J Clin Pathol: first published as 10.1136/jcp.s3-11.1.14 on 1 January 1977. Downloaded from J. clin. Path., 30, Suppl. (Roy. Coll. Path.), 11, 14-20 The biochemical consequences of hypoxia K. G. M. M. ALBERTI From the Department of Chemical Pathology and Human Metabolism, General Hospital, Southampton Unlike plants which can utilize the energy obtained and creatine phosphate which is impaired. The rest from the sun through photosynthesis man requires of this review will be concerned with the production to oxidize substrates in order to obtain energy. This of ATP aerobically and anaerobically, examining energy is required for the many energy-utilizing or first glycolysis, then the tricarboxylic acid cycle, exergonic reactions which take place in the body and and finally the cytochrome system. The biochemical are necessary to sustain life (table I). Energy exists in consequences of hypoxia to the whole organism will then be discussed using two models in particular: Syntheses exercise where oxygen delivery remains normal but Active transport Muscular contraction tissue requirement greatly exceeds delivery and Nerve function hypoxia in relation to the whole organ. The final section will deal with possible methods of assessing Table I Energy-utilizing (exergonic) reactions tissue hypoxia in clinical situations. the body in several forms. First there is potential energy contained in macromolecules and second Glycolysis there is a group of compounds which have been copyright. termed high-energy phosphates which act as the The three parts of the carbohydrate oxidation system energy currency ofthe body. Thephrase 'high energy' take place in three different parts of the cell. is used because the free energy of hydrolysis of the Glycolysis is a cytoplasmic process: the tricarboxylic terminal phosphate is - 7000 cal/mol compared acid cycle is localized within the matrix of the mito- with - 4000 cal/mol for ordinary ester phosphates. chondrion while the electron transport chain occupies The major high energy phosphates are shown in the inner mitochondrial membrane. Glycolysis table II. These include ATP and the other nucleo- itself thus functions primarily as a preliminary step in the breakdown of glucose, although it should be http://jcp.bmj.com/ Nucleotides Others remembered that it also has important functions as a Adenosine triphosphate (ATP) Creatine phosphate means of converting an acute energy source, glucose, Guanosine triphosphate (GTP) I ,3-diphosphoglycerate (I ,3-DPG) into a storage form of energy, triglyceride, Compared Inosine triphosphate (ITP) Phosphoenolpyruvate (PEP) Cytidine triphosphate (CTP) with the tricarboxylic acid cycle the energy yield of Uridine triphosphate (UTP) glycolysis is small but it does have one unique Table II High-energy phosphates function. This is the ability to produce ATP anaerobically. on September 25, 2021 by guest. Protected tide phosphates. ATP in particular is ubiquitous The major energy-utilizing and producing steps and is involved in exergonic reactions through- of glycolysis are shown in the figure. It can be seen out the body. Its energy is used, for example, in that a high-energy phosphate is used up in admitting muscular contraction, in nervous excitation, for glucose to the pathway. A further ATP molecule is active transport and in the synthesis of compounds required to phosphorylate fructose-6-phosphate to such as fatty acids. Other high-energy phosphates fructose-1 ,6-diphosphate. Thereafter, however, energy with more limited roles also exist (table II). These is produced rather than used. ATP is produced include creatine phosphate whose role is restricted at two stages: the breakdown of 1,3-diphosphogly- purely to muscle, and 1,3-diphosphoglycerate and cerate and of phosphenolpyruvate. As two of each phosphenolpyruvate which are parts of the glycolytic of these molecules is made from one glucose molecule pathway. It should be remembered that other four ATP molecules will be produced per passage of biologically important compounds also fall into the one glucose molecule down the pathway. This gives high energy class. These include acetyl CoA, certain a net yield of two ATPs between glucose and amino acid esters, S-adenosyl methionine and pyruvate. This is equivalent to 15 000 cal/mol of uridine diphosphate glucose. glucose and in many senses is trivial in that it In hypoxia it is the synthesis particularly of ATP represents just over 2 per cent of the total potential 14 J Clin Pathol: first published as 10.1136/jcp.s3-11.1.14 on 1 January 1977. Downloaded from The biochemical consequences ofhypoxia 15 Glycoqen If all reactions in the shuttle were at equilibrium the pyridine nucleotides would equilibrate between Glucose _tGlucose - b- phosphate ATP ADP 4 cytoplasm and mitochondria. This would be un- Fructose- 6 - phosphate favourable in that a high cytosolic NAD is required jATP to drive glyceraldehyde 3-phosphate dehydrogenase ADP in the direction of glycolysis while in the mitochon- Fructose -1,6- diphosphate dria a high NADH is required to drive the electron Dihydroxy- acetone phosphate transport chain. This state is achieved by making one (2) Glyceraldehyde-3-phosphate of the reactions of the malate shuttle irreversible. NAD (2) This is either malate transport into the mitochondria (NADH (2) or aspartate transport out (Newsholme and Start, (2) 1,3-Diphosphoglycerate 1973). In hypoxia the transport system will rapidly HADP (2) break down. NADH will accumulate within the mitochondrion. Malate and NAD will rapidly (2) 3 - Phosphoqlycerate accumulate and malate will no longer get in. As a (2) Lactate result cytosolic malate and NADH will accumulate (2) NAD and the reaction sequence will cease. (2) NADH4'Jj Obviously glycolysis would cease entirely ifNADH (2) Pyruvate.e Phosphoenolpyruvate (2) accumulated in an uncontrolled way in the cyto- ATP(2) ADP(2) plasm in that no NAD would remain for glyceralde- Fig Energy-utilizing andproducing reactions of hyde 3-phosphate dehydrogenase. If this were the glycolysis. case even the anaerobic formation of ATP would cease and cells would die. The NAD is in fact restored by the reduction of pyruvate with NADH energy of glucose (686 000 cal/mol). This amount of yielding lactate and NAD. Thus an anaerobiosis energy is, however, sufficient to sustain certain lactate will accumulate in large amounts and will copyright. cells in the resting state when hypoxia or anoxia is spill out of the hypoxic cells into the circulation to be present. re-used when oxygen is again available. It can be seen that NADH is also produced during Regulation of glycolysis occurs at one main step: glycolysis. This represents a form of potential energy the conversion of fructose-6-phosphate to fructose and if fully oxidized will yield six further ATPs per 1,6-diphosphate. The enzyme, phosphofructokinase, mole of glucose. For this to occur, the NADH must is inhibited by ATP and citrate as well as by hydrogen first enter the mitochondrion. Mitochondria are, ions. Inhibition by ATP and citrate means that when http://jcp.bmj.com/ however, impermeable to NADH and a rather energy is plentiful and the tricarboxylic acid cycle roundabout route is followed, termed the 'malate- is saturated glycolysis will cease so that substrate oxaloacetate shuttle' (equations 1-4). First of all the is not passed unnecessarily down the glycolytic reducing equivalent of NADH is transferred to pathway. The inhibition by hydrogen ion is interest- oxaloacetate yielding malate. ing in that this will limit lactic acid production during (1) IN CYTOPLASM: exercise and will prevent intracellular pH dropping Oxaloacetate + NADH-vMALATE + NAD+ to unacceptably low levels. In hypoxia glycolysis is on September 25, 2021 by guest. Protected Malate then traverses the mitochondrial membrane accelerated due to the stimulation of phosphofructo- and is oxidized back to oxaloacetate restoring kinase by AMP and lack of ATP (Newsholme and NADH: Start, 1973). (2) IN MITOCHONDRION MALATE + NAD+ -vOxaloacetate + NADH Tricarboxylic acid cycle The oxaloacetate is then converted to aspartate. (3) IN MITOCHONDRION This forms the final common pathway for all oxida- Oxaloacetate + glutamate -*aspartate + 2- tive energy-yielding reactions. The glycolytic end oxoglutarate product pyruvate enters the cycle as acetyl CoA by Both aspartate and 2-oxoglutarate diffuse out of condensing with oxaloacetate. During the passage of the mitochondrion. the acetyl CoA round the cycle two CO2molecules are (4) IN CYTOPLASM generated so that there is no net gain of carbon 2-Oxoglutarate + aspartate -) oxaloacetate + compounds. There are, however, several steps at glutamate which energy is unleashed, generally in the form of The net result therefore is the translocation of NADH. These energy yielding steps are shown in NADH from the cytoplasm to the mitochondrion. equations 5-10. J Clin Pathol: first published as 10.1136/jcp.s3-11.1.14 on 1 January 1977. Downloaded from 16 K. G. M. M. Alberti (5) Pyruvic acid + NAD+ + CoA.SH-*acetyl CoA + NADH + H+ (6) Isocitric acid + NAD -+ ADP ATP ADP ATP Oxalosuccinic acid + NADH + H+ (11) NAD(H) < 4 flavoprotein - CoQ -p cytochromne b cytochime It (7) 2-Oxoglutaric acid + NAD+ + CoA.SH--÷ (ubiquinone) l Succinyl CoA.SH (8) Succinyl CoA + GDP -+ succinic acid + 02: cytochrome a3 cytochromc a - cytohrome c GTP + CoA.SH (cytochrome oxidase) ATP ADP (9) Succinic acid + FAD -+ Fumaric acid + FADH2 It is known that three ATP molecules are formed (10) Malic acid + NAD+ -+oxaloacetic acid from the free energy released during passage of the + NADH + H+ electron. It has been calculated that there must be a Each NADH formed will yield three ATP mole- free energy change of approximately 9000 calories cules. There is also one ATP equivalent formed in between two components of the chain if an ATP reaction 8 in the shape of GTP and the FADH2 molecule is to be formed at that point. Four sites produced in reaction 9 will yield two ATP molecules. fulfil these requirements and in fact three are used as There will thus be 15 ATP molecules formed from shown in equation 11.