WO1997041848A1 - Pharmaceutical compositions containing alpha-keto carboxylates - Google Patents

Abstract

Novel pharmaceutical compositions comprising as an active phosphorylation potential enhancing substance, a pharmaceutically-acceptable salt of an alpha-keto carboxylic acid, method of making and use thereof.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING ALPHA-KETO CARBOXYLATES

I. GOVERNMENT INTEREST

This invention described herein may be manufactured, licensed and used by or for the United States Government without the payment of any royalties to us thereon. The Federal Government has a nonexclusive nontransferable, irrevocable, paid-up license to practice or have practiced for or on behalf of the United States any subject invention throughout the world.

LA. CROSS REFERENCE

This application is a continuation in part of U S Serial No. 08/239,635 filed May 9, 1994.

II. TECHNICAL FIELD OF THE INVENTION

The invention is in the field of protecting, preserving and restoring normal cell functions More specifically it is in the field of using alpha-keto carboxylate compositions as prophylactic and therapeutic agents to prevent the deterioration or promote the restoration and preservation of normal cell functions. HI. BACKGROUND OF THE INVENTION

Pyruvate is the key glycolytic intermediate of all mammalian cells As discussed in more detail below, this substance and pharmaceutically acceptable derivatives thereof are useful as biological stimulating agents

1) Pyruvate compartmentalization and cytoplasmic phosphorylation potential: Intracellular pyruvate is usually derived from glucose, i e it is a key glycolytic intermediate of all mammalian cells. It can also be formed from extracellular lactate via the lactate dehydrogenase reaction. In situations where pyruvate is employed as an exogneous metabolic substrate, i.e where its extracellular concentration is sufficiently raised, pyruvate functions as a precursor of lactate by reversing the lactate dehydrogenase reaction. Further, in contrast to alternative metabolic fuels such as acetate and also lactate, pyruvate has recently been established by applicant as an agent that consistently improves key indices ofthe cytoplasmic phosphorylation potential of creatine phosphate (ratios ofthe concentrations of creatine phosphate (CrP) to inorganic phosphate (P,), to that of creatine (Cr), or to the product ofthe concentrations of creatine and inorganic phosphate, [CrP]/([Cr]*[Pi]); a formally similar concentration ratio is the phosphorylation potential of ATP, [ATP]/([ADP]*[P,]), which is coupled to and in most cases in equilibrium with [CrP]/([Cr]*[P,]), an effect mediated by the powerful magnesium- and pH -dependent enzyme creatine kinase, this enzyme is present in high concentrations in striated and smooth muscle (heart, vascular smooth muscle, skeletal muscle) and brain, but not in liver and kidney [ATP]/([ADP]*[P,]) is a major determinant ofthe actual free energy available from cellular ATP hydrolysis according to the following equation

-G_ATp = 'G°_ATP+R-T-ln(\ADF\-\P. t\ATP])

in which *G° _ΛTP is the (relatively constant) standard free energy change of ATP hydrolysis under conditions prevailing in vivo (-32 35 kJ/mol, pH 7.2, free cytoplasmic magnesium concentration < 1 mM), R = gas constant (8 314 J/K*mol) and T - absolute temperature in degrees Kelvin (K) Thus, during alterations of physiologic states and under many pathophysiological states investigated so far, [ATP]/([ADP]*[P,]) can change considerably, whereas the *G°_ATP-term changes relatively little

Pyruvate, administered in doses between 2 to 10 mM, has recently been demonstrated by applicant to raise the phosphorylation potential in a dose-dependent manner in normal, but especially in reversibly damaged (ischemia/reperfusion protocols) heart models of guinea pig , dog and pig. Thus, Pyruvate administration can somewhat (by about 4 to 6 %) improve the free energy available for cellular phosphorylations and energy consuming ion transporters as well. Pyruvate is centered at the compartmental interface between cytoplasma and mitochondria; applicant has recently shown that it is linked via the cytoplasmic NAD7NADH system (which is under the joint control of two major cytoplasmic dehydrogenases, the lactate dehydrogenase and the glyceraldehyde-3 -phosphate dehydrogenase) to the cytoplasmic phosphorylation potential [3,3a]. Thus, pyruvate is coupled to [ATP]/([ADP]*[P,]) in its capacity as substrate of lactate dehydrogenase, which can affect the NAD7NADH system which in turn is stoichiometrically coupled the combined glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction, the latter enzyme system involves ATP, ADP, and P, as reactants, i.e is linked directly to the cytoplasmic [ATP]/([ADP]*[P,]) rather than the CrP phosphorylation potential, [CrP]/([Cr]*[P,]). In practice, [ATP]/([ADP]*[P,]) is usually determined using the [CrP]/([Cr]*[P,]), but applicant has demonstrated that it also can be estimated using the reactants of the glyceraldehyde-3-phosphate dehydrogenase combined with those ofthe lactate dehydrogenase.

2) Pyruvate dehydrogenase: Pyruvate is also the immediate substrate ofthe powerful mitochondrial pyruvate dehydrogenase enzyme complex (PDH), the main mechanism that controls entry of carbohydrate and lactate carbon into the citric acid cycle for end-oxidation (formation of water and carbon dioxide) coupled with oxidative phosphorylation (formation of ATP from ADP and inorganic phosphate) In addition, pyruvate, not lactate or acetate, is auto-catalytically active at the PDH enzyme complex, thus pyruvate stimulates covalent modification (dephosphorylation) ofthe interconvertible PDH complex, which results in increased activity ofthe PDH, this in turn stimulates oxidative decarboxylation of pyruvate to acetyl-CoA and carbon dioxide and hence facilitates complete conversion of cellular glucose- and lactate-carbon to water and carbon dioxide (see below) The net effect of these changes is an increased availability of NADH in the mitochondria, thereby improving the ability ofthe cell to adapt promptly to changing energy demands. 3) Pyruvate Carb^'oxylase: Another important feature of pyruvate only (not of other substrates such as lactate or acetate), is that it functions as the immediate substrate of the CO₂-fixing-enzyme pyruvate carboxylase This enzyme is present in relatively small amounts in liver and heart and probably other organs as well, but it is important, since it assimilates metabolic CO₂ by adding it to the carbon-3 -skeleton of pyruvate, thus providing the mammalian cell with an "anaplerotic" mechanism, the overall effect is the net synthesis of mitochondrial carbon-4-skeletons, which helps to adequately maintain the concentration ofthe rather small but absolutely vital carbon-4-oxaloacetate pool in the mitochondria Oxaloacetate is crucial for the mitochondrial condensing enzyme (citrate synthase) which catalyzes the aldol condensation between the methyl group of acetyl-CoA (generated in the PDH reaction or derived from ketone body or fatty acid metabolic pathways) and the carbonyl group of oxaloacetate resulting in the formation of citrate and coenzyme A Oxaloacetate, an alpha-ketoacid like pyruvate and alpha-ketoglutarate, is normally present in the mitochondrial matrix in only very small concentrations, it is clear therefore that maintenance of its mitochondrial concentration can become crucial for adequate citric acid cycle turnover, the mechanism which provides the necessary reducing equivalents (NADH₂, FADH₂) for the respiratory chain which in turn ensures maintenance of ATP synthesis (oxidative phosphorylation) and hence cellular energy status (phosphorylation potential)

4) Lactate dehydrogenase and cytoplasmic NADH₂: At physiological pH of 7 0 to 7 4 pyruvic acid, because of its relatively low pK value of 2 49, is virtually completely dissociated into the negatively charged pyruvate anion and the positively charged H" cation It is known that the pyruvate anion (but probably also the undissociated free pyruvic acid), if administered in sufficient quantities, lowers the cytoplasmic [NADH]*[H⁺]/[NAD⁺] ratio in cellular systems that contain lactate dehydrogenase This effect is often referred to as the oxidizing effect of pyruvate It has been recently demonstrated by applicant that this effect of pyruvate can prevent the normal accumulation of cytoplasmic NADH, duπng experimental cardiac ischemia [1 ] This special oxidizing mechanism of pyruvate is potentially of great clinical significance, as extramitochondrial NADH₂ has been found by others to be hazardous for isolated heart mitochondria (not for isolated liver mitochondria); in isolated heart mitochondria NADH₂ lowers respiratory control and impairs energy coupling (ATP/oxygen ratio decreases) during readmission of oxygen; simultaneously the anoxic/reoxygenated NADH₂-exposed cardiac mitochondria begin to double the generation ofthe pathological superoxide anion (O₂-) [2]; the superoxide anion belongs to a special class of reactive oxygen species usually called oxygen-derived free radicals, these molecular species have been implicated in the pathogenesis of oxidative stress during ischemia/reperfusion conditions of several organs such as heart, kidney and liver [3-6] Both the impairment of energy coupling and the pathological superoxide anion formation were only observed when the isolated heart mitochondria were incubated with NADH₂, not during acidosis or anoxia per se, i e. in the absence of NADH, Considering these new findings from isolated cardiac mitochondria, the recent demonstration by applicant that pyruvate can prevent large accumulations of NADH₂ during ischemia [1 ], it becomes evident that pyruvate - via its oxidizing effect p_eι ≤g - could be instrumental in maintaining adequate mitochondrial energy coupling during subsequent reperfusion According to the above rationale this would additionally be associated with a lower production rate of potentially dangerous oxygen-derived free radicals (superoxide anions) It is readily seen that these redox-effects of pyruvate may lead to improved reperfusion energetics and hence functional recovery in any organ that contains sufficient amounts of lactate dehydrogenase As for the heart, anticipated improvements include reduced myocardial stunning, reduced probability of arrhythmias and ventricular fibrillation, reduced accumulation of sodium and hence calcium, attenuation of cytoplasmic calcium accumulation is particularly important, since unphysiologically high concentrations ofthe calcium ion can activate dangerous autolytic enzymes such as phospholipases (membrane lipid damage), proteases (cytoskeletal protein and enzyme damage), and endonucleases (DNA strand breaks [7]) 5) Fenton reaction, Fe²⁴ (ferrous Ion) and reducing equivalents: There is yet another reason why the cytoplasmic redox effects of high concentrations of pyruvate could be beneficial during ischemia-acidosis/reperfusion situations, this mechanism concerns the formation of cytotoxic oxygen-derived free radicals via Fenton-type reactions This is the case because the Fenton reaction is the mechanism responsible for generation ofthe particularly cytotoxic hydroxyl radical, the reaction requires catalytic amounts of free Fe²⁺ (ferrous Ion) which interacts with hydrogen peroxide or the superoxide anion to yield three products Fe^v plus hydroxyl ion (both relatively inert) and the extremely reactive and therefore cytotoxic hydroxyl radical [8] Normally most of cellular iron is complexed in the form of Fe^v by ferritin or stored as haem-type-iron in proteins, enzymes, and cofactors These complexes usually contain iron but are not themselves substrates or catalysts in Fenton-type reactions However, during accumulation of reducing equivalents (FADH, FMN₂, NADH₂) in ischemic/anoxic/infarcted organs, reduced flavins can bring about the reductive release of iron from ferritin [9], this raises the concentration of free Fe2+ (ferrous Ion) which may then be available to react with hydrogen peroxide/superoxide anion of, e g , mitochondrial origin during subsequent reperfusion Indeed, during rat cardiac ischemia, accumulation of free Fe²⁺ (ferrous Ion) has recently been demonstrated, especially when ischemia was associated with significant cellular acidosis [0] Since pyruvate treatment can help to metabolically neutralize cellular H^* (as opposed to direct chemical buffering by, e g , bicarbonate) and also has been shown by applicant to prevent accumulation of reducing equivalents during ischemia by "clamping" the cytoplasmic redox status at the normoxic level [ 1 ], it is easily seen that acute parenteral pyruvate has the potential to attenuate or perhaps even completely prevent the intracellular reductive liberation of free Fe²' (ferrous ion) during acidotic ischemia/anoxia Applicant proposes that this special redox-effect of pyruvate has the potential to attenuate/prevent Intracellular Fenton-type reactions, which in turn would diminish or perhaps even completely eliminate the formation ofthe cytotoxic hydroxyl radical from hydrogen peroxide or superoxide anion Consequently, oxidative stress due to reperfusion/reoxygenation can be expected to be minimized, if adequate pyruvate therapy can be timely implemented to minimize the accumulation of reducing equivalents during prereperfusion ischemia/anoxia conditions. This particular and novel pyruvate mechanism will likely apply to all mammalian organs that contain lactate dehydrogenase and ferritin iron (e.g., brain, heart, kidney, lung), probable exceptions are liver and spleen since these organs do not appear to have substantial amounts of ferritin (for review see ref [0])

6) Intracellular hydrogen ion balance and metabolic removal of H⁺: Applicant also proposes that pyruvate can also influence favorably the cellular hydrogen ion balance Therapeutically applied pyruvate stimulates hydrogen ion removal by metabolic consumption as opposed to direct chemical buffering or neutralization as meditated by, e.g., bicarbonate or other cellular buffers; thus pyruvate can enhance metabolic removal (cause covalent sequestration of) intracellular hydrogen ions. Virtually all major mammalian cell types have at least four major enzymatic mechanisms at their disposal by which pyruvate metabolism contributes to this type of metabolic consumption of H⁺ a) during reduction of pyruvate to lactate via lactate dehydrogenase one H⁴ ion is consumed to form the lactate anion, lactate anion can then be washed out (transported across the cell membrane) by a specific process which comprises obligatory co-export with another H⁺ ion (H⁺-symport); this export system is the powerful monocarboxylate transport system ofthe cell membrane, b) during oxidative decarboxylation of pyruvate by pyruvate dehydrogenase , prior mitochondrial H'-import removes cytoplasmic H' , i.e for one pyruvate decarboxylated one H^J ion is stoichiometrically consumed turning up ultimately in the water generated by the mitochondrial respiratory chain, c) during CO₂-fixation by maleate dehydrogenase (malic enzyme, decarboxylating NADP^{^}-dependent malate dehydrogenase) maleate anion is formed and again one H⁺ ion is stoichiometrically consumed to form malate and NADP~ d) Furthering the oxidative metabolism of pyruvate plus one H⁺ is also the already mentioned anaplerotic pyruvate carboxylase ofthe mitochondria Thus, the pyruvate anion functions as natural hydrogen ion remover, gently alkalinizing cells and blood without depending on external buffers like bicarbonate Thus the applicant contends that pyruvate can possibly function as an alternative to traditional and perhaps less gentle treatments of metabolic acidosis by bicarbonate. The advantage of pyruvate cellular alkalization would be that systemic pyruvate application drives hydrogen ions out ofthe cells, whereas bicarbonate drives hydrogen ion into the cells (exacerbating intracellular acidosis) until the excess CO₂ is eliminated via the lungs.

A moderate alkalization is desirable for all mammalian cells or organs that recover from damage associated with intracellular acidification; this would apply to situations where the residual metabolism has become acidotic and must be restarted to reestablish normal ion homeostasis simultaneous with replenishment of crucial cellular metabolite pools (especially that of mitochondrial oxaloacetate) and energy stores (phosphorylation potential). The potential for reductive release of hazardous free iron from cellular complexes (ferritin, myoglobin, cytochromes) will also be diminished by pyruvate, since Fe2+ (ferrous Ion) release under intracellular conditions requires the above described accumulation of reducing equivalents in combination with H^* (acidosis). Clearly, applicant can claim that pyruvate has the potential to influence favorably cellular redox and hydrogen ion balances, via its effects on the cytoplasmic [NAD⁴]/[NADH]*[H⁺] ratio and its H^*- consuming metabolic pathways; these features appear to be particularly efficacious in states of partial and reversible cell damage and/or recovery from damage or from extreme stress: reoxigenation after hypoxia, reperfusion after ischemia and myocardial infarct, reestablishing coronary circulation after cardiopulmonary bypass, reperfusion after percutaneous transluminal coronary angioplasty, reperfusion after enzymatic recanalization of thrombotic vessels (streptokinase-type interventions), recovery from excessive catecholamine stress or physical exertion, recovery from probably all types of circulatory shock, if they were associated with hypoxia/ischemia and acidosis.

7) Protection of essential -SH groups: If during cellular damage reductive release of free Fe2+ (ferrous Ion) occurred, triggering Fenton-type reactions to produce free hydroxyl radicals, this oxidative stress could still be limited by exploitation of features of pyruvate other than those already discussed. The mechanism is another feature that applicant contends is unique to pyruvate It has been recognized that the cytotoxicity of oxygen-derived free radicals includes oxidation of labile -SH groups Optimum functioning of vital enzymes such as Na7K⁴-ATPase and glyceraldehyde-3 -phosphate dehydrogenase or metabolite transporters (e g , the specific mitochondrial pyruvate transporter) appears to depend on such labile -SH groups, other effects ofthe free radicals include the relatively unspecific peroxidation of membrane lipids which is thought to disturb normal membrane function, possibly, free radicals can also oxidize protein-thiols thus causing direct damage to structural proteins ofthe cytoskeletal apparatus [17] which can jeopardize the physical integrity and sturdiness ofthe cell

Applicant proposes that pyruvate has the potential to strengthen the intrinsic cellular tolerance against this type of oxidative stress on labile but essential -SH groups This latter mechanism probably operates via the well-known pyruvate-induced citrate accumulation, citrate is an allosteric inhibitor of phosphofructokinase, the main enzyme regulating glycolytic flux Inhibition of phosphofructokinase leads to an accumulation of glucose-6-phosphate (G-6-P), the immediate phosphorylation product of glucose (hexokinase) G-6-P is also the substrate ofthe G-6-P dehydrogenase, the first and rate- limiting enzyme controlling the metabolic throughput ofthe pentose phosphate cycle, it has been shown that increased levels of G-6-P. increase the rate of the pentose phosphate pathway in the heart [12] This metabolic pathway produces reducing equivalents in the form of NADPH, which are normally used for reductive syntheses and, importantly, also to keep the glutathione system in its physiologic reduced state The glutathione system is considered the main cellular defense against sudden oxidative stress due to oxygen-derived and possibly other free radicals Thus, applicant points out that pyruvate, via an allosteric effect on glycolysis at the level of phosphofructokinase, has the potential to strengthen the reductive capacity ofthe glutathione system, which will likely improve cellular tolerance to acute oxidative stress Of significance in this context is that reduced glutathione (GSH) likely prevents/minimizes oxidation of labile protein-SH groups, maintenance ofthe cellular GSH/GSSG redox status is therefore likely important for maintaining protein-thiols and enzyme-thiols in their physiologic reduced state. Several powerful enzymes that are instrumental for normal cell function contain labile -SH groups; known examples are the Ca^+÷-ATPase ofthe sarcoplasmic reticulum (excitation-contraction coupling in heart, skeletal and smooth muscle), the creatine kinase (maintaining the cytoplasmic phosphorylation potential in heart, skeletal and smooth muscle and brain), glycogen phosphorylase (mobilization of liver and kidney glycogen to stabilize blood glucose levels), and the already mentioned Na+/K+-ATPase (ubiquitous cellular Na7K' homeostasis, affecting also that of calcium via the Na^'/Ca^{2 " '}- and NaVH^* -exchangers) (for review see ref. [11]). Disabling these enzymes by oxidizing their labile -SH groups is incompatible with long-term cellular survival, not to mention the maintenance of their vital cell- and organ specific functions.

8) Non-enzymatic interaction of pyruvate with hydrogen peroxide: Another novel feature of pyruvate that applicant considers is its capacity to directly neutralize hydrogen peroxide on a 1 to 1 molar basis. Under cellular conditions this interaction is spontaneous and does not require enzyme catalysis; it is an interaction between pyruvate's carbonyl group (alpha-keto group) and hydrogen peroxide yielding carbon dioxide and acetate. This reaction is probably enhanced by the presence of free Fe2+( ferrous Ion). The released carbon dioxide is highly diffusible across all cellular membranes and can thus immediately be washed out from cells, organs or removed from the body via the lung; acetate, the other product ofthe pyruvate-hydrogen peroxide interaction, can be readily activated by mitochondrial acyl-coenzyme A synthases yielding acetyl-CoA, the main substrate for the citric acid cycle. Applicant points out that this non-enzymatic mechanism of H₂O₂-pyruvate interaction could conceivably mitigate the sudden oxidative stress experienced by cells/organs previously compromised by lack of oxygen, lack of circulation, metabolic acidosis, iron overload, extreme metabolic stress.

9) Effect on blood oxygen transport: Pyruvate has also advantageous effects on blood, erythrocytes and their capacity to release oxygen at the specific oxygen tension in the microcirculation It is well established that pyruvate can increase erythrocyte 2,3- diphosphoglycerate (2,3-DPG) levels [13], 2,3-DPG is important for the position ofthe oxygen dissociation curve and hence the release of oxygen from hemoglobin at the partial pressures of oxygen prevailing in the microcirculation. This effect of pyruvate will likely improve the oxygen supply to parenchymal cells; the effect may become especially important in situations where oxygen supply is precarious This effect of pyruvate requires the presence of adenine or inosine as well as relatively high concentrations of phosphate as additional substrates The feature has been exploited in blood banking for decades, but applicant points out that this is an additional important argument for novel systemic pyruvate applications in conditions of oxygen deficiencies such as restriction of circulation (ischemia), high altitude (hypoxia), hemodilution (severe external or internal blood loss), severe anemia. However, under high altitude conditions, respiratory alkalosis may develop, if such a conditions exists, pyruvate application may not be justified, since it could aggravate the alkalosis, an effect that may offset the beneficial effect of 2,3-DPG accumulation on the oxygen dissociation curve

In order to provide for a clearer understanding ofthe instant invention, applicant provides the following differentiating discussion on phosphorylation potential versus protein phosphorylation

PHOSPHORYLATION POTENTIAL VS. PROTEIN PHOSPHORYLATION

The phosphorylation potential is independent of second messengers and influences the ATP hydrolysis energy (ATPases) It thus determines the cellular distribution of ions such as sodium and calcium Protein phosphorylation is controlled in most cases by hormone-dependent second messengers which stimulate phosphate transfer reactions by kinases (not ATP hydrolysis by ATPases), altered protein phosphorylation often alter rates and affinities of reactions, not the distribution of compounds and ions The phosphorylation potential is important for all endergonic reactions providing the requisite energy Protein phosphorylation alters velocities of reactions as long as the level ofthe phosphorylation potential remains within limits that are compatible with normal physiologic function.

PHOSPHORYLATION POTENTIAL

The metabolite ratio [ATP]/([ADP]*[Pj), termed the phosphorylation potential, determines cellular ionic pump/gradients and the output of mechanical muscle energy (cross-bridge cycling). These reactions always produce free inorganic phosphate thereby providing chemical energy due to hydrolysis of ATP. [ATP]/([ADP]*[P,]) influences the extent of such endergonic reactions, not the rale or velocity of these reactions. The phosphorylation potential is effective via the hydrolytic energy available from A TP. which in turn is greatly influenced by mitochondrial function in the form of oxidative phosphorylation. [ATP]/([ADP]*[P,]) it is not dependent upon second messengers such as cyclic nucleotides or inositol phosphates.

ATP is the immediate energy source of chemical hydrolysis energy for all major cellular activities which combine to sustain cellular sodium and calcium homeostases and specific cellular functions. For example in muscle the most important physiologic function is contraction and relaxation, in neurons and glandular cells these functions are electrical and secretory activities, in liver hepatocytes they are protein/hormone synthesis and detoxification, in kidney it is the sodium and glucose transport against concentration gradients. The actual amount of energy available from ATP under a given cellular milieu, i.e. the free energy change of ATP hydrolysis is dependent upon the energy level of ATP. This energy level is mainly determined by the phosphorylation potential, i.e. the metabolite ratio [ATP]/([ADP]*[P,]) according to the formula for the Gibbs-free energy of

ATP hydrolysis termed ΔG_ATP:

AG_lTP = ΔG° _ATp+R 'T 'In(\ADP\'\P_!\/\ATP]) where ΔG°_AT!, is the standard free energy change (-7.73 kcal/mol=-32.3 kJ/mol at 38°C, ionic strength = 0 25) which is nearly constant under most physiologic conditions and likely also under many reversible pathological conditions provided intracellular free magnesium level and intracellular pH are also near normal R=gas constant (1 98 cal/mol*K), T=absolute temperature =273 °C -37°C=310 K for normal body temperature

ΔG°_ΛTP is normally between 55 to 60 kJ/mol in mammalian cells, while

[ATP]/[ADP]*[P,]), the phosphorylation potential, varies greatly as a function of the physiologic or pathologic states with values, in the myocardium, ranging between about 5,000 per mol during extreme stress and deenergization and 50,000 to 100,000 per mol during rest and the absence of physiologic work loads

The phosphorylation potential, [ATP]/([ADP]*[P_]]), is stoichiometrically involved in the chemical reactions of vital cellular ion pumps, especially the sodium pump (Nap,Kp-ATPase) and the calcium pumps (Ca²' -ATPases at sarcoplasmic reticulum and cell membrane) These reactions are not dependent on second messengers, instead they are governed by mass-action relationships The [ATP]/([ADP]*[P,) is thus crucial for ion homeostasis as its value is a determinant ofthe distribution of sodium across the cell membrane and that of calcium across the SR membrane as well [ATP]/([ADP]*[P,]) does not determine the rates or velocity of these ion pump reactions, rather it determines the distribution ofthe ions across the cellular boundary at which they are strategically located [ATP]/([ADP]*[P]]) thus determines the extent of these endergonic processes thereby creating the ionic gradients across cellular membranes

[ATP]/([ADP]* [P,]) is also stoichiometrically involved in muscular contraction providing the energy for cross-bridge cycling and hence the energy for contractile force and hydraulic work output ofthe muscle

PROTEIN PHOSPHORYLATION

Protein phosphorylation in contrast affects cellular metabolism and function mainly as a consequence ofthe activity second messenger-dependent kinases These kinases alter the state of phosphorylation of target regulatory proteins (tropin 1, phospholamban), key 'interconvertible ' metabolic enzymes (glycogen phosphorylase, glycogen synthetase, phosphofructokinase, pyruvate dehydrogenase complex), ion channels, receptors and contractile proteins (troponin T, myosin light chain). These kinase-dependent protein/enzyme/receptor/channel phosphorylations are not accompanied by liberation of inorganic phosphate or free energy and, except for pyruvate dehydrogenase phosphorylation, require for maximal activity the presence of a second messenger. Phosphorylation of target proteins/key metabolic enzymes/channels/receptors usually leads to altered rates of reactions via changes in affinities (K_M) and maximum velocities (V_MAX). Thus troponin I and phospholamban phosphorylations increase the rate of contraction in heart muscle whereas pyruvate dehydrogenase phosphorylation decreases (V_MAX) of oxidative decarboxylation of pyruvate, i.e. the rate of entry of carbohydrate carbon into the citric acid cycle. As for reasons of stoichiometry the degree of phosphorylation is also partially influenced by the level ofthe [ATP]/([ADP] ratio, which in turn is an integral constituent of [ATP]/([ADP]* [P,]), it is possible, if not likely that the phosphorylation potential can influence protein phosphorylation independent of second messengers. This mechanism simply follows from the mass-action equation of kinase reactions (however, to date there is only very sporadic evidence for this mechanism in the literature).

INDICATORS OF PHOSPHORYLATION POTENTIAL

[ATP]/([ADP]* [P,]) cannot be readily and directly measured because the concentrations of ADP in the cytoplasma is below the detection limit of, e.g., nuclear magnetic resonance technology, but also below the detection limit of enzymatic-optic and HPLC techniques. Further complicating the direct measurement of [ATP]/([ADP]* [PJ) is the fact that at least in muscle most of ADP is bound to actin. Measurements of total muscle ADP thus grossly overestimates the thermodynamically relevant free ADP concentration, the term that appears in the [ATP]/([ADP]* [P,]). However, there are at least four readily measurable indicators of [ATP]/([ADP]* [PJ) that can be used to assess the level and/or directional change of [ATP]/([ADP]* [PJ) These indicators are briefly discussed below

1) [CrPj/i fCrrrP-l ratio

[ATP]/([ADP]* [PJ) is stoichiometrically linked to the [CrP]/[(Cr]*[PJ ratio via the powerful creatine kinase reaction. This is true for muscle, brain and endothelium, i e cellular systems that contain the creatine kinase enzyme system Therefore, in these tissues, [ATP]/([ADP]* [PJ) can be assessed by measuring the reactants of creatine kinase (creatine phosphate, creatine, H^~) and inorganic phosphate

2^ [GAP1*[fPYR]/(r3PG1*[LACl ratio

[ATP]/([ADP]* [P,]) is also stoichiometrically linked to glycolysis, specifically the combined GAPDH/PGK reaction Thus, [ATP]/([ADP]* [PJ) can be assessed from the measured [GAP]*[PYR]/([3PG]*[LAC]) ratio provided glycolytic flux is relatively small and the key enzymes (glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, lactate dehydrogenase) can therefore be assumed to catalyze a near-equilibrium reactions This is known to obtain for heart muscle in low-metabolic rate Langendorff hearts Further, the [GAP]*[PYR]/([3PG]*[LAC]) ratio can be applied to assess [ATP]/([ADP]* [PJ) in tissues devoid of creatine kinase such as liver and kidney, if near equilibrium conditions obtain

3) Extracellular potassium

[ATP]/([ADP]* [PJ) is stoichiometric linked to the distribution of sodium and potassium ions across the cell membrane, the reaction being catalyzed by the Na\K⁺-ATPase Therefore, a fall in [ATP]/([ADP]* [PJ) would result in an increase in extracellular K⁺ concentration which can be easily measured Increased myocardial potassium release indicates a decrease in the [ATP]/[ADP]* [PJ) 4) Extracellular purine nucleosides

[ATP]/([ADP]* [P,]) can also be judged from the formation and release of ATP degradatives, adenosine and inosine and further degradatives This is true because the [ATP]/([ADP]* [P,]) ratio, an integral part ofthe [ATP]/([ADP]* [PJ), is stoichiometrically linked to cellular free AMP (via the adenylate kinase reaction) the immediate precursor of adenosine Thus, increased production and release of extracellular purine nucleosides like adenosine and inosine signals a fall in the [ATP]/[ADP] ratio which is usually the consequence of a fall in the phosphorylation potential [ATP]/([ADP]* [PJ)

IV. SUMMARY OF THE INVENTION

It is the object of this invention to provide a pharmaceutical composition which contains as an active phosphorylation potential enhancing substance an alpha- ketocarboxylic acid compound or a pharmaceutically-acceptable salt thereof in an amount sufficient to prevent the deterioration or promote the restoration and preservation of normal cell functions

V. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Energetically exhausted postischemic heart

In a series with 45 isolated perfused working guinea pig hearts subjected to 45 min ischemia followed by 20 min reperfusion hearts were treated with various substrates and monocarboxylates Figure 1 shows that only pyruvate anion treatment, infused in presence of glucose throughout ischemia and reperfusion, enabled the heart to nearly completely recover left ventricular function at essentially normal phosphorylation potential as judged by the [CrP]/([Cr]*[PJ) ratio Other monocarboxyaates like b-hydroxy butyrate, acetate or lactate were much less effective Also glucose alone was much less effective than its combination with pyruvate anion Non-oxidizable substrate such as deoxyglucose was completely ineffective in restoring phosphorylation potential and reperfusion function Figure 2 Energetically exhausted non-ischemic heart

In a series with 12 isolated perfused working guinea pig hearts, were energetically exhausted by substrate-free perfusion until left ventricular failure occurred This failure was considered established when cardiac output or left ventricular pressure volume work had declined to about 30% of its initial control value Monocarboxylates such as octanoate, b- hydroxy butyrate, lactate, and pyruvate were then infused with stepwise increasing concentrations to test whether hearts would recover from energetic exhaustion The medium chain length fatty acid octanoate produced recovery to about 80% in the range up to about 1 mM, but at concentrations greater than 2 mM octanoate produced cardiac failure b-hydroxy butyrate was essentially ineffective in restoring function ofthe energetically exhausted heart Lactate, the reduced congener of pyruvate, restored only 50% of ventricular function and proved to be toxic at concentrations above 10 mM Only the pyruvate anion restored cardiac function to about 90% in the concentration range between 2 to 20 mM No toxicity of even extremely high doses were noted when pyruvate was applied

Figure 3. The stunned dog heart

In a series with 13 dog hearts in situ which were stunned by five repeated ischemia of three min duration, ventricular segment shortening in the region of the left anterior descending coronary artery (LAD) was measured as an index of left ventricular global function Also the [CrP]/([Cr]*[PJ) ratio was measured as an index ofthe phosphorylation potential The stunned hearts were treated with 5 to 10 mM pyruvate anion infusion intracoronarily The data showed that the dog hearts recovered left ventricular function in parallel with the instantaneous [CrP]/([Cr]*[PJ) ratio as predicted from Fig 1 Moreover, the data showed that pyruvate treated dogs had considerably higher [CrP]/([Cr]*[PJ) ratios than the controls in six ofthe eight cases tested Overall, pyruvate raised significantly the [CrP]/([Cr]*[PJ) ratio in the stunned dog heart

Fig 4. Purine release during low-flow ischemia In a series with 14 isolated working guinea pig hearts, hearts were subjected to low flow ischemia (coronary flow reduced from 8 ml/min to 1 ml/min) for 45 min and perfused with either the physiological level of pyruvate anion (0.2 mM) or therapeutic doses of pyruvate (ImM). Glucose was the co-substrate The data showed that increased levels of pyruvate attenuated the ischemic rise in production of adenosine plus inosine. This suggested that pyruvate when applied at therapeutic levels reduced the energetic depletion during moderate ischemia. Similarly, pyruvate infusion also attenuated the production of lactate during low flow ischemia, suggesting again that pyruvate raised the [ATP]/([ADP]*[PJ) ratio according to the GAPDH/PGK reaction described above

The data also showed that pyruvate at the 1 mM dose significantly reduced the basal nucleoside production in normoxia, again demonstrating that pyruvate can raise the [ATP]/[ADP] ratio and hence the [ATP]/([ADP]*[PJ) ratio also in normal normoxic heart

VI. DETAILED DESCRIPTION OF THE INVENTION

A Biological activity has been discovered for a pharmaceutical composition whose dominate function is to enhance the phosphorylation potential and to reduce hydrogen load within the cell thereby preventing the deterioration or promoting the restoration and preservation of normal cell functions More precisely, applicant has discovered a pharmaceutical composition method of making and use thereof with the following attendant itemized features

FEATURES 1. A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

2 The method in accordance with Feature 1 wherein said cation is an alkali or alkaline earth metal, --

3 The method in accordance with Feature 2 wherein the alkali metal is sodium —

4 The method in accordance with Feature 3 wherein R is an alkyl group containing 1 to 12 carbon atoms —

5 The method in accordance with Feature 4 wherein the alkyl group is methyl ~

6 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a parenteral fluid containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation 7. A method according to Feature 6 wherein the parenteral fluid is selected from the group comprising total parenteral nutritional fluids, kidney and peritoneal dialyses fluids, volume and plasma expanding fluids, pyruvate/acetate near-isotonic solutions, lactate/acetate-free pyruvate isotonic solutions, normal saline solutions, hemoglobin-substitute containing solutions, vitamin supplement product, and cardioplegic solutions

8 A method according to Feature 6 wherein the amount of active ingredient is effective in reducing and/or ameliorating intracellular acidosis

9 A method according to Feature 6 wherein the amount of active ingredient is effective in neutralizing hydrogen peroxide through hydrogen peroxide-alpha-ketocarboxylate interaction to inhibit the formation of toxic-free radicals

10 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a rehydration fluid, which may contain electrolyte balances, containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

1 1 A method according to Feature 10 wherein the rehydration fluid contains electrolyte balances 12 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a topical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

13 A method according to Feature 12 wherein the topical composition is selected from the group comprising medicinal soaps, medicinal shampoos, sunscreens, medicinal ointments, vitamin capsules, dentrifice, mouthwash, douche solutions, and medicinal baths

14 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition selected from the group comprising an antibiotic and antiphlogistic containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, within the alkylene chain, halogen amino, alkylamino of 1 to 4 carbon atoms dialkylamino of 1 to 4 carbon atoms in each alkyl, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

15. The method in accordance with Feature 14 wherein said composition is administered by intramuscular injection.

16 The method in accordance with Feature 15 wherein said composition is an antibiotic

17 The method in accordance with Feature 16 wherein said composition is an antiphlogistic

18 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition for the treatment of local skin disorders, selected from the group comprising an antibiotic and antiphlogistic having as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl; phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

19 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof an aerosolized pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O)(CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, (carboxyalkylene of 1 to 20 carbon atoms within the alkylene chain, halogen amino, alkyl amino of 1 to 4 carbon atoms, dialkylamino of 1 to 4 carbon atoms in each alkyl group or phenyl); alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 atoms; benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl; phenyl; naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation alone or in combination with a bronchodilating agent

20. A method in accordance with Feature 19 resulting in the amelioration or prevention of the onset of abnormal respiratory conditions caused by a reactive airway disease

21. A method in accordance with Feature 20 wherein said reactive airway disease is selected from the group comprising asthma and bronco-pulmony dysplasia

22. A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising perfusion of a mammalian organ in need thereof with pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl; phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

23. The method in accordance with Feature 22 wherein said mammalian organ is selected from the group comprising heart, liver, kidney, brain, spleen vessels, arteries, endothelium, pancreas and glands

24. A method for enhancing the phosphorylation potential within bacterial or viral cells in culture or cloning media in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising adding to the incubation solution for said cells a pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

25 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions thereby enhancing physical endurance or refreshment comprising administering to a mammal in need thereof a food product containing a pharmaceutical composition having as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O)(CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cyloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

26. The method in accordance with Feature 25 wherein said food product is a beverage drink

27. The method in accordance with Feature 26 wherein said food product is a confectionery food

28 The method in accordance with Feature 27 wherein said food product is selected from the group comprising candies and pastries

29 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition containing (1) a thiamine (B l ) vitamin capsule and (2) a therapeutically-effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation 30 A composition of matter for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

31. A parenteral fluid useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically-effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 carbon atoms, benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to

4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

32. A composition according to Feature 31 wherein the parenteral fluid is selected from the group comprising total parenteral nutritional fluids, kidney and peritoneal dialyses fluids, volume and plasma expanding fluids, pyruvate/acetate near-isotonic solutions; lactate/acetate-free pyruvate isotonic solutions, normal saline solutions, hemoglobin-substitute containing solutions, vitamin supplement product, and cardioplegic solutions

33 A rehydration fluid, which may contain electrolyte balances, useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically-effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

34 A composition according to Feature 33 wherein the rehydration fluid contains electrolyte balances

35 A medicinal composition useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

36 A composition according to Feature 35 is selected from the group comprising medicinal soaps, medicinal shampoos, sunscreens, medicinal ointments, vitamin capsules, dentrifice, mouthwash, douche solutions, and medicinal baths

37 An antibiotic or antiphlogistic composition useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, within the alkylene chain, halogen amino, alhylamino of 1 to 4 carbon atoms dialkylamino of 1 to 4 carbon atoms in each alkyl, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

38 The composition according to Feature 37 wherein said composition is administered by intramuscular injection

39 The composition according to Feature 38 wherein said composition is an antibiotic

40 The method in accordance with Feature 39 wherein said composition is an antiphlogistic 41. An aerosolized pharmaceutical composition for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, (carboxyalkylene of 1 to 20 carbon atoms within the alkylene chain, halogen amino, alkyl amino of 1 to 4 carbon atoms, dialkylamino of 1 to 4 carbon atoms in each alkyl group or phenyl), alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation alone or in combination with a bronchodilating agent.

42 A perfusion fluid for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(0)(CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthvl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation 43 An incubation solution for enhancing the phosphorylation potential within bacterial or viral cells in culture or cloning media in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

44 A food product for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions thereby enhancing physical endurance or refreshment comprising a pharmaceutical composition having as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

45 The food product in accordance with Feature 44 wherein said food product is a beverage drink 46 The food product in accordance with Feature 45 wherein said food product is a confectionery food

47 The food product m accordance with Feature 44 wherein said food product is selected from the group comprising candies and pastries

48 A vitamin supplement product for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions hereby enhancing physical endurance or refreshment comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O)(CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono- di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino diethylamino, and M is a cation

VII. EXAMPLES

The herein offered examples of compositions provide methods for illustrating without implied limitation, formulations contemplated within the scope of this invention for activating the phosphorylation potential of cells

The representatives compositions are exemplary in nature to illustrate these compositions within the scope of this invention

All temperatures not otherwise indicated are in degrees Celsius (°C) and parts of percentages are given by weight A intravenous solutions Example 1 i v Ringer's lactate augmented with pyruvate (near-isotonic) per 100 ml solution

600 mg sodium chloride USP

30 mg potassium chloride USP

310 mg sodium lactate

310 mg sodium pyruvate

20 mg calcium chloride USP pH 6 5 (6 0 - 7 5) lactate 28 mEq/1 pyruvate 28 mEq/l osmolarity about 300 mOsmol/1 (calculated) purified (quartz-double-distilled and sterilized) water

Example 2 I v lactate-/acetate-free Ringer's fortified with pyruvate (isotonic) per 100ml solution

600 mg sodium chloride USP 30 mg potassium chloride USP 310 mg sodium pyruvate 20 mg calcium chloπde USP pH 6 5 (6 0 - 7 5) osmolarity about 273 mOsmol/1 (calculated) purified (quartz-double-distilled and sterilized) water

Example 3 i v 5% Dextrose solution fortified with pyruvate (hypertonic) per 100ml solution 5 g Dextrose hydrous USP 287 mg sodium chloride USP 310 mg sodium pyruvate pH 4 (3 5 - 6.5) hypertonic osmolarity about 406 mOsmol/1 (calculated) purified (quartz-double-distilled and sterilized) water

Example 4' i v 5% Dextrose in water fortified with pyruvate (isotonic). per 100ml solution' 5 g Dextrose hydrous USP 310 mg sodium pyruvate pH 4 (3 5 - 6.5) hypertonic osmolarity about 280 mOsmol/1 (calculated) purified (quartz-double-distilled and sterilized) water

Example 5 I v 0 45% sodium chloride solution fortified with pyruvate (hypotonic) per 100ml solution 450 mg sodium chloride USP 310 mg sodium pyruvate pH 4 (3 5 - 6.5) hypotonic osmolarity about 182 mOsmol/1 (calculated) purified (quartz-double-distilled and sterilized) water

B Peritoneal Dialysis (PD) solutions

Example (5) PD solution fortified with pyruvate (isotonic) final concentrations in dialysate sodium 132 mEq/1 calcium 2 mEq/l magnesium 1 0 mEq/1 chloride 105 mEq/1 pyruvate 30 mEq/1 Example (6) PD solution fortified with pyruvate and 1 -4% dextrose (slightly hypertonic) final concentrations in dialysate. sodium 132 mEq/1 calcium 2 mEq/1 magnesium 1 0 mEq/1 chloride 105 mEq/1 pyruvate 30 mEq/1 glucose (Dextrose) 1 -4 g/ 100 ml

C Hemodialysis (HD) solutions

Concentrations of anions and cations similar to PD solutions, fortified with sodium pyruvate and glucose

D Cardioplegic solutions

Example (7) University of Wisconsin solution augmented with pyruvate (near-isotonic) solution contains high potassium (30 mEq/1) all major extracellular electrolytes in normal concentrations plusl mmol/1 adenosine

5 mmol/1 pyruvate pH adjusted to 7 4 equilibrated with 95% oxygen/5% carbon dioxide purified (quartz-double-distilled and sterilized) water

E Oral Rehydration

Example (8) for an oral rehydration fluid augmented with pyruvate

(modified world health organization (WHO) solution) glucose 2 g/dl pyruvate sodium salt 1 g/dl sodium 90 mEq/1 potassium 20 mEq/1 chloride 80 mEq/1 bicarbonate 30 mEq/1

F Oil/Water Ointment

Example (9) for a universal oil/water ointment augmented with pyruvate calcium citrate 0 05 g sodium alginate 3 00 g

Methylparaben 0 20 g

Glycerin 45 OOg sodium pyruvate 0 05 g* double distilled water to make 100 00 g pH not known, probably about 6 0

*= use pyruvic acid, if pH is alkaline

G. Emulsifiable Ointment

Example (10) for a water-removable, emulsifiable ointment augmented with pyruvate polyethylene glycol 4000 50 0 g polyethylene glycol 400 40 0 g

Sorbitan Monapalmitate (Span 40( Atlas)) . 1 0 g sodium pyruvate 0 05 g * double distilled water 9 0 s pH not known, probably about 6 0

*= use pyruvic acid, if relatively low pH is desired

H Injectable Antibiotic

Example (1 1) for an injectable antibiotic augmented with pyruvate ceftriaxone sodium (Rocephin) 250 mg water 0 9 ml sodium pyruvate (final cone 4 5 mmol/1) 0 5 mg vial should contain these ingredients and should then be reconstituted with water

I Medicinal Aerosol

Example (12) for a Medicinal aerosol for relief of asthma, augmented with pyruvate particle size 3 to 6 micron water/ethanol 1/1 by volume epinephrine HCl or isoproterenol HCl sodium pyruvate to final concentration of 0 5 mg/ml propellant 3 to 15 %

J Scientific Perfusion Solution

A method of using the composition of Feature 1 as scientific perfusion solution for isolated animal organs comprising the heart, liver, kidney, brain, spleen, any vessel, pancreas and other endocrine glands

Example (13) for a modified Krebs-Henseleit solution augmented with pyruvate sodium chloride 1 16 mmol/1 sodium bicarbonate 26 mmol/1 potassium chloride 3 5 mmol/1 potassium dihydrogen phosphate 1 2 mmol/1 calcium chloride 1 0 mmol/1 magnesium sulfate 0 6 mmol/1 glucose (dextrose) 5 0 mmol/1 sodium pyruvate 5 0 mmol/1 solution equilibrated with Oxygen/Carbon dioxide = 95%/5% temperature 37 Celsius pH 7 4-7 45 osmolarity 280 mosmol/1 double distilled water must be used

K Scientific Incubation Medium

A method of using the composition of claim 1 as scientific incubation medium for cells isolated from heart, liver, kidney, brain, spleen, any vessel, endothelium, pancreas and other endocrine glands

Example (14) for a electrolyte incubation solution augmented with pyruvate sodium chloride 1 16 mmol/1 sodium bicarbonate 26 mmol/1 potassium chloride 3 5 mmol/1 potassium dihydrogen phosphate 1 2 mmol/1 calcium chloride 1 0 mmol/1 magnesium sulfate 0 6 mmol/1 glucose (dextrose) 5 0 mmol/1 sodium pyruvate 5 0 mmol/I solution equilibrated with Oxygen/Carbon dioxide = 95%/5% temperature 37 Celsius pH 7 4-7 45 osmolarity 280 mosmol/1

Albumin, essential amino acids, trace amounts of ferrous ion and copper salts and vitamins will have to be added to prevent growth limitation due to lack of essential nutrients and minerals Antibiotics may have to be used to prevent unwanted bacterial growth

Osmolarity increases due to addition of amino acids and vitamins will be balanced by appropriate iso-osmolar reductions in sodium chloride L: Scientific Cloning Medium

A method of using the composition of claim 1 as incubation medium for cells used in scientific cloning studies or as a superfusing solution of cells plated on Petri dishes or seeded on latex particles

Example (15): for a cloning/superfusing solution augmented with pyruvate sodium chloride 1 16 mmol/1 sodium bicarbonate 26 mmol/1 potassium chloride 3 5 mmol/1 potassium dihydrogen phosphate 1.2 mmol/1 calcium chloride 1 0 mmol/1 magnesium sulfate 0 6 mmol/1 glucose (dextrose) 5 0 mmol/1 sodium pyruvate 5 0 mmol/1 solution equilibrated with Oxygen/Carbon dioxide = 95%/5% temperature 37 Celsius pH 7 4-7 45 osmolarity 280 mosmol/1

Albumin, essential amino acids, ferrous ion and copper salts and vitamins will have to be added to prevent growth limitation due to lack of essential nutrients Antibiotics may have to be used to prevent unwanted bacterial growth Osmolarity increases due to addition of amino acids and vitamins will be balanced by appropriate iso-osmolar reductions in sodium chloride

M Diagnostic Agar Culture Media

Example (16) for a method of using the composition of Feature 1 as diagnostic agar or culture media for bacterial growth

Agar or culture media will be fortified with 5 mM glucose plus 5 mM sodium pyruvate N Metabolic Acidosis

Example (17) for a clinical method of reducing or ameliorating the level of metabolic acidosis in a patient using an effective dose ofthe composition of claim 1

Use of an i v solution fortified with pyruvate, examples are given in claim 3, "intravenous solutions"

O Preventing or Reducing Formation of Hydrogen Peroxide-Dependent Formation of toxic free radicals

Example (18) for a clinical method of preventing or reducing the formation of hydrogen peroxide-dependent formation of toxic free radicals (superoxide anion, hydroxyl radical) in a patient recovering from an ischemic insult (stroke, thrombosis, myocardial infarct) using an effective dose or infusion ofthe composition of Feature 1

Use of an i v solution fortified with pyruvate, examples are given in Feature 3, "intravenous solutions"

P Refreshments and Energizing Drinks

Commercial refreshments and energizing drinks usually contain sugar, protein, fat and a number of essential vitamins and minerals

Example (19) meal replacement drink (milk-shake-type) A 12 Fl OZ (355 ml) can containing additional

200 mg sodium pyruvate (about 5 mmol/1) Example (20) thirst quencher drink which does not contain fat and protein (Gatorade- type)

A 32 Fl OZ (946 ml) bottle containing additional

533 mg sodium pyruvate (about 5 mmol/1)

Example (21 ) nutritional water-soluble energizing powder which does not contain fat and protein

A 21 OZ (6g) bag (for 6 OZ (178 ml) water) containing additional

825 mg sodium pyruvate (about 7 4 mmol/178 ml = 42 mmol/1)

with the following standard ingredients vitamin C 2000 mg, potassium 400 mg, sodium 120 mg, calcium 100 mg, magnesium 40 mg, manganese 3 mg, zinc-ascorbate 4 mg, chromium-picolinate-ascorbate 20 micro g, vitamin Bl 0 75 mg, vitamin B2 0 85 mg, niacin-niacinamide 10 mg, vitamin B6 20 mg, vitamin B12 50 mg, pantothenic acid 5 mg Fructose (better glucose) as a sweetener in a base of citric, tartaric, aspartic, and malic acid Lemon flavors added plus potassium phosphate to adjust pH to near normal

Q Cereal Bars and Freeze-Dried Food Products

Commercial cereal bars and freeze-dried food products often contain complex carbohydrates, simple sugars, protein, saturated and unsaturated fats and a number of essential vitamins and minerals as well

Example (22) breakfast replacement bar (cereal-type) A 40 g bar containing additional

5 5 g sodium pyruvate (about 50 mmol) Example (23) freeze-dried action food for endurance hikers and military in the field A lOOg bag containing additional

14 g sodium pyruvate (about 126 mmol)

R. Vitamin Capsules

Commercial vitamin capsules are widely available Especially vitamin B 1 , thiamine, is absolutely essential for oxidative decarboxylation of pyruvate by pyruvate dehydrogenase in mammalian as well as yeast cells (alcoholic fermentation) Thus, for a composition according to Feature 1 to work efficiently, the water-soluble vitamin B 1 must be present in sufficient concentrations To enhance the effect of Feature 1 compositions, pyruvate and congeners will be combined with thiamine preparations Accordingly applicant contemplates the use of pyruvate capsules that contain vitamin Bl or a multi vitamin B system where thiamine is a main constituent

Example (24) pyruvate vitamin Bl

A capsule containing 250 mg vitamin B plus additional

550 mg sodium pyruvate (about 5 mmol)

Example (25) pyruvated vitamin B complex

A capsule containing all major vitamin B's plus additional

550 mg sodium pyruvate ( about 5 mmol)

S Dentrifice Products

Pyruvate added to toothpastes may help robotize the gingiva, especially when suffering from gingivitis or other tooth-decaying diseases Accordingly toothpastes enhanced with compositions according to Feature 1 are claimed Example (26) pyruvated toothpaste without vitamin Bl A tooth paste, 5 g, containing additional

550 mg sodium pyruvate (about 5 mmol)

Example (27) pyruvated toothpaste with vitamin Bl A toothpaste, 5 g, containing 250 mg vitamin Bl plus additional 550 mg sodium pyruvate (about 5 mmol)

T Hair Products

Hair shampoos containing pyruvate compositions may strengthen hair health and growth by roborizing the hair follicles A shampoo fortified by pyruvate is claimed

Example (28)- pyruvated hair shampoo without vitamin Bl A hair shampoo, 5 g, containing additional

550 mg sodium pyruvate (about 5 mmol)

Example (29) pyruvated hair shampoo with vitamin B 1 A hair shampoo, 5 g, containing 250 mg vitamin Bl plus additional 550 mg sodium pyruvate (about 5 mmol)

The above-mentioned compositions illustrate the advantageous use of pyruvate over presently known agent where pyruvate applications/supplementations/substitutions appear to be superior to or could markedly enhance current practices and clinical routines

VIII. CONTEMPLATED CLINICAL APPLICATIONS FOR Pyruvate A. ADVANTAGES OVER PRESENTLY KNOWN AGENTS

1) Cardiac ischemia/reperfusion damage, heart transplantation, and cardiopulmonary bypass: Pyruvate improves and accelerates recovery of cellular phosphorylation potential and ventricular function and inotropic state In contrast, traditional clinical lactate/glucose drips (infusions) are either without effect or likely even damaging to the phosphorylation potential and reperfusion function, this is the case because the recovering but still damaged cell needs to release, not take up, lactate in an effort to remove intracellular H^* and to reduce the concentration of NADH In the blood- perfused heart in situ, for example, a net release of lactate is usually a sign of hypoxia, ischemia, extreme metabolic stress Thus, infusing lactate into an organ that is attempting to remove its own endogenous lactate waste is clearly not optimal

With regard to acetate, this compound is known to lower the phosphorylation potential in experimental hearts and has also been found to impair reperfusion recovery in experimental situations As for clinical solutions containing aspartate or glutamate, both of which have been reported to be beneficial under some conditions, their mechanism is far from understood or proven, in particular, there are no known well characterized transporters on the plasma membrane that would allow efficient movement of these highly polar dicarboxylates into the cell In contrast, there is a high-capacity monocarboxylate transport system for pyruvate (and lactate) which, at least in heart and liver, has the capacity to transport pyruvate into and out ofthe cell and the mitochondria at rates that are more than sufficient under most, if not all conditions in health and disease

2) Post-surgical clinical stunned myocardium: Pyruvate likely improves the prolonged dysfunction and low-contractility state ofthe postischemic ventricle via enhancing the phosphorylation potential and possibly via removing intracellular H⁺ Also the specific anaplerotic (replenishing) effect on mitochondrial malate and oxaloacetate pools can only be considered desirable for the stunned myocardium. Pyruvate, unlike the clinically used adenosine (University of Wisconsin solution has 1 mM adenosine; adenosine is routinely injected to treat supraventricular tachycardia and other forms of arrhythmias), has no known serious hypotensive or bradycardic effects, pyruvate unlike adenosine is not a potent vasodilator and hence does not dangerously lower peripheral resistance ofthe circulation. Adenosine, in contrast to pyruvate, does not replenish the crucial mitochondrial metabolite pools. Pyruvate, in contrast to the clinically widely used inotropic "support" by adrenergic agonists (epinephrine, dobutamine) or vagus blockade by atropine, is not forcing restoration of postischemic function at low-ischemic phosphorylation potentials. Experimental data from guinea pigs and pigs show that norepinephrine, calcium-agonists (BayK 8644), hypercalcemia, and dobutamine as well, all normalize reperfusion function of the stunned heart, but this improvement consistently occurs without restoration or enhancement ofthe phosphorylation potential, which is in marked contrast to pyruvate therapy. In guinea pig myocardium reperfused after a 45 min low-flow ischemia, these traditional inotropic interventions restored function only at the expense ofthe cytoplasmic phosphorylation potential

Pyruvate can thus be seen as a novel class of inotropes, the METABOLIC INOTROPES, which produce a gentle and yet robust improvement of postischemic function, and that as permitted by or in accordance with the real-time cellular energy state. This is the principal difference with respect to current clinical adrenergic (inotropic) drug routines which force normalization of function ofthe damaged and/or disease-weakened heart (which, without the drugs, would be in a state of hemodynamic failure or stunning); but these inotropic regimens do not reenergize the cells nor do they create an anabolic situation to replenish crucial myocardial metabolite pools depleted by the prior damaging stimuli. Such forced restoration of performance can therefore occur at a potentially dangerous cost, a deleterious change in key metabolite levels resulting in a further fall in cellular energy level (phosphorylation potential); this will likely have obligatory but adverse consequences in cellular sodium homeostasis and calcium handling, which eventually likely combine to accelerate the development of acute and complete, and perhaps also essentially irreversible failure

Current clinical "inotropic support" for postoperative cardiac patients (who usually have aged or pre-damaged/weak hearts which probably function already at below- physiologic phosphorylation potentials) is essentially only a symptomatic or "cosmetic" treatment of ventricular function, without appropriate concern for correcting the underlying cause ofthe precarious energy balance and/or the associated Na⁺ and Ca²⁺ ionic imbalances and/or the reduced Ca²⁴ sensitivities ofthe excitation-contraction process Indeed, adrenergic agonists have long been recognized to lower the Ca²⁺-sensitivity ofthe myofilaments in the myocardium, a situation which makes it virtually impossible to rationalize the use of adrenergic support in stunned heart, as stunning is typically associated with exactly this type of reduced calcium sensitivity at the myofilament level [14-16] It comes therefore as no surprise that many cardiac surgeons view customary inotropic drug regimens, when applied to the stunned or spontaneously failing human heart, with concern and skepticism

Pathophysioiogically it is important to understand that inotropic drug therapy in the cardiac patient shifts the energy demand/supply balance toward higher demand, in aged hearts this may well occur in the presence of preexisting energy depletion/ion imbalance and/or a compromised coronary circulation (chronic ischemic coronary disease ofthe aged heart) This could put more myocytes at risk, at a moment when their recovery process has not yet begun or not yet been completed In marked contrast, pyruvate metabolic inotrope therapy shifts the energy demand/supply balance in favor of larger supply, the ensuing functional improvements are spontaneous, a natural consequence of improved cellular energy status, redox status, and ion homeostasis, all subsequent improvements of function are fully commensurate with and supported by the existing intrinsic energy supply status ofthe heart

Again, adrenergic inotropic drugs can cause desensitization towards calcium ofthe contractile elements, a shift toward the left in the tension/pCa⁺⁺ curve ofthe contractile elements [14]; myocardial stunning also is often associated with a similar calcium desensitization [15, 16] Consequently, it would not seem justified to continue the practice of indiscriminate use of adrenergic inotropic "support" in the post-surgical cardiac patient with the stunned heart syndrome Pyruvate as a metabolic inotrope would seem the more appropriate choice, even if it were only used in combination with classical adrenergic support in order to reduce the requisite dose of adrenergic agents

3) Metabolic acidosis The unique and special aspects of cellular pyruvate-H^* symport and metabolic pathways will help lower the size ofthe intracellular H^* ion pool Since all vital organs have substantial amounts of pyruvate transporters, lactate and pyruvate dehydrogenases and also mitochondria, pyruvate can be expected to counteract cellular acidosis body-wide, especially in heart, liver, lung, kidney, brain, and skeletal muscle This antiacidotic effect of exogenous pyruvate principally occurs according to the following mechanism when one pyruvate anion enters the cell, it will be obligatorily accompanied by one hydrogen ion, this hydrogen ion will then be used in the lactate dehydrogenase reaction to form the lactate anion (without H⁺), the lactate anion will then be exported from the cell together with one hydrogen ion, this latter hydrogen ion comes of course from the global cellular hydrogen ion pool, thus reducing cellular acidification The net effect is removal of one intracellular hydrogen ion per one pyruvate taken up and reduced to lactate or oxidized to carbon dioxide and water

Conversely, lactate infusion is contraindicated during systemic metabolic acidosis because lactate is a metabolic waste product under these conditions and produces rather than removes intracellular hydrogen ions The mechanism is as follows when one molecule of lactate enters the cell, it takes one hydrogen ion with it (much as pyruvate), then lactate will be oxidized to pyruvate generating rather than consuming another intracellular hydrogen ion (lactate dehydrogenase reaction) Thus, lactate oxidation to pyruvate via lactate dehydrogenase generates cytoplasmic hydrogen ions, whereas pyruvate reduction to lactate by reversal of lactate dehydrogenase removes cytoplasmic hydrogen ions Lactate infusion can therefore only exacerbate a preexisting cellular acidosis, while pyruvate infusion will likely ameliorate it This beneficial effect of pyruvate can be established of course only when there is some residual organ/cellular perfusion 4) Diabetic ketoacidosis and/or coma: Pyruvate infusion will ameliorate the metabolic acidosis as explained. Pyruvate will also directly improve cellular oxidative carbohydrate metabolism. The pyruvate dehydrogenase is inhibited in ketosis due to the high blood concentration of beta-hydroxy butyrate [17], This mitochondrial enzyme inhibition can be overcome simply by raising blood pyruvate concentration, the mechanism being the allosteric effect of pyruvate on PDH phosphorylation as explained above.

Pyruvate infusion during diabetic ketosis will not have the complications of insulin therapy: 1) Pyruvate's half live in blood is on the order of minutes, i.e. much shorter than that of insulin (order of 1/2 to 1 hour), as virtually all organs readily metabolize pyruvate. 2) Pyruvate will also not drastically lower blood sugar levels, as the glucose transport per se into skeletal and heart muscle as well as liver and kidney is not stimulated or inhibited directly by pyruvate. 3) Thus, dangerous hypoglycemia will not be a complication of systemic pyruvate administration to keto-acidotic diabetics.

Alternatively, if insulin treatment proves indispensable for some keto-acidotic or comatose patients, it would appear that the dose of insulin could be lowered by co- application of sufficient pyruvate. Thus pyruvate has the potential to substantially lower the dose and hence the risks of acute insulin administration during emergency medical care situations involving the diabetic patient.

5) Hypovolemic shock (auto accident, combat casualty, extensive internal or external bleeding): Hypovolemic shock is often associated with or progresses to systemic metabolic acidosis and a general deenergization of all organs; this will eventually lead to multiple organ failure and hardly manageable end-stage situations. Pyruvate as a natural alkalinizer that simultaneously enhances recovery of rephosphorylation ofthe cell and stabilizes the physiologic reduced state of vital -SH enzymes and transporters, can be expected to be much more effective than the traditional glucose, gluconate, lactate, or calcium drips alone. Combined with human full blood, pyruvate supplementation can be expected to enhance all known parenteral drip regimens.

6) Cardiogenic shock: The acutely or chronically failing heart is likely deenergized (low phosphorylation potential) and pyruvate metabolic inotrope therapy has the potential to bring about and/or expedite recovery from failure by reestablishing the cytoplasmic phosphorylation potential, the ion homeostasis and by mitigating any existing residual acidosis

7) Other forms of shock: Anaphylactic shock, endotoxin shock. Pyruvate is an ideal agent to improve oxidative energy and hydrogen metabolism ofthe heart and all major organs that contain lactate dehydrogenase and more than trace amounts of mitochondrial pyruvate dehydrogenase/carboxylase (heart, brain, lung, liver, kidney, skeletal muscle, smooth muscle)

8) Hemosiderosis: Pyruvate could be beneficial here too because it would be expected to possibly reduce the concentration of free iron in the cells, this can take place because pyruvate would reduce the concentration of [NADH]*[HJ which would attenuate, if not completely blunt the already mentioned reductive release of iron from binding sites such as ferritin, haem-protein or myoglobin Consequently, the damaging Fenton-type reactions could possibly be attenuated thus reducing the chronic overall cellular damage by toxic oxygen-derived free radicals

9) Strenuous exercise, physical exertion, endurance: Probably skeletal muscle and heart in particular, but perhaps also other organs, suffer from energetic exhaustion (low phosphorylation potential) due to prolonged strenuous/excessive physical stress Such a condition appears to be ideal for application of pyruvate metabolic inotropic therapy, since restoration ofthe phosphorylation potential and anaplerotic replenishment of key mitochondrial metabolites would be expedited and is of primary concern in such conditions Pyruvate, by effectively competing with lactate for transport into heart, liver, skeletal muscle and likely other organs will additionally alleviate intracellular hydrogen load thus enhancing the recovery process

10) Acute sickle cell crisis: Systemic hemolysis and local microembolism with subsequent ischemia are wide-spread The resulting anemia could favorably respond to pyruvate because, when applied in combination with adenine or inosine (two degradation products of ATP), levels of 2,3-diphosphoglycerate would increase in the remainder but still intact red cells, such a mechanism will certainly improve oxygen delivery to the tissues suffering from acute anemia combined with multiple microembolism and micro infarctions Thus, the need to immediately infuse donor blood or red cell concentrates with its associated problems (blood group incompatibilities) and risks of pathogens (e.g hepatitis, AIDS) may well be reduced.

The other complication, multiple systemic and organ-wide microembolism, will also probably be responsive to pyruvate, because pyruvate reduces the complications of ischemia itself and also those of subsequent reperfusion (after dissolution/organization/ recanalization ofthe micro emboli) as discussed above

11) Kidney dialysis (inpatient, outpatient, home) and peritoneal dialysis: Combat acidosis and maintain cells functional by optimizing energy status and hydrogen ions homeostasis in face of pathological concentrations of urea, creatinine, etc

12) Organ preservation and transplantation: Immediately after organ harvesting an initial perfusion with pyruvate-containing salt solutions/plasma expanders/hemoglobin substitutes instead of current pyruvate-free solutions (to remove cellular elements and clotting factors) would be superior, this is the case because pyruvate would reduce the amount of intracellular NADH, raise the phosphorylation potential, and optimize cellular ionic homeostasis combined with a stabilization ofthe membrane potential Also, since the procedures to collect and store donor organs usually create an ischemia/reperfusion-type condition which is typically followed by hypothermic storage and metabolic arrest, pyruvate therapy would be useful, since it is also directed at hydrogen peroxide-dependent hydroxyl radical damage Further, during cold storage of the organs, the presence of high levels of pyruvate would further minimize gradual accumulation of reducing equivalents, which in turn would minimize the reductive release of ferritin Fe²⁴ and hence reduce the probability of rewarming and reperfusion damage due to Fenton-type reactions. In this regard the currently often used University of Wisconsin-Solution and St Thomas Solution could be enhanced by adding, e g , 2-5 mM pyruvate

13) Medical emergency resuscitation: Shock-like situations, hypotonia, blood loss, extensive burns and similar conditions can be expected to respond favorably to pyruvate metabolic and anaplerotic treatment Quick reestablishment of cellular energy states, of crucial metabolite pools and transmembrane ion gradients, combined with gentle reduction of intracellular acidosis would be the main beneficial mechanisms Conventional lactate or acetate containing solutions cannot effectively combat intracellular acidosis, adenosine containing solutions would be contraindicated because ofthe danger of adverse cardiovascular side effects, pyruvate solutions would also avoid the potentially hazardous lowering of myocardial energy state that is likely to occur with catecholamine containing injectates. At the minimum, pyruvate therapy would promise to reduce the dosage of adrenergic inotropic drugs and also of bicarbonate and thus lower their potentially damaging side effects on cellular energetics and ionic homeostasis

14) Status asthmaticus: Any bronchiolar smooth muscle spasm is prone to cause secondary lung ischemia and certainly ventilation/perfusion imbalances in the lung This can lead to systemic hypoxemia and respiratory acidosis in severe cases. Current standard therapy is inhalation of aerosols usually containing a b-agonist such as albuterol or other congeners of isoproterenol, all of which are broncho dilators (smooth muscle relaxants). It seems possible that aerosolized pyruvate might also be efficacious, as pyruvate would help maintain the smooth muscle's metabolic phosphorylation potential and hence also its electrical membrane potential. The combined effect of these changes could favor bronchiolar relaxation (a complete relaxation of precontracted smooth muscle via this mechanism appears not impossible at this time, but remains purely speculative). Pyruvate could also be beneficial in combination therapies with albuterol, perhaps allowing the patient to inhale smaller amounts of adrenergic drugs at reduced frequency; the mechanism of such a pyruvate effect could again be the overall metabolic and energetic improvement ofthe bronchial smooth muscle

15) Spinal cord trauma and recovery therefrom: The enzymatic and metabolic machinery of neuronal tissue is not principally different from that of heart and muscle Consequently it can be expected that current surgical and medical treatment of spinal cord injury and rehabilitation will be enhanced by pyruvate administration To make pyruvate effective it should be injected intrathecally and intraventricularly. It seems possible that pyruvate treatment of spinal injury would help maintain the neuronal phosphorylation potential and hence also its electrical membrane potential This stabilizing effect could favor recovery of injured or stunned neurons and may even assist in dendritic, axon and synapse repair or growth Improved local neuronal function and recovery can theoretically be expected. Pyruvate could also be beneficial in combination with traditional surgical, medical and chiropractic therapies, perhaps allowing a faster and more complete recovery Whether severe spinal cord traumas will favorably respond to local intrathecal or intraventricular pyruvate doses only be speculated upon at the time of writing this disclosure

16) Nutritional cancer prevention or attenuation ?: Pyruvate as a major key to metabolic energy could conceivably improve the immune system's performance and defense against alien cells or microorganisms including cancerous cells A pyruvate dosage could exist that favor the human immune system over the cancerous cells especially if the latter have a degenerated monocarboxylate transport system with low affinity for pyruvate or lactate. Those cancerous cells metabolizing mainly lactate could be starved by pyruvate infusion, since pyruvate would be competing for lactate for transport into the cancer cells The overall effect could lead to substrate-deficiency ofthe cancer cells which in turn would reduce their capability to grow and become invasive In some cases pyruvate could perhaps prevent or at least delay the growth or appearance of cancerous developments, and in some instances it could even perhaps greatly strengthen the immune system's capability to eliminate completely the uncontrolled spread of metastases

With regard to all cells and organs that contain mitochondria, pyruvate is also an anaplerotic agent, since it helps maintain crucial cytoplasmic and mitochondrial metabolites at levels required for maintenance of normal function and metabolic/functional reserves This feature would seem to be important in situations during and after non-lethal, reversible injury Pyruvate also acts as a natural antidote for hydrogen peroxide (and hence the formation of oxygen-derived free radicals) which can produce wide-spread intracellular and extracellular damage Some major clinically relevant examples are presented in, but are not limited to those conditions discussed in section A 1 ) to A 14) above

C. KNOWN AND POSSIBLE USES FOR Pyruvate AND SIMILAR ALPHA- KETOACID CONGENERS

1) Antihypertensive

2) Examples of section A 1 ) to A 14)

3) Radiation overdose since radiation often produces free radicals and is associated with generation of hydrogen peroxide (nuclear power plant accident, X-ray overdose, Radiation sickness, Space radiation) pyruvate therapy, topical or intravenous, may prove beneficial as adjunct to current well tried treatment regimens

4) Incubation media and perfusion media in biomedical and agricultural research here pyruvate acts as a metabolic stabilizer in studies with isolated cells, subcellular organelles, microorganism etc , especially when these systems depend on oxidative phosphorylation and could easily become unduly acidotic or subject to spontaneous free radical damage

5) Blood banking high concentrations of pyruvate combined with adenine, inosine, and phosphate can raise red cell 2,3-DPG and rejuvenate stored blood

6) Antidote to hydrogen peroxide in cases where iron-and H,O,-dependent Fenton- type reactions are involved (reperfusion oxygen free radical damage) pyruvate would be expected to reduce, if not completely abolish the formation of cytotoxic oxygen-derived free radicals

7) Refreshments and commercial sport drinks any type of refreshment could possibly be enhanced by the inclusion of pyruvate replacing currently used lactate, fructose, sucrose or other poorly metabolizable carbohydrates Electrolyte-balanced drinks could very likely be enhanced by inclusion of pyruvate Pyruvate used as an enhancement of oral rehydration therapy would also fall into this category 8) Emergency fluids in submarines/airplanes/hot -air balloons/simulated high-altitude devices and decompression chambers/space flight and station- if a (sudden) drop in oxygen partial pressure occurs, readily available pyruvate drinks could improve the body's high altitude adaptation via stimulating 2,3-DPG synthesis in the red blood cells This type of pyruvate drinks should probably also contain adenine and or inosine in appropriate amounts There is a potential for acute flatulence, since the hydrochloric acid ofthe stomach will produce free pyruvic acid which may not be completely absorbed into the portal system; the intestinal bacterial flora will likely quickly convert pyruvate to water and carbon dioxide or alternatively perhaps decarboxylate it to acetate

9) Cosmetics and dermatology¹ Any application that targets the skin aging process, skin turgor, finger nail appearance, hair follicle growth or and hair shininess, might be enhancable by inclusion of pyruvate as a natural cellular energizer Eczema, seborrheic conditions, and other chronic skin irritations may be amenable to topical pyruvate therapy (ointments, shampoos, lotions) Sunscreens could possibly also be improved by addition of pyruvate, since it could act to absorb UV rays (maximum absorbance is at a wavelength of 210 nm) simultaneous with its anti-free radical effect which could counteract UV ray induced premature skin aging Perhaps even the incidence of skin cancer in populations with long-lasting exposure to sunlight and UV rays could be reduced by regular applications of ointments containing pyruvate as a supplement, if not as the main active ingredient

9) Antiobesity Diets Supplement to dietary food formulations

10) Psychotic crises Since the brain has substantial amounts of PDH and lactate dehydrogenase and because the blood-brain barrier may not be completely tight for pyruvate (nothing concrete is known about the latter issue), acute psychiatric disorders may be treatable by parenteral or better intrathecal application of pyruvate-enhanced solutions Certainly, classical lactate containing solution are contraindicated, since a brain that is out of its fine-tuned electrical and transmitter balance would probably have one or multiple foci of neurons and/or supporting glia whose energetic and ionic status is abnormal. Such hypothetical foci would be the target of metabolic pyruvate treatment Pyruvate applied intrathecally may also be considered in such situations

1 1 ) Total parenteral nutrition (TPN) Pyruvate appears a reasonable supplement to current TPN solutions, since it furthers replenishment of mitochondrial key metabolites, improves virtually universally cellular energy status, removes inhibition of carbohydrate utilization by allosterically relieving PDH inhibition caused by ketosis and high blood fatty acid concentrations

12) Broncho-pulmonary dysplasia ofthe premature infant The hyaline membranes impair alveolar oxygen diffusion and create a condition of systemic hypoxia, especially when this is combined with metabolic acidosis, then there are two conditions in which pyruvate therapy can be expected to be beneficial

13) Disseminated intravascular coagulation The consequences of the diffuse loss of blood, hypotonia, anemia, all can be expected to respond favorably to pyruvate treatment

IX. SUMMARY OF NOVEL FEATURES

The general aim is to improve both the basal status of a living cell or organ as well as its cell- or organ-specific functions without jeopardizing cellular energy status and without resorting to drugs that shift the energy demand/supply balance toward increased demand; the goal is to stabilize or if possible to enhance the cellular phosphorylation potential, ionic homeostasis acid-base status, and membrane potential, which automatically would normalize or restore normal cellular function. Another goal is to strengthen intrinsic defense against and tolerance towards sudden or chronic oxidative stress due to endogenously generated toxic free radicals due to reperfusion-type situations and radiation exposures; a further goal is to minimize accumulation of reducing equivalents during organ damage/ischemia, as the latter metabolites can inhibit glycolytic (non-oxidative) energy production, produce damage of mitochondrial function upon reperfusion and also trigger the dangerous cycle of reductive release of free Fe²⁺ which ultimately leads to generation of toxic free radicals.

Thus, the major targets for biological/clinical pyruvate applications are: 1) the cytoplasmic phosphorylation potential, the parameter that ultimately controls ionic homeostasis and powers all endergonic processes (e.g., muscle contraction, sodium and calcium pumping in excitable and quiescent cells, sodium and water reabsorption in kidney, synthesis and transport of transmitters/ hormones in brain, detoxification cycles in liver). 2) the obligatory stoichiometry between all known specific (phosphorylating) protein kinases and the [ATP]/[ADP] ratio; since the [ATP]/[ADP] ratio is a key component ofthe phosphorylation potential {[ATP]/([ADP]*[PJ)}, it is likely that the potential is also involved in regulatory enzyme or protein phosphorylation and hence in fine-tuning processes such as control of cardiac contractility or calcium-sequestration by sarcoplasmic reticulum Ca⁺⁺ pump. 3) the cellular NALT/NADH, redox status; by keeping the cytoplasmic [NAD⁺]/[NADH₂ ] concentration ratio relatively oxidized during ischemia, which can be expected to minimize hazardous secondary effects of NADH accumulation on mitochondrial energy coupling and reductive release of damaging Fe²⁺. 4) the cellular GSH/GSSG redox status, by keeping the reductive potential ofthe glutathione system high, essential protein-SH , transporter-SH, and enzyme-SH groups can be better maintained in their physiologic reduced state during states of oxidative stress. 5) Strengthening and expediting the cellular metabolic recovery process, especially with regard to mitochondrial oxaloacetate. 6) Metabolic alkalization and removal of intracellular hydrogen ion loads 7) Hydrogen peroxide, by direct interaction with pyruvate the peroxide is spontaneously degraded to biologically benign compounds instead of serving as precursor for the cytotoxic hydroxyl radical in Fenton-type reactions 8) The red blood cells whose ability to release oxygen in the microcirculation can be improved by pyruvate via the 2,3-DPG mechanism

Novel is here the focus on the phosphorylative, energetic, and reductive potential ofthe cell to correct a problem caused by pathological deenergization and/or acidification Novel is the approach to use a metabolic intervention (by pyruvate) rather than customary clinical drug therapies. Novel is also that administration of pyruvate likely strengthens the intrinsic natural antioxidant defense, that pyruvate itself can act as a hydrogen peroxide antidote, that it "clamps" the cytoplasmic redox status thereby preventing excessive accumulation of reducing equivalents during ischemia, which subsequently could impair mitochondrial function and also induce the release of Fe²" thus initiating damaging Fenton- type reactions This concept shifts the focus away from conventional symptomatic medical therapies to the treatment of the underlying biochemical and metabolic disorder (acidosis, deenergization, oxidative stress)

It is not the goal to obtain quick "cosmetic" improvements of organ function with disregard for the metabolic status of cells, organs or the entire body, such therapies, while often temporarily producing improved performance, often also produce further metabolic derangements of a system that was already in a preexisting precarious metabolic state Experience has demonstrated that not rarely such therapies, after initial success, speed up ultimate cellular and organ failure The main principal goal is therefore to first improve the metabolic status ofthe system, then enhance the systems physiologic performance and that only as safely permitted and supported by the real-time cellular energetics

IX. REFERENCES

1 Mallet RT et al 1993 In Interactive Phenomena in the Cardiac System, Sideman S , Beyar R edts , Plenum Publishing Corp , in press

2 Nohl et al , Free Rad Res Comms 1993,18 127-137

3 Downey JM Annu Rev Physiol 1990 52 487-504

4 Paller MS et al J Clin Invest 1984, 74 1 156-1 164

5 Camporti M Lab Invest 1985,53 599-623

6 Salahudeen AK et al J Clin Invest 1991 ,88 1886-1893

7 Nicotera et al Drug Metabolism Rev 1989,20, 193-201

8 Cohen G 1985 In Handbook of Methods for oxygen radical research Greenwald RA edt , CRC Press, pp 55-64

9 Funk et al Eur J Biochem 1985,152 167-172

10 Voogd A et al J Clin Invest 1992,90 2050-2055

1 1 Reed DJ Annu Rev Pharmacol Toxicol 1990,30 603-631

12 Zimmer HG et al J Mol Cell Cardiol 1981 , 13 531-535

13 Duhm J Biochem Biophys Acta 1974,343 89-100

14 Holroyde MJ et al Biochem Biophys Acta 1979,586 63-69

15 Kusuoka H et al Circ Res 1990,66 1268-1276

16 Hofman PA et al Circ Res 1993,72 50-56

17 Bunger R et al Eur J Physiol 1983,397,214-219

Claims

What is Claimed is

1 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

2 The method in accordance with Feature 1 wherein said cation is an alkali or alkaline earth metal, —

3 The method in accordance with Feature 2 wherein the alkali metal is sodium —

4 The method in accordance with Claim 3 wherein R is an alkyl group containing 1 to 12 carbon atoms —

5 The method in accordance with Claim 4 wherein the alkyl group is methyl —

6 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a parenteral fluid containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 carbon atoms; benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl; phenyl; naphthyl; substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation.

7. A method according to Claim 6 wherein the parenteral fluid is selected from the group comprising total parenteral nutritional fluids; kidney and peritoneal dialyses fluids; volume and plasma expanding fluids; pyruvate/acetate near-isotonic solutions; lactate/acetate-free pyruvate isotonic solutions; normal saline solutions; hemoglobin-substitute containing solutions; vitamin supplement product; and cardioplegic solutions.

8. A method according to Claim 6 wherein the amount of active ingredient is effective in reducing and/or ameliorating intracellular acidosis.

9. A method according to Claim 6 wherein the amount of active ingredient is effective in neutralizing hydrogen peroxide through hydrogen peroxide-alpha-ketocarboxylate interaction to inhibit the formation of toxic-free radicals.

10. A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a rehydration fluid, which may contain electrolyte balances, containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms; substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 atoms; benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation.

11. A method according to Claim 10 wherein the rehydration fluid contains electrolyte balances.

12. A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a topical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl; phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethvlamino, and M is a cation

13 A method according to Claim 12 wherein the topical composition is selected from the group comprising medicinal soaps; medicinal shampoos, sunscreens, medicinal ointments, vitamin capsules, dentrifice, mouthwash; douche solutions, and medicinal baths

14. A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition selected from the group comprising an antibiotic and antiphlogistic containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, within the alkylene chain, halogen amino, alhylamino of 1 to 4 carbon atoms dialkylamino of 1 to 4 carbon atoms in each alkyl, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

15 The method in accordance with Claim 14 wherein said composition is administered by intramuscular injection

16 The method in accordance with Claim 15 wherein said composition is an antibiotic

17. The method in accordance with Claim 16 wherein said composition is an antiphlogistic

18 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition for the treatment of local skin disorders, selected from the group comprising an antibiotic and antiphlogistic having as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation.

19. A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof an aerosolized pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms; substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, (carboxyalkylene of 1 to 20 carbon atoms within the alkylene chain, halogen amino, alkylamine of 1 to 4 carbon atoms, dialkylamino of 1 to 4 carbon atoms in each alkyl group or phenyl); alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 atoms; benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl; phenyl; naphthyl; substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation alone or in combination with a bronchodilating agent

20. A method in accordance with Claim 19 resulting in the amelioration or prevention of the onset of abnormal respiratory conditions caused by a reactive airway disease.

21. A method in accordance with Claim 20 wherein said reactive airway disease is selected from the group comprising asthma and bronco-pulmony dysplasia.

22. A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising perfusion of a mammalian organ in need thereof with pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

23 The method in accordance with Claim 22 wherein said mammalian organ is selected from the group comprising heart, liver, kidney, brain, spleen vessels, arteries, endothelium, pancreas and glands

24 A method for enhancing the phosphorylation potential within bacterial or viral cells in culture or cloning media in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising adding to the incubation solution for said cells a pharmaceutical composition containing as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

25 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions thereby enhancing physical endurance or refreshment comprising administering to a mammal in need thereof a food product containing a pharmaceutical composition having as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms; substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl; phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

26 The method in accordance with Claim 25 wherein said food product is a beverage drink

27 The method in accordance with Claim 26 wherein said food product is a confectionery food

28 The method in accordance with Claim 27 wherein said food product is selected from the group comprising candies and pastries

29 A method for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising administering to a mammal in need thereof a pharmaceutical composition containing ( 1 ) a thiamine (B 1 ) vitamin capsule and (2) a therapeutically-effective amount of a salt of an alpha-ketocarboxylic acid having th formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted a.,- ι,t^' 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl; phenyl; naphthyl; substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-. or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation.

30. A composition of matter for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms; substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 carbon atoms; benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl; phenyl; naphthyl; substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation.

31. A parenteral fluid useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically-effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms; substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 carbon atoms; benzyl; substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl; phenyl; naphthyl; substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to

4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation.

32 A composition according to Claim 31 wherein the parenteral fluid is selected from the group comprising total parenteral nutritional fluids, kidney and peritoneal dialyses fluids, volume and plasma expanding fluids, pyruvate/acetate near-isotonic solutions; lactate/acetate-free pyruvate isotonic solutions; normal saline solutions, hemoglobin-substitute containing solutions, vitamin supplement product, and cardioplegic solutions

33. A rehydration fluid, which may contain electrolyte balances, useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically-effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl; phenyl, naphthyl; substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

34 A composition according to Claim 33 wherein the rehydration fluid contains electrolyte balances

35. A medicinal composition useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms; benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

36 A composition according to Claim 35 is selected from the group comprising medicinal soaps; medicinal shampoos, sunscreens, medicinal ointments, vitamin capsules, dentrifice, mouthwash, douche solutions, and medicinal baths

37 An antibiotic or antiphlogistic composition useful for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, within the alkylene chain, halogen amino, alhylamino of 1 to 4 carbon atoms dialkylamino of 1 to 4 carbon atoms in each alkyl, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbons atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl ammo, diethylamino, and M is a cation

38 The composition according to Claim 37 wherein said composition is administered by intramuscular injection

39 The composition according to Claim 38 wherein said composition is an antibiotic

40. The method in accordance with Claim 39 wherein said composition is an antiphlogistic.

41. An aerosolized pharmaceutical composition for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms; substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, (carboxyalkylene of 1 to 20 carbon atoms within the alkylene chain, halogen amino, alkyl amino of 1 to 4 carbon atoms, dialkylamino of 1 to 4 carbon atoms in each alkyl group or phenyl), alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 atoms; benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring); adamantyl; phenyl, naphthyl; substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation alone or in combination with a bronchodilating agent.

42. A perfusion fluid for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms; substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms; alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl; phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

43. An incubation solution for enhancing the phosphorylation potential within bacterial or viral cells in culture or cloning media in order to prevent the deterioration or promote the restoration and preservation of normal cell functions comprising a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation

44 A food product for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions thereby enhancing physical endurance or refreshment comprising a pharmaceutical composition having as an active ingredient thereof a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl, dimethyl amino, diethylamino, and M is a cation 45 The food product in accordance with Claim 44 wherein said food product is a beverage drink

46 The food product in accordance with Claim 45 wherein said food product is a confectionery food

47 The food product in accordance with Feature 44 wherein said food product is selected from the group comprising candies and pastries

48 A vitamin supplement product for enhancing the phosphorylation potential within the cells of a mammal in order to prevent the deterioration or promote the restoration and preservation of normal cell functions hereby enhancing physical endurance or refreshment comprising a therapeutically effective amount of a salt of an alpha-ketocarboxylic acid having the formula R-C(O) (CO)OM wherein R is alkyl of 1 to 12 carbon atoms, substituted alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 3 to 6 carbon atoms, benzyl, substituted benzyl (wherein the substituent is methyl, phenyl on the alpha carbon atom or the substituent is methyl, dimethyl, halo, dihalo, or ethoxy on the phenyl ring), adamantyl, phenyl, naphthyl, substituted phenyl or substituted naphthyl (wherein the ring is mono-, di-, or trisubstituted and the substituents are alkyl of 1 to 4 carbon atoms, halo, alkoxy of 1 to 4 carbon atoms, phenoxy, trihalomethyl. dimethyl amino, diethylamino, and M is a cation