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J. clin. Path., 30, Suppl. (Roy. Coll. Path.), 11, 14-20

The biochemical consequences of hypoxiaK. G. M. M. ALBERTI

From the Department of Chemical Pathology and Human Metabolism, General Hospital, Southampton

Unlike plants which can utilize the energy obtainedfrom the sun through photosynthesis man requiresto oxidize substrates in order to obtain energy. Thisenergy is required for the many energy-utilizing orexergonic reactions which take place in the body andare necessary to sustain life (table I). Energy exists in

SynthesesActive transportMuscular contractionNerve function

Table I Energy-utilizing (exergonic) reactions

the body in several forms. First there is potentialenergy contained in macromolecules and secondthere is a group of compounds which have beentermed high-energy phosphates which act as theenergy currency ofthe body. The phrase 'high energy'is used because the free energy of hydrolysis of theterminal phosphate is - 7000 cal/mol comparedwith - 4000 cal/mol for ordinary ester phosphates.The major high energy phosphates are shown intable II. These include ATP and the other nucleo-

Nucleotides OthersAdenosine triphosphate (ATP) Creatine phosphateGuanosine triphosphate (GTP) I ,3-diphosphoglycerate (I ,3-DPG)Inosine triphosphate (ITP) Phosphoenolpyruvate (PEP)Cytidine triphosphate (CTP)Uridine triphosphate (UTP)

Table II High-energy phosphatestide phosphates. ATP in particular is ubiquitousand is involved in exergonic reactions through-out the body. Its energy is used, for example, inmuscular contraction, in nervous excitation, foractive transport and in the synthesis of compoundssuch as fatty acids. Other high-energy phosphateswith more limited roles also exist (table II). Theseinclude creatine phosphate whose role is restrictedpurely to muscle, and 1,3-diphosphoglycerate andphosphenolpyruvate which are parts of the glycolyticpathway. It should be remembered that otherbiologically important compounds also fall into thehigh energy class. These include acetyl CoA, certainamino acid esters, S-adenosyl methionine anduridine diphosphate glucose.

In hypoxia it is the synthesis particularly of ATP

and creatine phosphate which is impaired. The restof this review will be concerned with the productionof ATP aerobically and anaerobically, examiningfirst glycolysis, then the tricarboxylic acid cycle,and finally the cytochrome system. The biochemicalconsequences of hypoxia to the whole organism willthen be discussed using two models in particular:exercise where oxygen delivery remains normal buttissue requirement greatly exceeds delivery andhypoxia in relation to the whole organ. The finalsection will deal with possible methods of assessingtissue hypoxia in clinical situations.

Glycolysis

The three parts of the carbohydrate oxidation systemtake place in three different parts of the cell.Glycolysis is a cytoplasmic process: the tricarboxylicacid cycle is localized within the matrix of the mito-chondrion while the electron transport chain occupiesthe inner mitochondrial membrane. Glycolysisitself thus functions primarily as a preliminary stepin the breakdown of glucose, although it should beremembered that it also has important functions as ameans of converting an acute energy source, glucose,into a storage form of energy, triglyceride, Comparedwith the tricarboxylic acid cycle the energy yield ofglycolysis is small but it does have one uniquefunction. This is the ability to produce ATPanaerobically.The major energy-utilizing and producing steps

of glycolysis are shown in the figure. It can be seenthat a high-energy phosphate is used up in admittingglucose to the pathway. A further ATP molecule isrequired to phosphorylate fructose-6-phosphate tofructose-1 ,6-diphosphate. Thereafter, however, energyis produced rather than used. ATP is producedat two stages: the breakdown of 1,3-diphosphogly-cerate and of phosphenolpyruvate. As two of eachof these molecules is made from one glucose moleculefour ATP molecules will be produced per passage ofone glucose molecule down the pathway. This givesa net yield of two ATPs between glucose andpyruvate. This is equivalent to 15 000 cal/mol ofglucose and in many senses is trivial in that itrepresents just over 2 per cent of the total potential

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The biochemical consequences of hypoxia

Glycoqen

Glucose _tGlucose - b- phosphateATP ADP 4

Fructose- 6 - phosphatejATPADP

Fructose -1,6- di phosphate

Dihydroxy- acetone phosphate

(2) Glyceraldehyde-3-phosphateNAD (2)

(NADH (2)

(2) 1,3-Diphosphoglycerate

HADP (2)

(2) 3 - Phosphoqlycerate(2) Lactate

(2) NAD

(2) NADH4'Jj

(2) Pyruvate.e Phosphoenolpyruvate (2)ATP(2) ADP(2)

Fig Energy-utilizing and producing reactions ofglycolysis.

energy of glucose (686 000 cal/mol). This amount ofenergy is, however, sufficient to sustain certaincells in the resting state when hypoxia or anoxia ispresent.

It can be seen that NADH is also produced duringglycolysis. This represents a form of potential energyand if fully oxidized will yield six further ATPs permole of glucose. For this to occur, the NADH mustfirst enter the mitochondrion. Mitochondria are,however, impermeable to NADH and a ratherroundabout route is followed, termed the 'malate-oxaloacetate shuttle' (equations 1-4). First of all thereducing equivalent of NADH is transferred tooxaloacetate yielding malate.(1) IN CYTOPLASM:

Oxaloacetate + NADH-vMALATE + NAD+Malate then traverses the mitochondrial membrane

and is oxidized back to oxaloacetate restoringNADH:(2) IN MITOCHONDRION

MALATE + NAD+ -vOxaloacetate + NADHThe oxaloacetate is then converted to aspartate.

(3) IN MITOCHONDRION

Oxaloacetate + glutamate -*aspartate + 2-oxoglutarateBoth aspartate and 2-oxoglutarate diffuse out of

the mitochondrion.(4) IN CYTOPLASM

2-Oxoglutarate + aspartate -) oxaloacetate +

glutamateThe net result therefore is the translocation of

NADH from the cytoplasm to the mitochondrion.

If all reactions in the shuttle were at equilibrium thepyridine nucleotides would equilibrate betweencytoplasm and mitochondria. This would be un-favourable in that a high cytosolic NAD is requiredto drive glyceraldehyde 3-phosphate dehydrogenasein the direction of glycolysis while in the mitochon-dria a high NADH is required to drive the electrontransport chain. This state is achieved by making oneof the reactions of the malate shuttle irreversible.This is either malate transport into the mitochondriaor aspartate transport out (Newsholme and Start,1973). In hypoxia the transport system will rapidlybreak down. NADH will accumulate within themitochondrion. Malate and NAD will rapidlyaccumulate and malate will no longer get in. As aresult cytosolic malate and NADH will accumulateand the reaction sequence will cease.

Obviously glycolysis would cease entirely ifNADHaccumulated in an uncontrolled way in the cyto-plasm in that no NAD would remain for glyceralde-hyde 3-phosphate dehydrogenase. If this were thecase even the anaerobic formation of ATP wouldcease and cells would die. The NAD is in factrestored by the reduction of pyruvate with NADHyielding lactate and NAD. Thus an anaerobiosislactate will accumulate in large amounts and willspill out of the hypoxic cells into the circulation to bere-used when oxygen is again available.

Regulation of glycolysis occurs at one main step:the conversion of fructose-6-phosphate to fructose1,6-diphosphate. The enzyme, phosphofructokinase,is inhibited by ATP and citrate as well as by hydrogenions. Inhibition by ATP and citrate means that whenenergy is plentiful and the tricarboxylic acid cycleis saturated glycolysis will cease so that substrateis not passed unnecessarily down the glycolyticpathway. The inhibition by hydrogen ion is interest-ing in that this will limit lactic acid production duringexercise and will prevent intracellular pH droppingto unacceptably low levels. In hypoxia glycolysis isaccelerated due to the stimulation of phosphofructo-kinase by AMP and lack of ATP (Newsholme andStart, 1973).

Tricarboxylic acid cycle

This forms the final common pathway for all oxida-tive energy-yielding reactions. The glycolytic endproduct pyruvate enters the cycle as acetyl CoA bycondensing with oxaloacetate. During the passage ofthe acetyl CoA round the cycle two CO2 molecules aregenerated so that there is no net gain of carboncompounds. There are, however, several steps atwhich energy is unleashed, generally in the form ofNADH. These energy yielding steps are shown inequations 5-10.

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K. G. M. M. Alberti

(5) Pyruvic acid + NAD+ + CoA.SH-*acetylCoA + NADH + H+

(6) Isocitric acid + NAD -+Oxalosuccinic acid + NADH + H+

(7) 2-Oxoglutaric acid + NAD+ + CoA.SH--÷Succinyl CoA.SH

(8) Succinyl CoA + GDP -+ succinic acid +GTP + CoA.SH

(9) Succinic acid + FAD -+Fumaric acid + FADH2

(10) Malic acid + NAD+ -+oxaloacetic acid+ NADH + H+

Each NADH formed will yield three ATP mole-cules. There is also one ATP equivalent formed inreaction 8 in the shape of GTP and the FADH2produced in reaction 9 will yield two ATP molecules.There will thus be 15 ATP molecules formed fromthe passage of each pyruvate around the cycle. Asone glucose molecule produces two pyruvates therewill be 30 ATPs formed, making a total of 38 for theoxidative breakdown of one glucose molecule. Thisis equivalent to 42 per cent of the total potentialenergy of glucose, the rest being released in the formof heat which is obviously a useful form of energy inmammals.

Fatty acids enter the cycle through acetyl CoA.They are first broken down by beta-oxidation, eachstep yielding an acetyl CoA. In the formation ofeach acetyl CoA one NADH and one reducedflavoprotein molecule is formed so that five ATPequivalents are produced per acetyl CoA. A further12 ATPs will be formed by passage of the acetyl CoAaround the cycle. Allowing for the initial activation,a typical fatty acid such as palmitate will yield980 000 calories which amounts to 41 per cent of thepotential energy of the molecule. In the absence ofoxygen no energy will be released from fatty acids.Thus when oxygen is available there is a vastly.greater energy yield than under anaerobic conditions.

The electron transport chain

IfNADH is to yield any ATP at all, then the reducingequivalents must be passed down the electrontransport chain, finally yielding water. The com-ponents of the chain are now fairly well establishedand comprise flavo-protein, coenzyme Q and thecytochrome system. For electrons to flow down thechain the components must be arranged in order ofincreasing redox potential with the terminalcytochrome a3 responsible for the combination ofthe reducing equivalents with molecular oxygen. Thecomponents and their order are shown in equation 11.

ADP ATP ADP ATP

(11) NAD(H) < 4 flavoprotein - CoQ -p cytochromne b cytochime It(ubiquinone) l

02: cytochrome a3 cytochromc a - cytohrome c

(cytochrome oxidase) ATP ADP

It is known that three ATP molecules are formedfrom the free energy released during passage of theelectron. It has been calculated that there must be afree energy change of approximately 9000 caloriesbetween two components of the chain if an ATPmolecule is to be formed at that point. Four sitesfulfil these requirements and in fact three are used asshown in equation 11. The coupling of ATP form-ation to the respiratory chain is known as oxidativephosphorylation. It can be seen that the passage ofreducing equivalents from NADH will result in theformation of three ATPs while electron transportfrom a reduced flavoprotein will result in the form-ation of only two ATPs.The actual mechanism whereby ATP formation is

coupled to the electron transport chain remainscontroversial. For the purposes of this review it issufficient to say that there are two main hypotheses:a chemical hypothesis postulating direct chemicalcoupling (Ernster, Lee, and Janda, 1967) and thechemi-osmotic hypothesis which invokes the trans-port of hydrogen ions across the mitochondrialmembrane (Mitchell, 1972).The localization of the electron transport chain

has attracted much attention. It is suggested thatrepeating units exist on the inner mitochondrialmembrane. These comprise a base piece which issaid to contain the electron transport chain, a stalk,and a headpiece containing the ATP synthetic system(MacLennan, 1970).The regulation of the electron transport chain is

obviously of interest when considering hypoxia. Onemajor point is that cytochrome oxidase has anextremely high affinity for oxygen which allows therespiratory chain to function even when the mito-chondrion is virtually anoxic. This is obviouslycrucial in protecting tissues against low oxygentensions. The activity of the cytochrome system isregulated by the availability of ADP, of substrateand of oxygen. Obviously when oxygen is totallylacking the system cannot operate. The closest reg-ulation will come from ADP concentration so thatwhen energy utilization is high or hypoxia is presentand ATP concentrations are low with a raised ADP

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The biochemical consequences of hypoxia

formation, then the activity of the chain will bestimulated. This will tend, therefore, to restoreATP formation. Build up of NADH will also tendto drive the chain. This could be relevant following aperiod of hypoxia or exercise when NADH con-centrations will be raised and ATP concentrationslow. Further information on biochemical mechanismscan be found in papers by Newsholme and Start,and by Harper, 1973; 1975).

Muscle and hypoxia

It is relevant to consider muscle first in relation tohypoxia in that this tissue is most often exposed tolow oxygen tensions in normal man. This occursduring exercise. Muscle is in fact one of the mostresistant tissues to hypoxia. In experiments in ourown laboratory we showed that total occlusion ofblood supply to an arm for five minutes gave nobiochemical evidence of hypoxia (Braybrooke et al,1976). Only if the arm was exercised at the same timewas the effect of hypoxia apparent. This resistance tohypoxia is well known to surgeons who use tour-niquets for lengthy periods during amputations oflimbs.Muscle has indeed several mechanisms whereby

it is protected against mild hypoxia. First, oxygen isstored attached to myoglobin, particularly in musclesdesigned for sustained contraction such as the pos-tural muscles. The oxygen dissociation curve formyoglobin is a rectangular hyperbola which isplaced to the left of that for haemoglobin. Myoglobincan take up oxygen from haemoglobin and willrelease it only when oxygen tensions within themuscle cells become very low. It therefore serves asan emergency reserve. In terms of whole bodyoxygen utilization myoglobin plays only a minorrole, but it is said to be important for cardiacmuscle.

Muscle also contains important energy reserves inthe form of glycogen and creatine phosphate.Considering first creatine phosphate, this is formedfrom ATP and creatine when ATP supplies areplentiful.(12) Creatine phosphate + ADP creatine

+ ATPThe relative reserves of ATP, creatine phosphateand glycogen have been calculated by Hultman andBergstrom (1973). They have shown that there are10 ,umol of high-energy phosphate per gram dryweight of muscle as ATP, 61 ,umol as creatinephosphate and 1060 ,umol as glycogen for anaerobicmetabolism, and 14 200 ,xmol high-energy phosphateper gram dry weight from glycogen for aerobicmetabolism. It is obvious that the amount ofcreatine phosphate is relatively small, but this is

nonetheless extremely important at the beginning ofexercise and for short periods of severe exercise.In 90 per cent maximal exercise the creatinephosphate has virtually all disappeared at betweentwo and five minutes (Karlsson, 1971; Hultman andBergstrom, 1973). At 50 per cent maximal exercisethere is an initial drop in creatine phosphate toabout half of resting values but then the concen-tration remains constant, suggesting that sufficientATP is being produced to re-phosphorylate creatine.Most of the body's glycogen is to be found in

muscle. This represents a total energy reserve ofsome 1000 kilocalories. It should be rememberedthat this glycogen can only be used for muscles andtheir requirements in that glucose-6-phosphatase islacking in muscle so that glucose cannot be releasedinto the circulation. If exercise is totally anaerobicthen this glycogen reserve would last for approxi-mately 30 minutes.The role of fat in providing fuel for muscle should

not be underestimated (Felig and Wahren, 1975).At rest fatty acids provide 90 per cent of energyrequirements for muscle. About three quarters ofthis comes from endogenous triglyceride and therest from circulating free fatty acids. During exercisethere is a triphasic sequence of fuel utilization.Initially there is a rapid phase of glycogenolysis inwhich muscle glycogen is used. In the second phaseof exercise blood glucose provides substrate, thiscoming from liver glycogen. In the third phase freefatty acids provide most of the fuel. However, ifexercise is severe then the muscle will becomeanaerobic, fatty acids cannot be used and musclebecomes entirely dependent on anaerobic metabolism.The amount of exercise will then be determined bythe ability of muscle to go on producing sufficientATP and many authorities have shown that thisdepends on the starting concentration of glycogen.Experiments in which muscle glycogen has beenincreased by feeding regimens before exercise haveshown that this increases exercise tolerance (Rennieand Johnson, 1974; Bergstrom et al, 1967).During severe exercise muscle lactate concentra-

tions rise to very high levels, amounting to as muchas 25 mmol/kg wet weight. Concentrations withinmuscle may be several fold higher than those in thecirculation where maximum levels achieved are inthe order of 20 mmol/l, but more usually nearer10 mmol/l. Pyruvate concentrations do not riseproportionately and may increase by as little astwofold in muscle tissue. This means that the[lactate]/[pyruvate] ratio becomes very high (seebelow).The rate at which muscle becomes hypoxic will

depend not only on the rate of oxidative metabolismwithin the tissue but also on the supply of oxygen

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K. G. M. M. Alberti

in the circulation. The circulation is fully adapted toincreasing blood flow to exercising muscle (Mitchelland Blomqvist, 1971). Thus cardiac output increasesfrom 6 to 24 litres/min with blood flow to activemuscle increasing from 0-65 litres to 20-9 litres/min.At the same time pulmonary ventilation increasestwelve-fold from 10 litres/min to 120 litres/min withoxygen uptake going up from 0 3 to 3-8 litres/min.Extraction in the tissues is also greatly increasedso that the arteriovenous oxygen difference risesfrom 5 6 to 15 8 ml/100 ml blood. This serves notonly to increase oxygen and substrate supply tomuscle, but also to remove lactic acid and to expireincreased amounts of C02 thus helping to preserveacid-base balance. It is obvious that any diseasewhich interferes with any of these processes willrender muscle hypoxic more rapidly. Thus severebronchopulmonary disease or myocardial diseasewill be associated with a greatly reduced exercisepotential. Similarly anaemia will cause a decrease inexercise capacity. This is true as well for any diseaseassociated with diminished tissue glycogen stores.One special aspect of limited oxygen supply is that

of high altitude. This could be re-defined as a stateof chronic hypoxia. It is known that persons livingall the time at high altitude have a decreased abilityto produce lactic acid during exercise and a decreasedexercise capacity. It is thought that this decreasedability to accumulate lactic acid is due to a diminishedalkali reserve, which implies less buffering capacityso that pH will fall more quickly per mole of lacticacid produced. This will then inhibit glycolysis andATP production will be diminished. It is probablynot due to hypoxia per se in that acute replacementof oxygen has little effect on this process (Cerretelli,1967). In contrast, experiments with acute hypoxiahave shown no impairment in the production oflactic acid.The regulation of muscle metabolism in hypoxia

may be summarized as follows. Initially creatinephosphate and ATP concentrations will fall. AMPconcentrations will rise. This will result in accelera-tion of glycolysis from glycogen through stimulationof phosphofructokinase. Lactic acid will accumulatein order to regenerate NAD. The fall in intracellularpH will have two effects: first, phosphofructokinasewill tend to be inhibited but also muscle permeabilityto glucose will be enhanced (Randle and Smith,1958). Hypoxia will also have a more distant effectin that catecholamine secretion will be stimulated.This will directly increase glycogen breakdown inmuscle thus further enhancing glycolysis (Cain,1969).(The reader is referred to Pernow and Saltin, 1971,

for further discussion of muscle metabolism duringexercise.)

Other tissues and hypoxia

The ability of tissues to resist hypoxia appears todepend on their glycogen content. Brain cells containlittle glycogen while their supporting cells, the astro-cytes, contain only sufficient for some 15 seconds'anaerobic ATP production. Brain cells are notorious-ly sensitive to hypoxia with irreversible changesoccurring within minutes. In contrast a tissue suchas liver may withstand anaerobiosis for lengthyperiods. Woods and Krebs (1971) have shown thiselegantly in experiments with livers perfused underanaerobic conditions. They showed that livers fromwell fed rats perfused anaerobically could functionnormally for two to three hours with stoichiometricconversion of glycogen to lactate. They were alsoable to use glucose from the perfusion medium. Incontrast livers from starved rats were much moresensitive showing cellular oedema and decrease inbile flow in an hour or so. These livers were unable touse added glucose, presumably because glucokinaseactivity was decreased due to the starvation. In lesssevere hypoxia, liver probably continues to obtainmost of its energy supply from fatty acid degradation,thus allowing the various essential synthetic processesto continue.

Clinical assessment of tissue hypoxia

It is easy to discuss theoretical or experimentalaspects of hypoxia. It is more difficult to convertthis into clinically useful diagnostic methods. Anobvious method for assessing hypoxia has been tomeasure blood lactic acid concentration. This can bedone easily and rapidly so could be available in mostcentres. Certainly in severe exercise or severehypoxia, the increase in lactic acid concentration inthe blood is proportional to the degree of hypoxia.Thus, lactic acidosis is a known complication ofshock, low cardiac output states, septicaemia andpoor tissue perfusion (Cohen and Simpson, 1975).In most of these situations, however, the diagnosisof what is known as type A lactic acidosis is obviousand chemical confirmation does little to help thepatient. In milder degrees of hypoxia, measurementof lactate alone is of questionable value. There aremany reasons for an elevation in blood lactate otherthan hypoxia. These include liver disease, therapywith drugs such as biguanides, ingestion of alcoholor intravenous feeding with substances such asfructose. Lactate also rises in physiological circum-stances (Huckabee, 1958) such as in eating mealswhere the resultant insulin secretion acutely inhibitsgluconeogenesis and the liver and lactate uptake bythat organ is temporarily prevented. Thus a small risein blood lactate concentration is not specific

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The biochemical consequences of hypoxia 19

(Krebs et al, 1975).One possible method for measuring whole body

hypoxia of a temporary nature would be to measurethe so-called oxygen debt. It is well known thatafter exercise, oxygen consumption remains elevatedfor a considerable period. This is purported to bethe oxygen necessary for metabolism of the lacticacid which has accumulated anaerobically duringthe hypoxic phase, of, for example, exercise.Whether this is the correct explanation remainscontroversial. Its use clinically would only be oflimited value in that it would be helpful only fortemporary periods of hypoxia.The most commonly used assessment method is

that based on equations 13 and 14

(13) Lactate + NAD+ _ pyruvate + NADH + H+

(14) [Lactate] = [NADH] . 1 . (H+][Pyruvate] [NAD] K

Thus by re-arranging the lactate dehydrogenasereactants into the mass action equation one can seethat the ratio of lactate to pyruvate is proportionalto the ratio NADH to NAD. A rise in the NADH:NAD ratio should therefore be reflected by a rise inthe lactate:pyruvate ratio. It should be rememberedimmediately that this refers to events in the cellcytoplasm and is a reflection only of the cytoplasmicredox state. Nonetheless measurement of lactate:pyruvate ratios in blood has been used to assesshypoxia. The various assumptions inherent in thismeasurement must be remembered. These includethe assumption that lactate and pyruvate are freelydiffusible; that lactate and pyruvate are producedand used at the same rate; that the lactate:pyruvateratio is the same in all tissues; that the cytoplasmicredox ratio will reflect whole cell redox state; andthat the reaction has reached equilibrium. Exceptionsto each of these assumptions have been recorded.In the absence of a better index, however, it is stillpossible to use this ratio as a crude guide to hypoxia.Two further points must be remembered. The first isthat even if the L :P ratio is raised one has no indica-tion which tissue is hypoxic. The second is that theratio is influenced by pH. Examination of equation14 shows that the hydrogen ion is involved and that adoubling of hydrogen ion concentration, which isequivalent to a fall in pH from 7-4 to 7-1, couldresult in a doubling of the lactate:pyruvate ratiowithout any change in redox state being implied. Inmost hypoxic states, some degree of acidaemia ispresent so that interpretation of the L:P ratio mustalways be done in conjunction with pH measurements.The normal L:P ratio in blood is about 10 and thiscan rise to approximately 40 in a severe acidaemia.

Values above this level probably do reflect someintracellular oxygen deficit.The 3-hydroxybutyrate:acetoacetate ratio has

been used in asimilar wayto give an indication of themitochondrial redox state. It can be useful whenmeasured together with the L:P ratio but all theassumptions made for the latter must also be appliedto this ratio.One can only suggest that newer and better

methods for assessing tissue hypoxia are stillrequired.

Summary

The various phases of energy production have beendescribed. These include glycolysis which is uniquein its ability to produce ATP anaerobically, thetricarboxylic acid cycle with its major contributionto ATP production coming through the generationof NADH, and the cytochrome system at whichreducing equivalents are converted to water, thereleased energy being incorporated into high-energyphosphates. The regulation of these pathways hasbeen briefly described and the importance of thesmall amount of ATP generated anaerobicallyemphasized. The adaptation of muscle to periods ofhypoxia through the presence of myoglobin, creatinephosphate and large amounts of glycogen is thendiscussed. The role of pH in limiting anaerobicglycolysis in muscle and the importance of thecirculation in providing oxygen for exercising muscleare outlined. The effects of hypoxia on certain othertissues such as liver and brain have been detailed andfinally methods for assessment of tissue hypoxia inman such as the measurement of the lactate :pyruvateratio in blood are presented.

References

Bergstrom, J., Hermansen, L., Hultman, E., and Saltin, B.(1967). Diet, muscle glycogen and physical performance.Acta Physiologica Scandinavica, 71, 140-150.

Braybrooke, J., Lloyd, B., Nattrass, M., and Alberti,K.G.M.M. (1975). Blood sampling techniques for lactateand pyruvate estimation: A reappraisal. Annals of ClinicalBiochemistry. 12, 252-254.

Cain, S. M. (1969). Diminution of lactate rise during hypoxiaby PCO2 and beta-adrenergic blockade. American Journalof Physiology, 217, 110-116.

Cerretelli, P. (1967). Lactacid °2 debt in acute and chronichypoxia. In Exercise at Altitude. Edited by R. Margaria,pp. 58-64. Excerpta Medica Foundation, Amsterdam.

Cohen, R. D., and Simpson, R. (1975). Lactate metabolism.Anaesthesiology, 43, 661-673.

Ernster, L., Lee, C. P., and Janda, S. (1967). The reactionsequence in oxidative phosphorylation. In Biochemistry ofMitochondria, edited by E. C. Slater, Z. Kaniuga, andL. Wojtczak, pp. 29-51. Academic Press, New York.

Felig, P., and Wahren, J. (1975). Fuel homeostasis in exercise.New England Journal of Medicine, 293, 1078-1084.



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Harper, H. A. (1975). Review of Physiological Chemistry,15th edition, chs 9 and 13. Lange, Los Altos, California.

Huckabee, W. E. (1958). Relationships of pyruvate andlactate during anaerobic metabolism. 1. Effects of infusionof pyruvate or glucose and of hyperventilation. Journal ofClinical Investigation, 37, 244-254.

Hultman, E., and Bergstrom, J. (1973). Local energy-supplying substrates as limiting factors in different typesof leg muscle work in normal man. In Limiting Factors ofPhysical Performance, edited by J. Keul, Thieme, Stuttgart.

Karlsson, J. (1971). Lactate and phosphagen concentrationsin working muscle of man. Acta Physiologica Scandinavica,Suppl. 358, 1-72.

Krebs, H. A., Woods, H. F., and Alberti, K. G. M. M. (1975).Hyperlactataemia and lactic acidosis. Essays in Biochemis-try, 1, 81-103.

Maclennan, D. H. (1970). Molecular architecture of themitochondrion. In Current Topics in Membranes and

Transport, vol. 1, edited by F. Bronner, and A. Kleinzeller,pp. 177-232. Academic Press, New York.

Mitchell, J. H., and Blomqvist, G. (1971). Maximal oxygenuptake. New England Journal of Medicine, 284, 1018-1022.

Mitchell, P. (1972). Chemiosmotic coupling in energytransduction: a logical development of biochemicalknowledge. Journal of Bioenergetics, 3, 5-24.

Newsholme, E. A., and Start, C. M. (1973). Regulation inMetabolism, Wiley, Chichester and New York.

Pernow, B. and Saltin, B. (1971). Muscle metabolismduringexercise. Advances in Experimental Medicine and Biology,11.

Randle, P. J., and Smith, G. H. (1958). Regulation of glucoseuptake by muscle. Biochemical Journal, 70, 490-508.

Rennie, M. J., and Johnson, R. H. (1974). Effects of anexercise-diet program on metabolic changes with exercisein runners. Journal of Applied Physiology, 37, 821-825.

Woods, H. F., and Krebs, H. A. (1971). Lactate productionin the perfused rat liver. Biochemical Journal, 125, 129-139.



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