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1981

The effects of moderate altitude on patients withCOPD : the role of 2, 3-diphosphoglycerateThomas KleemanYale University

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Recommended CitationKleeman, Thomas, "The effects of moderate altitude on patients with COPD : the role of 2, 3-diphosphoglycerate" (1981). YaleMedicine Thesis Digital Library. 2799.http://elischolar.library.yale.edu/ymtdl/2799

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Thomas Kleeman

THE EFFECTS OF MODERATE ALTITUDE ON PATIENTS WITH COPD:

THE ROLE OF 2f3-DIPHOSPHOGLYCERATE

A Thesis Submitted to the Yale University School of Medicine in

Partial Fulfillment of the Requirement for the degree of Doctor

of Medicine - 1981

INTRODUCTION

The limits of Man's homeostatic capabilities are determined

primarily by his ability to derive oxygen from the environment.

He can survive without food for weeks, without water for days,

but deprived of oxygen, a chain reaction of events takes place

that will bring about his demise in a matter of minutes. The

limitations of altitude and depth are determined not by distance

but rather by pressure. A normal man can survive as high as

6000 m. above sea level, and a few have even climbed Mt. Everest

(8800 m.) without additional oxygen. On the other hand, the

descent by a diver breathing 20% oxygen is limited at only a

few hundred feet by oxygen toxicity. This is of course due to

the increased pressure generated by water as opposed to air.

The condition found at altitude is called hypoxic hypoxia

to differentiate it from the other forms of hypoxia.^ In

hypoxic hypoxia the reduced 02 shows up as a diminished P02 in

the arterial blood. This distinguishes it from anemic hypoxia

where the diminished hemoglobin causes the hypoxia, stagnant

or ischemic hypoxia where diminished blood flow compromises

tissue perfusion, or histotoxic hypoxia where a toxic agent

such as cyanide inhibits the tissue cells from utilizing the

02. Hypoxic hypoxia is a complication found not only at al¬

titude but in several common disease states. The most common

condition is hypoventilation where for one reason or another

insufficient 02 is reaching the alveoli. Other causes for hyp-

(2)

poxic hypoxia include alveolar-capillary diffusion block, un¬

even ventilation-perfusion ratios, or total bypass of the lung

by venous blood.

In Chronic Obstructive Pulmonary Disease (COPD), several

of these conditions are present at the same time. There is hypo¬

ventilation due to increased airway resistance as well as de¬

creased elastic recoil of the lungs. There is also an abnormal

ventilation-perfusion ratio due to loss of functional alveoli,

resulting in underventilation and overperfusion. The end result

of these factors is decreased arterial P02 or hypoxic hypoxia.

Much work has been done on Man's adaptation to altitude

both in short term and long term exposure.^ On immediate ascent

to 3000 m. a normal individual will begin hyperventilationg,

a mechanism induced by hypoxic stimulation to the peripheral

02 receptors. This has the effect of raising the partial pres¬

sure of oxygen in the alveolar spaces. Respiratory alkalosis

results as the arterial PC02 drops. This development acts as

an inhibitor of the ventilatory response by altering the pH

of the CSF which has an effect on the medullary H4 receptors.

Within a day or so there is movement of the bicarbonate out

of the CSF restoring the pH. At the same time, renal excretion

of bicarbonate is lowering the arterial pH to normal so that

within 2 or 3 days the baseline pH is restored and the hypoxic

stimulus cam proceed unopposed. This results in sustained

hyperventilation.

A second adaptation to altitude is an increase in the red

blood cell concentration of the blood. This is thought to be

secondary to the hypoxia induced release of erythropoietin

(3)

from the kidney. This effectively raises the hemoglobin concen¬

tration and therefore the 02 carrying capacity of the blood.

Thus the diminished aterial P02 and 02 saturation are partially

offset by the increased 02 carrying capacity.

A third adaptation involves the C>2 saturation as seen by

changes in the oxygen-hemoglobin dissociation curve. A "shift

to the right" is seen at altitude which is thought to enhance

the unloading of 02 to the tissue. The mechanism of this shift

is complex and not completely understood, byt is thought to be

mediated in part by an increase in the level of 2,3-diphospho-

glycerate (2,3-DPG), a byproduct of the glycolytic pathway.

The character, action and control of 2,3-DPG will be taken up

in some detail in the subsequent section.

Other adaptations that are seen at altitude include in¬

creased capillary density, increased concentration of myoglo¬

bin, and an increased maximum breathing capacity. There is also

a pulmonary vasoconstriction in response to alveolar hypoxia.

This in turn leads to pulmonary hypertension. The additional

work of the right heart results in hypertrophy with characteris¬

tic ECG changes. Unlike the aforementioned adaptations the

right sided hypertrophy takes longer to develop and is more

a characteristic of chronic exposure to altitude. Heath and

Williams (1977) ^^escribe other effects of chronic exposure

to altitude including development of a full chest and enlarge¬

ment of the carotid bodies due to hyperplasia of the chief

(Type 1) cells. This increase in the size of the chemorecep-

tors is associated with a blunted ventilatory response to hypo¬

xia.

(4)

In COPD the onset is more insidious and the adaptive mechanisms

are developed more slowly.^ As it is an ongoing disease pro¬

cess there is considerable variation in the number and degree

of these features, but almost all of the elements described for

altitude adaptation are seen in COPD. Due to the disease pro¬

cess, which affects primarily the lung tissue, the ventilatory

response of hyperventilation is often ineffective. Respiratory

alkalosis is also not a predominant feature of COPD, again due

to the insidious nature of its development. In fact, as the

ventilatory response becomes less effective, a respiratory aci¬

dosis will often develop which may or may not be adequately

compensated by renal excretion. Patients with COPD will often

develop full chests with an increase in the A-P diameter. Un¬

like altitude dwellers however, there is a decrease in the

vital capacity rather than an increase. The increased chest

volume is therfore composed of the increased functional residual

capacity (FRC) and residual volume (RV). These are a result of

the diminished elastic recoil and airway obstruction which to¬

gether cause premature closing of the airways and airtrapping.

With an insufficient ventilatory response to chromic hy¬

poxia, the COPD patient must rely on other compensatory measures

and these are the same ones seen on prolonged exposure of

normal subjects to altitude. There is an increase in the hema¬

tocrit and hemoglobin concentration in a degree proportional

to the extent of the hypoxia. There is also a shift seen in the

oxygen-hemoglobin dissociation curve (ODC). It is generally

agreed that there is also an increase in 2,3-DPG, although as

will be discussed in the next section, this is not a consis-

(5)

tent finding.

Other vascular changes are seen in COPD, including in¬

creased capillary density, incresed myoglobin, and pulmon¬

ary hypertension. Bight-sided cardiac hypertrophy cam also be

demonstrated, which earn lead to right-sided heart failure, a

condition called Cor Pulmonale. Hyperplasia of Chief cells has

also been shown in the carotid bodies of these patients with

am associated diminished CO2 response.

Thus it can be shown that the adaptations to chronic hy¬

poxic hypoxia are very similar in the altitude dweller and the

COPD patient. It is of further interest to note that the results

of decompensation are very similar in the two groups with pul¬

monary edema, C02 retention, acidosis, coma, and death being

the course of events if medical intervention is not instituted.

There is a considerable volume of information concerning

the effects of altitude on normal individuals in terms of their

response to hypoxia. Much is also known about the pathophysio¬

logy of the hypoxic response at sea level of persons with COPD.

There is very little known, however, about the response of

persons with COPD when they are exposed to moderate altitude.

It would seem likely that an individual with an initial im¬

pairment of the hypoxic compensatory mechanism might react to

a mildly hypoxic environment more dramatically than a normal

individual. In a modern age of rapid air travel, there is an

increasing number of people going from areas close to sea

level to areas such as Denver, Colorado which has an altitude

in excess of 1600 m. Furthermore, the jet aircraft in which

they travel have cabins that are pressurized at 1830 to 2440 m.

I (6)

Exposed to this relative altitude for up to 10 hours might

have a potentially deleterious effect on these individuals.

It was for this reason that Dr. William Graham undertook a

study to observe the reactions of 8 patients with COPD.^y

These volunteers were transported from Burlington, Vermont

(50 m.) to the summit of Mt. Washington (1920 m.) in New Hamp¬

shire for a period of 4 days. A number of clinical and physio¬

logical parameters were evaluated, and comparisons made be¬

tween sea level and moderate altitude. This initial study

done using patients with mild-to-moderate COPD (average rest-

in PaP02 of 66 mm Hg) who were symptomatic but not disabled

at sea level, demonstrated their ability to tolerate levels

of arterial hypoxemia (51.5 mm Hg) without notable symptoms.

Normal subjects at altitudes producing equivalent hypoxemia

(4270-5182 m.) would often demonstrate symptoms of Acute

Mountain Sickness (dizziness, headaches, nausea, pulmonary

edema), yet no such symptoms were observed in these patients

nor were there any ECG changes. Alveolar hyperventilation oc¬

curred within three hours of ascent producing a lowered PaC02

and respiratory alkalosis. Despite this alkalosis, there was

no change seen from the normal baseline levels of 2,3-DPG when

samples were taken at 16 and 42 hours after ascent. During

their 4 days at altitude, the average Pa02 rose to 54.5 mm Hg.

Pulse rates at rest and exercise did not change from sea level

values, in contrast to what has been observed in normals at

altitude. The conclusion was made that patients With mild-to-

moderate COPD were able to tolerate several days of altitude

suprizingly well, and were at no significant risk for air travel.

(7)

Dr. Graham felt that the study warranted repeating with more

severe COPD patients. One aspect of that repeat study was to

look more closely at the 2,3-DPG levels and why they should

or should not change at altitude. That investigation will be

the subject of this paper.

(8)

2.3-DPG - STRUCTURE AND FUNCTION

2,3-Diphosphoglycerate (2,3-DPG) was first isolated and

identified by Greenwald in 1925.He reported that it was a

major component of the organic phosphate in human, pig, and

dog erythrocytes. In 1939, Rapoport and Guest showed that

2,3-DPG was pH dependent in experimental conditions.^ Between

1950 and 1952, Rapoport and Luebering demonstrated the path¬

way (which bore their names) by which 2,3-DPG metabolism by¬

passes the phosphoglycerate kinase reaction in glycolysis.g

They demonstrated that 2,3-DPG is synthesized from 1,3-DPG and

3-phosphoglycerate by the action of diphosphoglyceromutase

and metabolized to 3-P-glycerate and Pi by 2,3-DPG phosphatase.

Nearly twenty years elapsed until in 1967 two independent

groups, Chanutin and Curnish, and Benesch and Benesch, simul¬

taneously reported that 2,3-DPG lowered the oxygen affinity

of hemoglobin.^ ^g Benesch and Benesch further reported that

2,3-DPG increases the heme-heme interaction whic gives the

ODC its sigmoid shape. This is expressed as n the exponent in

the Hill equation: (Hb02)/(Hb)=K(P02)n. Plotting n and the

02 affinity measured as log P^Q vs 2,3-DPG they showed that

the effect of the phosphate on altering the 02 affinity or

heme-heme interaction on dialyzed hemoglobin had reached a

maximum value at concentrations within the normal physiological

range.

In 1968, Benesch and Benesch produced two papers confirm¬

ing their earlier findings and added some new information,

quantifying the effect of 2,3-DPG on the O^-Hb affinity. They

estimated that the affinity was lowered by 30 times in the

presence of either 2,3-DPG or ATP. The concentration of ATP

was too small in the red cell to be a factor however. They

found that the concentration of 2,3-DPG was approximately the

same on a molar basis as hemoglobin (5X10”^M) and that 2,3-

DPG bound the deoxy form of Hb preferentially in a ration of

1 mole DPG/1 mole Hb. By using hybrid forms of Hb, they found

that isolated alpha chains did not react with DPG in either the

oxy or deoxy form whereas beta chains bound both forms equally

in both isolated and tetramer form.

Using the data of Perutz^ on the structure of hemoglobin,

and the fact that DPG and Hb bind on a 1:1 ratio, they postu¬

lated that DPG was bound in the central cavity along the diad

axis of symmetry. This cavity changes its width during oxy¬

genation, being too small to accommodate the molecule in that

form, but having sufficient width in the deoxy form.

This was finally verified in 1972 by Arnone.^ Using an

X-ray crystallographic study of deoxyhemoglobin crystals con¬

taining 2,3-DPG, he confirmed the one to one complementation

of the two molecules. DPG takes a stereochemically complemen¬

tary position on the two-fold symmetry axis "plugging" the

entrance to the central cavity. The anionic groups of the beta

chains which include Valine 1, Histadines 2 and 143, and Ly¬

sine 82. Because the complementation is specific for the deoxy

form, the 2,3-DPG stabalizes the deoxy form of Hb at the ex¬

pense of the oxy form. This shifts the allosteric equilibrium

toward the form with the lower 02 affinity. On oxygenation,

(10)

the alpha amino groups of the two N-terminal valines move apart

and the 2,3-DPG molecule is extruded as the H helices close up.

These observations confirmed one mechanism of the action of

2,3-DPG but did not explain the observation made by Benesch and

Benesch that 2,3-DPG exerted its maximum effect at levels within

the physiological range.^g Duhm in 1971 pointed out that there

was another mechanism involved that could only be appreciated

using intact cells.^ Benesch and Benesch had used dialyzed

hemogolobin. 2,3-DPG has several properties that enable it to

have its own Bohr effect independent of the existing pH within

the cell. First, it has 4 negative charges per molecule. It is

also unable to cross the erythrocyte membrane, which sets up a

transmembrane gradient of negative DPG ions. This in turn al¬

ters the Donnan equilibrium causing a decrease in intracellular

pH and a resulting right shift of the ODC. This will take place

even if there is no appreciable change in the plasma pH. Duhm

showed that at concentrations of 2,3-DPG above normal, this

latter mechanism predominates in altering the ODC. The amount

of this DPG-linked Bohr effect can be quantified using a for¬

mula developed by Duhm relating intracellular pH to organic

phosphate concentration in ju.moles/gram of hemoglobin:^

pHi = 7.306 - 0.0083 X P org (yumoles/g)

Baur et al showed in 1969 that DPG affects other modi¬

fiers of the ODC as well.2g ^ He specifically looked at the

Bohr effect (defined as A log P^Q/ApH). Using dialyzed Hb

solutions, he found that in the absence of C02» there was an

insignificant rise in Bohr effect in the presence-of low levels

of 2,3-DPG vs no 2,3-DPG. In the presence of C02 however (40 mm Hg)

(11)

there was a significant increase of Bohr effect when 2,3-DPG

was added to the solution. Bauer postulated that this pheno¬

menon was due to the effect of 2,3-DPG on carbamate formation.

It had been shown by Rossi-Bernardi et al.^ that carbamate

formation decreased the Bohr effect. In fact, in dialyzed

hemoglobin solutions (no carbonic anhydrase), C09 decreased

the Bohr effect by about 50# by this mechanism. Carbamate was

known to be formed at the alpha-amino groups of the N-terminal

valine residues of each hemoglobin chain. Bauer, knowing

that 2,3-DPG was bound only to beta chains, postulated that

the terminal valine must be a site of attachment for the mole¬

cule. 2g This was confirmed by Arnone in 1972.^ Thus the bind¬

ing of 2,3-DPG to hemoglobin interferes with carbamate forma¬

tion resulting in an increased Bohr effect.

Other functions have been discovered for 2,3-DPG, most

of them involving modulation of the enzymatic reactions occur¬

ring in the erythrocyte. The full tabulation of these effects

has been done by Chiba et al. (1978).^ Most of these effects

are inhibitory and are caused by reacting with the enzymes

themselves or by chelating Mg** and eliminating its participa¬

tion in the reactions. Most of the enzymes involved in the

glycolytic pathway are inhibited by 2,3-DPG, especially the

kinase reactions (hexokinaee, phosphofructokinase, and pyru¬

vate kinase). The enzyme involved in the formation of 2,3-

DPG (diphosphoglycerate mutase) is also inhibited by the pro¬

duct through a feedback inhibition. In the reaction which con¬

verts 3-PG to 2-PG, 2,3-DPG acts as an activator or cofactor.

This appears to maintain a balance between the formation of

(12)

2,3-DPG from either of its two precursors 1,3-DPG and 3-PG.

2,3-DPG also has an inhibitory effecto on AMP deaminase, the

enzyme responsible for metabolizing AMP to IMP and NH^. This

increase in AMP would inturn stimulate phosphofructokinase

which as has been pointed out is inhibited by 2,3-DPG. The

meaning of these seemingly opposing effects is most likely

related to the balance of glycolysis and adenine nucleotide

metabolism.

One interesting aspect of 2,3-DPG function has been the

discovery by Iatridis et al. (1975) that it inhibits platelet

aggregaion by an unknown raechanism.gr, It has been speculated

therefore that it plays some role in maintaining hemostatic

homeostasis.

REGULATION OF 2.3-DPG

The synthesis of 2,3-DPG is under the control of many

complex factors which can be roughly divided into three groups:

regulatory factors on glycolysis, on the two enzymes diphospho-

glycerate mutase and diphosphoglycerate phosphatase, and factors

that alter the amount of 2,3-DPG bound to hemoglobin.

The factors controlling erythrocyte glycolysis include

pH changes, inorganic phosphate, and oxygen. Rapoport showed

in 1939 that acidosis decreased the concentration of 2,3-DPG

in vitro.^ He later demonstrated that this took place at the

phosphofructokinase step resulting in a general inhibition of

glycolysis.g0 Acidosis also has an inhibitory effect at the

diphosphoglyceromutase step, altering the equilibrium between

(13)

1.3- DPG and 2,3-DPG. Alkalosis has been shown in several stu¬

dies to stimulate glycolysis and raise the 2,3-DPG level.^

This is also thought to occur at the phosphofructokinase level.

Inorganic phosphate also stimulates glycolysis independent of

any pH changes. The effect of hypoxia on glycolysis seems to be

the result of the hyperventilation that is brought about in

hypoxic states.^ ^ ^ This causes a respiratory alkalosis which

will in turn stimulate glycolysis

The two enzymes involved in the Rapoport-Leubering shut¬

tle are each under control by their own modulators. Diphospho-

glyceromutase is under feed back inhibition from its product

2.3- DPG as discussed earlier. It is also inhibited by inorganic

phosphate, however this effect is insignigicant in view of the

overall stimulatory effect of Pi on glycolysis. Diphosphoglycerate

phosphatase which catalyzes the conversion of 2,3-DPG to 3-PG

is activated by inorganic phosphate and inhibited by 3-PG and

2-PG. Although it was originally thought that these were sep¬

arate enzymes, it has recently been shown that there is one

multifunctional protein capable of catalyzing two irreversible

reactions. In fact, it appears that both reactions are cataly¬

zed at a common site on the protein and that products of either

reaction can be effectors of the other reaction.

The third mechanism for the regulation of 2,3-DPG invloves

its target molecule hemoglobin. As stated earlier, 2,3-DPG

binds preferentially to the deoxy form of hemoglobin. In states

of hypoxia, the concentration of deoxyheraoglobin will increase.

As the 2,3-DPG binds to these molecules, the concentration of

free 2,3-DPG decreases. This will activate dephosphoglycero-

(14)

mutase and result in increased 2,3-DPG production. In addition,

deoxyhemoglobin is more alkaline than the oxy form, and this

rise in pH will induce the production of 2,3-DPG by stimulating

glycolysis.6l

As discussed earlier, an isolated increase in 2,3-DPG- will

lower the 02 affinity of hemoglobin causing a shift to the right

of the oxygen-hemoglobin dissociation curve (ODC). Similarly,

a decrease in 2,3-DPG has the opposite effect of raising the C2

affinity and causing a shift to the left of the ODC. However,

it is well established that other factors alter the 02 affinity

of hemoglobin as well. An increase in hydrogen ion concentration,

carbon dioxide, or temperature will also cause a shift to the

right. In the many physiological and pathophysiological condi¬

tions that alter the concentration of 2,3-DPG , there is usually

an accompanied change in one ore more of these other factors.

The importance of these changes can best be appreciated by

looking at the individual conditions.

ALTITUDE

The effects of altitude on the 02 affinity of hemoglobin

have been used as the prototype for studying chronic hypoxia.

The effect of hypoxia on the ODC can best be appreciated by

looking at the P^Q, the P02 in mm Hg at which there is 50$

saturation of the hemoglobin molecule. An increase in the P^Q

is equivalent to a shift to the right, indicating a decrease

in 02 affinity. Keys et al. (1936) were the first to demon¬

strate an increased P^Q in people living at high altitude.^

(15)

It was not until 1968 however that Lenfant and his co-workers

observed elevated levels of 2,3-DPG with associated changes

in people living at high altitude (4500 ®.).2o They showed that

lowlanders ascending to altitude reached values of 2,3-DPG and

shifting of the ODC equal to the highlanders within two days.

These changes were maintained as long as the subjects remained

at altitude. When people at altitude returned to sea level,

there was a rapid decrease in and 2,3-DPG level. This was

found to occur both in lowlanders and in highlanders who had

lived permanently at altitude. In this original study, Lenfant

was unable to show any correlation between pH and changes in

2,3-DPG.

Astrup and Rorth (1970), studying patients with acid-base

disorders and people ascending to 3500 m. demonstrated a clear

correlation between pH and 2,3-DPG.^ They showed that at 3500 m.

the arterial blood pH increases from 7.40 to 7.43-7.44 as a

result of hyperventilation. This results in a 12-16$ rise in

2,3-DPG. They concluded that the lowered 02 saturation of the

blood at altitude will furthermore increase the mean red cell

pH about 0.10 without any change in the plasma pH. Their in¬

vestigation also showed increased levels of inorganic phosphate

in subjects whose 2,3-DPG levels were higher than might be

expected from the blood pH values alone.

In a repeat study in 1971, Lenfant et al. confirmed the

correlation between hyperventilation-induced alkalosis and

increased 2,3-DPG levels. They found that by administering

acetazolamide (a carbonic anhydrase inhibitor) to subjects

prior to ascent, they could prevent the alkalosis by inducing

a metabolic acidosis. The group pretreated with acetazolamide

had no increase in 2,3-DPG or P^Q on exposure to altitude.^

They also showed a better tolerance to exercise at altitude and

had fewer symptoms of mountain sickness (headache, nausea, vomit¬

ing, dizziness). Wallman et al. (1968) had shown that respira¬

tory alkalosis caused a decrease in cerebral blood flow and it

was felt that this might be related in some way to the symptoms

of mountain sickness. Lenfant's conclusion was that 2,3-DPG was

the most potent factor affecting the affinity of Hb for 02*42

However, based on a study of the changes of all the 02 transport

parameters at various altitudes, he concluded that the adaptive

role of the right-shifted ODC was insignificant. The results of

the subjects made acidotic prior to ascent were felt to be

definate proof that the ODC plays little or no role in adap¬

tation to altitude, and that an increase in ventilation was the

most efficient immediate adaptation.

This feeling was shared by R8rth (1972) who found in con-

rolled studies using simulated altitude, that exercise was a

necessary condition for inducing am elevated 2,3-DPG or rise

in P^q.£2 He felt that some oxidant such as pyruvate was per¬

haps responsible for the elevated 2,3-DPG. In any case, he

agreed with Lenfant that changes in respiration were more im¬

portant as adaptive measures.

Astrup (1970) pointed out that the effect of 2,3-DPG on

the ODC is only one phase of a biphasic phenomenon which re¬

sults from hypoxia.Upon ascent to altitude, the hypoxic

stimulus results in hyperventilation, an immediate reaction

which in turn leads to an immediate respiratory alkalosis.

(17)

This will cause a Bohr effect on the ODC, shifting the curve

to the left. The benefit of the increased ventilation is there¬

fore partially offset by the diminished unloading ability of

the hemoglobin. Over the course of 24-48 hours however, the

increased pH and deoxyhemoglobin concentration result in in¬

creased 2,3-DPG production which shifts the ODC in the opposite

direction by both stabalizing the deoxyhemoglobin and by lower¬

ing the pH. The end result may be an ODC that appears to be

unchanged or only slightly shifted to the right. The sustained

effect of the increased 2,3-DPG is to allow for increased ven¬

tilation without compromising on 02 unloading to the tissues.

There is therefore a complex balance of metabolic adjust¬

ments that are made in response to the hypoxia of altitude.

The cost of these adjustments at times outweighs the benefits,

and complications arise. Hyperventilating has the problem of

increased work load creating its own metabolic demands. Poly¬

cythemia has its own limitations as the viscosity of the blood

increases proportionally to the hematocrit. Eventually the

blood can't move fast enough through the capillaries to deliver

the 02 or for that matter to protect itself from stasis. This

"sludging" predisposes to thrombosis and embolic phenomenon,

a not uncommon tragedy at high altitude.

A shift to the right of the ODC has its own limitations.

Once the upper part of the curve has moved off the plateau, at

around a P02 of 60 mm Hg, the increased 02 unloading is offset

by the diminished loading in the lungs. In other words, the

arterial-venous difference remains unaltered.

The balance of all of these variables in providing op-

(18)

timal homeostasis seems to vary from individual to individual.

Cultural and sex differences have been noted, which raises the

question of genetic predisposition. Eaton et al. (1969) for ex¬

ample, found abnormally high levels of 2,3-DPG in men at alti¬

tude who had excessive polycythemia.^ In the women in his group

he found a much higher elevation of 2,3-DPG on the average than

in the male population. In addition, the women seemed to be

much less susceptible to excessive polycythemia. Only 5 of the

49 individuals experiencing chronic mountain sickness in his

group were women, (chronic mountain sickness defined as excess

polycythemia). In addition, all of these 5 women were post¬

menopausal. The reasons for these discrepancies were unclear,

but a hormonal etiology was suggested by the data.

COPD

Of the disease states that have been studied with respect

to 2,3-DPG, the studies on COPD patients have yielded the most

variable results. Most studies have demonstrated elevated levels

of 2,3-DPG^0 ^ 82* one stud-y showe(i levels that were normal.^

The disparity in results from these studies reflects the com¬

plexity of disease. A patient can develop a clinical picture

anywhere along a spectrum between a Type A (emphysematous) and

Type B (bronchitic).If a patient has more of a emphysematous

component, the Pa02 and PaC02 may remain close to normal due

to hyperventilation. This will result in less acidosis, but

also a smaller cardiac output secondary to the extensive lung

destruction.

(19)

A bronchitic patient will be more likely to have an en¬

larged heart, recurrent episodes of congestive right heart

failure, a diminished sensitivity to C02. They will have lower

Pa02 and higher PaCC>2 values as well as acidosis. Compensation

will come in the form of erythrocythemia but with a normal car¬

diac output.^

In the study that showed normal 2,3-DPG levels despite

severe hypoxemia, there was an associated acidosis (mean pH 7.34)

that was felt to be partly responsible for the lack of 2,3-DPG

response.^ These patients did have an increase in their in

vivo P^0 to approximately 30 torr, due to the Bohr effect of

their acidosis.

In another study done by Weiss et al. (1972) COPD patients

were compared to asthmatics as well as normal controls. The

COPD patients had significantly higher 2,3-DPG levels than both

the normal controls and the asthmatics. A correlation was obser¬

ved between 2,3-DPG levels and pH. The average arterial pH in

that study was 7.41.^

Keitt et al. (197*0 demonstrated a correlation between

2,3-DPG and PaC>2 in 12 COPD patients whose disease was in a

stable state. He was not able to correlate 2,3-DPG with pH in

the stable state. After 7 of these patients who had been on

continuous 02 for 4-12 months, volunteered to discontinue the

02, a rise in 2,3-DPG was noted over a 72 hour period that

correlated with pH but not with PaC>2. He concluded that changes

in arterial pH were prime mediators of 2,3-DPG control in acute

hypoxemia but that arterial P02 or oxyhemoglobin saturation

was responsible for mediating levels in the stable state of

(20)

chronic hypoxemia from COPD. It should be noted that in his

group of stable COPD patients, only one patient had an arter¬

ial pH below 7.37. This patient with a pH of 7.30 also had

the lowest 2,3-DPG level.g2

The collective data seems to indicate that in COPD the

2,3-DPG concentration will depend upon the clinical picture.

In patients with predominantly a bronchitic picture of hyper¬

capnia and severe acidosis, the 2,3-DPG synthesis will be re¬

tarded and any alteration in the ODC will be secondary to a

direct Bohr effect from the acidosis. In a more emphysematous

condition, or in a state of acute hypoxia superimposed on COPD,

conditions favoring a more normal or elevated pH, an elevated

2,3-DPG is seen which correlates with the arterial pH. Other

factors such as Pa02 also have an effect especially in a

steady state where an equilibrium has time to develop.

Klocke (1972) points out that in the presence of abnormal

02 loading where the arterial 02 tension moves toward the steep

portion of the ODC, the effect of a shift in the curve will

not be the same in every tissue.^ Fig.2 shows how the 02 deli¬

very is gained or lost as a function of arterial P02. Since

there are receptors in several organs which are sensitive to

changes in Pa02 it follows that organs will be affected dif¬

ferently. Fig 1 shows that in arterial hypoxemia (Pa02*43 mm Hg),

the brain and especially the heart suffer from a right-shifted

curve, while the kidney benefits from the same shift.

Thus in COPD, the complexity of the regulatory mechanism

plus the diversity of the disease process itself, make it

difficult to predict the appropriate 2,3-DPG level.

*

(21)

ANEMIA

Anemia causes a reduced flow of 02 to tissues by lowering

the 02-carrying capacity of the blood. In order for an aniemic

person to tolerate this reduction in 02 content, several mech¬

anisms are brought into play. Lowering the 02 tension in the

tissues and increasing the cardiac.output are two possible adap¬

tive mechanisms. Duke and Abelman (1969) have shown however,

that these mechanisms are unable to fully account for the tol¬

erance seen in anemic patients.^

A third mechanism for dealing with tissue hypoxia in anemia

is a reduction in the 02 affinity of hemoglobin. Richards and

Strauss (1927) were the first to show a right shift of the ODC

in anemia.2 This was repeatedly confirmed in later studies.^ ^

Many studies have shown an inverse relationship between hemo¬

globin concentration and 2,3-DPG in both normal subjects and

patients with various types of anemia.2^ ^2 ^ It has been pro¬

posed by Torrance et al. (1970) that although the 02 saturation

of arterial blood is normal in anemia, the 02 saturation of ven¬

ous blood is reduced, and this is responsible for the increase

in 2,3-DPG and P^0*42 This has feeen substantiated by Thomas et

al. (1974),83

An interesting study undertaken by Oski et al. (1971) de¬

monstrated the role of 2,3-DPG in anemia quite dramatically.^

Two patients with similar degrees of anemia (hemoglobin approx.

10 mg/100 ml) but with oppositely shifted ODCs were exercised

on a bicycle ergometer. One patient had a hexokinase deficien¬

cy resulting in a low concentration of 2,3-DPG, a left-shifted

(22)

curve, and a of 19mn Hg. The other patient had a pyruvate

kinase deficiencty resulting in elevated levels of 2,3-DPG.

This was accompanied by a right shifted curve and a P^Q of

38 mm Hg. Upon exercise, the patient with the hexokinase defi¬

ciency demonstrated a prompt fall in central venous 02 tension

to 22.6 mm Hg. and was unable to progressively desaturate her

blood at the maximal work load (40.2# sat.). Her compensation

was in the form of doubling her cardiac index with exercise o

from 4.7 1/m per m to 10.0. The other patient with the pyru¬

vate kinase deficiency and right shifted curve demonstrated

a gradual fall in central venous 02 tension to 25.8 mm Hg. and

was able to progressively desaturate his blood at maximal work

load (22.5# sat.). He did not demonstrate the same degree of

cardiac compensation, showing only a 48# increase in the car¬

diac index from 5.7 to 8.4. Neither patient had any acute change

in 2,3-DPG as would be expected . This study demonstrated that

a shift to the right of the ODC allowed for increased 02 ex¬

traction without maximal demands on cardiac output.

ACID-BASE DISTURBANCES

The role of experimental acid-base changes in relation to

2,3-DPG was first demonstrated by Rapoport in 1939.^ He showed

that experimental acidosis resulted in reduced levels of 2,3-

DPG. He also reported a dramatic fall in red cell 2,3-DPG in

ketoacidosis. The pH dependency of 2,3-DPG has been well estab

lished.^0 ^ It is known to take place at the phosphofructo-

kinase step where acidosis reduces erythrocyte glycolysis with

(23)

an additional inhibitory action on diphosphoglyceromutase

As discussed earlier the effects ofpH and DPG are related

but dissimilar in time course and direction. The Bohr effect

occurs instantaneously whereas the opposing change resulting

from alterations in 2,3-DPG are delayed by several days. Thus

in an acute acid-base disturbance, striking shifts of the ODC

may occur from the Bohr effect. In contrast, a chronic or slow¬

ly changing acid-base abnormality will show little change in

the ODC as the rise in 2,3-DPG counteracts the Bohr effect.

This has clinical relevance in terms of correcting the body

pH to normal in diabetic ketocidosis. Bellingham et al.(1971)

cautioned against sudden corrections in body pH. They calcu¬

lated that the ODC was often in a normal position in keto¬

acidosis due to the offsetting effects of increased hydrogen

ion and reduced levels of 2,3-DPG. However, if the acidosis

was corrected too rapidly, there would be a marked shift to

the left as the reduced 2,3-DPG effect went unopposed. A

slow pH correction would allow the erythrocytes time to rep¬

lenish their 2,3-DPG and avoid the complication of cellular

02 deprivation.^

Much work has been done on regulating factors on 2,3-

DPG during changing metabolic states. Ditzel (1973) concluded

that the inorganic phosphate concentration was the priciple

factor determining the red cell 2,3-DPG content during these

states»y^

(24)

INORGANIC PHOSPHATE DISTURBANCES

As indicated earlier, inorganic phosphates have a role in

influencing the rate of synthesis of 2,3-DPG. Elevated concen-

rations of plasma inorganic phosphates are seen in patients

with uremia. Although there has been some conflicting data,

it appears that when there is no associated acid-base change

to confuse the picture, both ATP and 2,3-DPG levels show a pos¬

itive correlation with inorganic phosphate levels.^ gg

Low levels of inorganic phosphates have been reported in

patients receiving intravenous hyperalimentaion or during

oral administration of aluminum hydroxide gels in patients on

maintenance hemodialysis.These hypophosphatemic states have

been associated with decreased red cell 2,3-DPG concentrations

and decreased P^Q values.gy

EFFECTS OF HORMONES

There is some disagreement concerning the effect of tri¬

iodothyronine (T^) and thyroxine (T^) on red cell 2,3-DPG.

Most of the studies have shown a correlation between the levels

of these hormones and red cell 2,3-DPG.^Qg The evidence points

to a direct stimulation of diphosphoglyceromutase by both T^

and T^. There is, however, evidence that there is no effect

of T^ or T^ on 2,3-DPG levels.^ g^

Other hormones that have been found to cause an increase

in 2,3-DPG include androgens, prostaglandin E2, cortisol, and

aldosterone.gg g^ ^ There has also been a report of normal

levels of 2,3-DPG in patients with panhypopituitarism despite

a marked decrease in red cell mass. This is in contrast to

high levels of 2,3-DPG found in red cell mass deficiencies of

other origins.^ The explanation given for this discrepency

was the decreased consumption in these patients.

OTHER DISEASE STATES

Elevated 2,3-DPG levels have been found in many other

disease states. De la Morena (1979) found increased 2,3-DPG

in cancers of breast, ovary, lung, and colon, but nor rectum.^

Higher values increasing with age, were also found in Hodg¬

kin's disease and other lymphomas. The mechanism was thought

to be an increase in anaerobic and aerobic glycolysis by the

turaerous tissues.

Sagone (1973) found unaltered levels of 2,3-DPG in smokers

despite increased hemoglobin concentration and hematocrit. He

postulated that since there was a marked increase in carboxy-

hemoglobin (COHb) and no increase in reduced hemoglobin, that

no alteration of 2,3-DPG would be expected in absence of pH

changes. This is because the binding of DPG to COHb is similar

to that for oxyhemoglobin.^

Astrup (1973) and others have reported an increased 2,3-DPG

and rightward displacement of the ODC associated with cirr¬

hosis of the liver.^ This could be due to the anemia, acid-

base abnormalities, and occaisional desaturation of arterial

blood which are all associated with cirrhosis.^

Several non-disease states have also been associated with

alterations in 2,3-DPG. In normal primigravidae women, elevated

2,3-DPG levels have been observed from the third month of preg¬

nancy. These levels remained elevated untildelivery and then

returned to normal. The stimulus for this elevation was thought

to be the increased desaturation of blood by the fetal circula¬

tion and placenta.

Several studies have looked at 2,3-DPG in relation to ex¬

ercise. Most of these studies seem to show no alteration in

DPG levels during exercise, but a significant mean decrease

during the postexercise period has been seen, which was inversely

correlated with lactate levels.^ This suggested that 2,3-DPG

may not be a compensatory mechanism when the workload requires

a preponderance of anaerobic metabolism promoting lactoacidemia.

One study demonstrated a 10# increase in mean 2,3-DPG concen¬

tration during a six month physical training program. This was

seen concomitantly with a mean 16# increase in the predicted

maximal 02 uptake (V02 max).1Q0

BLOOD STORAGE AND TRANSFUSION

Valtis and Kennedy (195*0 were the first to report that

the P*jq of blood stored in acid citrate dexrose (ACD) decreased

markedly over the first week of storage.^ They also found that

the P^0 of recipient blood was lower after the transfusion with

this blood. Bunn et al. (1969) demonstrated that the increased

02 affinity of stored blood is related to an accompanying de¬

crease in 2,3-DPG concentration.^ ATP was also found to be

lower in stored blood. Since that time, voluminous material

(27)

has accumulated on the subject of blood transfusion and 2,3-

DPG. Several excellent review articles have encapsulated the

present knowledge of this topic.The drop in 2,3-DPG

can be avoided by using frozen or fresh blood, or by adding

inosine to the preservation media. The use of citrate-phosphate-

dextrose (CPD) has been shown to slow the rate of 2,3-DPG de¬

terioration. Following storage in ACD or CPD preservative, the

DPG concentration can be returned to normal within 30 minutes

by incubation of the cells with inosine, pyruvate and inor¬

ganic phosphate,^ la most clinical situations requiring blood

transfusion, the level of 2,3-DPG is probably of little sig¬

nificance, as the level returns to normal within several hours

following transfusion. There are some situations, however, where

studies have shown increased morbidity that can be prevented

by the use of 2,3-DPG enriched (150# of normal) blood.g^

These include massive blood loss, open-heart surgery (use of

extra-corporeal circulation), and septic shock. The use of

2,3-DPG enriched blood has also been shown to be of benefit

in exchange transfusion in infants, replacing fetal hemoglobin

with blood containing adult hemoglobin and above normal levels

of 2,3-DPG.^

(28)

METHODS

Ten volunteer patients were chosen for this study. They

were all nonasthmatic patients who were dyspneic following

minimal exercise and who showed moderate degress of airway

obstruction by standard spirometric test results. They also

exhibited mild-to-moderate arterial hypoxemia at rest or ex¬

ercise. Patients were excluded from the study if they dis¬

played (1) ECG or roentgenographic evidence of pulmonary

hypertension, (2) history of respiratory failure with raised

PaC02 values, (3) systemic hypertension, history of angina

pectoris, arteriosclerotic heart disease, or cerebrovascular

disease.

All patients were examined in Burlington, Vt. (altitude 50 m.)

during the week before ascent. Pulmonary function studies and

blood gas data were collected at rest and exercising on a

calibrated treadmill. To minimize blood drawing, no 2,3-DPG

samples were drawn at sea level as numerous studies have con¬

firmed that there is no immediate change in 2,3-DPG on ascent

to altitude.

Patients were driven to the summit of Mt. Washington, N.H.

(altitude 1920 m.) where they remained for 4 days. Living accomo¬

dations were provided at the summit building. Pood and fluid

intake were not controlled, and the patients were allowed un¬

restricted activity, although few did much in the way of exer¬

cise other than the predetermined protocol. This consisted of

two six minute exercise periods on the treadmill at the same

calibration used at sea level.

(29)

Blood samples were drawn via arterial sticks into heparin¬

ized syringes for blood gases and 2,3-DPG values. Four samples

were collected on each patient: (1) immediately upon ascent, (2)

morning of day 2, (3) morning of day 3, (4) morning of day 4.

Arterial blood gas tensions and pH were analyzed in glass elec-

rodes. Blood samples for 2,3-DPG determinations were measured

for hematocrit using a centrifuge technique, and then spun up to

10,000 RPM for removal of plasma and buffy coat. The red cells

were washed in cold saline and then lysed using .56 M perchloric

acid. The sediment was spun off, again at 10,000 RPM, and the

supernatant was neutralized with 5.62 M K2C0^. The sample was

spun again and the final supernatant separated and frozen at 0°C

for later analysis. This was done at Yale Medical School in the

lab of J.F. Hoffman using a fluorometric assay described by

Keitt (1970).^ This assay involves the stoichiometric conver¬

sion of 2,3-DPG to glyceraldehyde-3-phosphate with measurement

of the resulting change in reduced nicotinamide adenine dinuc¬

leotide (NADH) absorbance or fluorescence.

Control samples were taken from thirteen healthy volunteers

at sea level and four healthy volunteers at altitude on day 4

of the study. These were venous samples, from which the hema¬

tocrit and 2,3-DPG values were derived. Several studies have

shown that the arterial-venous difference in 2,3-DPG is within

statistical error.^

(30)

RESULTS

During the stay at altitude, there were few adverse symp¬

toms exhibited by the patients. A few experienced transient head¬

aches. One patient had some mild pulmonary congestion on day 2

with audible rales on lung exam. Many of the patients complained

of insomnia which was partly attributable to the high winds that

blew on Mt. Washington at night. Many of the patients also com¬

plained of fatigue during the four day stay. There were no com¬

plaints of angina, palpitations, nausea, vomiting, or loss of

appetite. The overall cooperation and morale were excellent.

Table 1 shows the hematocrit and 2,3-DPG levels for the

12 normal volunteers at sea level. The average concentration was

5.55/Mmoles/ml red cells ♦ 1.19. This value compares well with

other established normal values, being approximately equal to

the concentration of hemoglobin (5/^moles/ml red cells). ^

There was a significant correlation between the 2,3-DPG levels

and the hematocrit in these subjects (P<.05). Also shown in

Table 1 are the four normal subject's 2,3-DPG levels after four

days at altitude. The average of 5.13/*moles/ml red cells is

essentially unchanged from the sea level values.

Arterial blood gas data and pH values are given in Table 2.

Baseline values are given in the first column followed by con¬

secutive days at altitude. As stated earlier, day 1 values for

2,3-DPG represent baseline values for the patients. The average

baseline Pa02 of 62.6 mm Hg and PaC02 of 38.3 mm Hg demonstrate

the degree of hypoxemia and ventilatory status of these patients

at sea level. On ascent to altitude there was an immediate stim-

(3D

ulus to hyperventilate resulting in the lowering of the PaC02

significantly (P <.05) to 34.9 mm Hg. The PaC02 was maintained

at 34.2 mm Hg on the fourth day indicating continued hyperven¬

tilation. The Pa02 dropped significantly (P<.05) to 46.8 mm Hg

despite the hyperventilation. By the fourth day however, it had

risen slightly to 51.2 mm Hg (sig. at P<.05).

The hyperventilation resulted in a respiratory alkalosis

which was apparent within an hour after ascent. From baseline

levels averaging 7.43, there was a significant rise (P <.05) to

7.47 within the first hour. By day 4 however, it was almost down

to baseline at 7.44, despite the continued hyperventilation.

This indicated some form of metabolic compensation. The hemato¬

crit showed no significant change from the baseline level of 44.2$.

The 2,3-DPG levels are also shown in Table 2. The average

baseline level of 7.771/tmoles/ml red cells is significantly

elevated from the normal average of 5.55/unioles/ml (P<.005).

Although a significant increase could not be shown using the

Hotelling T square test^a trend can be seen showing a slight

rise of 2,3-DPG at 48 hours (8.14 yzmoles/ml) increasing to

8,53/<.n»oles/ml at 72 hours.

No significant correlations could be shown between 2,3-DPG

and Pa02, PaC02, or hematocrit. However a significant correlation

could be demonstrated between 2,3-DPG and arterial pH (P<.05).

(32)

DISCUSSION

This study offered a good opportunity to evaluate the

role of 2,3-DPG in adapting to hypoxic hypoxia. In order to

maintain homeostasis, the bottom line is energy production

in the form of ATP. For the production of this energy how¬

ever, oxygen is needed. The steps involved in making this

oxygen available to the cells include: (1) ventilating the

lungs with environmental air, (2) extracting the O2 from the

air across the alveolar and capillary walls, (3) picking up

the 02 by the red cells and hemoglobin (where most of the

02 is transported), (4) transporting the 02 to the target

tissues via the vascular system, (5) releasing the 02 from

the hemoglobin, and (6) picking it up by the cells of the

target tissue where it can be used for glycolysis. Any block

along this pathway must be compensated for or homeostasis is

lost.

Altitude places a limitation at the first step, the air

itself. If there are no other abnormalities concurrent with

this hypoxia, the body has adaptive mechanisms at each of the

steps below, to compensate for the diminished environmental 02.

These have been discussed earlier in the section on altitude.

Of these mechanisms, hyperventilation appears to be the most

important immediate compensation.

COPD involves a block in that organ responsible for ex¬

tracting the 02 from the air. The type and extent of the dis¬

ease will dictate the effectiveness of hyperventilation as a

compensatory mechanism. With advanced disease invloving obs-

(33)

truction and diminished surface area, there will actually be

hypoventilation. This places a greater burden on other compen¬

satory factors such as increasing the hemoglobin concentration

or altering the 02-Hb affinity.

The question remains as to the exact role of 2,3-DPG in

adaptation to hypoxic hypoxia. It has been stated many times

that the primary immediate response is hyperventilation which

allows more oxygen to be extracted from the environment. The

alteration of the ODC and thereby the 02 affinity of hemo¬

globin is thought to be less significant than was originally

thought. The reasoning for this assumption was that when ace-

tazolamide was administered prior to ascent, there was no in¬

crease in 2,3-DPG, little alteration of the ODC, and fewer

symptoms of altitude sickness.^ If the major effect of 2,3-DPG

was the stabalization of deoxyhemoglobin and shifting of the

ODC to the right, the significance of this adaptation could

indeed be questioned.

Another way to look at 2,3-DPG however, is not just in

terms of its primary effect on 02-Hb affinity, but also the

secondary effect on the acid-base status of the red cell and

the circulating plasma. As hyperventilation proceeds, the res¬

piratory alkalosis that results has an inhibitory effect on

the ventilatory drive by supressing the central ventilatory

center in the medulla that is sensitive to C02. This alkalosis

also causes a shift to the left of the ODC which makes it more

difficult to unload 02 to the tissues. The body must have a

mechanism for allowing this critical adaptive ventilatory res¬

ponse to continue without being suppressed, and also to balance

(34)

the effect on the ODC. The most effective way of doing this

is to lower the pH of the blood but also within the red cell

itself. The body has a mechanism for doing this on a vascular

level through renal excretion of bicarbonate.

However there is also a mechanism on a cellular level and

this is the Rapoport-Luebering shuttle. This cellular alteration

of pH protects the hemoglobin from the deleterious consequences

of the left shift which was shown by Oski et al. (1971) to be

quite limiting on exercise tolerance.The net result may be

a normal curve or one that is only slightly shifted to the right,

but the significance is important.

The other effect of 2,3-DPG on lowering the pH of the blood,

will work in conjunction with renal excretion of bicarbonate

to allow the ventilatory response to continue unimpeded.

These effects of 2,3-DPG explain why the administration of

acetazolamide works so well by itself, without the necessity of

an increased 2,3-DPG. The acidosis created by the carbonic

anhydrase inhibatory effect does the same thing that 2,3-DPG

does i.e. it protects the red cells and the central ventila¬

tory stimulators from the effects of the respiratory alkalosis

brought on by hyperventilation.

The role of 2,3-DPG can not be thought of as a separate

adaptive mechanism, but rather an integral part of a complex

system that includes pulmonary, hematolgic, renal, and meta¬

bolic components. These all have to balance each other to al¬

low for the optimal homeostasis. I have summarized this hypoxic

response in Table 3. It stresses the importance of pH homeo¬

stasis as well as 02 delivery, since the production of energy

(35)

is as much dependent on enzymatic activity as it is on oxygen

delivery, and pH determines the activity of these metabolic

enzymes.

The patients in this study illustrated the importance of

these factors through their ability to tolerate levels of ar¬

terial hypoxemia that would constitute respiratory failure by

some criteria.^ Their ascent to 1920 m. is roughly the equi¬

valent of a normal resident of Denver, Colorado ascending to

an altitude of 4000-5000 m. This rapid an ascent would produce

symptoms of high altitude sickness in many normal subjects.

Although there were some transient headaches and one incident

of mild pulmonary congestion, with some other mild complaints

of fatigue and insomnia, these were all mild in nature and

seemed to subside after the second day.

The baseline blood gas values for these patients (Pa02

62.6 mm Hg, PaC02 38.3 mm Hg, pH 7.43) demonstrate that des¬

pite their hypoxemia at sea level, there was adequate ability

to compensate through mild hyperventilation. This accounted

for the alight alkalosis, which in turn resulted in the ele¬

vated 2,3-DPG (7.71 julmoles/ml) . This would be expected from

previous observations which have showed the lack of 2,3-DPG

response only in C0PD patients with acidosis.^

On ascent to altitude, these patients demonstrated the

same adaptive mechanisms seen in normal subjects at higher

altitudes. There was an immediate alveolar hyperventilation,

probably the result of the drop in the Pa02 ^rom 62.6 mm Hg

to 46.8 mm. Hg stimulating the peripheral 02 receptors. The

immediate drop in PaC02 from the baseline 38.3 mm Hg to 34.9

(36)

mm Hg was the first evidence of this hyperventilation. This,

in conjunction with the expected increase in deoxyhemoglobin,

resulted in the observed respiratory alkalosis (pH 7.4?).

The rise in 2,3-DPG seen by the third day, was probably

the result of the respiratory alkalosis stimulating glycolysis

as well as the decrease in free 2,3-DPG (secondary to the in¬

creased deoxyhemoglobin) stimulating its own production.

The rise was more delayed than those seen in other studies,

but this could most likely be due to the baseline condition

of this patient population. As they were already producing

greater than normal levels of 2,3-DPG, it would take longer to

increase this level even further. This delay of 48 hours in

achieving a perceivable rise in 2,3-DPG helps explain why Dr.

Graham (1976) did not see a rise in 2,3-DPG in his previous

study,^^ as he only took samples at 16 and 42 hours. The ex¬

planation for his normal baseline values is more difficult to

explain as there was no associated acidosis in that group to

inhibit the production of the phosphate. One can only specu¬

late that differences in collection or assay technique were

responsible for the discrepancy in findings.

The 2,3-DPG rise seen in this study correlates well with

the study done by Astrup and Horth (1970) which showed a 12-

16# increase in 2,3-DPG with a .54# increase in pH in normals

ascending to 3500 m. Our study showed an 11# increase in 2,3-

DPG also with a .54# increase in pH. This indicates that the

COPD patient with an elevated baseline 2,3-DPG, still has the

ability to adjust to an acute hypoxic stress in much the same

manner as a normal person.

(37)

A significant correlation was seen between the arterial

pH and the 2,3-DPG levels in this group of patients. This is

in agreement with most of the studies that have looked at this

relationship, including the original article by Bapoport and

Guest (1939).^ No other correlations were seen in the patient

group, which is also in agreement with other studies.^ This

information further demonstrates the critical interrelation¬

ship between pH and 2,3-DPG during periods of hypoxic stress.

The effect of 2,3-DPG on lowering the pH without inter¬

fering with the ventilatory stimulus, was evident on day 4 of

the study. The PaC02 was still quite low at 3^.3 nun Hg, indi¬

cating that the hyperventilation was being maintained, however

the pH was almost back down to baseline (7.44), showing that

compensation was going on for the respiratory alkalosis of

the first three days. It is impossible to quantify the rela¬

tive importance of 2,3-DPG versus renal excretion in returning

the pH to normal as they both have the same time delay in pro¬

ducing their effect. It would have helped to have urine bicar¬

bonate levels in order to quantify the renal component.

In our normal control group, a significant correlation was

observed between hematocrit and 2,3-DPG. This was an inverse

correlation, i.e. the greater the hematocrit, the lower the

2,3-DPG concentration. This is in agreement with other studies

such as one done by Brewer (1971), where he demonstrated the

same inverse correlation in normal individuals as well as in

anemic patients.^ The same correlation would not be expected in

COPD patients as there are many other variables related to the

lung pathology that would affect the hemoglobin concentration

(38)

and therefore the hematocrit.

It is interesting to note that in the normal group, there

was only one individual who was in exeptional physical condition,

beint a regular long distance runner. This same individual had

the highest normal baseline 2,3-DPG (8.60yu.moles/ml) as well as

the lowest hematocrit (39$). In fact, this 2,3-DPG level was

higher than the average baseline level for the COPD group. Al¬

though this is one isolated case, it is in agreement with a

study done on normal athletes during a six month physical

training period in which a 10$ increase in mean 2,3-DPG was

seen.ioo

Thus it appears that in health as well as in disease, 2,3-

DPG may play an important and integral role in adjusting to hypoxic

hypoxia and maintaining ventilatory and metabolic homeostasis.

(39)

TABLE 1 - 2,3-DPG levels in Normal Controls

Subject - sea level Hct 2,3-DPG (umoles/ml)

CM 40 6.50 DS 49 5.77 CR 49 5.23 JM 45 5.74 KR 4l 6.28 DW 43 4.86 wc 41 5.81 JH 41 5.52 BK 39 8.60 TK 48 3.99 SA 45 4.39 TC 44 4.39 BI 47 5.03

Subject - altitude

RO 41 4.34 WG 45 5.36 RU 47 4.70 GR 47 6.12

TABLE 2 - Average values for COPD Patients

Day 2.3-DPG EH Pa02 PaC02 Hct

Base 7.43 62.6 38.3 44.2

1 7.71 7.47 46.8 34.9 41.2

2 7.69 7.46 48.2 34.2 45.0

3 8.14 7.47 51.7 34.2 44.6

4 8.53 7.44 51.2 34.3 44.5

(40)

TABLE 3 - The Hypoxic Response

l-'icriu 1. Ovyccn delivery to various 11"■:• ^ in art

tuia i. lkne. — -1 i min Hu1 will, a normal 1 l':.„

ili'Mu'i.itnm r tir\r.

F,(.,-he 2 .The < n«M t of arterial IV on <'r. m oxvRcn delivery to various ti-sfles produced by an increase

in 1’-,, to 30.5 mm Hu from 2d 5 mm lie Ai tenovenous

oxygen differences of 13.0. Hi and 1.7 sol lucent for (he

heart, brain and kidney, respect,veb. are taken from Kahn

and Fenri.’n pH is held eon-taut in these calculations. Hemn-

Ulobin concentration and o\\c< n capacitx an lo.O cm, percent

and 20.0 vol | sen cut. Fractional saturation (y) for the normal

and rinht-shifted i nrve is niven b> loc !> ' ( 1-> ‘ i — 11 '(,K

where n — 2 75.

BIBLIOGRAPHY

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