is done either in a closed chamber by gassing with pure nitrogen [Mach, M. et al., Experimental modeling of hypoxia in pregnancy and early postnatal life, Interdisc. Toxicol. 2009, Vol.2 (1): 28-32] or by immersion in a tempered water bath [Hoeger, H. et al., Long term effect of moderate and profound hypothermia on morphology, neurological, cognitive and behavioral function in a rat model of perinatal asphyxia, Amino Acids, 2006, Nov., 31 (4): 385-96. Epub 2006 Aug 31].

Immediately after this ischemic period, the experimental animals are placed in a special ventilation chamber and charged with the respiratory gas. The judgment criterion is the number of experimental animals that survive this treatment unscathed. The test with pure air is used as reference value. The breathing gas, usually normal room air or a mixture of 65% nitrogen and 35% oxygen comes from a pressure bottle or an air pump. It passes a flow meter with control and then passes into a gas washing bottle. The wash solution contains one or more antioxidants dissolved in water or other suitable solvent. If the antioxidant is not soluble in water, it is, as such or in a non-polar solvent, dissolved over the water. The breathing gas enters the bottom of the washing bottle and bubbles up to the surface of the washing liquid. It absorbs the vapors of dissolved or over-coated antioxidants in the water and forwards them to the aeration chamber and to the experimental animals. The breathing gas used is usually room air or an oxygen-nitrogen mixture which has a higher oxygen content. In some cases, 1 - 4% of hydrogen is also known as the ° OH radical scavenger is known. In this test arrangement, a gas flow of 500 liters per hour has been proven. After aeration time of 10 -60 minutes, the test animals are removed from the aeration chamber and after counting the surviving animals are left to their normal living conditions and later subjected to more detailed examinations.

As described in the introduction arise in Reoxigenation various ROS. Of particular importance are ° OH and H2O2. An ideal shielding gas and the entire treatment should be effective against both ROS and also inactivate already formed peroxides in the cell membranes and re-activate oxidized -SH groups (-S-S-) of proteins and other biomolecules. The enzymes glucose oxidase, lipoxygenase, cyclooxygenase, myeloperoxidase and xanthine oxygenase, which play a role in inflammation, should also be adjusted for their optimal biological concentration and activity.

This diverse action can be achieved by a mixture of the various active substances. As described below, it is convenient to combine the different dosage forms - via the breath for volatile drugs and by intravenous or intra-arterial injection for non-volatile drugs.

The active substances

1. dimethyl sulfide (DMS)

CH3-S-CH3 A colorless, strong-smelling liquid, bp. 37 ° C, insoluble in water. It is produced by the bioplankton of the oceans and reaches the earth's atmosphere in relatively large quantities. It is also a natural component of coffee, cabbage, radish and other flavors. Use in a gas washing bottle as a 1% solution in benzyl benzoate or DMSO.

2. dimethyl sulfoxide (DMSO)

CH3-SO-CH3 A colorless and odorless liquid, bp. 189 ° C, soluble in water and organic solvents except paraffins. For details see: [Stanley W. Jacob, Don C. Wood, Dimethylsulfoxide, Full. Basic Concepts of DMSO, Marcel Dekker, Inc., New York 1971]. Use in the wash bottle as a 1 - 10% aqueous solution.

3. Methyl mercaptan (methanethiol)

CH3-SH A strong-smelling Ga, bp. 6 ° C, part of the smell of cabbage, cheese, milk, coffee, oysters, onion and others. It was also detected in the breath. It is G.R.A.S. and has the FEMA No 2716. Its odor threshold is at a concentration of 0.02 ppb / 1 application as a 1% solution in benzyl benzoate or DMSO.

4. Ethyl mercaptan (ethanethiol)

CH3-CH2-SH Highly volatile liquid, bp. 37 ° C, properties and application similar to methylmercaptan.

5. Beta-mercaptoethanol (2-thioethanol)

SH-CH 2 -CH 2 OH A liquid with bp. 157 ° C, soluble in water and many organic solvents. It can reduce the -S-S bridges from oxidized proteins to -SH. In immunology, it is used to block antigen - antibody reactions.

6. 2, 3-dimercapto-1-propanol (dimercaprol)

HS-CH 2 -CHSH-CH 2 OH A colorless, oily liquid, decomposed with water. It acts as a thiol group supplier and has a complexing effect on metals. Its use in the gas washing bottle is carried out as a 1 - 2% solution in benzyl benzoate. 1,4-dimercapto-2,3-butanediol (dithiothreitol, Clelands reagent) works similarly. It reduces disulfide compounds to thiols and is therefore used for mucolysis.

7. Other sulfur-containing molecules which theoretically have a protective action are: natural extracts of lily plants such as the onion and garlic and some of the isolated molecules (cepaenes, thiosulfinic esters, allyl isothiocyanate, diallylsulfide, allicin, dimethyl disulfide, propan-gallial S-oxide and others) have an inhibitory effect on lipoxygenase and cyclooxygenase. Thiophene, 2-mercaptothiophene and other ingredients of coffee and cocoa, natural or synthetic coffee oil, garlic oil, onion oil and radish extract may also give good results in the described respiration test.

8. Natural or synthetic essential oils and flavors with antioxidant activity or inactivation of oxidative enzymes are: Bergamot, Eucalyptus, Rosemary, Vetyver, Clove, Thyme, Grapefruit, Wintergreen and their individual ingredients, as well as Methyl Salicylate, Ethyl Salicylate, Amyl Salicylate, Vanillin, Ethyl Vanillin. They are dissolved in benzyl benzoate and float in the wash bottle as a topcoat on the wash water.

9. Biogenic polyamines

Spermine and other polyamines are important biological antioxidants. Spermine stabilizes the DNA in the nucleus. Since they are water-soluble, they are dissolved directly 1 -5% in the washing water.

Administration by aerosol mist or intravenous or intraarterial injection

Many biological antioxidants are non-volatile and therefore can not be solved via the wash bottle in the air. The same applies to many reducing agents and ATP. With many of these active ingredients it is possible to spray their aqueous solutions with ultrasound so finely that the particles reach the lungs as an aerosol and are absorbed there. For molecules that are not absorbed via the lungs, administration is by parenteral injection into veins or arteries. The amount of absorbed antioxidants and biomolecules should be such that their concentration in the body of the patient or the experimental animal reaches the simple to double concentration of a healthy body.

10. nicotinamide (niacinamide)

The water-soluble amide of nicotinic acid belongs to the group of B vitamins. The vitamin effects of nicotinamide and nicotinic acid are identical, but they have different pharmacological properties. Nicotinamide, in contrast to nicotinic acid, has no influence on blood pressure, heart rate or body temperature. Nicotinamide has the following properties: Antioxidant, immunomodulator, inhibits phosphodiesterases and therefore has anti-inflammatory, protection against necrosis. Its cytostatic effect in vitro is greatly increased by vitamin C, vitamin E and carotenes. [N. Otte, C. Borelli, FI.C. Körting, Nicotinamide - biologic actions of an emerging cosmetic ingredient, Int. Journ. of Cosmetic Science, 2005, 27, 255-261]. Nicotinamide is the hydrogen acceptor part of NAD and NADP.

11. NAD (nicotinamide adenine dinucleotide) and NADP (~ phosphate) They act as hydride ion carriers and are the major coenzymes for hydrogen transfer reactions. The redox system consists of the oxidized form NAD (P) +, which is converted into the reduced form NAD (P) + H + by absorption of hydride ions. NAD + FT »transfers reduction equivalents (1e <-> / H <+>) to the respiratory chain and thus serves to generate ATP. It acts to reduce more than ascorbate and tocopherol and can protect cell membranes from ROS. NADPH + H + is the most important reducing agent in biosynthesis.

12. ATP (adenosine triphosphate)

ATP is known as the most important source of energy in the organism. It is also vasodilating and reduces the formation of peroxides in the laboratory [Mocan H. Saruhan et al., The effect of ATP-MgCl 2 on lipid peroxidation in ischemic and reperfused rabbit kidney, European Journal of Pediatric Surgery, 9 (1): 42. 6, 1999].

13. Folic acid

A vitamin from the water-soluble group of B vitamins. Folic acid in its active form as tetrahydrofolate (THF) is a methyl group donor and inhibits xanthine oxygenase. It is stable in neutral solution. In addition to inactivating free radicals, THF can repair already oxidized thiol groups and despite its water solubility inhibit the oxidation of cell membrane lipids. THF is formed from folic acid by the enzyme folate reductase. Therefore, the biological functions of folic acid depend on a sufficient presence of reducing equivalents provided by NADH + H +.

14. Taurine

Taurine (aminoethanesulfonic acid, H2N-CH2-CH2-SO3H) is synthesized in the liver and brain from methionine via cysteine. In the young, growing brain his concentration is particularly high. It is enriched in millimolar concentration in tissues with high membrane content and high activity such as nerves, retina and heart. In the brain it acts as an antagonist against excitatory stimuli of the glutamate NMDA receptor. Taurine lowers the intracellular calcium level and thus the formation of NO (nitric oxide). NO is oxidized by superoxide to ONOO- (peroxynitrite). This nitrosated aromatic amino acid, oxidizes -SH groups and inactivates SOD (Mn) [Wolfram Kersten; Paradigm shift in the understanding of chronic civilization diseases, complement. Integr. Med.-04/2009]. Taurine therefore protects the cell membrane against peroxidation, calcium overload, glutamate overactivity and against hypochlorite (OCF) which is formed by the myeloperoxidase from hydrogen peroxide and chloride ions. Taurine is present in breast milk, but is missing in milk powder from cow's milk. NO itself is a radical and can react with other radicals. Therefore, it is a radical scavenger or antioxidant especially against hydroxyl radicals. Its reactivity - toxic or protective - depends on its concentration. In relatively high concentrations, it reacts with oxygen or superoxide to form peroxynitrite and other cell-damaging molecules. However, if NO is produced continuously in small amounts, it has cell-protecting properties [Manfred K. Eberhardt; Reactive Oxygen Metabolites, Chemistry and Medical Consequences, CRC Press, Boca Raton, 2001].

15. Ghitathion

Glutathione, a tripeptide (gamma-glutamylcysteine glycine) is one of the most important biological reducing agents. Thanks to its thiol group, reduced glutathione (GSH) can transfer electrons to oxygen radicals and render them harmless. Two GSHs combine to form glutathione disulfide (G-S-S-G). Thanks to a bidirectional transport mechanism, it is absorbed into the cell. Its concentration in human tissue is between 0.1 - 10 m mol [Ingrid Arnold, Reinhard E. Geiger, Lorenz Gesswein; Orthomolecular Therapy, Part 2: Glutathione against Cancer, space & time 167/2010, 36-41]. S-acetylglutathione is used as an active pharmaceutical ingredient against a number of diseases. N-acetyl-cysteine (NAC), a precursor of GSH, has an expectorant and anti-inflammatory effect.

16. S-adenosyl-L-methionine (SAMe)

SAMe is omnipresent in all cells and plays a central role in many biological reactions as one of the best methyl group donors. It is formed from L-methionine and ATP with the help of the enzyme SAMesynthetase. Cofactors for this reaction are vitamin B] 2 and folic acid. A concentration in the cytoplasm of 20-50 micrograms / ml is indicated as favorable [Maria Elena Carabetta; S-adenosyl-L-methionine: Nuova applicazione nutrazeutica nelle sindromi depressivi, L'Integratore Nutrizionale 12 (4) 2009, 33-39].

17. Choline

Choline, an amino alcohol and a constituent of lecithin, is an omnipresent methyl group donor in the body. It is particularly enriched in the brain, in the blood of newborns and in breast milk. Its aqueous solution reacts strongly basic. It acts vasodilator, reduces the accumulation of body fat, causes uterine contraction and regulates the intestinal movement. In the form of its chloride, it is used as a salt substitute. Biochemical functions: lecithin and sphingomyelin formation, acetylcholine formation methyl group donor, it is necessary for the synthesis of methionine from homocysteine and is oxidized to betaine.

18. Betaine

Betaines are intramolecular salts of quaternary onium bases, e.g. Ammonium belonging to the group of biogenic amines. The two best known are glycine betaine (N-trimethylglycine, (CH3) 3N <+> - CH2-COO <->), commonly referred to as betaine, and carnitine (amino-beta-hydroxybutyrobetaine). Betaine occurs in the amniotic fluid. It can protect proteins and membranes against osmotic shock at high salt concentration (Osmoprotectant). Carnitine is widely used in all cells, especially in the heart, liver and brain. The heart muscle derives its energy mainly from the beta-oxidation of the fatty acids in the mitochondria. Carnitine acts in the form of its fatty acid ester as a carrier for these fatty acids from the cytosol into the membrane of the mitochondria where release occurs by hydrolysis. It is therefore indispensable for fat burning. Betaine and carnitine as well as S-adenosylmethionine and phosphatidylcholine act as suppliers of electrophilic methyl groups for transmethylation. They have been proposed to balance redox status in oxidative stress [Ghyczy M., Boros M .; Electrophilic methyl groups present in the diet ameliorate pathological states induced by reductive and oxidative stress: ahypothesis; Br. Journ. Nutr. 2001; 85: 409-14].

19. uric acid

Uric acid is the most important reducing agent in human plasma. Its concentration of 160 - 450 micro mole is significantly higher than that of ascorbate. Blood contains 5 mg / 100 ml of uric acid. It is formed by the xanthine oxidase from xanthine. It mainly inactivates hydroxyl and peroxyl radicals, but not the superoxide anion radical, which also occurs in the xanthine oxidase reaction. Uric acid is oxidized to allantoin, which has anti-inflammatory properties. It is noteworthy that uric acid does not react with NO but with ONOO <-> the peroxynitrite. So it does not interfere with the biological functions of NO but protects the cells from the harmful ONOO <->. Uric acid complexes free metal ions. As a result, it protects many other biomolecules from metal-catalyzed oxidation.

20. Vitamin C (ascorbic acid)

Ascorbic acid is one of the most important water-soluble reducing agents and has two slightly ionizing H atoms. In aqueous solution it is present as ascorbate anion (AH2 = AH <-> + H +). Ascorbate is oxidized to dehydroascorbate, losing its antioxidant action. It is a cofactor of hydroxylase enzymes that allow the synthesis of collagen. The concentration of ascorbate in human plasma is between 27-51 microMol. In brain, heart, liver, kidney, pancreas and spleen, its concentration is much higher and goes up to 0.8 milliMol. Ascorbate in plasma and leukocytes from premature births and the elderly is considerably reduced. Also in cancer, arthritis, tuberculosis, after operations with subsequent inflammation, stroke, trauma and smokers [Hans-Anton Lehr, Rainer K. Saetzler, Karl E. Afors; Effect of Vitamin C on Leukocyte Function and Adhesion to Endothelial Cells, in Vitamin C in Health and Disease, ed. By Lester Packer, Jürgen Fuchs; Marcel Decker Inc. New York 1997].

In the presence of free metal ions, ascorbate can form ROS and act as a pro-oxidant. It should therefore be used in combination with complexing agents such as EDTA. At concentrations up to 5 milliMol, no pro-oxidizing effect was observed [Manfred K. Eberhardt; Reactive Oxygen Metabolites, Chemistry and Medical Consequences, CRC Press, Boca Raton, 2001]. Ascorbate helps to regenerate the consumed vitamin E (tocopherols) and indirectly stabilizes the cell membranes. In aqueous solution, ascorbic acid is rapidly oxidized. Far more stable are sodium ascorbate, sodium ascorbate phosphate and magnesium ascorbate phosphate. The lipophilic ascorbyl palmitate is recommended as being particularly cell-compatible [M. Pokorski et al., Ascorbyl Palmitate as a Carrier of Ascorbate into Neural Tissues, J. Biomed. Sei., 2003; 10: 193-198].

21. Vitamin E (tocopherols)

Natural vitamin E is usually a mixture of 4 tocopherol isomers and 4 tocotriene isomers. The latter having unsaturated side chain are far more effective as biological antioxidants than the most commonly used alpha tocopherol. Tocopherols are oil-soluble but have a hydrophilic OH group on the chroman ring. This amphiphilic molecular structure allows for good confinement in the cell membrane where it affects its fluidity. The antioxidant effect of tocopherols takes place mainly in depot fat. Most antioxidant experiments with tocopherols were performed in vitro. Ascorbate or ubiquinol (coenzymeQ) regenerate the tocopherol radical, which is formed during oxidation, back to tocopherol. In the rat experiment, tocopherol acts neuroprotectively after ischemia / reoxygenation [Mitsuo Yamamoto et al .; A Possible Role of Lipid Peroxidation in Cellular Damages Caused by Cerebral Ischemia and the Protective Effect of Alpha-tocopherol Administration, Stroke, vol 14, No6, nov./dec. 1983, p. 977-982].

22. Melatonin

Melatonin (N-acetyl-5-methoxytryptamine) is a hormone that is mainly produced in the pineal gland. The influence of light via the optic nerve reduces production, so its concentration in the tissue is highest at night. Melatonin synthesis is reduced in old age. Melatonin is lipophilic and well soluble in the cell membranes, especially of the brain. This allows its penetration into the cell and cell organelles. Melatonin inactivates hydroxyl radicals, singlet oxygen and hypochlorite. This preserves the enzyme catalase from inactivation by hypochlorite. In vitro, melatonin inhibited the autoxidation of catecholamines (neurotransmitters) and the intracellular influence of calcium ions.

23. Mannitol

Mannitol is a mannose-derived sugar alcohol (hexitol) and is widely used in plants, fungi and algae. In the literature, mannitol is indicated as a scavenger for the hydroxyl radical. Due to its pronounced water solubility, it theoretically acts only in an aqueous environment and not in cell membranes.

24. Carnosine

Carnosine is a dipeptide (beta-alanine-L-histidine). It is enriched in relatively high concentrations in skeletal and cardiac muscle and in the brain. Human cerebrospinal fluid contains up to 50 micromoles of homocarnosine, brain substance contains 0.3 - 0.5 mM carnosine, homocarnosine and anserine. The imidazole ring of histidine requires its reactivity with the plydroxyl radical and singlet oxygen. It also acts as a complexing agent for copper ions and inactivates the aldehydes (malondialdehyde) formed by oxidation of the membrane fatty acids. In addition to its antioxidant effect carnosine is also important as a pH buffer with increased acid production.

25. Ubichinone (Coenzyme Q)

Ubiquinones (2,3-dimethoxybenzoquinone) are widely used agents in animals, plants and microorganisms. They are particularly found in the lipoprotein fraction of the mitochondria. The reduced form (ubiquinol or ubihydroquinone) is active outside the cell membranes as a biological reducing agent. It reduces peroxy radicals and helps to regenerate oxidized tocopherol. As a lipophilic side chain tocopherols contain an isoprene radical of 6 to 10 dehydroisoprene units which accomplishes the anchoring in the lipid membrane. Because of their involvement as coenzymes in the respiratory chain for ATP synthesis, acting as carriers of reduction equivalents, they are also called Coenzyme Q. The most common form in mammals contains 10 isoprene units in its lipophilic side chain. Hence the name Coenzyme Q10. This hydrophobic quinone diffuses rapidly within the inner mitochondrial membrane. Ubiquinone can regenerate the alpha-tocopherol radical in lipoproteins and membranes to tocopherol [Barry Halliwell, John M.C. Gutteridge, Free Radicals in Biology and Medicine, 4.ed. 2007, Oxford Univ. Press, ISBN 978-0-19-856868-1, standard work in this field].

26. Lipoic acid

Lipoic acid (1,2-dithiolene-3-pentenoic acid, thioctic acid) is a coenzyme in the synthesis of acetyl coenzyme A from pyruvate. This reaction establishes the link between glycolysis and the citrate cycle. The pyruvate formed in glycolysis is converted into acetyl-coenzyme A the fuel of the citrate cycle. The redox couple is formed from lipoic acid (disulfide) / dihydrolipoic acid (dithiol). Both forms show antioxidant action. The thiol form reacts with all known biological ROS and also binds harmful heavy metal ions. Dihydrolipoic acid, although present at a very low concentration, is a physiological antioxidant which maintains the biological redox potential of the cell and the membrane potential of the mitochondria. In experiments on the isolated rat heart and rat bone and mitochondria in suspension dihydrolipoate was able to partially compensate for the negative consequences of ischemia and subsequent reoxygenation [G. Zimmer et al .; Ischemia and Reperfusion: Effect of Antioxidants on the Function of Working Rat Hearts, Council Extremities, Mitochondria and ATP Synthesis, 2nd Int. Thioctic Acid-Workshop, Schmidt / Ulrich (Editor), pmi Verlag 1992].

27th amino acids

Proteinogenic and non-proteinogenic amino acids play an important role in the formation of biologically active molecules. For the brain and nerves are important: L-Ornitin reduces the concentration of free ammonia in the blood and brain. (Ornitin cycle: This prevents hepatic encephalitis caused by decreased levels of ammonia in the liver, and the amino acids leucine, isoleucine, and valine can also reduce free blood ammonia. From ornithine, the polyamines spermidine and spermine are formed. Ornitin also participates in the regulation of the NMDA receptor. This is probably due to the formation of spermine. The forms of application in pharmacy are the L-ommitin hydrochloride, L-ornithine-alpha-ketoglutarate and L-ornitine-L-aspartate. L-cysteine is needed for the production of GSH and is usually the limiting factor for it. Its application is as N-acetylcysteine [Terhi Ahola, Preventive potential of N-acetylcysteine in oxidative stress-related complications of prematurity, ISBN 952-91-7212-5, Helsinki 2004].

Example of a test procedure

Acidosis, energy deficiency, excitatory amino acids and ROS are the major cause of death of the rat baby after an ischemia-reoxygenation period [Barbara Lubec et al., Decrease of heart protein kinase C and cyclin-dependent kinase precedes death in perinatal asphyxia of the rat , The FASEB Journal, Vol. 11, May 1997, p. 482-492]. 100% of the rat babies survive an ischemic period of a maximum of 16 min. At 19-20 min. survive 64%, at 20-21 min. 40% and at 21 - 22 min. only 5% more. [Elisabetta Dell'Anna et al .; Delayed neuronal death following perinatal asphyxia in rat, Exp. Brain. Res. (1997) 115: 105-115].

Reoxygenation with antioxidants

10 rat fetuses are described according to the method described in the above literature an ischemic period of 20 min. exposed. Immediately thereafter, they are placed in the breathing chamber and 30 min. long fed with an air - antioxidant mixture. Air supply 500 liters per hour, filling the wash bottle with 100 ml of water overlaid with 40 g of a 1.0% solution of antioxidant (e.g., dimethyl sulfide) in benzyl benzoate.

As a result of the experiment, the number of surviving rats is evaluated.

Claims (10)

1. A method for reversing or reducing the negative consequences of oxidation of the cell membranes, in particular the mitochondria and other cell organelles of human organs, characterized in that the person concerned during or shortly after the occurrence of the triggering event molecules provide the biochemical energy molecules that a reducing Have molecules that have an antioxidant effect, molecules that act as a methyl group supplier and molecules with complexing action for free metal ions are administered via the air. 2. The method according to claim 1, characterized in that these molecules are intensively mixed with the respiratory air as gas, vapor or fine aerosol and are absorbed in the lung (pulmonary). 3. The method according to claim 1 and 2, characterized in that the pulmonary absorption is supplemented by oral or parenteral administration of these molecules. 4. The method according to claim 1, 2 and 3, characterized in that these molecules are administered alone or in combination with each other. 5. The method of claim 1, 2, 3 and 4, characterized in that the energy-yielding molecules ATP, GTP, NADH and NADPH are. 6. The method of claim 1, 2, 3, 4 and 5, characterized in that the molecules having a reducing effect dimethyl sulfide, dimethyl sulfoxide, methyl mercaptan, ethyl mercaptan, furfuryl mercaptan, beta mercaptoethanol, 2,3-dimercapto-1-propanol, N-acetylcysteine, L-cysteinamide, methionine, methionine sulfoxide, S-methylcysteine sulfoxide, dithiothreitol, glutathione, ascorbic acid as ascorbate, sodium ascorbate phosphate and magnesium ascorbate phosphate, esters of ascorbic acid, thioglycolic acid and thiodipropionic acid and extracts of lily plants such as onion and garlic and biogenic polyamines in particular Spermidine and spermine, as well as spermine diphosphate. 7. The method of claim 1, 2, 3, 4, 5 and 6, characterized in that the molecules with antioxidant natural and synthetic essential oils and their individual ingredients, in particular bergamot, eucalyptus, rosemary, Vetyver, clove, thyme, grapefruit, wintergreen and their individual ingredients as well as methyl salicylate, ethyl salicylate, amyl salicylate, vanillin and ethyl vanillin. 8. The method of claim 1, 2, 3, 4, 5, 6 and 7, characterized in that said agents are mixed prior to use with substances that cause improved uptake through the cell membrane of the Lungenveveolen, hereinafter called "Carrier solvents" , 9. The method according to claim 8, characterized in that are used as "carrier solvents" dimethyl sulfide, Dimetylsulfoxid, methylsulfonylmethane, limonene, benzyl benzoate, dihydrojasmonate, cyclopentadecanolide, phospholipids, in particular phosphatidylcholine, sphingolipids, especially sphingomyelin. 10. The method according to claim 1 to 9, characterized in that said substances are administered by means of a breathing mask to the patient.