THE SYSTEM OF THE ERYTHROCYTE

by

Charles Prévost

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science.

Department of Biochemistry, Mc Gill University, Montreal. September 1961 i

ABSTRACT

The writer has developed an as say for estimation of optimal

hexokinase activity in stroma-free hemolyzate, and shawn that

hexokinase remains stable and active for at least 30 days in human

erythrocytes preserved in CD or ACD at 4 ° C.

Addition of ADP or DPN to the assay medium could increase

the 1s activity; nicotinamide or alloxan depressed it.

It is suggested that failure of glycolytic activity during preservation

is not therefore due to the formerly supposed lability of hexokinase,

but to inhibition of hexokinase attributable to falling pH and ATP, and

conditions favouring glucose-6-P accumulation such as decrease of , DPN and ADP.

Pyruvate produced in cells during the first 15 days of storage tended to be expelled into the external medium.

Cyclic adenylate was utilised by the human red cell, as evidenced by conversion of its ribose moiety into lactic acid with concomitant esterification of inorganic phosphate into the stable phosphate fraction. ii

AC KNOWLEDGE MEN TS

I am most grateful to Dr. O. F. Denstedt for his sympathetic

guidance and understanding throughout the course of this investigation.

The kindness and spirit of my colleagues are greatly appreciated

and I wish to express my thanks to Mrs. Marina van Ermengen, Miss

Anne Hemphill and Miss Arlene Maximchuk for assistance in some

of the experimenta, and to Miss Maximchuk for proofreading the manuscript.

Many thanks to my friend Samuel Refetoff for completing the colored diagrams in the final copies.

I am most indebted and thankful to my wife, Francine, for assistance in experimenta which ran into the night and for her meticulous care in typing the thesis.

Grateful acknowledgement is made also for financial assistance from a grant to Professer O. F. Denstedt from the Defence Research

Board of Canada (Grant Number 9350-01). iii

PREFACE

The work presented here is part of a larger programme

of research on the preservation of blood under the direction

of Dr. O. F. Denstedt.

The main object was to examine the validity of a current hypothesis that the key factor in the metabolic failure of the erythrocyte during preservation in the cold (4"C} is the progressive inactivation of hexokinase.

In order to understand how the loss of cell viability cornes about during the preservation of blood, it is helpful to review the properties of the hexokinase system and also per'tinent aspects of the structure of the erythrocyte and properties of the membrane in relation to the metabolic activity of the cell. lV

TABLE OF CONTENTS

ABSTRACT. • • • . i ACKNOWLEDGEMENTS .• . . ii PREFACE • • • • iii LIST OF TABLES • .vi LIST OF FIGURES • . • vii LIST OF ABBREVIATIONS. . . viii

INTRODUCTION. 1

ERYTHROCYTE COMPOSITION AND STRUCTURE 1 PERMEABILITY . 4 Glucose. 6 Sodium and potassium 8 Inorganic phosphate • 13 METABOLISM OF THE ERYTHROCYTE. 15 Hexokinase 17 Phosphoglucoisomerase . 20 Phosphofructokinase . 21 of the metabolism of triose phosphates 21 Enzymes connected with the metabolism of high energy phosphate compounds 23 Other enzymes 26 Metabolism of red cells during preservation at 4°C. 29

EX PERIMENT AL 36

I. MA TE RIALS AND REAGENT5. 36 A. Substrates 36 B. Buffers 36 C. Enzymes . 37

II. METHODS 38 A. Procedure for collection and preservation of blood 38 B. Analytical procedures • 39 Glucose, 39 Pyruvic acid 40 Lac tic acid. 40 Pentose. 40 Phosphate fractions • 40 Hemoglobin 41 Hematocrit. 41 v

C. General method of taking samples for analysis 41 D. Electrophoretic separation of nucleotides 42 E. Assay of hexokinase activity . 43

III. EXPERIMENTAL RESULTS • 44 A. Optimum conditions for hexokinase activity 44 1. The influence of hydrogen ion concentration . 44 2. Influence of added ATP and ADP on glucose utilisation and lac tic acid production • 46 3, The influence of other glycolytic enzymes and the optimal concentration of DPN • 51 4. Inhibition of glucose utilisation and lactic acid production by nicotinamide and alloxan 55 B. The activity of hexokinase during preservation of blood at 4°C 58 C. Influence of added 3 1, 5'-cyclic adenylic acid on the metabolic activity of blood during storage in ACD at 4°C. 73

DISCUSSION AND CONCLUSIONS 83

SUMMAR Y . 94

CLAIMS TO ORIGINALITY • 96

BIBLIOGRAPHY. 97 Vl

LIST OF TABLES

I. The influence of ATP and ADP on hexokinase activity 47

II. Hexokinase activity in the SFH from two specimens of blood during storage in CD and ACD respectively at 4°C • 60

III. Changes in pH, pyruvic acid, glucose and lactic acid concen­ tration in two specimens of blood during storage in CD and ACD respectively at 4°C . 62

IV. Partition of glucose and lactic acid between cells and plasma in two specimens of blood during storage at 4°C 65

V. Changes in the concentration of phosphate fractions in two specimens of blood during storage in CD and ACD respectively at 4 oc 66

VI. Partition of inorganic phosphate between cells and plasma in two specimens of blood during storage at 4 oC 69

VII. Partition of pyruvic acid between cells and plasma in two specimens of blood during storage at 4 oC 71

VIII. Changes in pH and ribose concentration in blood during storage in ACD at 4°C to which nucleoside was added on the 25th day 74

IX. Influence of added nucleoside on pyruvic acid concentration in blood during storage at 4°C, and partition of pyruvic acid between cells and plasma in the inosine-containing sample . 77

X. Influence of added nucleoside on the concentration of inorganic and stable phosphate, and partition of inorganic phosphate between the cells and the plasma during storage at 4°C. 79 vii

LIST OF FIGURES

1 . The glycolytic and pentose metaholic pathways in the red cell . 16

2. The influence of the hydrogen ion concentration on hexokinase activity • 45

3. The influence of ATP and ADP on hexokinase activity 48

4. Nucleotide composition of the SFH during hexokinase assay 50

5. The influence of added SFH and DPN on the activity of yeast hexokinase • 53

6. The influence of nicotinamide on the metabolic behaviour of intact red cells and SFH • 56

7. The influence of alloxan on the metabolic behaviour of intact cells and SFH . 57

8. Hexokinase activity of SFH from blood during storage at 4°C . 61

9. Changes in pH, glucose and lactic acid concentration during preservation of blood at 4°C 63

10. Changes in the phosphate fractions during storage of blood at 4°C 67

11, Partition of inorganic phosphate between cells and plasma during storage of blood at 4 oC 70

12.. Changes in the concentration of pyruvic acid and its partition between tells and plasma during storage of blood at 4°C 72

13. The influence of added 3 1, 5'-cyclic adenylic acid or inosine on the utilisation of ribose by blood during storage in ACD at 4°C 75

14. {a) Theillfluen'ce ofadded 3',5'-cyclic adenylic acid or inosine on the pyruvic acid concentration during storage of blood in ACD at 4°C 78 (b) The influence of added inosine on the partition of pyruVié. acld between cells and plasma during storage of blood in ACD at 4 oC 78

15. The influence of added 3 1, 5 1 -cyclic adenylic a cid or inosine on the concentration of inorganic and stable phosphate of blood during storage in ACD at 4" C . 80

1 16. The influence of added 3 , 5 '-cyclic adenylic ac id or inosine on the partition of inorganic phosphate between cells and plasma during storage of blood in ACD at 4 oC 81 viii

LIST OF ABBREVIATIONS

AMP, ADP and A TP adenosine mono-, di-, and triphosphate

CD! ACD citrate-dextrose medium, acid citrate-dextrose medium. Suffixes +CA or + 1 indicate the 1 1 medium to which had been added 3 , 5 -cyclic adenylic acid or inosine respectively.

1, 3-DPG, 2, 3-DPG 1, 3- and 2, 3-diphosphoglyceric acid

DPN, DPNH diphosphopyridine nucleotide and reduced DPN

GTP, UTP g:uanosine triphosphate and uridine triphosphate

G-6-P glucose-6-phosphate

Hb h;emoglobin

IMP, ITP inosine mono- and triphospha.te

molar, millimolar concentration

inorganic phosphate, labile phosphate, and stable phosphate

SFH stroma-free hemolyzate

TCA trichloroacetic acid

TPN, TPNH triphosphopyridine nucleotide and reduced TPN w/v, w/w weight/volume, and weight/weight

1 Cyclic AMP 3 , 5 1-cyclic adenylic acid INTRODUCTION

ERYTHROCYTE COMPOSITION AND STRUCTURE

Of the cells that make up a complex organism such as the animal

body, the erythrocyte plays a peculiarly important role, since it

carries oxygen from the lunga to every cell in the body and on its

return journey to the lunga picks up and transports carbon dioxide.

Its hemoglobin also is one of the most important buffers in the

regulation of the hydrogen ion concentration of the blood and the tissùes.

Knowledge of the detailed structure of the erythrocyte still is incomplete. The red cell of the mammal is a nonnucleated biconcave disk with a diameter of about O. 008 millimeter (8Jl) and a thickness around O. 0005 millimeter (O. S)l). It is a highly specialized cell which in the process of its maturation loses most of its organelles.- nucleus, mitochondria, microsomes, etc. , and becomes filled with a respiratory pigment, hemoglobin. The cellular membrane comprises

3 o/o of the cell mate rial; the rest of the cell is made up of hemoglobin

32o/o and water 65o/o (1 ).

The red cel! stroma, or ghost, as the isolated cellular membrane is sometimes called, contains about lOo/o lipid (2). Prankerd (3) -12 lists the lipids of the stroma as follows: cholesterol, 1. 0- 1. 4 x 10 , 12 2 and phospholipids, 1. 2 - 1. 6 x 10- mg/}l of c eU surface. The phospholipid fraction contains 37o/o cephalins, 35o/o lecithins and Z8o/o sphyngomyelin. The fatty acids found in the neutral fat and phospholipid 2

extracts range from clo-capric to cl9-arachidonic acids.

The carbohydrate of the stroma, according to an analysis by

Ludewig (4), was comprised of hexoses 2% (w/w), of which one-fifth

was found to be in the form of a lipid-carbohydrate complex,

hexosamines l. 2% and sialic acid 1. 2%. No change occurred in the

composition of the membrane of the red cells stored for 45 days

in the cold. The hexose fraction consisted mainly of galactose but

included smaller amounts of glucose and mannose, and a trace of

fucose. Rankerd and Altman (5} found ATP and 2, 3-DPG in the

stroma and argued they could not be contaminants since labelled

ATP and 2, 3-DPG extracted from the stroma did not show a specifie

activity parallel to that of these substances in the cytoplasm.

Three complex proteins have been isolated from the stroma.

Hemoglobin is a definite constituent of the membrane. It appears to

be firmly bound since it cannat be removed even with severa! washings.

Lipids are removable by this treatment. A stroma globulin called

11 the "S-protein , with an isoelectric point at pH 6. 8 has been isolated.

Moskowitz and Calvin (6} used ether to isolate a fibrous conjugated protein which they named elanin; it is a lipid-carbohydrate-protein complex containing the A, B, and Rh blood-group substances.

Estimates of the membrane thickness va.ry according to the methods used. Mitchison (7), in birefringence studies, obtained a value of

0 5, 000 A for the wet membrane. Fricke (8), by means of electrical conductivity measurements, obtained a value of 50 Â for the dried membrane. Estimates made with electron microscopy on various 3

dried preparations of the membrane ranged from 50 to 1, 000 Â.

The classical concept of the membrane structure pictures it as a bimolecular film of lipid with polar groups linked by metallic ions

such as calcium, sandwiched between two protein layers. Mitchison {7)

considers the membrane to be composed of stromatin {protein)

arranged as bundles with the long axis randomly situated in the plane of the membrane surface, but with the main parts of the protein

chains within them orientated at right angles. Such an arrangement, he postulates, would account for the birefringence observed. From

electron microscope pictures, Hillier and Hoffman ( 9, 10) described the membrane as being covered with structures which they called

11 "plaques •

The plaques are pictured as disk-like components approximately

0 0 30 A long with a diameter between 100 and 500 A. The plaques are

supposed to be arranged in random-like sequences like links in a chain, or singly, forming a single-layer mosaic. Moskowitz and Calvin (6) have proposed a picture in which elanin comprises the main fibrillar framework of the cell associated with hemoglobin and the S-protein.

The fact that the plaques are readily removable but not soluble on treatment with lipid solvents suggests that they are composed mostly of protein, perhaps elanin, the molecules of which could be joined together by lipid, Parpart and Ballentine (li), on physiological grounds, believed that the membrane contains pores in number comparable to the number of plaques found. They proposed further that the pores are 4

lined with lipid molecules. From Hillier 1 s evidence the pores would have

0 a diameter around 35 A, which would be too small to let hemoglobin

pass through.

The ideas concerning the nature of the surface of the membrane also

are conflieting. Fonder and Fonder (12), by blocking surface charges

with uranyl ions, came to the conclusion that the dominant surface charge

on the membrane is due to the presence of phosphate radicals. Harris (13)

has suggested that the membrane has a coating of albumin. Feher and

Matkovics (14} used dinitrofluorobenzoic acid to tag the exposed amino

groups on the membrane and obtained evidence that arginine and serine

were the only amino acids tagged. The arginine is present in the proteins

while the serine could be part of serine-cephalin in a lipoprotein.

These workers do not consider their evidence to be compatible with the

concept of an uninterrupted lipoid membrane.

Although the nature of the red cell membrane still is obscure, the newer physical aids and chemical methods are providing much interesting new information.

PE;lRMEABILITY

Studies on the permeability of the membrane to various substances have shed further light on the nature of the red cell membrane and its dynamic properties.

It is well known that certain substances, e. g. glucose, pass through the membrane with relative ease by passive diffusion. Certain others, such as sucrose, citrate, tartrate will not pass through. Anions such 5

as Cl and HC0 3-, lactate, and some others rapidly become equilibrated

on the two sides of the membrane while organic phosphate esters do not pass either into or out of the cell. Between these extremes there are

substances like inorganic phosphate and potassium that will enter the

cell only with a high activation energy provided by metabolic activity,

and others like sodium that will similarly be kept outside the cell.

Still others will find their way in, but with a little more difficulty than by mere passive diffusion.

Since there are present in the cell some nondiffusible charged particles, there results an osmotic pressure build-up. The red cell, however, unlike its ghost, devia tes from true osmotic swelling.

Teorell (15) suggests that this is attributable to the bound water

associated with hemoglobin. According to Koefoed-Johnsen and Ussing (16), the diffusion of water in red cells is best explained by the presence of pores in the membrane. The values of the gradients for lactate and pyruvate across the membrane and the changes in the gradients during in vitro experiments suggest that lactate moves passively and freely through the membrane, while the pyruvate gradient is determined by the metabolic activity of the cells and the relatively high resistance of the membrane to the passive diffusion of this metabolite·. (11).

Davson and Danielli {18) suggest that the impermeability of the red cell membrane to non-electrolytes is controlled by factors such as the lipid solubility, molecular volume, specifie surface sites of the cell and specifie molecular groups of the penetrating substances. 6

Glucose

The mechanism of glucose entry has been studied widely and many

mechanisms have been propos ed: free diffusion, active transport,

facilitated transport, and altered diffusion.

Laris (19} has noted that the addition of glucose to the plasma

resulted in an increase in the glucose content of the red cell. Mawe (20) has observed that glucose penetration into the human erythrocyte obeys

Fick 's law of diffusion, which means that the rate of penetration of

glucose is a linear function of the difference between the concentrations

of glucose on the two sides of the membrane.

Klinghoffer (21) observed on the contrary that increasing the glucose

concentration in the plasma reduced the rate of penetration. Kashket (22) found that the rate of penetration did not exceed the rate of utilisation by the cell. Since, in low concentrations (O. 03M}, the rates of entry in decreasing arder were glucose, mannose, galactose, arabinose, sor bose, whereas in high concentrations the or der was revers ed,

Wilbrandt (23) postulated a transport system.

It is well known that the temperature coefficient is relatively high for the passage of sugars through the membrane but since the red cells do not concentrate hexoses against a concentration gradient, the re is little evidence for an active transport system. That transport of sugars may depend on an enzyme transport system is a possibility in view Of the observation that the rate of sugar penetration is temperature-dependent and has a 0 10 of the same arder as to be expected from an enzymatic 7

process.

Glucose penetration through the red cell membrane is inhibited by

Hg++ ions, phloretin and p-chloromercuribenzoate, and a functional

carbonyl group appears to be essential for the entry of glucose.

LeFevre (24) demonstrated that the property of phloretin as a competitive

inhibitor is shared only by molecules resembling phloretin in respect

to the spacing between phenolic-OH groups. He postulated, therefore,

that groups capable of reversibly binding or associating with phenolic-OH

groups may be distributed in a repeating pattern over part, or all, of

the red cell surface, and that the operation of the sugar-transfer system

involves the same loci. Thus, it would appear that glucose penetration is an example of facilitated diffusion and is limited by the availability

of certain specifie sites at the cell surface.

The observations by Rosenberg and Wilbrandt (25) with phloretin and by Bowyer and Widdas (26) with dinitrofluorobenzene, that the outward passage of glucose was much more inhibited than its inward pas sage may signify that different pathways are involved in the movement of this sugar into and out of the cell. The hypothesis of the latter authors is that a glucose ester may be transported across the lipid membrane by diffusion and at the inner interface the molecule would become at­ tached to an acceptor site on the enzyme and be hydrolyzed. Thus, the glucose would be liberated to the inside of the cell and the phosphate, for example, would become attached to another acceptor molecule.

Another pos sibility would be that enzymic sites may be located at inter vals 8

in the lipid membrane (polar poles) and the glucose phosphate could

either diffuse or be transferred directly or via intermediate sites to an ac­

cepter site at the inner interface. In these suppositions, circulation

of phosphate would be necessary either across the membrane or in a

local circuit between intermediate sites in the pore. Hillman (27)

obs erved that by increasing the chain length of the aliphatic substituent

on carbon 3 of glucose, from methyl to butyl, thus enhancing its

solubility in lipid, the rate of its penetration into rabbit red cells was

greatly accelerated.

On the other hand, Parpart and Ballentine (11) and Danielli (28)

have postulated the existence of aqueous channels in the plasma

membrane which are supposed to be spiral or convoluted in shape thus

presenting a barrier to free diffusion which is overcome by thermal

agitation. Faust (29) has proposed a modified ditffusion mechanism

with spiral- shaped aqueous channels in which the sugar molecule, its

size and charge, if any, and hydrogen bonding are the factors that

determine the rate of sugar penetration.

Sodium and sium

The osmotic pressure inside the red cell and that of the surrounding plasma are equal (30)~ In fact, Parpart and Green (31) have noted that when the cation permeability of the membrane increases, the volume also increases.

There is no doubt that sodium is kept out of the cell since the 9 concentration in the plasma is ten times that in the cell. R>tassium, on the other hand, is concentrated inside the cell, the concentration being thirty times higher than in the plasma. Harris and Maizels (32) have measured a rate of transfer for sodium which was always ten times greater for the outward transfer than for the inward.

Results obtained by Glynn (33) suggest that the rate of potassium influx includes a small passive component, which is proportional to the external potassium concentration, and a large active component. The latter is controlled by a mechanism which becomes saturated at high external potassium concentrations. The removal of glucose has no effect on the rate of sodium efflux in the absence of potassium, but abolishes about 75% of the increased efflux that occurs in the presence of potassium. The active component is closely linked to the potassium influx and is driven by energy obtained from glucose consumption; the passive component is not linked to potassium influx and is unaffected by the glucos~ concentration. The fact that removal of glucose leads to a decrease of roughly the same magnitude in the potassium influx and sodium efflux suggests that the connection between the active movements of the two cations involves a mole-for-mole exchange of sodium for potassium.

Post and J ally (34). on the other hand, have demonstrated a transfer ratio of two atoms of potassium to three atoms of sodium. They have shawn furtherm.ore that either the absence of potassium outside the cell or the absence of sodium inside stops the active transport of bath ions. 10

Since stoichiometric relationships between reactants and products are

a characteristic of simple chemical reactions, the demonstration that

the movements d sodium and potassium across the human erythrocyte

membrane are numerically related gives support to the view that active

transport proceeds by way of a chemical combination of the cations with

specifie binding sites. It further suggests that a chemical group must

pas s from the carrier of one cation to the carrier of the other at sorne

spacific stage in the transport process in order to keep the two

systems in step with each other. Such a transfer could take place at

either or both surfaces of the membrane and could be a transfer from

either the potassium or the sodium carrier.

Rabbit red cells exposed ton-butyl alcohol, as studied by Parpart

and Green (31), become more permeable to cations and a disorientation

of such surface components as the Hpoprotein complexes may account

for such an effect. Green (35), using u.v. light, concluded that two

types of changes may occur: one in which there is an equivalent exchange

of sodium for potassium and another in which there is an unequal

exchange. The former occurs usually when glycolysis is not interfered

with and the latter when glycolysis is inhibited. This generalization

implies that agents or conditions that modify the red cell surface but

not the metabolism of the cell produce equivalent cation exchange.

Exposure of red cells to glycosides is known to inhibit potassium influx to the extent of about 80o/o, and this would indicate that there

are two separate processes of potassium influx. Salomon et al. (36) 11 propose that the action of g1ycosides on potassium transport can be accounted for on the basis of competition of g1ycosides and potassium for a which is limited in amount or possib1y a site on the cell's surface. Solomon (37), studying the effect of ouabain, which is an inhibitor of potassium transport, has estimated the distribution of sites at one in every 1 o6- 10 7 A2.

If the membrane is pierced by aqueous channels through which only small neutra! or negatively-charged particles can pass, potassium would have to be conveyed by sorne other route open to lipid-soluble molecules either large or small, which may enter the red cell by dissolution in the membrane. Hoffman, Tosteson and Whittam (38) have noticed that the ghost may preferentially retain potassium upon incubation in an NaCl medium, thus suggesting that after osmotic hemolysis ghosts may regain sorne degree of the low permeability to potassium which is characteristic of erythrocytes. It is of inter est that Solomon, Lionetti and Curran (39) have extracted an heterogeneous material from blood that can discriminate between sodium and potassium ions, preference being given to the latter, and have shown that it is soluble in lipid solvents and dissociates in water. They suggest that this substance may possibly be a carrier for potassium transport. It seems likely that the material is of a lipoid nature and possibly contains phosphatide.

It is evident that the transport system is linked with glycolysis in sorne way. Eckel (40) has observed that the addition of O. 005M sodium fluoride gradually .!5lowed the potassium uptake but the addition of pyruvate 12

almost comp1etely abolished the net potassium 1oss. The uptake and

outgo remained ne arly equal at about 70% of the normal fluxes. The

addition of O. 025M sodium fluoride, however, seemed to cause the

potassium uptake to divide into two fractions, namely a slowly exchanging

and a rapidly exchanging component. The existence of these components

would imply the presence of two compartments or pools of potassium

within the cell. However, it must be borne in mind that the cell

population includes cells of various ages and stability, and that individual

cells therefore may differ in the cation exchange rate.

Whittam (41) suggests that of the enzymes of the glycolytic system

with which potassium influx might be linked in the absence of glucose,

enolase, pyruvate and phosphoglycer1ic kinase are the most

probable ones. To the writer, it seems reasonable to regard potassium

influx and sodium efflux as being due to the operation of sorne energy­

requiring carrier mechanism that utilizes ATP. Solomon et al. (36) have calculated that 7% of the free energy available from glucose

consumption would be required to effect potassium transport. Whittam (41) has shown that active movements against cationic gradients would require

only about 75% of the energy of hydrolysis of ATP.

In cases of failure of cationic transport, only ATP, among the glycolytic cofactors, was found to be depleted. In glucose-free cells, the glycosides, ouabain and digoxin, have been found to exert a protective action against the hydrolysis of ATP. This inhibition of hydrolysis by agents that are known to eliminate active fluxes of sodium and potassium 13

strongly suggests that ATP is utilis ed in the active transport of cations.

This observation offers added support for the ATP-Iinked carrier

hypothesis for the transport of thes e cations.

These results are consistent with the view that ATP is intimately

involved in the coupling of the glycolytic system and an active transport

of potassium. Post et al. {42) postulate that an ATPase in the membrane

is involved in the transport of potassium and sodium. It is probable that

ATP, an anion with a strong negative net charge, plays a specifie role

in many functions of the membrane. It is essential also in the maintenance

of the membrane structure since this maintenance requires the expenditure

of energy from metabolism.

Inorganic phosphate

As radioisotopic experimenta have demonstrated, there is relatively

little exchange between the small concentrations of inorganic phosphate

inside and outside the cell. Tabechian et al. (43) have demonstrated an uptake and a release of phosphate by the cell. It is suggested that the inorganic phosphate in the cell is derived from the hydrolysis of phosphate

esters within the cell. Normally, the inorganic phosphate uptake by the

cell seems to be effected through a mechanism requiring a large amount

of energy. Gourley (44) has reported an activation energy of 16,000-

19, 000 calories per mole as compared to 4, 000 calories per mole for a process of simple diffusion.

Prankerd and Altman (5) have noted that the uptake of phosphate is entirely dependent on glycolysis since inhibitors of glycolysis or depletion 14 of the cell's glucose supply will inhibit the phosphate transport. Since the entry of orthophosphate into the cell is accompanied by an activation energy analogous to an enzymatic reaction, these workers, and Bartlett (45) as well, have suggested that the triose phosphate dehydrogenase present in the cell membrane or cell coat picks up the phosphate which is transferred to carbon 1 of 1, 3-DPG. The latter worker defines the ''cell coat" as a framework of insoluble fibrous proteins and lipids, containing its own cytoplasm and separated from the interior of the cell and from the plasma by phase boundaries that are called inner and outer surfaces; its thick-

0 ness would vary between 50 and 250 A.

Further evidence obtained by Kahn (46) and by Levy et al. (47) and by Lionetti et al. (48) suggests the existence of another pathway, which becomes dominant when nucleosides are present. If there is already inorganic phosphate present inside the cell, as after a period of cold storage, the addition of the nucleoside will inhibit the transport of phosphate to the cell. In this case, one mole of intracellular phosphate will be used for each mole of ribose split from the nucleoside to produce ribose phosphate which can be further metabolized to replenish the energy stores of the cell. In fresh cells, however, the re is little inorganic phosphate and the addition of nucleosides will cause an increas ed uptake of inorganic phosphate by the cell.

If the activation energy of phosphorolysis of the nucleoside is added to the activation energy for phosphate uptake by the cell without nucleosides, the sum thus obtained agrees with the activation energy value found for 15 phosphate uptake by erythrocytes with nucleosides. These additive magnitudes suggest at least two independent mechanisms for the phosphate transfer in erythrocytes when nucleosides are present.

Altman (49), studying the metabolism of red blood cells from patients with hemolytic anemias, observed that sodium fluoride, instead of decreasing the rate of entry of phosphate, increased it. This could perhaps be caused by alteration in the ratio of ATP to ADP which could lead to a temporary acceleration in the rate of formation of triose phosphates. If the transport of orthophosphate is mediated through the action of the triose phosphate dehydrogenase in the membrane of 32 the celland if the rate of passage of orthophosphate-P in and out of the red blood cell can be regarded as an index of the metabolic activity of the membrane at the surface of the cell, then it may be postulated that metabolic abnormalities are directly related to changes in the structure of the cell membrane and to the disposition of the enzymic units therein. Such altered cells would be less viable and probably be li able to a more rapid rate of destruction than normal cells.

METABOLISM OF THE ERYTHROCYTE

It is well known that the mature erythrocyte obtains its energy entirely from the metabolism of glucose to lactate mainly by way of the glycolytic pathway (50-56). Warburg and Christian (56), Brin and Yonemoto (55) and others had provided evidence of the presence of a pentose phosphate metabolic "shunt" as an alternate of glucose metabolism to lactate.

The glycolytic and pentose metabolic pathways are pictured in Figure 1, and all the reactions are numbered and from now on may be referred to 16

FIGURE 1

The glycolytic and pentose metabolic pathways in the red cell.

Reaction Step Enzyme

1 Phosphorylase 2 Phosphoglucomutase 3 1-phosphoglucokinase 4 Glucose-1, 6-diphosphatase 5, 7 Hexokinase 6 Phosphoglucoisomerase GLYCOLYTIC 8 Phosphofructokinas e 9 Aldolase PATHWAY 10 Triosephosphate dehydrogenase 11 Phosphoglyceric kinase 12 Diphosphoglyceric mutase 13 Glycerate-2, 3-diphosphatase 14 Phosphoglyceromutase 15 Enolase 16 Pyruvic phosphoferase {kinase} 17 Lactic dehydrogenase

18 Nucleoside phosphorylase 19 P4osphoribomutase . PENTOSE 20 Glucose-6-P dehydrogenase 21 Gluconolactonase PHOSPHATE 22, 23 6-phosphogluconic dehydrogenase PATHWAY 24 Pentose phosphate 25 Ketopentose phosphate isomerase 26, 28 Transketola se 27 Transaldolase

Reaction Steps 3 and 4 have not been elucidated but are included to account for the relatively high glucose-1, 6-diP concentration in the red cell. The equilibrium in Steps 5, 7, 8 and 12 is such as to make the reaction virtually irreversible. No appreciable phosphatase activity has be en detected in Steps 5, 7 and 8. FIGURE

GLYCOLYTIC AND PENTOSE METABOLIC PATHWAYS PUI!IIr-!Wtl

I·I·P+PUIIII

_____!!!!QJ!!!+-, :, ....+Ali' ,

1 ~-•DPfl'",..------1 ~ 1 ADP•-1-+1,3-DPG-.;::-" '------.. DP~H+H+ l 1" 11 ;'::Y.t3-DPG Ali' 1 1 AfP.-""-+3-PG•-- AfP ~ • 1 u 1 ,.t :------·-'~ .------; z!J.G•--.ll---•2-P-IIIOLPYIUYATI PYIUYATI lACTATE

Pentose metabolic pathway represented by solid lines and glycolytic pathway by broken lines. 17

1 1 in the text as ' Step X' •

The presence of the shunt was established by Brownstone (57) in our

laboratory in 1959. Due to the absence in the red cell of a complete

oxidative system for the reoxidation of TPNH, this pathway apparently

is relatively inactive in the mature red cell except in the reduction of

methemoglobin.

Dische (58), in 1938, showed that nucleosides, like adenosine, can

enter the metabolism of the red cell directly in the pentose phosphate

shunt (Steps 18 and 19). Warburg (59) observed that the addition of

methylene blue to a red cell suspension caused a marked increase in

oxygen uptake. In the years that followed other workers showed that

methylene blue brings about the reoxidation of TPNH, and the reduced

methylene blue in turn is oxidized by molecular oxygen. For further

information on the mechanism of the pentose metabolic pathway and its

relation to the glycolytic system, the reader is referred to Brownstone (57)

who has covered the literature on the enzymes of the pentose phosphate

metabolic pathway and to Blostein (60) who has reviewed the more recent literature on the enzymes of the glycolytic pathway in the erythrocyte.

Hexokinase

Kashket (ZZ) has reviewed the literature on hexokinase (Step 5). As yet the hexokinase of the erythrocyte has not been extensively purified and most of the studies on the enzyme in the red cell have been done on the stroma-free hemolyzate (SFH).

Kashket (22) gives the following data for the hexokinase of rabbit red 18

-3 d cells: the KMgATP and Kg1ucose have the values 1. 5 x 10 Man 4 2. 8 x l0- M respectively. The activation energy for the phosphorylation

of glucose by the hexokinase in the SFH was found to be of the order of

11, 500 calories /mole. The enzyme, according to Kashket, has two

activity peaks: one prominent maximum at pH 7. 8 and a lesser one at

pH 6. 0 with much- reduced activity between the two at the minimum at pH 6. 6. The enzyme was found not to be inhibited by fluoride but

inactivated by lipase, pepsin and by taurocholate. These observations

suggest that the red cell hexokinase is lipoprotein in nature. AMP a:r:1d

glucose-6-P also were found to inhibit the enzyme. Kashket was unable

to demonstrate the presence of the enzyme in the cell stroma and he

suggests that it may be distributed over the inner surface of the membrane.

Valuable information concerning hexokinase of the red cell has been anticipated by inference from the studies of other workers on hexokinas e from other sources. The crystalline hexokinase prepared by Kunitz and

McDonald (61) from bakers' yeast was found to have a molecular weight of 96, 600 and to require the presence of magnesium ions for its activity.

Melchior et al. (62) have shown that hexokinase from yeast, if incùbated with fluoride before the addition of ATP, is inhibited, and that the effect is attributable to removal of magnesium ions by the fluoride with formation of a magnesium-fluoride complex. According to Boser (63), the hexokinase in yeast is a glycoprotein composed of 52% carbohydrate made up exclusively of mannose residues.

For purified rat-brain hexokinase, Crane and Sols (64) found that the 19

K and KATP were lx 10 -SM and l. 3 x lo- 4M respectively. g~oe1 8 - - Gamble and Najjar (65), with crystalline yeast hexokinase, found that it catalyzed the conversion of glucose-6-P to glucose much less strongly (l/50th) than that of glucose to glucose-6-P.

Kashket (22) observed that the hexokinase of the rabbit red blood cell phosphorylated various sugars in decreasing order as follows: D-glucose,

D-mannose, D-fructose, D-galactose at pH 7. 8 while at pH 6. 6 the decreasing arder was mannose, fructose, glucose, galactose.

It may be inferred that the above-mentioned sugars have the required structural configuration for phosphorylation by the hexokinas e. Sols and

Crane (66) have established that the pyranoid form of the hexoses is one of the essential structural features. They further concluded that the formation of the active glucose-hexokinase complex must involve the hydroxyl groups at carbons 1, 3, 4, and 6. Lange and Kahn (67) found that hexokinas es from intestine, kidney and rat liver were completely inactive towards sugars which do not have the same configuration of hydroxyl groups as glucose at carbons 2 and 3 or at carbons 3 and 4, with the exception of D-gulose which has the same configuration as glucose on carbon atoms 4, 5 and 6.

Crane and Sols (64) found that the hexokinase from rat brain is inhibited by AMP, and sometimes by ADP, depending on the affinity as compared to that for ATP and glucose-6-P. Colowick and Kalckar (68), with the enzyme from bakers' yeast, observed inhibition of the activity with glucose-6-P and reversal of the inhibition on addition of phosphofructokinase. The 20

competition was ci the noncompetitive type and only the pyranoses with the

essential configuration of the hydroxyl groups at carbons 2 and 4, and

phosphorylated on carbon 6, were inhibitory towards brain hexokinase.

The evidence indicates that brain hexokinase possesses, in addition to

the two binding sites for substrates and ATP, a third specifically

for glucose-6-P.

Colowick and Kalckar postulate furthermore that the metabolism of hexoses in animal tissues is characterized by a certain "steady state 11 in which the rate of phosphorylation of sugars is limited at all times by

the conditions existing within the hexokinase-G-6-P-phosphofructokinase

system. Since ATP is converted to ADP in the reaction, it is also possible that the ratio of ATP:ADP in the cell, like that in the glucose-6-P system,

may be the main factor in controlling the activity of hexokinase, and hence of glucose utilisation and of the metabolism of the cell.

Fhosphoglucoisomerase

Kahana et al. (69) have measured the equilibrium of various purified specimens of phosphoglucoisomera'l>e (Step 6), including that of the erythrocyte, and found that the equilibrium in all cases tends slightly to favor the formation of glucose-6-P, this tendency being accentuated as the temperature is lowered. ATP and inorganic phosphate were found to inhibit the enzyme at a concentration of O. 4 x 1 o- 3M and 1. 7 x 1 o- 3M respectively. Tsuboi (70), with a purified sample of the enzyme, estimated the activation energy to be about Il, 000 calories. The phosphoisomeras e appears to be a "nonsulfhydryl 11 enzyme. 21

Phosphofructokinas e

This enzyme catalyzes the interconversion of fructos e-1, 6-diP and fructose-6-P (Step 8). The reaction is endergonic and, like the hexokinase

reaction, is coupled with the conversion of ATP to ADP from which

reaction the required energy is derived. The equilibrium of the reaction is far towards fructose-!, 6-diP formation. Fructose-!, 6-diP is broken clown with the help of a1dolase to triose phosphates {Step 9), and although the equilibrium is in favor of fructose-!, 6-diP, the reaction rate is about thirty times more rapid than the rate of glycolysis {71).

Enzymes of the metabolism of triose phosphates

The triose phosphate dehydrogenase reaction along with the hexokinase reaction is considered by Krebs and Kornberg (72) to be the rate- controlling step of the glycolytic system. The enzyme is DPN-linked and catalyzes the oxidation of 3-phosphoglyceraldehyde to 1, 3-diphosphog1ycerate, requiring DPNH in the reaction along with inorganic phosphate and g1utathione {Step 1 0).

The 1, 3-DPG can be metabolized by way of at 1east three different pathways. The usual pathway is that cata1yzed by with the formation of 3-phosphoglycerate and regeneration of ATP (Step 11).

A second pathway is that cata1yzed by a mutase and glycerate-2, 3-diphosphatase with the formation first of 2, 3-DPG {Step 12) and then 3-phosphoglycerate

(Step 13). No ATP is formed in this sequence. The pathway is difficult to differentiate from the third, name1y the one-way direct conversion of

1, 3-DPG to 3-phosphog1ycerate by the action of an acyl phosphatase (73). 22

The question as to which of these pathways is the dominant one in red

cell glycolysis remains unsettled. The exceptionally high concentration of

2, 3-DPG found in the human erythrocyte (74) points to the second alternative

as being important in the erythrocyte. The addition of labelled orthophosphate

to blood gives rise to labelling first on the terminal labile phosphate group

of ATP and later in the 2, 3-DPG molecule (3, 45, 75). Y et, Bartlett and

Marlow (76) have demonstrated that 90o/o of cl 4 -glucose goes through

2, 3-DPG (Step 12). On the other hand, Rapoport (77) has observed that

fluoride, which inhibits enolase, causes an accumulation of 3-phosphoglycerate

which is known to stimula te the mutase- catalyzed reaction (Step 12}. The re

results an accumulation of 2, 3-DPG since the 3-phosphoglycerate is known also

to inhibit the phosphatase (Step 13) that converts 2, 3-DPG to 3-phospho­

glycerate (78, 79}. Once the inhibition is removed, however, the 2, 3-DPG

ceases to accumulate. A low concentration of ADP (at Steps 11 and 16)

could also preferentially favor the conversion of l, 3-DPG to

2, 3-DPG (Step 12). Rapoport (80) has pointed out that the great drop of free energy in the diphosphoglyceromutase reaction (Step 12) makes it practically irreversible. Therefore it would seem that if glucose is

metabolized through 2, 3-DPG, the potential ATP that would otherwise be produced through the phosphoglyceric kinase, is irrevocably lost.

As the hexokinase reaction regulates the rate of glucose utilisation,

Rapoport (78) and Grisolia (81) have suggested that this whole system might constitute a self-regulatory cycle and be an important means of regulating the storage of energy in the cell. 23

The 3-phosphoglycerate is converted to pyruvate viaS teps 14, 15 and

16 and the last reaction (Step 16) regenerates ATP. As the final step in

glycolysis, pyruvate is converted to lactate by the enzyme lactic dehydro­

genase which requires DPNH as . In this reaction oxidized DPN

is regenerated. Bartlett (45) suggests that the triose phosphate dehydro­

genase (Step 10) in the red cell probably is situated close to the lactic

dehydrogenase (Step 17) on one side, and to 3-'lJhosphoglyceric kinase

(Step 11) on the other, perhaps in the "cell coat 11 (see P. 14). He suggests

further: "The property of movement in living cellular cytoplasm is so

universal, where it can be seen, that it would be reasonable to assume

its validity for the red cell until proved otherwise. It should not stretch

the imagination unduly to have the enzymes in the cell coat in a more

active motion than that due to molecular collision. There is one more

of the ATP splitting reaction, namely energy. To complete our

scheme we postulate that the principal use of this energy is to impart

vigorous motion to the cytoplasm. 11

Enzymes connected with the metabolism of high energy phosphate comp"ounds.

The metabolic processes in the red cell which require ATP include the maintenance d the ion concentration gradients across the cell membrane,

and phosphorylation reactions; ATP also is the substrate of the ,

ATPase. The ATP level is the main energy reservoir of the celland its concentration, along with that of phosphorylated intermediates, which in the course of their metabolism give rise to the regeneration of ATP, constitute 24

the metabolic potential of the cell.

Addition of nucleosides to fresh blood has been found in our laboratory

not to increase the ATP concentration.

However, Bartlett (45) claims that addition of inosine, even to fresh

erythrocytes, increases the ATP content to 1t times the initial value.

The formation of ATP in rabbit erythrocytes has been studied by Lowy

et al. (82), who estimate that the half-life of the adenine moiety of ATP

is about 13 days. They obtained evidence, furthermore, that ATP can be

produced from adenine and phosphoribose pyrophosphate. Adenosine also

was found to be a precursor of ATP: either from adenosine to adenylic

aci d to ATP, or from adenosine to inosinic acid to hypoxanthine to adenylic acid to

ATP. More recently (83), these authors have shown that, while adenine

can be a precursor of ATP in the red cell, xanthine, guanine and guanosine

analogously can be precursors of GTP. They demonstrated that hypoxanthine,

inosine and adenosine can be converted to both ATP and GTP in vitro in

the red cell. They suggest further that the newly formed red cells are

endowed with a certain quantity of ATP which thereafter continuously

decreas es. This downward trend in energy res erve leads to gradua! decline in the metabolic capacity and activity with aging, and ultimately to loss of viability and to destruction.

Aside fran the glycolytic enzymes that participate in the regeneration and in the utilisation of ATP, the re are two other main ones which may greatly influence the ATP level in the cell. These are: and ATPase. The first catalyzes the interconversion of 2 moles of ADP to 25

one mole of ATP and one mole of AMP. The second probably repres ents

more than one enzyme with the property of hydrolyzing ATP to ADP and

in certain conditions the hydrolysis of ADP to AMP.

Adenylate kinase plays an important role in the production of A TP

and the critical equilibrium between ADP and ATP. Grisolia (81) has

observed that a lack of ADP at the pyruvic phosphoferase· level (Step 16) cân'

cause an accumulation of 3-phosphqglycerate.

The presence of adenylate kinase in the erythrocyte has been reported

by Kashkèt (84) and Tatibana (85). Kashket noted the strong activity of

this kinase both in the membrane and in the cytoplasm (SFH). He observed

further that ADP, when added to a suspension of washed erythrocytes,

caused an increase in the rate of glucose utilisation and that it gave rise

also to ATP in the external medium as demonstrated by the phosphorylation

of glucose in the external medium, in the presence of added yeast hexokinase which has a specifie requirement for ATP. The ADP evidently can enter the cell membrane where it is converted by adenylate kinase to equimolar concentrations of AMP and ATP. The ATP thus formed evidently can pass both into the cell and into the external medium.

Herbert (86) identified an ATPase in the red cell stroma which is active towards ADP and ATP and is activated by both magnesium and calcium ions; the effective concentration for magnesium was lower than that for calcium. Calcium was observed to antagonize the action of magnesium by what appeared to be·a process of competitive inhibition. Caffrey (87), on the other band, has expressed scepticism about sane of the findings of 26

previous workers (86, 88, 89} with stroma. He claims that the ATPase

activity can be obtained only with the stroma-free hemolyzate. The

enzyme is magnesium-activated and is inhibited by all other divalent

cations in the presence of magnesium. The ATPase is said to hydrolyze

the high energy phosphate compounds: ATP, ADP, ITP, UTP. It will

not hydrolyze AMP. The pH optimum of Herbert's and of Caffrey's

preparations was found to be the same: Herbert's peak activity occurred

between pH 7. 3 and 7. 6 while that found by Caffrey was between 7. 0 and

7. 4. Furthermore, the two enzyme preparations were observed to be

protected by cysteine. It would appear that the ATPases in the two

preparations, notwithstanding the difference in their preparation, were

identical. The fact that Herbert obtained the activity with the particulate

fraction would suggest that the enzyme is somewhat associated with the stroma.

Pyrophosphatase activity was obtained with a nonpurified ATPase preparation. Malkin (90} in our laboratory observed that the activity is

confined to the cytoplasm of the cell. The question of whether there is

more than one ATPase and whether it is present in the strana or cytoplasm

only, or in both, retnains unanswered.

Other enzymes

Of the remainder of the enzymes, known to be present in the erythrocyte, a few have a more or less direct relation with the metabolism of the cell while others are occupied only with the actual functions of the red cell.

Lowy et al. (91}, in their studies on the metabolism of various nucleosides in the intact human erythrocyte, showed that only the ribose moiety of the 27

riboside is utilised and that most of it is metabolized to lactic acid. This

can be taken to indicate that, although the equilibrium is towards the

formation of the nucleosides, the ribose phosphorylase does not occur

in the membrane. Rubinstein et ai. (92) found over 90% of the nucleoside

phosphorylase activity in the cytoplasm. Lionetti et al. (93, 48) however

report that they have prepared ghosts which utilise the ribose of the nucleosides, which brings about an increase of the phosphate ester

content of the ghosts. Prankerd and Altman {5) have also isolated a number of glycolytic intermediates from the stroma. It seems likely

therefore that some of the enzymes of the pentose phosphate and glycolytic pathways are 'situated in, or closely associated with, the membrane in the

erythrocyte.

Alivisatos (94) of our laboratory, in 1951, demonstrated the presence

of a DPNase on the ruter surface of the erythrocyte membrane. Whenever hemolysis occurs, the DPN inside the cell is rapidly destroyed upon contact with the DPNase on the membrane. Concentrations of nicotinamide

{2 x lo- 2M) that completely inhibited the DPNase in whole hemolyzates were found also to inhibit partially the DPN- and TPN-linked dehydrogenases (95).

Certain enzymes in the red cell play a role in maintaining hemcglobin in the reduced state in the cell. Catalase also may play a role in protecting hemoglobin from oxidation {96). Two enzyme systems are known which can reduce hemoglobin directly: one DPNH- (97) and one TPNH-linked reductase (98). Still another enzyme appears to re duce hemoglobin indirectly through the intermediation of glutathione. This reaction can be demonstrated 28

in vitro. The glutathione reductase, either the DPNH.. or the TPNH-linked

(99) form, maintains glutathione in the reduced state and thus protects

SH groups of the enzyme from oxidation and inactivation. Carbonic

anhydrase (lOO) catalyzes the formation and breakdown of carbonic acid

and plays an important role in the efficiency of the red cell regarding the

transport of C02 from the tissues to the lungs.

The erythrocyte contains a number of enzymes of unknown function:

acetylcholine esterase (1 01) and certain proteolytic enzymes capable of

digesting denatured hemoglobin (102).

Relatively little information has been gathered regarding changes in

the activity of enzymes during aging of the erythrocyte. Recently Marks

et al. (103) have succeeded in separating the younger cells fran older ones by a technique of seria! hemolysis, the young cells being the more resistant to osmotic lysis. The glucose-6-P dehydrogenase, 6-phosphogluconic

dehydrogenase and phosphoglucoisomerase were found to be of higher

activity in the young er cells (1 04).

Hoffman (105) and Prankerd {106), labelling the cells with, Fe59, observed that due to a decreased content of lipids the cell density increased with age. It is possible that the loss of lipid associated with age is an important factor in the survival, since cells artificially denuded of lipid survive but a short time after transfusion. The total phosphate ester and glutathione content were not different in old and young cells. The young er cells contained a higher concentration of potassium and less sodium than the older ones. Chalfin (107} corroborated these observations and observed 29

that in mature rabbit erythrocytes the total cation concentration is the same in young and older cell fractions.

Metabolism of red cells during preservation at 4°C.

The practice of preserving human blood for clinical use in hospitals has be en in vogue for more than a quarter of a century. A very large number

of studies has been carried out on the changes that occur in the cells during

storage in the cold. The re ader is referred to Pappius (1 08) for the his tory on earl y studies regarding blood preservation. Many workers in this laboratory have contributed to the present knowledge of the metabolism of blood during storage. Gabrio and Finch (109) have established that little relationship exists between the behaviour of red cells during aging in vivo and in vitro.

Greig and Gibbons (llO) have suggested that phenothiazine derivatives may be effective in the preservation of blood, since these agents are supposed to inhibit a lecithinase which in turn is claimed to release choline esterase from the cell membrane. It was supposed that this removal weakens the membrane and may alter the properties of the membrane.

However these agents and other allegedly beneficia! compounds such as o(-tocopheryl di sodium phosphate ( 111) were found to have no special me rit.

In view of the presentation to follow later it may be of help to review briefly the general metabolic behaviour of red cells during storage in the cold. As glucose is converted to lactic acid by glycolysis the hydrogen ion concentration increases, and the pH falls from 7. 6 at the start to 6. 5 in

ACD and from 7. 4 to 6. 9 in CD within about 4 weeks' time during preservation 30

of the cells at 4°C. With the fall in pH the glycolytic activity becomes progressively retarded. When the pH falls below 7. 0, activation of phosphatase

occurs and 2, 3-DPG and other phosphate esters undergo hydrolysis with

liberation of inorganic phosphate. By the end of the third week the rate

of glucose utilisation by the cells has become negligible and the ATP content

is reduced by 55% or more. The decrease in the ATP tends to be least with

the ACD preservative solution. Pyruvate also accumulates from the beginning

of storage in ACD but not until about the 12th-15th day in CD.

The behaviour of the cation movements in the cold has been studied.

extensively. During storage the cell potassium slowly escapes into the plasma while sodium enters in exchange. These cations move by passive

diffusion along the respective concentration gradient, and at a rate which

may be influenced by the hydrogen ion concentration (112} and other factors.

Recent studies by Blostein et al. (113) in our laboratory have established that active metabolic control over cation transport against the respective ionie gradients is not operative or negligible at 4°C. The cation concentrations or movements are not ù.nfluenced by endogenous metabolism or by ATP concentration of the cell, but are influenced by the inorganic phosphate concentration and probably by the hydrogen ion concentration. They further obtained evidence of the dependence of the cells on the metabolic activity and

ATP reserve for the restoration of the cation concentrations towards the normal when the preserved cells are restored to a temperature of 37 oc.

These observations are essentially in agreement with the findings of

Whittam (41), Post (42), and Eckel (40}. 31

Rubinstein et al. (114) believe that the most important condition for

the maintenance of viability in preserved erythrocytes is the maintenance

of the ATP res erve of the cells. Kashket (115) found that. one of the

conditions that tend to interfere with the maintenance of the ATP and

phosphory1ated intermediates during storage is the increase of the hydrogen

ion concentration, owing mainly to the accumulation of lactic acid. The

hydrogen ion concentration is then far from the optimum pH of hexokinase

and thus the phosphorylation of glucose to glucose-6-P cannot take place.

Furthermore, the increase in the hydrogen ion concentration to about

pH 7. 0 causes activation of the phosphatase, 2, 3-DPGase (115) which brings

about rapid hydrolysis of the 2, 3-DPG and a rise in the inorganic phosphate

of the cell.

Even though one of the phosphate groups may be lost through hydrolysis

by phosphatase during storage of blood the phosphate on carbon 3 of the

diphosphoglycerate is readily utilised for the formation of ATP through the

catalytic action of enolase and pyruvic phosphoferase (Steps 15, 16).

It is interesting that although the rate of synthesis of ATP is retarded

in cells preserved in the ACD medium, the rate of breakdown of the endogenous

reserve also is retarded at the lower pH and thus the energy reserve is

better maintained during storage. However, the biochemicallesion due to

starage appears to be rapidly reversible to a greater or lesser degree upon

introduction of the erythrocytes into the circulation. This is evidenced by

·-the replenishment of the phosphate esters (116).

Bartlett and Shafer {11 7) have shown a definite correlation between the 32

ATP content of the preserved cells and the capacity to survive in the

circulation after transfusion.

Brownstone (118) and Huennekens et al. (119) have conclusively

established that the enzymes of the pentose phosphate pathway and the

system itself remain active in the red cells even after pro1onged storage

in the cold.

Pappius (120) has noted that the capacity of the cells to utilise glucose

became progressively impaired during storage in the cold and that the

addition of more glucose did not stimulate the rate of utilisation significantly.

If the glucose were used up, the capacity to utilise added glucose was greatly

reduced.

Blanchaer (121} showed that, during storage, intact erythrocytes pro-

gressively lose the ability to reduce added pyruvate to lactate, whereas the

whole hemolyzate remains active for at least 33 days. He further established

that the triose phosphate dehydrogenase, the phosphoglyceric kinase and the

triose phosphate isomerase remain fully active for at least three weeks at

4°C, He ccncluded that the blockage in the reduction of pyruvate in the intact cell probably lies higher in the system where glucose is converted to fructose-cliP. {Steps 5, 6, 7, 8). Later, he found that the phosphofructokinase activity in cells and hemolyzates decreased progressively during storage and that by the 25th day the activity had fallen to 20 or 30% of the original (122}.

Following the discovery of Gabrio et al. (115} of the dramatiè effect of added adenosine in 11 rejuvenating 11 the metabolic capacity of preserved . erythrocytes, many workers (92, 97, 113, 123-128) became interested in 33

the phenomenon. Rubinstein and Denstedt (92) were the first to show that

this "rejuvenation" occurs, as might be expected, also with added inosine

which proved much less toxic than adenosine. It has amply been demonstrated

that the added nucleosides effect a restoration of the ATP, 2, 3-DPG and

other phosphate esters. The restoration is accompanied by a decrease

in the cell volume, and return of the potassium:sodium ratio toward reestablishment of the normal osmotic stability (128, 129). However,

Weinstein et al. (111) have shown that the osmotic resistance of preserved

blood, as measured by "fragility" tests, is not a reliable criterion on which

to predict the capacity of the cells to remain in the circulation after

transfusion.

Arranged in decreasing order of activity in effecting regeneration of phosphorylated esters in preserved erythrocytes, the nucleosides are as follows : inosine, adenosine, guano sine and xanthosine ~ 131). The aminated nucleosides undergo deamination in the cells, and all of the nucleosides undergo phœphorolysis to yield ribose-1-P and a purine moiety. The

ribose-1-P is converted to ribose-5-P which is metabolized to lactate and yields one mole ATP. The base diffuses from the cell into the plasma, reaching an equilibrium with that in the cell (92, 128). Bucolo and Bartlett

(132), with column chromatography and differentiai elution, have quantitatively estimated the metabolites produced after the addition of inosine to fresh human red blood cells. They found that the ATP increased to 1t times the normal value. The AMP and ADP were found to decrease and an equivalent amount of inosinic acid was produced. The 2, 3-DPG concentration was 34 increased by about 50o/o, accornpanied by a srnall rise in the rnonophospho­ glycerate; concentration of fructose diphosphate was increased several-fold and was sufficient to account for the proportion of the ribose that was not converted to lactic acid. The ribose-5-P and the dihydroxyacetone phosphate concentration also were increased several-fold. The workers found that the inorganic phosphate concentration was a lirniting factor.

Rubinstein et al. (127) found that adenosine is more effective than inosine in rnaintaining the capacity of preserved red cells to utilis,e glucose during storage. This rnerit they attribute to the buffering effect of the arnrnonia liberated from adenosine on dearnination and the maintenance of the pH within the optimum range for hexokinase activity. Inosine, on the contrary, does net have the buffering effect, consequently as the pH falls the capacity of the cells to utilise glucose rapidly becornes irnpaired. It will be recalled that the enzymes of the pentose rnetabolic pathway rernain active over a wide range of pH whereas sorne of the enzymes, notably the of the glycolytic system, are readily inactivated by an increase in hydrogen ion concentration to pH 7. Ribose phosphate (from the nucleoside) therefore is rnuch more easily utilisable than glucose at the lower pH.

Berry and Chanutin (133), with the aid of an electrophoretic rnethod, have observed the presence, in the hernolyzate of red cells preserved with inosine, of an unidentified cornponent which tends gradually to disappear from the red cells after a rnonth of storage.

Brownstone and Blanchaer (134) have clearly dernonstrated the decreased phosphofructokinase activity during storage and its regeneration upon addition 35

of adenosine to the preserved cells. They found further that the addition

of adenosine, inosine or ribose-5-P to the fresh blood, and even to stored

hemolyzate, prevented the decrease in the phosphofructokinase activity

during storage.

Kashket (115), on the other hand, found that the hexokinase of the preserved

erythrocyte could not be reactivated by the addition of inosine.

However, when cells preserved with inosine frem the beginning of storage

are washed and resuspended in a glucose-containing medium, they show

greater ability to utilise glucose than specimens stored without inosine and to

which inosine was added later during storage. It is of interest therefore that the presence of the nucleoside tends to preserve the hexokinase and favor maintenance of the ATP reserve through the utillsation of ribose.

Chaplin et al. (135-136), Strumia (137) and others hav,e done much

experimentation in recent years on the preservation of blood at sub-zero temperatures in media containing lactose or other sugars. The storage period was extended to two years. The limited success with these methods appears to dep:end not so much on the provision of substrate for the cell as to introduce rpaterial with strong hydrogen-bonding properties to prevent the complete crystallization of water molecules and to create a more favorable external osmotic condition for the cells. Whatever merit the methods may have is seriously cffset by the cumbersomeness of the procedures. EXPERIMENTAL

I. MATE RIALS AND REAGENTS

A. Substrates

The substrates and biochemical reagents used throughout this investigation were obtained from Nutritional Biochemicals Corporation,

Sigma Chemical Company, and ether commercial sources.

Solutions of diphosphopyridine nucleotide and the sodium or potassium salts of adenosine di- and triphosphate were neutralized before use. Tests with paper electrophoresis showed that most of the preparations were very pure. Any that contained inorganic phosphate exceeding 5% of the total acid soluble phosphate were rejected.

B. Buffers

Phosphate and glycylglycine buffers were used but it was found necessary to use higher concentrations than were used by Kashket in his studies on hexokinase. Measurements of the pH of the preserved blood specimens were taken during the period of storage, and before and after incubation of samples. A 11 Radiometer Copenhagen pH 22 11 instrument was used for these measurements.

Since blood itself has strong buffe ring power, a fairly high concentration of buffer may be required to prevent shifting of the pH of a medium when blood is added to it, and in many instances it was necessary further to adjust the pH of the final solution to the desired level, after the procedure 37 of Rubinstein (127), by addition of a small amount of O. 3N HCl or KOH.

Usually, it is necessary to perform a trial to determine the exact quantity of acid or alkali required, and then repeat the preparation adding the acid or alkali to the buffered solution before adding the blood. This procedure avoids denaturation of the proteins.

C. Enzymes

Severa! unsuccessful attempts were made also to purify the hexokinase from human erythrocytes by removal of the hemoglobin from the stroma- free hemolyzate by means of the ethanol-chloroform precipitation (119).

However, the hemoglobin-free supernatant was found to contain no activity.

lt was soon realized that the whole hemolyzate was unsuitable to work with, due to the difficulty in reproducing resulta with a given hemolyzate.

The factors affecting the reproducibility of resulta could have been the presence of DPNase and the formation, during the incubation period, of a jelly-like material with the appearance of egg-white. Baker (138) has observed in hemolyzates of rabbit and rat erythrocytes the same phenome- non and that the material responsible for it is found in the stroma.

The stroma-free hemolyzate, which contains all the enzymes of the glycolytic pathway, was therefore used as the hexokinase preparation and was prepared as follows:

A sample of fresh citrated blood was centrifuged in the cold and the plasma including the white cell layer removed. To the packed red cells isotonie KCl (O. 154M) was added, the cella re­ suspended, and the sample centrifuged. Resuspension of the cella in the KCl and centrifuging was repeated three to four times. Finally to one volume of the washed and packed cells was added one volume of the KCl, and the cell suspension hemolyzed by alternate 38

freezing in a dry ice-acetone bath and thawing at 37°C, repeating the treatment three times. Another volume of cold KCl was added and the preparation became more turbid. It was then centrifuged in the cold for 30 minutes at 10, 000 x g. to remove the finely dispersed stroma.

It is very important that the temperature be kept very near the freezing point so as to inhibit the activity of the DPNase situated on the outer surface of the membrane (94) and minimize the loss of DPN liberated during the hemolysis of the cells.

Since the activity of the SFH varied linearly with dilution it was found convenient to use the concentration of hemoglobin as an index of the enzyme concentration.

Yeast hexokinase (Nutritional Biochemicals Corporation) with a specifie activity of 28,000 Kunitz-McDonald units per gram (60), at 30°C, was used to determine the relation of the glycolytic chain of enzymes to the hexokinase system.

II. METHODS

A. Procedure for collection and preservation of blood

Blood was collected by venipuncture from each of two donors into sterile neutral citrate-dextrose (CD) and the acidified citrate-dextrose (ACD) preservative solutions. The CD solution contained 1. 80% (w/v) sodium citrate and 1. 47o/o dextrose; the ACD solution contained 1. 32% sodium citrate,

O. 44% citric acid and 1. 47l/o dextrose. The proportion of b1ood to preservative medium was 4:1. The samples were stored immediately at 4 ± 1 oC. 39

B. Analytical procedures

Glucose

"Glucostat", a commercial preparation of glucose oxidase obtained from Worthington Biochemical Corporation, Freehold, New Jersey, was used for the estimation of micro-quantities of glucose. The particulars of the reagent as described by the supplier are as follows:

"Glucostat is a coupled enzyme system suitable for the rapid, quantitative, colorimetrie determination of true glucose. It is based on the specifie enzymatic oxidation of glucose by glucose oxidase; gluconic acid and hydrogen peroxide are the end products. The hydrogen peroxide reacts with a chromogenic hydrogen donor in the presence of peroxidase and the color formed at a given time is proportional to the concentration of glucose originally present. No heating is required and the reaction can be run at room temperature~ 11

Glucose oxidase is specifie for "beta" glucose.

The protein-free filtrate was prepared according to the Somogyi method (139).

The glucostat method was applied as follows:

To 2 ml. of the protein-free filtrate containing from 10 to 60 pg. of glucose per ml. were added 2 ml. of the glucostat reagent. After exactly 10 minutes the reaction was stopped by the addition of a drop of 4N HCl and the resultant stable yellow color was read in a Coleman Junior spectrophotometer at 400 mp.. Standards were always run together with the unknowns.

The Nelson method (140) for the determination of glucose was occasionally used and the values obtained with this method were sometimes comparable to those obtained with the glucostat method but more often tended to be higher.

With "caramelized" solutions (such as CD after autoclaving) the glucostat method gave a 50% decrease in the glucose concentration while the Nelson method gave only a 25% decrease. The glucostat method for estimation of 40 glucose proved to be three times more sensitive and precise than the

Nelson method.

Pyruvic acid.

Pyruvic acid was determined by the method of Lu (141), as modified by Bueding and Wortis (142) and Elgart and Nelson (143).

Lactic acid.

Lactic acid was determined according to the method of Barker and

Summerson (144} as modified by Lepage (145).

Pentose

Ribose was determined by the method of Mejbaum (146).

Phosphate fractions

Inorganic ph0sphate was determined by the method of Fiske and Subbarow

(147}.

Organic phosphate esters were determined by a fractional analysis as described by Pappius et al. (148). The samples were deproteinized with trichloroacetic acid and the filtrates hydrolyzed with lN HCl at 100°C.

For convenience of discussion these fractions will be designated by the following symbols:

Pi: Inorganic phosphate initially present.

P 1 : "Labile" phosphate represents the fraction of phosphate esters

that undergo hydrolysis on heating with lN HCl at lOOoc for seven

minutes. During this interval the two terminal phosphate groups

of ATP and one of ADP and the phosphate on carbon 1 of hexoses

and pentoses undergo hydrolysis. Thus P 1 = inorganic phosphate,

determined after seven minutes hydrolysis, minus the inorganic

phosphate initially present. 41

P : This fraction represente the stable phosphate esters which s have resisted hydrolysis on treating for 100 minutes in lN HC1

at 100°C, The stable phosphate esters are composed mainly

of 2, 3-DPG.

Thus P s = inorganic phosphate present after total acid-soluble

phosphate hydrolysis carried out in 70o/o perchloric acid for

10 minutes at boiling temperature, minus the inorganic phosphate

present after the 1 00-minute period of hydrolysis. The inorganic

phosphate liberated between the 7- and lOO-minute hydrolysis is

derived from the phosphate attached to the terminal carbon of hexose

and pentose sugars.

Hemoglobin

Hemoglobin was estimated by means of a modification of the method of

King (149) as described by Brownstone (57). The procedure was as follows:

A sample of either O. 02 ml. or O. 1 ml. of blood or hemolyzate was added to 4 ml. O. 125N HCl, and water was added to make the volume to 5 ml. The sample was permitted to stand for 10 minutes, then 1 ml. of 6% NaCN was added and the tube shaken. The color density was read at 540 m}l with a Coleman Junior spectrophotometer.

Hematocrit

The hematocrit was determined with a micro-capillary centrifuge,

Internationàl Model MB.

C. General method of taking samples for analysis

During the preservation of blood, samples were withdrawn on the given days, after careful resuspension of the cells, with a sterile syringe and needle. Part of the blood was centrifuged for the analysis of the plasma. 42

The actual amounts used for the different analyses were as follows:

Metabolite Blood sample Total filtrate volume Nature of filtrate analysed (ml.) (ml.)

Glucose 0.02 8. 0 Ba(OH)2-ZnS04

Pyruvic acid Phosphate 1.0 12. 0 lOo/o TCA Ribose Lactic acid

The TCA filtrates for lactic acid were further treated with Ca(OH)2-CuS04 to remove interfering material.

At the given times during the assay oi hexokinase activity, which will be described later, the following samples were withdrawn for analysis:

Metabolite Sample Final filtrate volume Nature of filtrate analysed (ml.) (ml.)

Glucose 0.5 -0. 6 s. 0 Ba(OH)2-ZnS04 Lactic acid o. 01 -o. 05 5. 0 Ca(OH)2 -CuS04 Hemoglobin 0.01-0.1 6. 0 HCl, NaCN Nucleotides 0.2 o. 3 20o/o TCA

D. Electrophoretic separation of nucleotides

After deproteinisation with cold 20o/o TCA O. 02 ml. of the filtrate was applied three times in succession on the same spot to a paper strip in a

Spinco apparatus (Durrum type). Citrate buffer (O. OSM) at pH 4. 8 was used and a constant current of 5 ma. was applied for a period of 16-18 hours.

The paper strips were then dried and contact photographie prints made by exposure to a filtered ultra-violet light for 3 seconds. The photographie paper was developed with Kodak D-72 Developer for 1 minute, stopped with O. 6o/o glacial acetic acid solution and fixed with the usua1 fixing solution for 15 minutes. 43

The nucleotides can be eluted quantitatively from the paper strips, as follows: The paper is eut at the concentration bands or zones. Each of the pieces is eut further into small pieces (about 2 x 2 mm. } ; the pieces from each band are placed in a small volume (3- 5 ml.} of O. lN HCl and shaken for 3 hours at room temperature. The concentration of the nucleotide in the eluate can be estimated in a spectrophotometer at 260 mp., using silica cuvettes.

E. Assay of hexokinase activity

The estimation of hexokinase activity was done on the stroma-free hemolyzate (SFH} by determining the quantity of glucose disappearing from the medium. The activity is usually expressed as pg. or pmoles of glucose utilised per 100 mg. hemoglobin per hour.

The writer has designed a method which evolves from the findings presented in section A of the experimental results and which gave the maximal and most consistent values for the level of hexokinase activity.

The procedure is as follows:

Assay medium

Concentration of Quantity of Final concentration Ingredient solution added solution added of ingredient (M} (ml.} (mM)

P04 buffer o. 2 o. 2 13. 3 Glycylglycine buffer 0.4 0.4 53. 2 ADP o. 054 0.2 3. 6 ATP 0.075 0.2 5.0 SFH 1.0 MgC12 0,075 0.2 s. 0 DPN 0.03 o. 2 1.0 Glucose 0.29 o. 25 2.4 44

The medium made up to 2. 75 ml. with KCl (O. 154M) is incubated 10 minutes at 37°C without glucose. The medium is cooled in an ice bath and glucose is added, completing the volume to 3. 0 ml.. The pH (7. 75 ± O. 1 0) is verified and samples are withdrawn for analy sis (see p. 42). The medium is then incubated with agitation in a bath at 37. 0 ± O. 1 oc for 90 minutes. At the end of the incubation, the medium is rapidly cooled and samples are again withdrawn and the pH checked.

Magnesium chloride must be added after the SFH preparation so as to prevent the formation of insoluble magnesium hydroxide. The latter sometimes is formed at the lower hydrogen concentration which prevails before the addition of the SFH.

III. EXPERIMENTAL RESULTS

A. Optimum conditions for hexokinase activity

1. The influence of hydrogen ion concentration

The influence of the hydrogen ion concentration on hexokinase activity is indicated in Figure 2. The optimum activity lies between pH 7. 45 and

7. 85, being a little higher at 7. 85. The activity was found to decrease rapidly on the alkaline side of the optimum while on the acid side it dropped sharply until pH 7. 1 and from then on decreased more gradually. This picture differs slightly from that obtained by Kashket (22), who consistently obtained a second, but smaller, peak at pH 6. 0 in addition to the greater optimum around pH 7. 8. The minimum between these peaks occurred at pH 6. 6. The pH range for the optimum hexokinase activity, as found by the writer, is very similar to that reported for muscle, kidney and intestinal mucosa (150, 151). Very recent1y, Rap<;>port (152) has reported a value of pH 8. 1 for optimum activity of the enzyme in human red cells. Since we 45

FIGURE 2

The inftuence of the hydrogen ion concentration on hexokinase activity

Conditions: 2.0 ml. SFH (24 mg. Hb/ml. final concentration) were incubated with: Final concentration (mM) P04 buffer 15.0 Glycylglycine buffer 40.0 ATP 5.0 5.0 MgC12 Glucose 1.4 Final volume adjusted to 4.0 ml. with KCl ( 0.154M). Incubation at 37° C for 90 minutes, pH as indicated.

The variation was not grea ter than ± O. 07 in the lower range and les s than ± O. 04 in the higher range of pH. FIGURE 2

ct: ::::> 0 200 'l: ...... 0 'l: 0 E 0 150 Q ...... c ~ ..J i= ::::> 100 liJ (/) 0 0 ::::> ..J (!) 50 ô '

5 6 7 8 9 pH 46 have no values for the activity at pH 8. 1 and since the trend in the curve in Figure 2 is upward at pH 7. 85, it is quite possible that the activity may continue to increase to pH 8. 1 before falling off.

2. Influence of added ATP and ADP on glucose utilisation and lactic acid production

ATP or ADP was added, and in some cases they were added together, in various concentrations, to the SFH. The utilisation of glucose and production of lactic acid, as well as the changes in the concentration of the nucleotides, are recorded in Table I. As indicated in Figure 3a, the glucose utilisation was greatest where ATP and ADP {5. 0 and 3. 6mM final concentration respectively) were added together, and was smallest where ADP alone was added. It is not surprising that ADP promoted the phosphorylation of glucose in view of the finding by Kashket {84} and Tatibana (85) that the red cell membrane and cytoplasm of the red cell contain a powerful adenylate kinase which converts the ADP to ATP. The sugar utilisation, with added

ATP was lower than that with ADP alone. Addition of ADP to the ATP- containing sample increased the sugar utilisation, as illustrated in Figure 3a.

This finding is contrary to that of Kashket {84) who obtained the highest activity with ATP alone, and found that the addition of ADP tended to decrease the utilisation.

The glucose utilisation with time, as indicated in Figure 3b, followed a linear relationship in the ADP (Sample V) a.nd ATP +ADP (Sample III} samples, whereas in the ATP sample the utilisation was consistently slightly slower during the first 45 minutes, again during the last 45 minutes.

The lactic acid production averaged about 30% of that which theoretically 47

TABLE I

The influence of ATP and ADP on hexokinase activity

Glucose utilisation, lactic acid production, and changes in the concentrations of ATP, ADP and IMP during the assay period.

Conditions: as under Figure 3, p. 48.

Sample Sample

III III

IV IV

II II

v v

I I

>:< >:<

(mM (mM

To To

ATP ATP

A A

ATP ATP

the the

TP TP

express express

Nucleotide Nucleotide

final final

glucose glucose

(5. (5.

(5. (5.

(2. (2.

ATP ATP

ADP ADP

Solution Solution

0) 0)

0) 0)

5) 5)

concentration) concentration)

the the

+ +

+ +

+ +

(5. (5.

{3. {3.

ADP ADP

ADP ADP

A~P A~P

value, value,

percentage percentage

0) 0)

added added

6) 6)

(3. (3.

(O. (O.

(3. (3.

in in

9) 9)

6) 6)

6} 6}

possible possible

Duration Duration

incubation incubation

of of

(Min.) (Min.)

135 135

180 180

135 135

180 180

135 135 135 135

180 180

135 135

180 180

180 180

the the

90 90

45 45

90 90

45 45 45 45

90 90

45 45 45 45

90 90

90 90

0 0 0 0

0 0

0 0 0 0

equivalents equivalents

lactic lactic

of of

TABLE TABLE

acid acid

ATP ATP

Nucleotide Nucleotide

4.2 4.2

4. 4.

1.1 1.1

s. s.

1. 1.

6. 6.

1.6 1.6

3. 3.

7~ 7~

(pmoles/ml.) (pmoles/ml.)

8 8

8 8

0 0

1 1

1 1

3 3

produced produced

of of

ADP ADP

0.2 0.2

0.4 0.4

0.6 0.6

0.2 0.2 o. o.

o. o.

1.4 1.4 1.4 1.4 0.5 0.5

lactic lactic

-

2 2 8 8

I I

found found

IMP IMP

0.4 0.4

2. 2.

2.4 2.4

o. o.

2. 2.

1.3 1.3

2. 2.

acid, acid,

-

in in

6 6

7 7

7 7

3 3

function function

(Jlmoles) (Jlmoles)

is is

Glucose Glucose

utilised utilised

. .

4.8 4.8 4.2 4.2

4. 4.

4. 4.

5. 5.

6. 6.

1..7 1..7

2.5 2.5

8.0 8.0

6. 6. 9. 9.

8.2 8.2

2.5 2.5

9. 9.

2. 2.

7. 7.

1.4 1.4

3. 3.

6. 6.

7. 7.

multip1ied multip1ied

2 2

6 6

6 6

5 5

2 2

1 1

7 7

1 1

7 7

1 1

8 8

7 7

of of

the the

Lactic Lactic

glucose glucose

produced produced

(pmoles) (pmoles)

by by

4.2 4.2

2. 2.

2.8 2.8

5. 5.

5. 5. 3. 3.

5. 5.

1.7 1.7

3. 3.

3. 3.

2. 2.

-

-

-

- - -

- -

- -

-

-

4 4

2 2

0 0

2 2

2 2

5 5

2 2

acid acid

utilised, utilised,

Glucose Glucose

Lactic Lactic

(o/o) (o/o)

32 32

33 33

29 29

28 28

29 29

35 35

21 21

28 28

32 32 32 32

·-

-

-

-

-

-

-

-

acid acid

(x (x

2)* 2)* 1 1 48

FIGURE 3

The influence of ATP and ADP on hexokinase activity

(a) Influence on glucose utilisation

(b) Glucose utilisation with time,

Conditions: 2. 0 ml. SFH were incubated with: Final concentration (mM) P0 buffer 13.3 4 Glycylglycine buffer 53.3 MgC1 5.0 2 Glucose 3.0 Nucleotide as in Table I, p. 4 7 Final volume to 6. 0 ml. with added KCl (0.154M). First, incubated at 37° C for 10 minutes withouTglucose. Then, incubated at pH 7. 8 at 37°C for 180 minutes. FIGURE 3

(a) ({) a:: :l 0 ~ ::t: tt') 9 1- - r--- 0 r--- --IA.I Cf) :J i= ,...-- :;:) 6 !- IA.I Cf) 8 :;:) .J (!) !-> Cf) 3 IA.I .J 0 2 =r....

:r II III nz: I Relative nucleotide concentration addtd

-Er; 3.0 4 .J i "\ 2 )( -z 0 !i 2.0 sa :::! 5 I ~ 8 :m: ::,:) .J (!) 1.0 45 90 135 180

TIME (MIN.) 49

would be expected on the basis of the glucose utilised.

The analytical resulta presented in Table I clearly indicate the inter­

conversion of nucleotides. As pictured in Figure 4, in the sample where

only ATP (Sample I} was added, the ATP content decreased and no ADP

could be detected while a very slight amount of IMP appeared at the end

of 180 minutes of incubation. This nucleotide arises presumably from the

AMP produced by the action of adenylate kinase, and subsequent deamination

by the nucleotide deaminase (153, 85}. Tatibana (85} has estimated that the

time required to complete the conversion of 5 p.moles of ADP to ATP and

AMP at 37°C was less than 20 seconds. The adenylate kinase activity was

found to remain unchanged during a 6-week-period of storage. That the enzyme

is extremely active is evident when one looks at the sample where ADP alone

(Sample V) was added: within 10 minutes of incubation, more than 5/6th

of the added ADP has been converted to ATP and IMP. Thereafter the

ATP and ADP levels decreased while IMP accumulated. This pattern of

events was even more accentuated in the case of the sample containing

ATP and ADP; the ADP concentration was higher in this sample at the

beginning of the incubation since the ATP added was present to establish an

equilibrium and inhibit the activity of the adenylate kinase.

The data may shed light also on the question of the glucose utilisation since,

from Kashket' s work (22), 5. 0 mM A TP is the optimal concentration of this

coenzyme in the hexokinase reaction. The low utilisation in Sample V might have been increased by the addition of larger amounts of ADP since the 50

FIGURE 4

Nucleotide composition of the SFH during hexokinase assay

Conditions: as under Figure 3, p. 48. 0 co

. . > ' . . ... Lt.l a. 0 ..J j't•• A. a ' . . en 2 <{ . . ~ (/)

0

. .,.. f • ....'0: 0 ~ co .:;- . ' .. '':'" a. - a .. ~ <{ 0 Lt.l .. .. \ ..J A. ~ .. :. t en + ' . . ~ 2 a. ~ ..... (/) <{

' ~ .· . ":..,~ 0 ,.. "" -: -- .. .. ~:$. '

0 co

a. 0 ..... en <{

0

a. a. z "":' Lt.l a. 0 z Q ~ a 1- <{ <{ 2 t= Q ~ 0 1.1.1 ID - 1.1.1 Q ::l 1.1.1 ..J Q 0 2 0 ~ z ::l 1- z 51

ATP formed was only Z.OmM, well below the optimal concentration.

The effect of added ADP in increasing the glucose utilisation in the presence of ATP is not readily explained since the ATP concentration was observed to increase from 4. 8 to 7 .lmM , a concentration which is well above the concentration for maximum activity. The effect of ADP would have to be one in which ADP is more directly involved. It must be borne in mind that only 30% of the glucose that disappeared appeared in the lactic acid. Unsuccessful attempts have been made to characterize the intermediate(s) but it was shown that pyruvate did not accumulate. It is possible that the lack of ADP due to the extremely active adenylate kinase may reduce the conversion of 1, 3-DPG through the 3-phosphoglycerate kinase reaction

(Step 10, Fig. 1, p. 16) and cause an accumulation of either 2, -3DPG or fructose 1,6-diP.

Such a control mechanism has been suggested by Rapoport (80) and

Grisolia (81 ). Therefore in such a system, increasing the ADP concentration, even slightly, would tend to stimula te the conversion of 1, 3-DPG to 3-phospho- glycerate and at the same time encourage glucose utilisation. The ADP concentration was highest in the ATP + ADP-containing medium (Sample III) and the glucose utilisation was greatest.

Another interesting feature of the results is that the rate of glucose utilisation was linear both in the case where ADP alone was present (Sample V) and that in which ATP and ADP were present together (Sample III).

3., The influence of other glycolytic enzymes and the optimal concentration of DPN

Crystalline yeast hexokinase was used to demonstrate the extent of 52

inhibition caused by glucose-6-P and the importance of the other enzymes

of the glycolytic sequence as -well as an optimal concentration of DPN in

removing this inhibition.

As indicated in Figure Sb, yeast hexokinase with all the necessary co­

factors failed to show any activity whatsoever. However, as illustrated

in Figure Sa, when a dialyzed SFH was added to the yeast hexokinase,

glucose phosphorylation was resumed and at a rate much greater than that effected by the hexokinase of the SFH preparation. An effect of com­ parative magnitude was observed when SFH prepared from blood stored for 60 days at 4°C,was added to yeast hexokinase. The effect of added SFH

in stimulating the phosphorylation of glucose by the yeast hexokinase ceased

when the final concentration of added SFH approached the equivalent of 12 to 15 mg. hemoglobin per ml.

The activity of the preparation was observed to decrease with time and further addition of SFH did not alter this relationship. However, addition of DPN (O. 35 mM) caused a 30o/o increase in the activity and the rate of glucose utilisation remained constant for an hour.

The yeast hexokinase catalyzed the phosphorylation of glucose but as glucose-6-P accumulated it caused progressive inhibition of the enzyme (66).

However, the addition of SFH to the system brought about the removal of glucose-6-P and the reactivation of the hexokinase. This phenomenon has been described also by Long and Thomson (154), who likewise demonstrated the reactivation of rat-brain hexokinase by the addition of erythrocyte hemolyzate or muscle extract. They attributed the effect to the removal 53

FIGURE 5

The influence of added SFH and DPN on the activity of yeast hexokinase

{a) Influence of added SFH and DPN

(b) Glucose utilisation with time.

Conditions: In Experiment (a): O. 02- 1. 0 mL SFH were added to: (See medium below)

In experiment (b): 1. 0 ml. SFH {Hb 25 mg. /mL final concentration) was added to: Final concentra ti on {mM) P04 buffer 20.0 Glycylglycine buffer 40,0 ATP 5.0 MgC1 5.0 2 Glucose 2.3 Where indicated, were added: O. 04 ml. yeast hexokinase (0. 8 mg. /mL ) O. 08 ml. DPN ( 0.013M) Final volume 3. 0 ml. adjusted with KCl (0.154M). Incubation at pH 7.8 at 37°C for one hour. -

The SFH preparation was dialyzed for 4 hours against cold running water.

The term "hexokinase" in the figure designates yeast hexokinase. FIGURE 5

(a) a: 5 :;) 0 :c SFH + HE:XOKINASE + DPN

SFH + HEXOKINASE

SFH

5 10 15 20 25 CONCE:NTRATION OF SFH (Hb mg.fml.)

5 SFH+ HEXOKINASI 0 1.&.1(/) 4 + DPN ::::; i= ::::> SFH + HEXOKINASI 1.&.1 (/) 3 0 0 ::::> ~ 2 (/) 1.&.1 ~ :t ~ HEXOKINASE

15 30 45 60

TIME (MIN.) 54

of the accumulated glucose-6-P. The effect of added DPN in stimulating

glycolytic activity is attributable to the provision of a sufficient amount

of coenzyme at the triose phosphate dehydrogenase level, which by its

increased activity in removing more intermediates at this step and also

indirectly at all the previous steps, increases the removal of the inhibitory

glucose-6-P. This phenomenon affords another example of the influence on

the hexokinase activity of the backing up of the glycolytic equilibrium because

of the impairment of a step situated at some distance away in the glycolytic

chain.

As a rule, the addition of DPN effected only a slight increase in the rate

of production of lactate (e. g. 43 to 49o/o} both with ..the SFH and SFH with

added yeast hexokinase. The ratio of lactic acid produced to glucose utilised

with the SFH and hexokinase was about the same as with the SFH alone.

This would indicate that, in the experimental conditions prevailing, the

equilibrium of the glycolytic system of the SFH preparation is not affected

by an increased turnover of glucose which causes further accumulation of

intermediate(s) since only a fraction of glucose is converted to lactic acid.

It was natural to expect a suboptimal amount of DPN from a dialyzed preparation. However, it was often found that even the SFH prepared from

fresh blood must be supplemented with DPN in order to achieve the maximum activity of the hexokinase. By this means the activity was increased by 10 to 30%. The DPNase on the outer surface of the membrane may be responsible for destruction of the endogenous DPN when cells are hemolyzed unless the precaution is taken of adding nicotinamide beforehand to inhibit the DPNase. 55

4. Inhibition of glucose utilisation and lactic acid production by nic otinamide and alloxan.

While nicotinamide can protect DPN from destruction by the DPNase the amide may at the same time be inhibitory to the glycolytic system, since Alivisatos (95) has demonstrated that nicotinamide can act also as a substrate for the DPN- and TPN-linked dehydrogenases and thus inhibit them. Alivisatos (95) demonstrated the inhibition of DPN-linked lactic dehydrogenase to the extent of 65o/o in the presence of 20 x 10-ZM nicotinamide.

Similar1y he obtained a 50o/o inhibition of the TPN-linked glucose-6-P dehydrogenase (153) with the same concentration of the amide. The same nicotinamide concentration added to SFH in our study, as indicated in

Figure 6b, caused a 40o/o inhibition in the glucose utilisation, and a corre ... sponding decrease in the lactic acid production. As shown in Figure 6a, the addition of nicotinamide to intact red cells has a negligible effect. on the glucose utilisation. There appeared however to be a slight decrease in the lactic acid production. The red cell membrane appears to be impermeable to nicotinamide. Henning and Sonakul (155), in our group, observed a slight decrease in glucose utilisation and lactic acid production when blood is preserved in CD or ACD medium with added nicotinamide.

As indicated in Figure 7a, alloxan in the medium had no effect on the metabolic activity of the intact cells, but it had a powerful effect on the glycolytic activity of the SFH. 3 As shown in Figure 7b, alloxan in 5 x 10- M concentration caused soo/o inhibition of glucose uptake and more than 80o/o inhibition of lactic acid production. Al1oxan is a powerful oxidizing agent and is capable of 56

FIGURE 6

The influence of nicotinamide on the metabolic behaviour of intact red cells and SFH.

Conditions : 1. 0 ml. of SFH or washed intact cells were incubated with: Final concentration (m.!;:!} P04 buffer 13.3 Glycylglycine buffer 53.3 ATP (for SFH} 5.0 or . ADP (for mtact cells) 5.0 MgClz 5.0 Glucose 2.4 Nicotinamide as indicated Final volume was adjusted to 3. 0 ml. with KCl (0.154M). Incubation at pH 7.5 at 37°C for one hour. ·a'V .:J

0 "' -0::: <( ..J 0 Ul ::E N 1 0 )ë l: LL (/) -f.IJ 0 c ~ <(z ;::: 0 1() 0z -.a 1() 0 . Ul 0 1(). CD t\Î "' 0 L&J ~nOH / qH -8wOOI/ S310W r( 0::: ::> C) LL

'V 0 g • 0 0• 0 a: ::) "' -C( ii ..J 0 (/) 1() ::E ..J ..J "' L&J 0 0 )( L&J -L&J ..J 0 c 0 l: ::E :r: <(z ;::: 0 2 1() z -a 1() 0 1(). ~ 1() N 0 ~OOH/"' qH '6WOOI / S310W r( 57

FIGURE 7

The influence of alloxan on the metabolic beha viaur of intact red cells and SFH

Conditions: as under Figure 5, p. 53.

Alloxan concentrations as indicated. Alloxan was first neutralized. FIGURE 7

(a) WHOLE CELLS ( b) SFH 1 2.5 2.5 a:: 0:: ~ ::l 0 :::r: ~ 2.0 -...... 0 '.0 :::r: :::r:

0 E 1.5 ~ 1.5 0 0 0 Glucose 0 -..... (/) 1.0 '(/) 1.0 Lù Lù ..J ..J 0 0 :E :lE :a... :a...0.5 0.5

Lactic acid

2 4 6 8 2 4 6 8 3 ALLOXAN {X 10- MOLAR) ALLOXAN (X 10-:3MOLAR) 58 inactivating many enzymes by oxidizing essential SH groups. In view of its lack of action on the glycolytic activity of the intact red cells, as indicated in Figure ?a, one may infer that the cell membrane is impermeable to alloxan.

An interesting observatïon was made with regard to the comparative glucose utilisation and lactic acid formation of intact red cells and the SFH of equal hemoglobin content. The intact cella were found to convert glucose to two moles of lactic acid whereas SFH yielded only about one mole. The two preparations produced the same amount of lactic acid but SFH utilised twice as much glucose. The explanation is not clear at present and this phenome­ non is being studied further in this laboratory. More commenta will be offered later in the discussion.

B. The activity of hexokinase during preservation of blood at 4°C

A procedure for the assay of hexokinase activity under optimal conditions for its activity having been worked out, the writer undertook to measure the activity of the enzyme in human red cells during storage in various preservative solutions at 4°C. As mentioned before, the currently held notion is that hexokinase is the most labile of the enzymes of the glycolytic system and that the failure of the metabolic system of the red cell is largely the result of progressive inactivation of hexokinase (115).

A specimen of blood was collected from each of two donors, a man

(Specimen A) and a woman (Specimen B). A portion of each donation was collected into the CD and ACD preservative mixtures and stored at 4°C 59

for 30 days. At various intervals during this period, samples were

withdrawn aseptically for analysis. A portion of each sample was used

for the measurement of pH and estimation of glucose, pyruvic acid, lactic

acid, inorganic phosphate and organic phosphate esters. Another portion

was used for the preparation of the SFH and the immediate assay of

hexokinase activity. Repeated assays on the SFH, which was kept frozen

at -lO"C for a week, showed virtually no change in the activity.

The results are presented in Table II and in Figure 8. The shaded

area in the figure gives the range of variation in the values obtained in the

hexokinase assays from day to day, and sometimes from sample to sample.

Thus, as evident from Table II, in most of the determinations the difference

between duplicate measurements was less than ?o/o and on the average only

3%. Furthermore, the activity within a range of variation of 20% was not

altered in the SFH from either CD or ACD specimen during the storage

period of 30 days.

The values for pH, pyruvic acid, glucose, and lactic acid concentration

for the corresponding intervals during storage are given in Table III. The

fall in pH, and the glucose utilisation and lactic acid production, are indicated in Figure 9. The values, expressed in p.moles per ml. of whole blood are obtained by multiplying the value from the analysis of the medium by the dilution factor, 1. 25.

The behaviour of the metabolites of blood preserved in CD and ACD during storage at 4"C followed the typical pattern previously described

(see pp. 29, 30). The pH fell from 7. 3 to 6. 7 in the CD-preserved blood 60

TABLE II

Hexokinase activity in the SFH from two specimens of blood during storage in CD and ACD respectively at 4°C

Preservative me dia

Specimen A Specimen B Storage- days CDA AGDA ' CDB ACDB

(p.g. glucose utilised/100 mg. Hb/hour)

0 380±11* 384 ± 14 390 ± 7 400± 2

3 372 ± 5 380 ± 2 396±4 412 ± 14

6 355 ± 5 373 ± 4 380± 9 380 ± 4

9 380 ± 14 370 ± 2 370± 4 375 ± 20

14 402± 2 375 ± 9 398± 7 385± 4

17 398 ± 14 358 ± 4

21 350 ± 2 340± 2 385 ± 4

30 404± 4 394 ± 5 346± 2 346 ± 7

Conditions: the hexokinase assay used is described on p. 43.

):c All values are the means with the range of deviation from two assays. 61

FIGURE 8

Hexokinase activity of SFH from blood during storage at 4°C

o -- Specimen A blood stored in CD ·-- Specimen A blood stored in ACD 0 Specimen B blood stored in CD ·-- Specimen B blood stored in ACD

Conditions: As described on p. 43. FIGURE 8

400

~ :::1 x0 >- ...... -x~ .D > c;. 300 ~ e (.) 0

5 10 15 2 0 25 30 DU RATION OF STORAGE-DAYS TABLE III

Changes in pH, pyruvic acid, glucose and lactic acid concentration in two specimens of blood during storage in CD and ACD respectively at 4°C

Preservative media Preservative media

Specimen A Specimen B Specimen A Specimen B Storage- Storage- Condition da ys Condition days CDA1ACDA CDBlACDB CDAIACDA CDB IACDB (p.moles/ml. whole blood)

ù 7. 33 7.04 7. 31 7.01 0 11. 0 -23. 2 11. 0 21. 3 3 7.28 7.00 7.24 7,00 3 9.9 21. 3 8.8 21.0 6 7.16 6.89 7. 14 6.90 6 8.5 20.0 7. 1 20.2 pH 9 7. 15 6.85 7. 10 6. 83 Glucose 9 7. 1 18.7 6.4 19. 4 14 6.95 6. 71 6.93 6.75 14 4.6 5. 1 18. 4 17 6.92 6.70 17 3.4 15.5 21 6,80 6,60 6. 80 6, 65 21 1.6 14.2 2.4 16.4 3.0 6.70 6.55 6. 72 6.56 30 0 12. 1 o. 6 14.2

(pmoles/ml. whole blood)

0 0.20 0.20 0.22 o. 16 0 2. 9 1.5 2. 5 l. 6 1 3 o. 19 o. 19 0.22 o. 19 3 5.0 4.2 5.0 4. 1 6 0.25 0,30 o. 29 o. 32 6 7. 8 7. 5 7. 4 5,4 Pyruvic 9 o. 32 0.36 o. 39 o. 31 Lac tic 9 9.5 10.2 7. 8 7. 1 a cid 14 0.53 o. 31 0.59 o. 19 a cid 14 14.9 12.9 12. 1 1 o. 0 17 o. 76 0.25 17 16. 4 15.4 0" 21 0.95 0.26 o. 84 o. 16 21 23. 8 21. 1 20.5 16. 8 N 30 l. 18 o. 31 0.86 0,21 30 29. 5 27.0 19. 9 --- ~-- 63

FIGURE 9

Changes in pH, glucose and lactic acid concentration during preservation of blood at 4 ° C

(a) pH changes vs. storage-days

(b) Glucose utilisation and lactïc acid production vs. storage-days.

o -- Specimen A blood stored in CD ·-- Specimen A blood stored in ACD o - Specimen B blood stored in CD ·- Specimen B blood stored in ACD FIGURE 9

(a)

7.2

1.0 pH 6.8

6.6

5 10 15 20 25 30 DURATION OF STORAGE-DAYS

(b) 30 0 0 0 ...J m 25 Lactlc acid LLI ...J 0 20 :I: 3: . 15 ·-//~/ / ... - E • 10 Cf) "LLI ...J 0 5 GlU COll ~ ~

5 10 15 20 25 30 DURATION OF STORAGE- DAYS 64 and from 7. 0 to 6. 5 in the ACD-preserved blood during the 30-day period.

Since donor A was male and B, female, the proportion of cells in specimen B was less than in specimen A, indicated by the hematocrit.

As the hexokinase activity in both specimens was comparable, the glucose utilisation and lactic acid production were less in blood specimen B. The glucose utilisation in both specimens was depressed in the ACD compared to that in the neutra! CD medium. In both preservative media, the rate of glucose utilisation was slowed down by the 30th day to about SOo/o of the initial value s.

The partition of glucose and lactic acid between cells and plasma during the storage is indicated in Table IV. The values obtained for the concentration of both were higher in the plasma during storage. However, if correction is made on the ba sis of the cell water, which amounts to 65- 70o/o, the value for the concentration in the cells is found to be very close to that found in the plasma. Evidently therefore, these substances were distributed equally on the two sides of the membrane throughout the period of storage.

In Table V are recorded the changes in the following phosphate fractions: inorganic phosphate (Pi}, stable phosphate (P s> and labile phosphate (P1).

As indicated in Figure 10, the decrease in stable phosphate was accompanied by an increase in inorganic phosphate. These concomitant changes began on the 6th day in the CD specimen and at the beginning of storage in the

ACD specimen. The labile phosphate decreased progressively in both specimens until the 30th day, but in the ACD specimen 30o/o of the original amount was still present while only 20o/o remained in the CD sample. TABLE IV

Partition of glucose and lactic acid between cells and plasma in two specimens of blood during storage at 4°C

Preservative media

Specimen A Specimen B

Storage- ACDB Metabolite CDA AGDA CDB da ys cells plasma cells plasma cells plasma cells plasma

(}lmoles/ml. cèlls or plasma)

0 7.. 1 10.0 17. 3 20. 1 8.4 14. 1 18. 8 3 6.7 8. 8 14.5 19. 1 3. 7 7.7 14.5 18.0 6 5.9 7. 5 14.0 17. 7 5.5 5.8 14. 1 17.2 Glucose 9 5.0 6. 3 12. 6 17.0 4. 3 5.5 16. 5 14 3.2 4. 1 13. 3 15.4 17 2.2 3. 1 10.8 13. 6 21 1.9 1.9

30 .. 8. 6 10. 6 o. 7 0.4 9.7 12. 3

/ 0 2.6 2. 1 1.2 1.2 1.8 2. 1 1.4 1.3 3 2. 6 5.0 3.4 3. 6 3. 6 4.2 4.2 2. 8 6 5. 1 7.0 6. 7 6. 0 6.7 5.5 4.0 4.5 Lac tic 9 7.2 9.2 7.2 9.2 6.4 6. 1 6.9 5. 0 a cid 14 1 o. 5 12. 2 11. 8 1 o. 5 7. 8 8. 1 17 11. 8 14. 1 10. 9 13. 6 0' Ul 21 16. 1 21. 1 15. 8 17. 9 16. 3 16.9 12.4 14. 1 30 18. 7 24.2 17. 3 22. 1 11. 5 16. 9 66

TABLE V

Changes in the concentration of phosphate fractions in two specimens of blood during storage in CD and ACD respectively at 4°C

Preservative media

- Specimen A Specimen B Phosphate Storage-1--· fraction days CDA AGDA CDB ACDB

~p.mo1es phosphate/ml. who1e blood)

0 1..8 2. 0 1.5 1.7 3 1.5 2. 6 1.6 2. 6 ' 6 1.8 4.0 1.9 3. 9 p. 9 2.2 5.6 2. 6 4.9 l 14 6. 2 '3.8 5. 5 17 4.4 6. 7 21 5. 4 7. 2 s.. 5 5. 9 30 7. 1 7. 3 6. 1 6.1

0 5. 0 5. 7 4.2 4.2 3 5. 7 4.5 4.4 3.8 6 5. 0 2. 8 3. 5 1.6 ps 9 1.7 2. 5 0.4 14 3. 4 1.2 1.8 0.4 17 2. 6 21 1.9 o. 6 o. 8 o. 3 30 o. 6 o_,5 o. 1 o. 3 ' 0 1.2 1.3 0.9 1.0 3 1.0 1.2 0.8 o. 8 6 1.0 1.2 0.9 o. 9 pl 9 1.1 o. 7 o. 7 o. 7 14 o. 9 o. 6 0.6 17 o. 7 21 o. 7 o. 5 o. 3 o. 3 30 o. 2 0.5 o. 1 0.3 67

FIGURE 10

Changes in the phosphate fractions during storage of blood at 4°C FIGURE 10

Q 0 (a) CDA p. 0_. 1 dl 6 L&J_. :a:0 3t . E 4 --L&J 1- <( :a: 0.. Cf) 0 :a: 0.. Cf) L&J_. Ps 0 2 pl :a... 5 10 15 20 25 30 DURATION OF STORAGE-DAYS

Q 0 (b) ACDA p. 0_. 1 dl L&J_. 6 0 J: 3t . E ...... 4 L&J 1- <( :a: 0.. Cf) :a:0 2 0.. Cf) L&J_. Ps 0 ~ ::&... pl 5 10 15 20 25 30 DURATION OF STORAGE-DAYS 68

The rise in inorganic phosphate, as indicated in Table VI and in

Figure 11, signifies the accumulation of inorganic phosphate within the cells, since the cell membrane at 4°C is almost impermeable to this anion.

The values for the concentration of substances within the cells were calculated from the following formula :

c = W-P(l-h) h

where C representa the concentration of the substance in the cells,

W, the total concentration in the whole sample,

P, the concentration in the plasma,

h, the hematocrit.

The accumulation of pyruvic acid in the two specimens is recorded in

Table VII and illustrated in Figure 12. Pyruvic acid progressively accumu- lated from the 6th day of storage in the CD sample while in the ACD specimen it started to increase on the 3rd day and continued to increase until the 9th day, after which it decreased. It is noteworthy that in the CD specimen, the pyruvic acid in the cells remained unchanged during the first 14 days, then it increased in the cells along with that in the plasma. The distribution of the pyruvate between cells and plasma in the ACD sample during storage followed a similar pattern. The observations are in agreement with those of Huckabee that a pyruvate concentration gradient is set up between the cells and plasma ( see p. 5 ). TABLE VI

Partition of inorganic phosphate between cells and plasma in two specimens of blood during storage at 4°C

Preservative media

Specimen A Specimen B

Storage- CDA ACDA ,-n..., B ACDB days cells plasma cells plasma cells plasma cells plasma

(pmoles phosphate/rp.l. cells or plasma)

0 2.2 D. 9 2.5 0,8 1.7 0.9 3. 1 0.9

3 1.8 0.8 3. 6 0,8 2.2 o. 8 4. 1 1.0

6 2. 3 o. 8 5.6 1.2 2.9 0.9 7. 0 0,8

9 3. 1 0.8 7.9 1.6 4. 1 1.1 7.9 1.6

14 8. 1 2. 3 6. 3 1.4 8. 1 2.2

17 6. 2 1.6 8.4 2. 8

21 7.4 2.3 8. 9 3. 2 8.4 2.4 7. 6 3.0 0' 30 8.4 3.8 7.7 4.2 8.5 3. 1 7. 0 3. 7 ....0 70

FIGURE 11

Partition of inorganic phosphate between cells and plasma during storage of blood at 4°C FIGURE Il

(a) (b) COA 1 ACDA 1 8 Ce lis

: ....: 8 E E ...... I&J 6 1- I&J <( 1- ::z::: <( 6 ::z::: a.. a.. (/) Cf) 0 0 ::z::: 4 ::z::: a.. ~ 0..4 (/) Plasma (/) I&J I&J ..J ..J 0 0 ::E 2 ::E ::s... ::s.....2

5 10 15 20 25 30 5 10 15 20 25 30 DURATION OF STORAGE-DAYS DURATION OF STORAGE-DAYS TABLE VII

Partition of pyruvic acid between cells and plasma in two specimens of blood during stora·ge at 4°C

Preservative media

Specimen A Specimen B ' Storage- CDA ACD CDB ACDB da ys A cells plasma cells plasma cells plasma cells plasma

(pm.oles pyruvic acid/ml. cells or plasma} 0 o. 06 o. 20 o. 11 0.21 0,08 0.23 0.04 o. 18

3 0.06 o. 21 0.09 0.24 o. 10 0.22 o. 10 o. 18

6 0.06 o. 27 0.09 o. 37 0.06 o. 31 0.05 o. 38

9 o. 07 o. 39 0.09 0.41 0.05 0.44 o. 01 o. 39

14 o. 10 o.. 64 o. 11 o. 37 o. 01 o. 70 0.05 0.21

17 0.28 0.84 o. 30

21 o. 31 1. 08 0.15 0.26 o. 14 0.92 0.04 o. 18

-J 30 0.57 1. 62 0.18 o. 31 o. 30 o. 89 0.08 0.23 ...... 72

FIGURE 12

Changes in the concentration of pyruvic acid and its partition between cells and plasma during storage of blood at 4°C FIGURE 12

(a) •Plasma 1.5 COA . E ...... LLJ 1- / . :::> Whole blood a:: >- • ~ ./ en LLJ 0.5 / Cella ...J 0 ::E • ::::1....

5 10 15 20 25 30 OURATION OF STORAGE-OAYS (b)

0.4 • Plasma

Whole blood

en LLJ ...J 0 ::E :1... o. 1

5 10 15 20 25 30 DURATION OF STORAGE- DAYS 73

C. Influence of added 3 1, 5 1-cyclic adenylic acid on the metabolic activity of blood during storage in ACD at 4°C

A preliminary study was made to find out whether 3 1, 5'-cyclic adenylic acid can be utilised by the erythrocytes at 4°C. 3', 5 1-cyclic adenylic acid is adenosine with a phosphate group attached to carbons 3 and 5 of the ribose moiety. The influence of cyclic AMP on the utilisation of ribose and on the metabolism of the cella was compared with that of inosine.

To 4 volumes of ACD-preserved blood, one volume of a 3', 5'-cyclic adenylic acid or inosine solution (200 mg. o/o final concentration) was added on the 25th day of storage. The substances were carefully dissolved in sterile KCl (0.154M) solution containing 450 mg. o/o inorganic phosphate.

The concentration of glucose, pyruvic and lactic acids, and ribose, were estimated before and after the addition of the nucleosides and subsequently at various time intervals for 25 days.

As indicated in Table VIII and Figure 13, the ribose moiety of the cyclic AMP was utilised as rapidly as that of inosine during the first 4 days following the addition, but at a lesser rate thereafter.

The quantity of glucose utilised during the 9 days following the addition of the nucleosides was 2. 2 pmoles/ml. whole blood with added cyclic adenylic acid (ACD+CA) and 1. 2 )lmoles/ml. in the specimen with added inosine (ACD + I). The corresponding lac tic acid production was respectively

7. 8 and 11. 2 pmoles/ml.

The quantity of lactic acid produced exceeded that which would be expected from the amount of glucose utilised. It is evident therefore that part of the lactic acid was derived from the ribose moiety of the cyclic 74

TABLE VIII

Changes in pH and ribose concentration in blood during storage in ACD at 4°C to which nucleoside* was added on the 25th day

Preservative media Storage- Condition days ACD+CA 1 ACD+I 21 6.60 6.65 25b 6 .. 53 6.61 25a 6. 55 6. 70 pH 29 6.60 6. 75 34 6.57 6.67 41 6. 60 6.60 51 6.38 6.43

(}lmoles/ml. whole b1ood)

21 2.4 1.9 25b ?. 5 2. 0 25a 19. 1 21. 6 Ribose 29 10.5 11. 9 34 11. 2 5.0 41 8. 7 2. 3 51 3.4 o. 6

*The nucleoside added was 3 1, 5 1-cyclic adenylic acid (ACD +CA) or inosine (ACD + I). The subletters "b" and 11 a 11 on day-25 indicate respective1y before and after the addition of the nucleoside. 75

FIGURE 13

1 The influence of added 3 , 5 '-cyclic adenylic acid or inosine on the utilisation of ribose by blood during storage in ACD at 4 oC FIGURE 13

RIBOSE

Q 20 0 0 ...J m 1 w ...J 1 0 15 l: 1 ~. -E 1 ...... w 1 tn 10 0 m 1 a= 1 tn w 1 ...J 5 0 1 :5E ACD+CA :a.. d * t Nucleoside added ACD+I 20 25 30 35 40 45 50 DU RATION OF STORAGE-DAYS

1 * The nucleoside added was 3 1, 5 -cyclic adenylic acid (ACD+ CA) or inosine (ACD+ I). 76 adenylate. The greater utilisation of ribose in the ACD + I specimen accounts for the lower glucose utilisation and higher lactic acid production.

The influence of added nucleoside on the pyruvic acid concentration in cella and plasma is indicated in Table IX and in Figure 14. The addition of inosine was accompanied by an abrupt faU of the pyruvate concentration in the plasma and in the cells in the ACD + I specimen, but this did not occur in the other specimen on addition of the cyclic adenylic acid. The pyruvate in the ACD + I sample continued to decrease until the 34th day, when it increased abruptly. The rise in pyruvate occurred when about 75o/o of the ribose of the nucleoside bad been utilised.

The influence of the added nucleoside on the inorganic and stable phosphate fractions is indicated in Table X and in Figure 15. The changes · in the inorganic phosphate of the cella and of the plasma are shown in

Figure 16. The addition of 3 1,5 1-cyclic adenylic acid and inosine to the respective specimens caused an immediate decrease of the inorganic phosphate in the cells, which continued for 4 days. From then on it increased rapidly. The fall in the cellular inorganic phosphate was more pronounced in the ACD + I specimen. The inorganic phosphate in the plasma decreased in both cases.

The initial fall and subsequent rise in the inorganic phosphate after the 4th day in the whole ACD+ CA sample merely reflect the changes that took place in the inorganic phosphate in the cella. 77

TABLE IX

.,, Influence of added nucleoside''' on pyruvic acid concentration in blood during storage at 4°C, and partition of pyruvic acid between cells and plasma in the inosine-containing sample

Preservative media

ACD+CA ACD+I Storage- days wh ole wh ole cells plasma blood blood

(p.moles pyruvate/ml. whole blood, cells or plasma)

21 0.26 0.16 o. 12 o. 18

25b o. 27 0.25 0.18 o. 21

25 a 0.26 o. 17 0.04 0.13 29 0.41 . o. 12 0.03 0.05 34 0.55 0.08 0.03 0.05

41 0.60 0.45 0.08 o. 30

51 0.92 o. 85 o. 41 0.,58

):':The nucleoside added was 3 1, 5 1-cyclic adenylic acid (ACD +CA) or inosine (ACD + I). The subletters "b" and 11 a 11 on day-25 indicate respectively before and after the addition of the nucleoside. 78

FIGURE 14

(a)

The influence of added 3 1, 5'-cyclic adenylic acid or inosine on the pyruvic acid concentration during storage of blood in ACD at 4 "C

(b)

The influemce of added inosine on the partition of pyruvic acid between cells and plasma during storage of blood in ACD at 4°C FIGURE 14 la) 1.0 0 0 ACD+CA 0 ..J m 0.8 w ..J 0 J: ~ . 0.6 -e ...... UJ ~ > 0.4 * ;::) Nucleoside '\dtd a: a..>- (J) 0.2 1&.1 ..J 0 ::E ~ 20 25 30 35 40 45 50 DURATION OF STORAGE-DAYS

(b) . -e 0.6 ACD+I Plasma ...... w ~ >;::) 0.4 a: o.>-

Cf) w 0.2 ..J 0 ::E :a...

20 25 30 35 40 45 50 DURA Tl ON OF STORAGE-DAYS

* The nucleoside added was 3 1, 5 1-cyclic adenylic acid (ACD+ CA) or inosine (ACD+ I). TABLE X

Influence of added nucleoside;'< on the concentration of inorganic and stable phosphate, and partition of inorganic phosphate between the cells and the plasma during storage at 4 oC

Preservative media

ACD+CA ACD+I l p. p. Storage- ps 1 Ps 1. 1 da ys • wiwle · whole 1 cells plasma who le wh ole cells plasma blood blood 1 blood blood

ip.moles phosphate/ml. whole blood, cells or plasma)

21 o. 6 7. 2 7. 8 3.2 0.3 5. 8 6. 6 3. 0 . 25b o. 6 7.2 8. 3 3. 6 ). 3 ·S. 0 6. 9 3. 3

25 36. 1 6. 2 7 28. 1 . a 2.7 29.0 o. 34.4 1.4 29 3.2 32. 7 3. 6 27. 5 2.4 31. 5 1. 0 24. 5

34 3. 7 37.6 23.2 26.0 4.4 30.5 7. 2 21. 1

41 3. 2 37,0 25,8 22.7 5. 3 31. 2 15. 1 21. 0

51 2.4 39. 0 27.4 24.0 5. 1 35. 8 2L 1 21. 1

-.] '-'> ~:'Then'.lcleoside added was 3', 5'-cyclic adenylic acid (ACD+CA) or inosine (ACD+I). The subletters 11 b 11 and 11 a" on day-25 indicate respectively before and after the addition of the nucleoside. 80

FIGURE 15

The influence of added 3', 5 1-cyclic adenylic acid or inosine on the concentration of inorganic and stable phosphate of blood during storage in ACD at 4°C FIGURE 15

(a) ACD+CA c 40 0 0 m..J LIJ ..J 0x 30 1 ~ - 1 e 1 Lù '!:( 20 1 INORGANIC PHOSPHATE x Q. 1 f/) 0x 1 Q. f/) 10 1 Lù ..J 0 j :E :: :a.. Nucleosld:f {and phosphate) added

20 25 30 35 40 45 50 DURATION OF STORAGE-DAYS

(b) c 0 0 m..J 5 STABLE PHOSPHATE ACD+I LIJ ..J 0x :&: 4 -e ...... LIJ 3 !:( x Q. f/) 2 0x Q.

f/) Lù ..J 0 :E :l.. 20 25 30 35 40 45 50 DURATION OF STORAGE-DAYS * The nucleoside added was 3 1, 5 1-cyclic adenylic acid (ACD+CA) or inosine (ACDI-I). 81

FIGURE 16

The influence of added 3', 5'-cyclic adenylic acid or inosine on the partition of inorganic phosphate between cells and plasma during storage of blood in ACD at 4 oC ACD+I

50

45

added

40

(ACD+I).

phosphate)

STORAGE-DAYS

35

(and

inosine

PLASMA

OF

*

or

30

Nucleoside

1

t

25

DURATION

(ACD+CA)

~

acid

(b}

20

16

10

20l

30

::::1...

e

0 LIJ

UJ ..J 0 0..

:z: :z:

0..

:E ~

UJ

LLJ

......

adenylic

FIGURE

50

-cyclic

1

5

,

1

ACIJ-t-1

3

ACD+CA

45

was

40

added

STORAGE-DAYS

35

CELLS

OF

nucleoside

30

The

added

*

*

!

OURATION

25

__.

(a)

Nucleoside

20

10~

30

20

e

~

0 0.. f

UJ

c:( :z:

......

i 82

The addition of cyclic adenylate caused an abrupt and more pronounced increase in the stable phosphate fraction compared to that elicited by the addition of inosine in the other specimen. The inosine however stimulated to a greater degree the esterification of inorganic phosphate with production of stable phosphate fraction, mainly 2, 3-DPG.

The level reached a maximum on the !6th day after the addition of the nucleoside, and thereafter remained relatively constant. The stable phosphate increased also, though less rapidly, in the other specimen on addition of the cyclic adenylate, but from the 9th day the level again feU, as the rate of hydrolysis exceeded that of formation. DISCUSSION AND CONCLUSIONS

The current view, when the writer be gan this study, was that hexokinase is the most labile enzyme of the glycolytic system. The assignment was to study the stability of the enzyme with respect to the concentration of hydrogenions, and to other factors.

Kashket {22), in our laboratory, in his study of the enzyme had found that increase in the hydrogen ion concentration in the red cells during storage in the cold was largely responsible for the progressive slowing dawn and ultimate cessation of glucose utilisation, and failure of glycolytic activity. Pappius (1 08) at a still earlier time in our laboratory had shawn also that if the glucose concentration in the blood specimen were permitted to fall to zero, even momentarily, the prompt addition of glucose was not able to restore the previous rate of glucose utilisation. This finding was taken to indicate the extreme lability of hexokinase in the absence of its substrate and to signify the importance of maintaining an adequate level of glucose for the maintenance of cell viability.

It came as a surprise, therefore, that the writer 1s assays of the activity of hexokinase in the red cells during storage in the cold shÔwed virtually no alteration of the stability of the isolated hexokinase system over a period of 30 days (see p. 59, and Fig. 8, p. 61).

".rlle discrepancies between the findings in this study and those obtained by Kashket may be explained by the circumstance that the writer used the

SFH preparation whereas Kashket used the whole hemolyzate for the assay 84 of the enzyme. The decreasing concentration of DPN in the cell during storage and the presence of an active DPNase in the whole hemolyzate are factors which could possibly have influenced the determination of hexokinase activity (see pp. 38, 54).

Furthermore, the writer was not able to demonstrate the second, and minor, pH maximum at pH 6. 0 as obtained by Kashket.

It was necessary therefore to look for another explanation for the falling off in the activity of the glycolytic system of the red cell during preservation at 4" C.

As one studies the metabolic behaviour of the cell, one is forced to realize that the membrane cannot be considered merely as an envelope for the cell contents. It is now recognized that the membrane also is a dynamic component of the cell and intimately connected with the overall metabolism of the cell.

Lionetti, in his studies on the 11 ghosts 11 of red cells, has demonstrated the utilisation of nucleosides and the accompanying increase in the concentration of organic phosphate esters.

Prankerd has shown that sorne of the glycolytic intermediates are present in the stroma (see p. 27}. Many enzymes also have been found in the stroma, including ATPase and adenylate kinase, the two most important ones in the regulation of the levels of the nucleotide coenzymes in the cell (see pp. 25, 26). Kashket (22) suggests that the enzyme hexokinase may be distributed on the interior surface of the cell membrane.

No hexokinase activity has ever been found with a "hemoglobin-free 11 85 preparation (see p. 37}, thus suggesting that this and other enzymes are precipitated with the hemoglobin or denatured and inactivated. Bartlett has suggested also that the triose phosphate dehydrogenase, phospho­ glyceric kinase and lactic dehydrogenase may be situated in the "cell coat" (see p. 23).

The membrane should be considered as being continuous with the cytoplasm and thus containing many, if not all of the enzymes of the cell.

The membrane furthermore is nf:lt to be regarded as a rigid structure but rather as a dynamic sieve-like organ whose permeability and adaptability are controlled by the metabolic activity of the cell. It responds to changes in conditions and the metabolic needs of the cell. The membrane normally appears to be impermeable to organic phosphate esters. However,

Kashket (see p. 25) has observed that ADP, added to the external medium, apparently can enter the cell membrane and stimulate the glucose utilisation of the cell. AMP and A TP on the contrary apparently cannot enter the membrane.

The 3', 5'-cyclic adenylic acid apparently can gain entrance into the cell since there is evidence of the utilisation of the ribose moiety (see

Fig. 13, p. 75) and its conversion to lactic acid. The nucleotide further­ more brings about a decrease in the inorganic phosphate and an increase in stable phosphate. The effect of the cyclic nucleotide is similar to that of inosine though not as pronounced. (see Fig. 15, p. 80).

Our observation that glucose tends to be equally distributed on both sides of the red cell membrane confirma that of Laris (6). 86

Lactic acid also was found to be evenly distributed on both sides of

the membrane (see p. 64). Pyruvic acid, however, tends to accumulate

in the plasma for sorne time during storage before it begins to rise in the

red cell (Fig. 12., p. 72.). That a pyruvate gradient is set up by the

metabolic activity of the cell has been demonstrated by Huckabee {see p. 5).

It would appear that during the first 15 days or so during storage of blood

in the cold, sorne of the pyruvate formed in the cells is expelled into the

plasma. After this time the ability of the cell to exclude pyruvate appears

to be diminished and as a consequence, the pyruvate in the cell begins

to rise.

An accumulation of pyruvate resulting from the metabolic breakdown

of 2., 3-DPG would tend to cause inhibition of lactic dehydrogenase.

Ottolenghi {156) has shown that a pyruvate concentration of the order of

O. 0004M will inhibit the enzyme to a slight degree. As already mentioned, lactic dehydrogenase is present in the cell membrane. The expulsion of

the excess of pyruvic acid into the externat medium thus may afford a means of excluding a noxious substrate.

Even inorganic phosphate, when liberated in the cell, does not equilibrate equally between the cell and the plasma (see Fig. 11, p. 79).

The rate at which inorganic phosphate is admitted into the cell is regulated by the need of the cell and the external conditions (see pp. 13-15 ).

The cell membrane formerly was considered as normally being impermeable to sodium. In the light of present knowledge, the membrane is regarded as being permeable to both sodium and potassium, but the 87 sodium normally is virtually excluded from the cell by virtue of its metabolic potential, and likewise the potassium is retained in the cell.

Any condition such as l'owering of the temperature, change of pH, etc., which reduces the metabolic potential results in a movement of the cations along the respective gradients. It is known that radioactive sodium or potassium added to the external medium rapidly reaches a state of equilibrium between the cells and the external medium.

It is of interest that twice as much glucose was found to be utilised by the stroma-free hemolyzate as by fresh intact cells {see p. 58). However, the latter were observed to convert the glucose entirely to lactic acid while with the stroma-free hemolyzate, only about 50 o/o of the sugar was meta­ bolized to the end product.

Evidently, the conditions in the intact red cells are not optimal for the activity of the hexokinase.

Measurement of the activity of an isolated enzyme does not necessarily give a true picture of the level of activity of the enzyme in the cell. Factors such as the concentration of the coenzyme at the enzyme site, and the disposition of the enzyme in relation to other enzymes may limit the rates of coupled reactions and the rate of trans fer of metabolites from enzyme to enzyme.

However, the glycoiytic system seems to be organized so as to convert all the phosphorylated glucose into lactic acid. When the cells are broken, the organization of the system is disrupted and the enzymes may be thrown more or less randomly together. Presumably portions of 88 the structural units of the glycolytic system may still remain intact on the surface of the stroma fragments. However, the greater the degree of disintegration of the cell structure the more random and uncontrolled the overall system becomes. Thus, the conditions regulating the equilibria in the SFH, although favouring a higher glucose utilisation, do not favour a lOOo/o glucose conversion to lactic acid. In other words the controlling organization and direction à.re1argely lost in the SFH.

In the experimenta where ADP alone and ADP + ATP were added to a

SFH preparation (p. 46), the glucose utilisation followed a linear relation­ ship with time during an incubation period of three hours, but the rate was slower in the ADP sample. This interesting observation raises doubt as to whether the enzyme activity measured by the assay procedure deàcribed

(see p. 43) is actually the maximal activity of the enzyme. Thus, in a multi-enzyme system, such as is present in the SFH, the initial reaction, which is catalyzed by hexokinase, may be inhibited by accumulation of its own product. If the product is removed at a constant rate, the inhibition is not detectable. In our experimenta the conversion of glucose to lactic acid in the SFH was only 50o/o of the theoretical. Hence the intermediate(s) which accumulates must be disposed of so that it does not affect the equilibrium of the glycolytic system and interfere with the constant rate of glucose utilisation. The most likely eventuality may be the diversion of much of the product into the formation of 2, 3-DPG. Since the reaction

(Step 12, Fig. 1, p. 16) is considered to be virtually irreversible and its hydrolysis to 3-phosphoglycerate to occur only when the phosphatase becomes 89 activated at a lower pH, the accumulation of 2, 3-DPG would not affect the equilibrium of the main system.

The importance of the inhibition caused by glucose-6-P on hexokinase is very well illustrated in Figure 5 (p. 53). Not only was the intermediation of phosphohexoisomerase and phosphofructokinase for removal of the glucose-6-P and its inhibitory action necessary, but also an optimal activity of the triose phosphate dehydrogenase was necessary, as was evidenced by the stimulating effect of added DPN. This is illustrated further by the inhibition of glucose utilisation on addition of nico±inamide, via inhibition of the triose phosphate dehydrogenase. The activity of the phosphoglyceric kinase similarly may influence the rate of glucose phosphorylation by hexokinase (see p. 51).

The increase in glucose utilisation by red cell suspension on the addition of ADP to intact cells, as observed by Kashket (84), (see p. 25), could also be explained by a direct action of ADP as phosphate acceptor in the reaction catalyzed by phosphoglyceric kinase (see p. 25). ADP is essential for the forward reaction. The presence of adenylate kinase {see p. 49, andFig. 4, p. 50) in the erythrocyte may reduce ADP to the level where the forward reaction cannot occur. Thus adenylate kinase, by determining the ADP: ATP ratio available to the phosphoglyceric kinase (Fig. 1, p. 16, Step 11), may also regulate the activity of the preceding steps, including the hexokinase reaction. It has been suggested that when ADP accumulates, adenylate kinase couverts it immediately to AMP and ATP. The increase in ATP concentration then inhibits the enzyme, thus maintaining a constant ADP: ATP 90

ratio. Disruption of the cell structure by hemolysis or hpmogenization

may alter the organization of the unit enzyme system, its equilibria, and

the ADP: ATP ratio so that the production of lactic acid from glucose is less complete.

In view of the action of adenylate kinase and the effects of disruption

of cell structure, the interpretation of the findingsin previous studies on

the activity of the phosphoglyceric kinase and pyruvic phosphoferase in the

SFH, as reported in the literature, must be critically re-examined.

The influence of the hydrogen ion concentration on the activity of hexokinase showed only one maximum peak between pH 7. 4 and 7. 8 (see Fig. 2, p. 45). The activity of the enzyme in the red cells during storage therefore is progressively inhibited as the concentration of lactic acid increases.

The rate of utilisation of glucose by the cell gives no indication of the potential activity of the hexokinase within the cell. There is no doubt that the increase in hydrogen ion concentration in the cells during storage is greatly responsible at the start for the decrease in the rate of glucose utilisation. Rubinstein et al. (127) attribute the superior utilisation of glucose in the presence of adenosine, compared to that with inosine, to the higher pH in the case of the former, owing to the liberation of ammonia on deamination of the adenine moiety. This doubtless is true, However, the lower glucose utilisation in the inosine sample cannat be due only to an inhibition caused by higher hydrogen ion concentration~ The writer consider s that, at fir st, the hexokinase is slightly inhibited by the higher hydrogen ion concentration, allowing a greater ribose utilisation. Very 91 soon however, glucose-6-P accumulates and inhibits hexokinase activity entirely. Bartlett and Shafer (157) have shown clearly that this inter­ mediate is formed and accumulates in the presence of added inosine.

Prankerd (124) observed that red cells stored for 4 weeks at 4oc with or without added nucleoside (adenosine}, lose the ability to utilise glucose on incubation at 37°C. If, however, adenosine be added to the preserved sample, and the sample incubated at 37°C, the cells regain the ability to utilise glucose. The ATP concentration, in the course of the incubation, was increased to O. 54 ?mole/ml. cells. This finding is taken to indicate the requirement for a certain minimal level of A TP for hexokinase activity and glucose utilisation.

Meyerhof (158}, in 1948, postulated that the rate of glycolysis in tumour homogenates is controlled by the balance in the activity of the enzymes, hexokinase and ATPase. He suggested further that the effective concentration of ATP for the hexokinase reaction in the intact cells is regulated by the enzyme A TPase in the membrane.

The known ability of a proportion of preserved red cells to remain in the circulation after transfusion implies that the cells recover the capacity to utilise glucose in the normal manner. Moreover, it is known that a redistribution of cations toward the normal and resynthesis of phosphate ester intermediates occur after the cells are again placed in the circulation {see p. 31 ).

Rubinstein et al. (114) have observed that red cells preserved with inosine in the medium, on being washed and resuspended in a glucose-containing 92

medium at pH 7. 4 or 8. 0, were able to utilise glucose. The rate of

utilisation, however, tends to decrease with increasing age of the specimen.

It is noteworthy that as the rate of glucose utilisation decreased, there was

a parallel decrease in the A TP content of the cells. The sample in which

inosine had been added at the beginning of storage maintained a higher level

of ATP for a longer time, and simultaneously preserved for a similar

period of time the capacity of the cells to utilise glucose. It seems quite

evident that the decreasing capacity of the cells to utilise glucose was not

due to hexokinase inactivation, but rather to a decrease in ATP, which

affected the rate of utilisation of glucose by the cells.

In conclusion, the writer's view as to the sequence of events leading to

the failure of energy metabolism in the red cells during preservation in the

cold is as follows: As glucose is utilised and lactic acid produced, the

hydrogen ion concentration in the preserved cells increases and certain

enzymes, hexokinase in particular, become progressively inhibited.

Furthermore, the equilibrium of the phosphohexoisomerase reaction is

known to be in the direction of the formation of glucose-6-P and this is further accentuated at lower temperatures (see p. 20). The phosphofructokinase

enzyme has been shown to become inactive during storage (see p. 32}. The

decrease in DPN may lower the triose phosphate dehydrogenase reaction and the observed decrease in ADP may retard the phosphoglycerate kinase

reaction. All these conditions will slow down the rate of glucose utilisation in the intact cells since they have either a direct effect on the hexokinase itself through the decrease in the level of ATP, or an indirect one by causing 93

an accumulation of glucose-6-P.

Since blood representa a population of cells of ages between the

newly-formed and those that are at the limit of their life span, it is

evident that the proportion of resuscitatable cells and the degree of

resuscitation will decrease with the duration of storage. Doubtless with

aging sorne of the apoenzymes and essential structures become altered

and disintegrated.

It is evident that unless all conditions that cause an accumulation of

glucose-6-P are corrected and the ATP concentration is raised to a

sufficient level for optimal hexokinase activity and the hydrogen ion

concentration rectified, the cells will be unable to resume normal glycolytic activity. It so far has been impossible experimentally, in vitro, to

achieve these requirements, but all the conditions may be fulfilled readily by placing the cells in the circulation. SUMMARY

Contrary to the prevailing views, based on the work done by former

workers in our laboratory and elsewhere, that the hexokinase of the red

cell is an extremely labile enzyme in the absence of glucose, and that the

lability of the enzyme is largely responsible for the failure of the meta­

bolism of the red cell during storage, the writer has demonstrated, by

assay of the activity of the hexokinase system, that the enzyme remains

stable and active for at least 30 days in human red cells preserved in CD

or ACD solution at 4°C.

An assay procedure has been developed for the estimation of the

optimal hexokinase activity in the SFH. The addition of ADP or DPN

to the medium was found to increase the activity of the enzyme while

nicotinamide or alloxan depressed it.

A study of the distribution of pyruvate between the cells and the

external medium revealed the odd situation that pyruvate, produced in

the cells during the first 15 days of storage, tends to be expelled into

the external medium. Thereafter the capacity of the cells to maintain

the external pyruvate gradient decreases and the level of this intermediate in the cells progressively increases.

A preliminary study to find out whether 3 1, 5'-cyclic adenylic acid can be utilised by the erythrocytes showed that the nucleotide may enter the cells and that the ribose moiety, like that of added inosine, can be metabolized. The proportion of the ribose converted to lactic acid however 95 is less than with inosine, as with inosine the utilisation of the ribose was accompanied by the esterification of inorganic phosphate into the stable phosphate ester fraction.

The suggestion is made that the failure of the glycolytic activity in the red cells during preservation may be attributable to the fall in pH, the decrease in ATP and the conditions favouring glucose-6-P accumulation such as a decrease in phosphofructokinase, DPN, and

ADP. CLAIMS TO ORIGINALITY

The writer considers that the following observations have not so far been reported in the literature:

1. The evidence for the stability of hexokinase in the red cell during

storage for 30 days at 4 °C,

2. The observation of the behaviour of pyruvate distribution between

cells and plasma during storage.

3. The interpretation of the influence of a decrease in pH and ATP,

and the accumulation of glucose-6-P, as predominant factors in

the inhibition of glucose utilisation during storage of blood at 4°C,

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