The Theoretical Premises of the ICDR Method

The Role of Carbon Dioxide in the Vital Processes of the organism (Chemical and Biochemical Aspects).

1. Forms of Carbon Dioxide

It is well known from the results of many investigations that carbon dioxide in the organism of Man and animals is not only a component of buffer systems and a regulator of respiration but is also a factor in the control of cardiac activity and of the vascular, hormonal, nervous and digestive systems.

At present, it can be said that carbon dioxide takes part in the control of many of the most important physical functions in the organisms of humans and animals.

When carbon dioxide is mentioned, often only one of its forms is implied, that is, the gaseous CO2 or carbon dioxide gas.

When that is the case, what is being overlooked is that five different forms of carbon dioxide can be found in an organism:

1. dissolved carbon dioxide gas (dCO2);
2. carbonic acid (H2CO3);
3. bicarbonates (HCO3- );
4. carbonates (CO3)2-; {adapted}
5. carbamates (R - NH COOH); {adapted}

{The following equilibrium equations adapted from an equilibrium diagram}

CO2 <=> pCO2

pCO2 + H2O <=> H2CO3

pCO2 + H2O <=> HCO3- + H+

pCO2 + R_NH2 <=> R_N_(H2+)_COO-

pCO2 + OH- <=> HCO3-

H2CO3 <=> HCO3- + H+

HCO3- <=> (CO3)2- + H+

2. The Role of Carbon Dioxide in the Respiratory Process.

The main source of carbon dioxide in an organism is indigenous carbon dioxide. The intensity of CO2 formation in tissues in the processes of metabolism can vary in a considerable range depending on various factors, but the normal CO2 concentration in blood and other biological fluids in a organism is not subject to any major fluctuations. As a rule, it is maintained at a strictly, defined level and any excess is immediately removed. This removal is ensured by the function of a complex system of mechanisms controlling the concentration. Of these the most important is the processes of neutralisation and rapid removal of excess CO2 from the tissues into the environment. This mechanism can be understood as follows.

The CO2 which forms inside cells penetrates into the extracellular fluid and then into the bloodstream. The mechanism of CO2 transfer through cellular membranes has not yet been fully established. All that is known is that membranes are more permeable to CO2 and H2CO3 than to HCO3-. As the CO2 permeates the interstitial fluid and then enters into the bloodstream most of it is transformed into HCO3-. This process is catalysed by the enzyme, carboanhydrase, (CA) of erythrocytes:

CO2 + H2O <=(KA)=> H2CO3 {+} H+ + HCO3

As a consequence of this process the pH inside erythrocytes begins to fall. At certain pH values the glyoxaline imidazole group of histidine and the amino group of valine, both situated on the chains of hemoglobin, begin to incorporate H+ ions which leads to the acquisition of a positive (+) charge. This charge stabilises the salt bridges which have the function of preserving the desoxystructure of hemoglobin. Apart from this process, another process of replacement of Cl ions by carbon dioxide takes place in the net of electrobathic interactions, thus leading to an increase in the stability of the quaternary structure of hemoglobin. Further, the carbon dioxide influences the binding of DPH and binds to -amino groups, which also stabilises the desoxystructure of hemoglobin (the Verigo-Bohr effect).

On account of the difference in concentrations of oxygen in erythrocytes and tissues, the oxygen diffuses into the tissues. As a consequence of the cooperative O2-binding effect, the separation of succeeding oxygen molecules is made less difficult.

The summary results of the two processes are the maintenance of a practically constant pH, which aids the diffusion of oxygen and the retention within the erythrocytes of K+ ions, which were neutralised by HbO2 earlier and are subsequently neutralised by the forming HCO3- ions.

As a result, the major part of CO2 which diffused out of the tissues into the erythrocytes transfers from capillaries into veins in the form of HCO3- ions contained in erythrocytes.

Due to this isoacidic shift, the ratio of HCO3- in erythrocytes (e) to HCO3- in plasma (p) is altered as a result of the HCO3- increase in erythrocytes. No longer is the ratio of HCO3-e/CL-e maintained at a level equal to the ratio of HCO3-p/CL-p which had been established through the manifestation of the Gibbs-Donnan effect.

The tendency of HCO3- to leave the cells rises and the resulting loss of HCO3- ions is compensated by Cl- ions entering from the plasma until a new equilibrium is achieved. The final result of this process is the situation where a major portion of the total amount of CO2 which had entered into the erythrocytes in the capillaries, is then transferred in the form of HCO3- into venous plasma.

It should be noted that the transformation of HbO2 into HbH+ and the resulting buffer action of this transformation does not influence the osmotic pressure due to the hemoglobin of cells because the amount of hemoglobin does not change, but the total effect of the isoacidic and chloride shifts does result in an increase in the total quantity of anions and this does raise the effective osmotic pressure in cells. As a consequence, water is redistributed between the cells and the plasma.

This process takes a reverse direction in the lungs. Because of the difference in pressures oxygen penetrates into erythrocytes and forms acid HHbO2. As a result of its disassociation H+ ions are formed which react with HCO3- forming H2CO3. H2CO3 splits into CO2 and H2O under the influence of carboanhydrase:

H2CO3 => H2O + CO2

CO2 and H2O are released into alveolar spaces.

The release of CO2 leads to a shift in Bohr's reaction to the left side which results in a normalisation of pH and facilitates the binding of oxygen to hemoglobin.

While this process takes place in the lungs, the processes of carbonate splitting and of the transfer of proteins into a free state proceed at the same time.

Bicarbonate, which has entered the blood reacts with carbonic acid and forms the pair HCO3-/H2CO3 which is a component of the main buffer system in extracellular fluid. The need for the existence of precisely such a buffer is determined by many reasons:

1. There is considerably more HCO3- than other buffer compound in the extracellular fluid:
2. The intake of CO2 is not limited:
3. The physiological mechanisms which maintain the normal pH value of extracellular fluid do so by regulating the concentrations either of HCO3- or CO2. Van Slyke and then Warburg found that there is a linear relationship between pH,, dCO2 and HCO3- in blood described by the Henderson-Hasselbach equation of:
   pH = 6.1 + lg([HCO3-]/[pCO2])
4. the HCO3/H2CO3 buffer system functions in concert with the Hb system, as described above.

As in all buffer systems, the pH does not depend on the absolute concentrations of the buffer components but on their ratios (Tables 1, 2, 3 from the book: Robinson J.R. Grounds for the Regulation of an Acid-Alkaline Equilibrium. Moscow, Meditsina, 1969).

Table 1
The effect of dCO₂ on the pH of a solution containing 27m. - equiv. of bicarbonate per 1L, at 37^oC.

Table 2
The effect of changes in the concentration of bicarbonate.

dCO2 mm Hg	40	40	40
H2CO3- m.-equiv/l	1.35	1.35	1.35
HCO3- m.-equiv/l	54	27	13.5
HCO3-/H2CO3	40	20	10
pH	7.7	7.4	7.1

Table 3
Interaction between changes of dCO2 and HCO3

However, it should be noted in Table 3 that although the pH is kept constant, the buffer capacity of the plasma is decreased. The effectiveness of the HCO3- buffer system increase considerably in the presence of erythrocytes.

The described combination of buffer properties, where one of the components is a gas and where automatic self regulation is achieved by means of intracellular Hb, lead to a remarkably stable pH being maintained in blood plasma.

In addition, the organism uses two other stabilising systems, the respiratory system and the kidneys which correspondingly control (H2CO3) and (HCO3-), and thereby produce additional possibilities for the maintenance of a constant pH in extracellular fluid.

3. Control of CO2, HCO3- and H+ Levels in the Tissues of Animals.

As distinct from HCO3- (fixed anion concentration), (H2CO3) is determined solely by one parameter: the partial pressure of CO2 in the gaseous mixture in equilibrium with extracellular fluid as present in alveolar air. This pressure in turn depends on the rate at which the CO2 passing out of the blood in the lungs is diluted by atmospheric air and so it depends on the depth and the frequency of breathing. The character of respiration is regulated by a respiration centre in the nervous system; apparently this centre is sensitive to pH and dCO2 in the extracellular fluid. When the pH of the extracellular fluid falls below a normal value because of a decrease in HCO3-, respiration is stimulated which leads to a lowering of alveolar dCO2 and consequently of extracellular (H2CO3).

This restores the ratio of (HCO3-)/(H2CO3) to the normal value of 20: 1 and leads to the return of pH to the normal value. The resultant fall in CO2 pressure in plasma acts in an opposite direction upon the regulating nervous centre, so the compensation would never be completed if only this regulatory mechanism was functioning.

At high levels of plasma pH the frequency of respiration falls and alveolar dCO2 and therefore (H2CO3) in plasma rise which then shifts the pH down to a normal value. In this case full compensation is not achieved because the elevated plasma (H2CO3) acting on the respiratory centre produces an effect opposite to that of increased pH. if the frequency of respiration is considerably lowered, a decrease in dO2 will stimulate an increase in respiratory activity.

At the time when the respiratory mechanism compensates for deteriorations of acid-alkaline equilibrium by means of H2CO3 regulation in extracellular fluid, kidneys participate in pH control by regulating HCO3-. Kidneys counteract the decrease in extracellular pH, caused by the increase of alveolar dCO2 or by the decrease in HCO3-, by the two ways available to them, namely, by liberation of H+ in the form either of undissociated acid or NH4+.

Renal compensation, under the conditions which could bring about an increase in extracellular pHi is accomplished by means of a decrease in HCO3- of extracellular fluid.

There is evidence that simultaneously with these mechanisms which compensate for extracellular pH fluctuations the "potassium-sodium pump" also comes into action. Muscular tissues, epithelial cells of renal ducts, and, possibly, all other cells have an ion-exchange mechanism which carries out exchange of either K+ or H+ or both of these ions for Na+ through the cell membrane. Due to this exchange, the cell content can participate in maintaining extracellular pH.

With increase in extracellular HCO3-, Na+ enters the cells in exchange for H+ and K+. Protons react with extracellular HCO3- and the CO2 formed is removed with exhaled air, K+ is removed with urine together with the equivalent amount of HCO3-. With the decrease in pH, Na+ leaves the cell and H+, K+ ions enter it.

By direction of shift of active reaction of blood all changes of acid-alkaline balance are divided into acidosis and alkalosis, and due to the reason causing changes in pH of blood they are divided into metabolic and respiratory.

4. Shifts of Acid-Alkaline Equilibrium Respiratory Acidosis:

This is characterised by the following factors: dCO2> normal range; pH< 7.36; (HCO3-)..normal range or above it. These states are observed, with the inhibition effect on the respiratory centre by morphine, during inhalation of gaseous mixtures containing a high percentage of carbon dioxide, in severe cases of pneumonia, oedema and emphysema, during spontaneous arrest of respiration, with asthma and suffocation.

Respiratory Alkalosis: Factors:dCO27.44; (HCO3-) ... normal range or slightly lower than the normal range. This state is developed during hyperventilation of lungs, with severe inflammation of internal sex organs, hypoxia and impaired blood circulation. With prolonged respiratory alkalosis an increase in lactic acid, which forms due to the incompletely oxidised pyruvate, that is, in the absence of sufficient oxygen concentrations in tissues, is constantly observed.

Metabolic Acidosis: Factors: PH [ normal range; (HCO3-)[ normal range; dCO2 [normal range. Metabolic acidosis develops in cases of origination of excessive amounts of non-volatile acids in extracellular fluid which shows itself:

1. with the intake of food containing a large amount of non-volatile acids;
2. with the formation of large amounts of organic acids (lactic, acetoacetic, pyruvic, etc.) during oxygen insufficiency in tissues;
3. due to deterioration of the renal function in elimination of hydrogen ions;
4. with a relative excess of non-volatile acids, due to the loss of bases during vomiting, diarrhoea, fistula, etc.

Metabolic Alkalosis: Factors: increase in PH; (HCO3-)> normal range; dCO2 >normal range. Metabolic alkalosis develops in the case of loss of non-volatile acids or in the case of loss of K+ (this results in excessive elimination of H+ by the kidneys).

Extensive distribution of the above mentioned states of the organism of animals and man became a main reason for numerous investigations carried out since the end of the 19th century up to the present time. These have been devoted to the study of characteristic properties of metabolism in tissues during various conditions of alkalosis and acidosis. However, approaches to the study of this problem and explanation of obtained results, for a long time, in the majority of cases, were based solely on the role of change in concentration of hydrogen ions and hardly any attention has been paid to the important metabolic meaning of change of various forms of carbon dioxide in tissues under these conditions.

In recent years interest in clarification of the metabolic meaning of carbon dioxide has increased. It has been established that CO2 takes part in regulation of numerous key points of metabolism and respective physiological functions of an organism. In particular, it has been shown that the intensity of biosynthetic processes of basic organic components of animal tissue is directly proportional to the intensity of carbon dioxide fixation processes (reactions of carboxylation).

5. Reactions of Carboxylation

Only recently has it become known that processes of carbon dioxide fixation lie at the basis of all important metaboIic and primarily biosynthetic processes in tissues. Up to the present a number of reports has been published on these topics. Summing up accumulated data, it is possible to conclude that a great number of reactions exists in tissues of an organism, which depending on the source of used energy can be divided into three groups:

a) reactions taking place at the expense of energy of ATP(these are reactions of carboxylation of propionyl - CoA, pyruvate, acetyl CoA, -methyl-crotonyl-CoA, geranyl-CoA, urea and formation of carbomylphosphate). Here fixation of carbon dioxide is preceded by its activation, which is accomplished by its relationship with ATP. An important role in this case is assigned to biotin a prosthetic group of almost all carboxylases catalysing ATP - dependent reactions of carboxylation.

These process of carboxylation using biotin can be represented by the scheme: {Modified after scanning}

enzyme - biotin + HCO3- + ATP <==Mg2+==> enzyme - biotin + HO-CO-O-PO(OH)2; enzyme - biotin + HO-CO-O-PO(OH)2 <==> enzyme - biotin CO2 + ADP + Pi

The formed complex enzyme-biotin-CO2 is able to transfer carboxyl group;

b) reactions proceeding at the expense of the energy of the reduced forms of pyridinenucleotides (this is a reaction of carboxylation of alpha-ketoglutarate, pyruvate, ribulose-5-phosphate, serine, glutamate, acetate);

c) reactions not requiring energy from outside (5-aminoimidazole-ribotidecarboxylase, phosphoenylpyruvate-carboxylase, and phosphoenolpyruvate carboxy - transphosphorylase reactions belong to this group of reactions.)

Analysing the data on CO2 fixation in all three types of reactions, a number of factors influencing this process can be indicated. Of these, the main factors are the following:

1. variations of concentrations and magnitudes of the relationship of basic forms of carbon dioxide in tissues (HCO3- and dCO2);
2. change in concentrations of certain ions of metals which are activators and inhibitors of carboxylases;
3. change in concentrations of substrates subjected to carboxylation;
4. change in the degree of provision of cells by energy sources as well as by a number of other metabolit-allosteric effectors of carboxylases;
5. change in concentrations of carboxylasaes coenzymes in tissue.

The first is the most universal from all indicated ways of the effect on the carbon dioxide fixation because in all reactions of carboxylation dCO2 and HCO3- are substrates. Concentration of the substrate in enzymatic reactions can even play a certain role in intensity of the process of its transformation. It was shown in many experiments under the conditions of normal pH of incubative medium the intensity of carbon dioxide fixation in tissues IN VITRO increased as its concentration increased. Thus, it can be concluded that the intensity of carboxylation reactions in many instances is determined by concentration and by magnitude of dCO2 and HCO3- ratios in tissues. Under the conditions of constant pH the possibility exists for considerable increase in intensity of the carboxylation processes by increasing carbon dioxide concentration up to a certain level in tissues.

It has been established in many investigations that metabolic importance of carbon dioxide is not limited by the above mentioned suggestions. It was found that when changing carbon dioxide level in tissues, the activity of a wide variety of enzymes of glycolysis, tricarboxylic cycle and nitrogenous metabolism changed and the formation of some hormones, etc.increased.

6. Role of Carbon Dioxide and Regulation of Reactions of Tricarboxylic Acids Cycle (TAC).

{Diagram of the Tricarboxylic Acids Cycle omitted}

Considering the role of carbon dioxide in the regulation of reactions of tricarboxylic cycle the following should be noted. Many biochemists elaborated the erroneous idea that the cycle of tricarboxylic acids was only a mechanism of oxidation of acetyl-coenzyme A. For this reason, such names as "energy-mill", "kettle", etc. were secured for TAC, and almost all biochemistry textbooks and even in many monographs, TAC was presented as a sequence of reactions with the process occurring only in a clockwise direction.

In consequence often the question is raised: What is the role of carbon dioxide in providing normal functioning of the cycle, in the sense that in one cycle in the reactions of carboxylation two molecules of CO2 are formed, but only one CO2 is used in the formation of oxaloacetate (carboxylation of pyruvate). Further, because during a cycle the oxaloacetate is again regenerated, then it is sufficient that once a molecule of oxaloacetate has been formed it will constantly maintain the cycle in a functioning state. An impression can be had that the carbon dioxide is not practically required for the functioning of the cycle. Such suggestions are fundamentally erroneous. The error of these notions is as follows.

1) Almost all reactions of TAC are reversible. They proceed in both directions in a cell. At reverse direction (in an anticlockwise direction) the carbon of carbon dioxide will not be liberated but used in the reactions of carboxylation for build-up of a carbon frame of a number of organic compounds, this means then that carbon dioxide should come from outside. In this case, the concentration of bicarbonates and CO2 in a cell will greatly influence the rate of these reactions and as a consequence the state and function of TAC.

2) TAC plays an important role by providing the basis for oxidising and biosynthetic processes where it serves as a mechanism for generating numerous substrates used in the synthesis of many amino acids and of the nitrogenous bases of purine and pyrimidine nucleotides for carbohydrates and lipids.

3) A considerable number of TAC metabolites are used in the process of transmembrane transport of various anions and other substances. There is a view that about 40% of the TAC metabolic pool is consistently removed and used in various biosynthetic processes. If this view is right, then normal functioning of TAC is possible only when the means for continuous formation of its metabolites are not disrupted.

Because 4 of the 9 metabolites (oxaloacetate, malate, succinate, isocitrate) are formed as a result of the carboxylation of pyruvate and, phosphoenolpyruvate, propionyl-coenzyme A, alpha-ketoglutarate, functioning of the cycle is impossible without constant inflow of bicarbonate and carbon dioxide.

Summarising the results of various experiments in this respect (Adler et al., Ostberg et al.), it can be said that the activation of TAC is determined largely by the magnitude of pH of biological fluids and by the level of bicarbonates and CO2 contained. At the same time, under the conditions of constant pH of biological fluids, the activity of TAC also increases if their concentration of carbon dioxide increases. The experimental results of Berri, Kun and Werner are among convincing confirmations of the importance of carbon dioxide and the processes of its fixation in the regulation of oxidation processes of organic substances in the cell. These experiments showed that during incubation for 40 minutes of the isolated liver cells of rats in a medium containing bicarbonate, the concentration of malate increased approximately 7 times in comparison with the concentration in a medium without bicarbonate (phosphate buffer), although pH of the media were the same.

Thus it can be concluded that carbon dioxide, being one of the basic final products of metabolic reactions of TAC, appears as one of its powerful regulators. The results of the latest investigations confirm that the mechanism of regulatory effect of carbon dioxide on the functioning of TAC is not only in the fact that it is one of the components of carboxylation or decarboxylation reactions. The fact is that the activity of certain enzymes, which are not related to the processes of carboxylation and decarboxylation, can be substantially changed at constant pH of the medium depending on the changes in HCO3- and CO2 concentrations in the medium (even in the ranges of physiological fluctuations).

Apart from this the obtained data show that the level of carbon dioxide determines not only the intensity of functioning of the indicated reactions, but also influences the level of biosynthetic processes in an organism, which also are in continuous dependence on the functioning of tricarbonic cycle processes of metabolism of lipids, carbohydrates, proteins, nucleotides and nucleic acids belong to such processes.

7. The Role of Carbon Dioxide in Processes of Biosynthesis

Biosynthesis of Amino Acids: Synthesis of such amino acids as glycine, serine, citrulline, glutamic and aspartic acids and respectively of their amides, glutamine and asparagine, almost fully depends on the intensity of carboxylation of the corresponding substrates:  {diagram modified}

   pyruvate + CO2 + ATP ---------------->|
                                        |
   phosphoenolpyruvate + CO2 + GDP ----->|
                                        |
   pyruvate + CO2 + NADP.H2-------------->|
                                        |
   propionyl -CoA + CO2 + ATP----------->|
                                        |
 |<-------------------------------------| 
 |
 |-> oxaloacetate + NH3--> aspartate + NH3--> asparagine
        |
        | (acetyl CoA)
        |
        |---> citrate
               |
               |--> isocytrate--> succinate
                     |         -> glyoxylate-> glycine
                     |
   |-----------------|
   |
   |-> alpha-ketoglutarate--> glutamate--> glutatmine

serine + CO2 + H3 + NAD.H2  ------> 2 glycine + NAD+

  CO2 + H3 +ADP ---> carbomylphosphate
                       |
                       |===> citrulline ----> arginine
            ornithine-->

Biosynthesis of Nitrogenous Bases of Nucleotides

Biosynthesis of purine nucleotides: The first important data from biosynthesis of purine bases were obtained by Beaukenen who fed animals with various labelled precursors and determined spots of inclusion of the labelled atoms in the purine ring. These experiments were carried out on birds whose nitrogen is liberated mainly in the form of uric acid, which represents a derivative of purine. It was found that two nitrogen atoms N (3,9) of the purine ring were derived from the amide group of glutamine, the third nitrogen atom (1) was derived from aspartic acid and finally, the last (7) had its origin in glycine.    {diagram modified}

                                   8  
                               --- C ===
                               |       |
                               |       |
                             9 N     7 N      
(asparate)  (formate)          |       |     (CO2)
   === N ----- C ===== N ----- C ===== C ----- C ====
   ||  1       2       3       4       5       6    ||
   ||                                               ||
   ==================================================

                         3,9 amide nitrogen of glutamine
                         4+5+7 glycine

The fourth and fifth carbon atoms also had their origin from glycine. Thus, glycine molecule gave three atoms enclosed in a frame. The second and eighth carbon atoms originated from formate, and the sixth carbon atom came from CO2.

Biosynthesis of Pyrimidine Nucleotides

Carbomylphosphate + aspartic acid   <====> 

                                   -H2O

alpha carbomoylaspartic acid    <========>

                                   +H2O

                         NADH  H+

L-dihydro-orotic acid  <==========>  orotic acid

                         NAD+

            {Adapted from Diagram}

           [CO2]
    1       2      3      4        5      6
    NH ---- CO----HN3-----CO-------CH-----C(COOH)---
    |                                              |
     ----------------------------------------------

The second carbon atom of pyrimidine is formed only at the expense of carbon of the CO2 molecule. Thus, this suggests that without carbon dioxide, biosynthesis of such compounds is not possible. Considering that in biosynthesis of nitrogenous bases amino acids such as aspartate, glycine and glutamine participate, whose formation depends on the corresponding reactions of carboxylation, it can be said that biosynthesis of these bases, and also of nucleic acids is directly dependant upon the carboxylation reactions.

Biosynthesis of Lipids: When studying the destiny of the labelled carbon atoms of the CO2 molecule in liver extracts which synthesised fatty acids with a long acetate chain, Wakephil and other authors observed that carbon atoms were found in one of the carboxyl groups of malonyl-CoA.

Malonyl-CoA is formed during carboxylation of acetyl-CoA

COOH | CH3CO.CoA + CO2 + ATP <== Mg2+ ==> CH2 + ADP + P | CO.CoA

and is an immediate precursor of two carbon units in synthesis of fatty acids.

Biosynthesis of Carbohydrates:

The majority of stages of well studied biosynthetic course of the glucose formation from pyruvate is catalysed by enzymes of glycolytic cycle, and, thus, they represent reversal of the reactions forming in the process of glycolysis. However, three irreversible stages exist in the normal glycolytic course which cannot be used during conversion of pyruvate into glucose; the biosynthesis proceeds by by-passing these stages using alternative reactions which favour the synthesis thermodynamically.

One of them is the change of pyruvate into phosphoenolpyruvate. Usually this reaction does not proceed by means of direct application of pyruvatekinase reaction. Phosphorylation of pyruvate is achieved using an indirect succession of reactions:

pyruvate <--------------- phosphenolpyruvate<==>glucose
         |                 -->
         |                 |
     CO2 |                 |-------------------> CO2
         |                 | +Mg2+
         ----oxaloacetate--- 
                             +GTP       

It is very important to emphasise that carbon dioxide fulfils the catalytic function both in lipogenesis and gluconeogenesis because its carbon in these reactions is not directly incorporated into either fatty acids or phosphoenolpyruvate. However, the importance of these reactions is high because they are limiting (reactions) in the indicated processes.

Taking into account the participation of carbon dioxide in all basic biosynthetic and oxymethylic processes in a cell, it can be proved that without CO2 life is absolutely impossible.