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312 LIBRARY

,ai".

Michigan State
University

 

This is to certify that the

thesis entitled

ERYTHROCYTE OSMOTIC FRAGILITY AND AGING

presented by

Rose Marie Dougherty

has been accepted towards fulfillment

of the requirements for
MASTER OF SCIENCE

degree in

 

 

PATHOLOGY

Major professor

Date 7/?‘2/7?

0-7 639

 

ovmmua FINES ARE 25¢ PER DAY _
PER ITEM

Return to book drop to remove
this checkout from your record.

 

 

 

ERYTHROCYTE OSMOTIC FRAGILITY AND AGING

BY

Rose Marie Dougherty

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1979

ABSTRACT

ERYTHROCYTE OSMOTIC FRAGILITY AND AGING

BY

Rose Marie Dougherty

Erythrocyte osmotic fragility tests were performed on heparin-
ized venous blood samples from 44 hematologically normal subjects
who ranged in age from 18 to 82 years. Osmotic fragility measure—
ments were refined over previous studies of this kind in that
(1) an automatic pipetter was used to simultaneously dispense the
red cell suspensions and NaCl solutions, thus reducing sample varia-
bility; (2) the NaCl solutions were adjusted to a pH of 7.40 without
the use of buffers, which themselves may influence the integrity
of the red cell membrane; and (3) the resultant sigmoid osmotic
fragility curves of percentage of hemolysis versus NaCl osmolality
were treated as linear transforms to permit statistical evaluation
of the results.

The NaCl osmolalities (mOsm/kg) used were 300, 130, 125, 120,
115, 110, 105, and O (distilled, deionized water). The hemoglobin
content of the supernatant fluids was assayed spectrophotometrically
to provide a measure of hemolysis, and controls for hemolysis due

to mechanical damage during sample processing were employed.

Rose Marie Dougherty

The NaCl osmolality at which 50% hemolysis occurred (H50) was
determined for each subject from the linear regression of trans—
formed hemolysis against osmolality and plotted against the subject's
age in years. The slopes of the linearized osmotic fragility curves
were also determined and plotted against subject age.

Statistically significant (p<0.05) positive correlation
(r = +0.35) was demonstrated between erythrocyte osmotic fragility
and age in years. Fifty percent hemolysis occurred at osmolalities
varying from 116 mOsm/kg to 136 mOsm/kg.

Red blood cell variability, as expressed by the slopes of the
osmotic fragility curves, also increased significantly with age
(r = +0.50, p<0.01).

These results are discussed with reference to (1) factors
known to influence erythrocyte osmotic fragility and (2) various

theories of aging.

TO

ROSEMARY D. WOOD: my mother
DR. G. MARIAN KINGET

ELWOOD N. WHITBECK

ii

ACKNOWLEDGEMENTS

I wish to acknowledge the assistance of the members of my
graduate advisory committee: Dr. Anthony J. Bowdler of the
Department of Medicine, Mrs. Martha T. Thomas and Dr. John F.
Dunkel of the Department of Pathology; Dr. Robert W. Leader,
Chairman of the Department of Pathology; Dr. Garson H. Tishkoff
of the American Red Cross; the staff and senior citizens at the
Senior Citizens' Centers of Lansing and Ann Arbor, Michigan;

Noelle Bowdler; Louise Mueller and April A. Whitbeck.

iii

LITERATURE REVIEW . . . .

Aging. . . . . . .
Definition.

TABLE OF CONTENTS

Criteria of Age Changes .

Categories of Age Changes

Molecular Stochastic DisrUptions.
Theories of Aging . .

Biological Theories.
Genetic Theories.

Nongenetic Cellular

Single Organ System

Aging and Physiological Controls.
Endocrine Control System .

Nervous Control Mechanisms

Summary of Theories of Aging.
Effects of Aging in Body Systems.
A Model for Studying Aging:

Osmotic Fragility.

The Red Bl

Theories.
Physiological Theories of Aging. . .

Hypotheses. .

Clinical Significance of Osmotic Fragility
Measurements. . . .
The Time Course of Osmotic Hemolysis.
Red Blood Cell Membrane Components and Sug-
gested Models . . .

RBC Shape:Deformability:Surface

Relationship. . . .

MATERIALS AND METHODS . .

RESULTS . . . . . . . . .

DISCUSSION. . . . . . . .

LIST OF REFERENCES. . . .

iv

Area/Volume

ood Cell

Page

’..:

56

67

72

75

81

84

LIST OF TABLES

Table Page
1 Origins, Levels, and Categories of Age Changes . . . . . 8
2 Stochastic Causes of Aging . . . . . . . . . . . . . . . 9
3 Genetic Causes of Aging. . . . . . . . . . . . . . . . . 10

4 Effects of Aging in Body Systems . . . . . . . . . . . . 36

5 Comparison of Age with H50 and Slope of Osmotic
Fragility Curve. . . . . . . . . . . . . . . . . . .

LIST OF FIGURES
Figure Page

1 Effect of Ionic Composition of Suspending Medium on
Osmotic Fragility of Human Red Blood Cells . . . . . . . 48

2 Normal and Abnormal Osmotic Fragility Curves . . . . . . 49
3 Osmotic Fragility in Disease States. . . . . . . . . . . 50
4 Behavior of Normal Cell in Hypertonic and Hypotonic

Media. 0 O C O O O O O O O O O O I O O O O O O O O O O O 54

5 Diagram of the Sequence of Events in the Process of
Osmotic Hemolysis. . . . . . . . . . . . . . . . . . . . 55

6 H50 versus Age . . . . . . . . . . . . . . . . . . . . . 77

7 Slope of Osmotic Fragility Curve versus Age. . . . . . . 79

vi

L ITERATURE REVI EW

Aging

Definition

The terms "aging" and "senescence" are familiar, but unfor-
tunately are rather imprecise (103): there appears to be no general
agreement either in general usage or among gerontologists about how
they should be used. "Senescent" and "senescence" are used when
referring to changes which occur during the period of obvious func—
tional decline in the later years of an animal's life-span. Some
gerontologists use the term "aging" for the same processes and
period. Others use it in a much more general way, with "aging"
meaning simply "growing older", and aging changes being any changes
related to age, regardless of when in the life-span they occur.
There are advantages in using aging in this general way, but it is
difficult to do so, because in common language aging implies some-
thing more than simply getting older. Most biologists have accepted
these connotations and think of aging as occurring only after
maturity has been reached. It is evident from some of the defini-
tions given below that the terms "aging" and "senescence" are
frequently used interchangeably.

There have been many formal definitions of "aging" and

"senescence." Medawar (120) defines "senescence" as follows:

2

"Senescence. . .may be defined as that change of the
bodily faculties and sensibilities and energies which
accompanies aging, and which renders the individual pro—
gressively more likely to die from accidental causes of
random incidence. Strictly speaking, the word 'acci—
dental' is redundant, for all deaths are in some degree
accidental. No death is wholly 'natural'; no one dies
merely of the burden of the years."

Comfort, whose View is similar, has stated his definition in the
following terms (33):

"Senescence is a deteriorative process. What is being
measured, when we measure it, is a decrease in viability
and an increase in vulnerability. Other definitions

are possible but they tend to ignore the raison d'étre
of human and scientific concern with age processes.
Senescence shows itself as an increasing probability

of death with increasing chronological age. The study
of senescence is the study of the group of processes,
different in different organisms, which lead to this
increase in vulnerability."

Shock (170) defines aging thus: "Aging is the sum total of changes
during an individual's life span, which are common to all members
of his species or strain." Finch and Hayflick (59) state:

"Aging will be considered as any time-dependent change
which occurs after maturity of size, form or function
is reached and which is distinct from daily, seasonal,
and other biological rhythms."

According to Strehler (187),

"Senescence is defined as the changes which occur (1)
generally in the postreproductive period and (2) which
result in a decreased survival capacity on the part of
the individual organism. It must be clear from this
definition that different evolutionary lines might very
well decline in their survival capacities for entirely
different immediate reasons. It may also be, however,
that there are one or more dominant mechanisms of
aging, common to all higher forms of life. Such common
pathways of aging might well reflect similarities in
the developmental or evolutionary history of various
kinds of animals and plants."

Criteria of Age Changes

It should be emphasized that aging is a property of the organism
and not of its environment. Age changes due to the basic aging
process should occur even in the most propitious environments in
order to be considered as true age changes. It is reasonable that
changes ascribable to an aging process should occur in all indi-
viduals within a species rather than in occasional instances; for
in occasional instances the age-associated changes might be conse-
quences of accidents or of unusual genotypes. A primary objective
of aging research is the description of the nature of the basic
underlying changes in structure and function which lead to the
gradual dysfunction and, finally, to the death of the individual
organism. Much of the confusion which has surrounded the biological
concept of aging in the past is due to a failure to establish a
usable definition of aging and objective criteria through which the
results of specific research on animals at several different ages
can be evaluated.

Strehler (186) has suggested four criteria which are in essence
general, arbitrary definitions of what constitutes or is meant by
biological aging, as tentative guideposts in the search for basic
mechanisms of the aging process. It was proposed that the criteria
which any age-associated change should meet before being considered
a part of any basic aging process are four in number and include
the following concepts: (1) universality, (2) intrinsicality,

(3) progressiveness, and (4) deleteriousness.
The basic postulate underlying these criteria is that there

exist gradual changes in the structure of organisms which are not

4

due to preventable diseases or other gross accidents and which
eventually lead to the increased probability of death of the indi-
vidual as he grows older with the passage of time. Such changes

are consistent with the implied criteria stated by Medawar (120) and
Comfort (33). Strehler chose these criteria for the following
reasons.

(1) Universality - If a given observed phenomenon is part
of a natural aging process, it should occur in all older members of
the species. This criterion eliminates many specific, hereditary
aberrations as well as diseases which are dependent on a given
environment. True, underlying basic aging processes will occur in
all older individuals of a species whether they express themselves
as recognizable disease lesions or not.

(2) Intrinsicality - Like universality, this criterion is
designed to distinguish aging from age-correlated changes which are
due to the operation of factors outside of the organism. It is
included as an additional criterion because certain age changes may
be universal but still be a consequence of environmental effects on
living systems.

(3) Progressiveness - Aging is usually considered to be a
process rather than a sudden event. It follows, therefore, that
its onset should be a gradual and cumulative occurrence. Predis-
posing factors are very likely a part of the aging process, and it
is for this reason that the study of aging mechanisms is a key to
a greatly neglected area of biomedical research. Generally processes
are gradual in their occurrence because they are due to changes in
small subunits of structure, e.g., in cells or even in subcellular

organelles or molecules. The individual events at the molecular

5
level leading to dysfunction at the cellular level and ultimately
reaching up to the behavior of the organism do occur in a very short
time, but the large number of events necessary to produce gross
change tends to smooth out the statistical fluctuations occurring
at the micro level and to give the processes an appearance of
smooth-flowing uniformity.

(4) Deleteriousness - This final criterion is based on the
fact that the most characteristic change occurring during the aging
process is the decline in functional capacity which is reflected in
an increased mortality rate. Universal, intrinsic, and gradual
changes must also contribute to the increased probability of death
if they are to be considered as part of the basic biological aging
process. Some age-correlated changes which meet the first three
criteria may thus be eliminated because they may simply be the final
stages in adaptive processes and actually result in an improvement
in the organism's survival capacity. As such, they would be classed
as part of the developmental process rather than of aging. Not
all developmental processes, however, are necessarily eliminated,
for certain of them may become deadaptive in their final or extreme
expression. Thus, while certain aspects of development may indeed
have great relevance to the basic biology of aging, it is equally
apparent that this is only true of those developmental changes
whose ultimate expressions in time decrease the organism's capacity
to survive or which constitute a mixture of adaptive and deadaptive

effects.

Categories of Age Changes

 

It is Possible to classify the kinds of changes leading to the
decreased functional capacity of living systems. Generally, they
are divisible into two general categories: those which are the
result of the normal aging process and those which are the result
of environmental factors. Medawar (120) discussed this division into
the two kinds of causes in the following terms:

"Senescence, as it is measured by increase of vulnera-

bility, or the likelihood of an individual's dying, is

therefore of at least twofold origin. There is (a) the

innate or ingrained senescence, which is, in a general

sense, developmental or the effect of nature; and (b) the

senescence comprised of the accumulated sum of the

events of recurrent stress or injury or infection. The

latter is environmental in origin and thus, in a para-

doxically technical sense, the effect of 'nurture.'" (180)

Strehler (187) uses somewhat different terminology. He calls
those processes which are an intrinsic portion of the life history
of a species determinate processes; those which are the results of
unpredictable environmental influences he terms ancillary or
stochastic processes. Determinate (genetic) processes are those
which occur with certainty in all animals of a given species regardless
of their environment and experience. This definition includes the
definition of normal aging and such processes which are considered
to be a consequence of any combination of general features of the
physical world [Tables 1 and 3 (187)]. Ancillary (stochastic) processes
lead to the decreased vitality of old organisms and are considered
as consequences of gross accidental events which are not a result of
the structural organization of an organism per se. That is, ancil-
lary processes are a consequence of deleterious agents in the environ-

ment and are generally subject to modification, whereas determinate

processes by their very nature are an inherent part of the organism.

7
Tables 1, 2 and 3 illustrate the origins and relationships of
the various categories of age changes (184,186,187). These tables
illustrate that the change of most general interest, the decreased
survival capacity of individual animals with age, is based on a
hierarchy of events proceeding from elementary interactions of mole-
cules at the cellular and subcellular level to the very complex
interactions of the whole organisms, societies, and their environ-
ments. Thus, it becomes evident that each of the higher levels of
function or organization is dependent on less complex phenomena,

structures, or processes.

Molecular Stochastic Disruptions

 

Stochastic processes at the micro level, that is, those involv-
ing the reactions of small molecules or aggregates of molecules, may
also result in deleterious changes with the passage of time. The
rate of disruptive reactions is determined by two fundamental
factors. In order for a reaction to occur the atoms making up the
reactive molecules must become appropriately sited and the environ-
ment must furnish sufficient energy to overcome the activation
barrier to the reaction. Any structure will have a certain inherent
instability if it is subject to local energy fluctuations that
exceed the bond energies maintaining structure. The sources of
those potentially disruptive energy fluxes arise only from three
types of events. These are (1) the redistribution of heat energy,
(2) the absorption of radiant energy, or (3) the occurrence of
chemical or nuclear reactions which result in sudden local concen-
trations of heat energy. A brief description of each type of event

follows.

TABLE 1.

Origins. Levels. and Categories of Age Chenges‘

 

Level of
organization

Manifestation

Measure

 

Population

Individual

Integrated function
(organ systems)

Tissue

Cell

Subcellular ele-
ments: organelles
and molecules

Increased probability of death
(aging) derives from

Decreased adaptation which, in
turn, reflects

Decreased ability to perform
specific functions resulting from

Changes in tissues and/ or

Death of or changes In cells conse-
quent to

Changes in subcellular elements
or extracellular environments
(e.g., nuclei, DNA, polysomes,

Mortality rates

General performance,
morbidity
Physiological tests

Histochemlcal, electron
microscopic and chem-
Ical measurements

Histology, histopathology,
cell physiology

Histochemistry, immuno-
chemistry. biochemistry
molecular biology

membranes, mitochondria.
Golgi, lysosomes, collagen,
basement membranes. solutes.
stroma) which, in turn, arise
from

Changes in synthetic rates. rates
of utilization. diffusion. trans-
port, storage (formation of preci-
pitates. exhaustion of stored ma-
terials). all of which are the
results of failure of design (de-
ficiencies in genetic properties)
to take Into account the delete-
rlous effects of

Y
I 7

Accidents Genetics and development
(stochastic processes) (genetic processes)

 

Inadequacy of Omission of design Errors in design Contradictions in

design (Inability to (presence of design

(inability to repair effects of slow. but harm- (opposing effects
repair and/or microaccidents) full side-reac- of same design
avoid gross tions. catalyzed feature at differ-
damage) and uncatalyzed) ent times. In dif-

ferent cells, In
different environ-
ments)

 

' Reprinted from (187).

TABLE 2.

Stochastic Causes of Aging'

 

A. Macroaccidents

 

I

Physical agents Chemical agents

1
r n l I i 1
Bacterial Heat. cold, Radiation Nutrition Anoxia Toxic
infections cuts. bruises. substances

breaks. burns.

freezing.

wear and

tear

 

 

B. Microaccidents (such as mutations. dena-
turation) which result from disruptions
of molecular structure because of locally
high concentrations of energy arising from

I

 

 

1
Physical agents Chemical agents
F
Statistical local Absorption of Heat liberated locally as a result of
heat fluctuations radiant energy or energetic chemical reactions
high-energy
particles

 

'Reprinted from (187).

10

TABLE 3.

Genetic Causes of Aging'

 

A. Changes in Cellular Properties
1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

f l
Physical Chemical
1 I
F l l V I
Size Viscosuy Differentiation Size limitations Accumulation
(Surface/ of Side
volume) products 0r
deSchhon
of cellular
constituents
r fl
Loss Of Chem'ca' Growth cessa, EUOIS In
plasticny overspe- tion catalysis
Cialization Loss of
ability to
replace Uncatalyzed
parts reactions
i
Loss of ability to Loss of ability
replace subcellular to differenti-
elements ate into
replacement
cells
due I to
i l 1
Loss of Loss of Loss of
code readout conditions
device for
readout
8. Supracellular Changes
l
Physical interaction Chemical interaction
t
r 1
Geometry of Amount or kind
cellular of extracellular
relationships substances
4 —- Cross-feedin
F fl 0
Morpho- Cell loss Viscosity Physical '— Competition '0'
genesis suppon nutrients

Cell movement Diffusion barriers

(random)

L Loss of immune
tolerance

 

— Cross-toxicitity

 

'Fleprinted from ( 187).

11

(a) Heat - Random local fluctuations in energy about some
average value will occasionally result in local energy concentrations
sufficiently high to permit the alteration of structures in the
vicinity.

(b) Radiation - A second path by which molecules or
polymolecular structures may become unstable is through the locali-
zation of energy as a result of the absorption of radiant energy.
For a short time after the absorption of a quantum of light an
absorbing system exists in a so-called excited state. Energy stored
in this temporary form is shortly dissipated in one of four ways:

(1) through the reemission of light in the form of fluorescence or
phosphorescence; (2) through a chemical reaction between the
unstable (excited state) and some other molecule in the environment;
(3) by the breaking of a bond in the excited molecule before the
excitation energy can be dissipated; or (4) by the transfer of the
energy to some other molecule, resulting either in reaction or in
further redistribution of the excitation energy as heat energy. The
last three possibilities are capable of promoting disruptive chemical
reactions (61,187).

(c) Chemical reaction - The third major source of energy
capable of producing molecular disruption is the liberation of a
locally high concentration of thermal energy as a result of the
occurrence of chemical reactions. The energy available locally at
the end of a reaction may be considerably greater than the free
energy liberated by a reaction. This is a consequence of the fact
that a molecule must absorb thermal energy before it can react. A
catalyst will increase the total rate of reaction, and it will

decrease the amount of the total energy liberated at the instant

12

of reaction by the amount that it decreases the activation energy.
Thus, even though an intermediate in a reaction sequence might be
transformed noncatalytically into its product, there is an advantage,
so far as the long-term survival of the catalytic system is con-
cerned, to inserting a catalyst which will decrease the energy
liberated locally at the end of the reaction. Since catalyzed
reactions are characterized by lower activation energies, it also
follows that the energy possessed by the product at the moment of
its formation will be less for catalyzed reactions than for uncata—
lyzed ones. Enzymes thus function to decrease the probability of
damage per reaction as well as to speed up the rate of reaction.

It is not possible, at the present time, to evaluate the extent to
which uncatalyzed chemical reactions contribute to the overall
decline in function characteristic of senescence. However, it is
known that certain kinds of age change can be accelerated either by
raising the temperature or by exposing tissue to visible, ultra-
violet, or ionizing radiation (61,187).

(d) The sources of gene-determined damage (aging) - In addition
to the destructive effects of macroaccidents (Table 2) and the slow
degradation of living systems as a result of chemical reactions not
governed by genetically controlled catalysts, there is a third
broad category of age changes that is a part of the aging process
(Table 3). This consists of the changes in cells and in multi-
cellular structures, tissues, and organs, which occur as a result of
the operation of genetically determined factors. These changes are

either the direct or indirect result of the presence of catalysts.

l3

Aging as non-steady state - In a general sense, the chemical
constitution of cells will change if the rate at which each substance
appears is not equal to the rate of its disappearance due to trans-
port out of the cell or through its utilization. In this context,
aging may be viewed as change in constitution due to the inequality
of the rates of appearance and disappearance of specific chemical
structures, changes which are not directed toward the production of
a more effectively adapted organism.

Sources of non-steady state - At the cellular level, those
changes which are genetic in origin are detailed in Table 3. These
changes are produced by changes in one or more of the following
factors so as to yield a steady state which is incompatible with
continued survival: (a) rate of synthesis, (b) rate of utilization,
(c) rate of transport, (d) rate of storage or depletion of stores,
and (e) rate of entropy change, rearrangement, disorganization, or
organization.

The various possible theories of the mechanism of aging are
expressions of the above hypothesis in various degrees of refinement
and sophistication.

(a) Surface/Volume - Of particular importance in determining
the steady state are such factors as cell surface-to—volume ratio,
cytoplasmic viscosity, and the geometric arrangement of cells.

These factors would appear to be extremely important in determining
the ingress and egress of diffusible constituents. Intracellular
chemical changes also include by-products of catalyzed or uncatalyzed
reactions as well as continuations or extensions of the normal

differentiation process.

14

(b) Differentiation - The hypothesis of differentiation as a
prime causative factor in cellular aging has received support from
biologists. The possibility that senescence may actually be a
consequence of cellular overdifferentiation has been explored by
Strehler (183). Pearl (40) particularly favored the teleological
concept that senescence is the price which organisms pay for the
luxury of differentiation.

(c) Growth cessation - Comfort (33) has discussed in detail
the fact that many organisms attain a finite adult size, and the
consequences of this limitation, as regards repair processes and the
maintenance of chemical constancy.

(d) Damage to genome - During recent years a number of
theories (40) have been suggested which are based on the idea of a
deterioration of the genetic code or information content during the
aging of organisms. Such losses of code would be extremely diffi-
cult to repair once they occurred within a cell. However, they
might be circumvented by simply discarding damaged cells as they
are formed.

(e) Nongenetic damage - Another category of chemical changes
in cellular prOperties which are directly attributable to the
presence of genetically controlled catalysts is the occurrence, at
low rates, of improbable chemical reactions between components. Even
though such "side reactions" may be extremely slow compared to
catalyzed reactions, their cumulative effects are probably very large.
Since catalysts usually change the steady-state concentrations of
intermediates, they will indirectly promote the occurrence of
uncatalyzed reactions purely through the Operation of the law of

mass action. Such reactions might either involve the destruction

15

or alteration of cytoplasmic components or they might produce
insoluble or toxic products which cannot be removed efficiently from
the cells. "Unprogrammed" reactions might also occur because of
errors in catalysis, that is, because of nonabsolute specificity of
catalysts' activities or because of the occurrence of non-catalyzed
reactions between highly reactive intermediates produced metaboli-
cally. Such changes are to be expected in practically any organism
whether it be single-celled or composed of many cells. But, in addi-
tion, multicellular organisms, which possess certain advantages as
a consequence of their multicellular specialized organization, also
acquire certain disadvantageous instabilities as by-products of
multicellularity. Such instabilities result either from changes in
chemical or in physical relationships (interactions) between cells.
Physical and chemical interactions between cells are moderated by
the nature of the nonliving matter between cells, as well by the
cells' geometrical relationships. The geometrical factors which
result in coherent cell function will be altered by changes in rela-
tive cell size, by the elimination of certain cells through death,
by the random movement of cells with relation to each other, or
through a postdevelopmental continuation of various morphogenetic
processes. Of great potential importance in regulating the inter—
actions between cells are the gels, membranes, fibers, and particles
which make up intercellular substances. Because of their location
between living cells, such structures may well influence the rate
of diffusion of metabolites and waste products into and out of cells
and between the cells making up a tissue, organ system, or organism.

(f) Altered cell interactions - Chemical interactions between

the cells of a multicellular organism largely depend on the kind and

16
concentration of diffusible substances passing from one cell to
another (3,16). These interactions involve inhibition or stimula-
tion of cellular function by cross-feeding. They involve competition
for nutrients. They involve reaction to toxic or regulatory sub—
stances produced by one cell and utilized or detoxified by another.
They may involve autoimmune reactions between different cells of
the same organism.

It is not yet possible to decide which of these many alterna-
tives are the major contributing factors to the normal aging process.
However, it is clear that practically all these possibilities must
take place at some rate during aging. Therefore, the major general
scientific objective of aging research is the assessment of the
relative contributions of each of these processes and the definition

of detailed underlying mechanisms (187).

Theories of Aging

 

Theories play two major roles in science. The first is to
systematize a range of discrete observations into a single conceptual
pattern that will make possible a smaller number of generalizations.
The second role of theories is to develop rational explanations for
the mechanisms which will explain how and why a general phenomenon
occurs. Theories are important to a science since they will have a
marked influence on the nature of experiments that will be performed
(168). The following will outline in general terms current theories
which attempt to explain the biological and physiological changes

which occur with aging.

l7

Biological Theories. Ultimately all biological theories of

 

aging have a genetic basis. The fundamental assumption is that the
life-span of any animal species is ultimately determined by a program
which resides in the genes of the species and ultimately in the
information contained in the DNA molecules of the gene. One can
divide these theories into two categories. In the first category
the primary focus of the theories is on the way that information is
transferred from DNA molecules through a variety of steps to the
ultimate formation of proteins (enzymes) which are critical for the
continued function of specific cells in the animal. The second
category of theories, which are termed nongenetic, places emphasis
on the changes that may occur with the passage of time in the cellu-
lar proteins after they have been formed. These theories place
primary emphasis on changes that occur at the cellular and tissue
level.

Physiological theories, on the other hand, place major emphasis
on the interaction between cells, tissues and organ systems in
attempting to explain age changes in overall response of the intact
animal. Physiologists are aware of the fact that the ultimate
explanation of the phenomena with which they are concerned may well
be at the cellular level. However, their emphasis lies in investi—
gating the interrelationships between the performance of different
organ systems in maintaining the overall performance of the animal

(171) .

Genetic Theories

 

General genetic theory. There are marked differences

in average age of death or life-span between different species of

18
animals (33). It is therefore probable that there is some genetic
program which sets the upper limit of life-spans. Maynard Smith's
(117) experiments have shown that inbred strains of a species
usually have shorter life-spans than the original wild strain. It
has therefore been concluded that long life is related to many dif-
ferent genes and a large gene pool is advantageous for long life.
In humans, females live about eight years longer on the average than
males. The same sex difference in favor of females also appears in
a number of other species. Since the primary factor that determines
sex in the animal is the presence of XY chromosomes in males and
XX chromosomes in females, it has been postulated that the sex dif—
ference in longevity is due to the greater amount of X chromatin in
females. Support for theories of preprogrammed aging has also been
provided by studies which indicate that at least certain cells of the
body when maintained in tissue cultures are capable of only a limited
number of cell divisions (76). Furthermore, cells taken from old
animals can undergo fewer cell divisions in contrast to cell divisions
possible in cells taken from young animals. It is important to note
that although these studies involving cell division provide a poten-
tial model for studying aging of cells under defined conditions,
they may not reflect the aging characteristics of cells within the
intact animal.

The present evidence indicates that (1) there may be a genetic
program which sets the upper limits of life-span in a species, (2)
that there is some familial characteristic that influences differ-
ences in life-span among individuals of a single species, and (3) the
expression of the basic genetic program can be altered by environ-

mental factors (168).

19

Cellular genetic theories - DNA damage. Identification

 

of the DNA molecules as the ultimate source of all information
required by the cell for the production of the essential proteins
required to maintain life has led to the theory that death occurs in
the cell because of damage to the cellular DNA. The information for
formation of specific enzymes and other proteins depends on the
precise molecular structure of different parts of the very large DNA
molecules (197). Breaks in the chain of the DNA molecule or exchange
of positions of parts of the chain would alter the message and result
in the inability of the cell to manufacture essential enzymes. This

could result ultimately in death of the cell (168).

Somatic mutation - radiation. Exposure to nonlethal
doses of radiation substantially shortens life-span. These experi-
mental observations led to the develoPment of the somatic mutation
theory of aging (40). According to this theory, exposure to radiation
accelerates aging. It was postulated that even in the absence of
radiation exposure some cells undergo mutations, most of which are
deleterious.

The "hit theory of aging", proposed by Szilard, is a variation
of the somatic theory. Szilard postulated that over the lifetime of
the animal "hits" occur at random which render a gene or its DNA
molecules ineffective in providing the information necessary for
the formation of critical proteins in the cell. According to
Szilard's conception, a cell becomes ineffective when the two similar
chromosomes of a pair have each suffered a "hit" in the same place or
when one of the pair has been "hit" and the other carries an inherited

"fault" in this locus. A "fault" is regarded as a recessive gene

20

which renders the cell inviable or incapable of performing necessary
cell functions in the adult organism only if it occurs in both genes
of a pair. Death of the animal occurs when some critical fraction
of the cells initially present is rendered ineffective.

The somatic mutation theory has been abandoned for three reasons.
In the first place, the effects of radiation and mutations occur
primarily in dividing cells, such as red cell precursors. Effects
are seen after cell division when mutations occur. In contrast, age
effects are primarily centered in cell lines that no longer divide
in the adult animal, as for example neurons and muscle cells. The
second reason for abandonment of the theory is that calculations on
the rate at which mutations occur in the absence of exposure to
radiation indicate that the number of cells which are apt to go
through mutations in mammals is too small to account for overall age
changes (118). In the third place, recent studies have shown that
most cells contain mechanisms for the repair of damaged DNA molecules
(210). Consequently, it is doubtful whether damage to the DNA mole-

cule itself plays much of a role in cellular senescence.

Error theogy of aging - cellular. In any cell the

 

information stored in the genetic code, as determined by the structure
of the DNA molecule, must be transcribed and translated in various
steps until the final protein or enzyme molecules required by the

cell finally appear. The actual assembly of appropriate amino acids
to form specific proteins occurs at another location in the cell,
namely, on the ribosome. In order for this to occur, information from
DNA must be transferred to an appropriate location on the ribosome.

This transfer occurs by the assembly of RNA molecules at specific points

21
on the DNA molecule which then migrate to the ribosomes. Protein
formed depends upon what part of the very large DNA molecule is
decoded with the formation of an apprOpriate mRNA.

This general theory of biology has been transferred to geron-
tology as the error theory of aging (121). According to this theory,
aging and death of the cell are the result of errors which may occur
at any step in this sequence of information transfer resulting in the
formation of a protein or enzyme which is not an exact copy and
therefore is unable to carry out its function properly. The error
may be simply the incorporation of the wrong amino acid in the protein
or a slight error in the normal sequence.

Unlike DNA molecules, which are highly stable and are maintained
throughout the life span of a cell, RNA molecules have a half-life
of only eight or nine days. Consequently, new RNA molecules are
being formed continuously to provide replacement of enzyme molecules.
Orgel (142) has argued that the ability of a cell to produce func-
tional proteins, such as enzymes, depends not only on the correct
genetic information contained in the DNA molecules, but also on the
competence of the protein synthetic apparatus, i.e., the enzymes
necessary for the translation and transcription of the information
from the DNA to the final protein product. According to this theory,
aging is the result of errors in enzymes involved in translation and
transcription. Orgel claims that although the initial frequency of
errors in a cell may be low, in the absence of cell division as in
an adult animal there would be an exponential increase in the frequency
of errors in the proteins synthesized with the passage of time which

would lead to an "error catastrophe" and the death of the cell.

22
Although the error hypothesis is an attractive one, it has a
number of limitations. In the first place, there are many steps
between the information source (DNA) and the final protein product
and errors can conceivably occur at many points. Thus, the theory
is relatively non-specific (78,154). Furthermore, the details of
these transfer processes are still unknown so that the nature of

the error cannot be specified (168).

Nongenetic Cellular Theories. At the cellular level non-
genetic theories of aging presume that, with the passage of time,
changes take place in molecules and structural elements of cells

which impair their effectiveness.

Wear and tear theory. This theory is based on the

 

assumption that living organisms behave like machines. That is, from
repeated use parts wear out, defects occur, and the machinery finally
comes to a halt. However, the man-machine analogy is not a very
good one. Unlike machines which cannot repair themselves, living
organisms have developed many mechanisms which permit self-repair.
In some tissues, such as skin, the lining of the gastrointestinal
tract, and red blood cells, new cells are continuously formed to
replace old ones so that the effects of aging are minimal. In other
cell systems, such as nerve cells and muscle cells, which no longer
divide in the adult, repair mechanisms are present and there is a
constant turnover among many of the molecules within a cell.
Increasing the environmental temperature shortens the life-span
of cold-blooded animals. At lower environmental temperatures life-
span is lengthened. Since a rise in temperature increases metabolic

rate in cold-blooded animals, these experiments have been interpreted

23
as evidence to support the "wear and tear" or "rate of living"
hypothesis about the mechanism of aging. In contrast to cold-
blooded animals which show a reduction in metabolism as environmental
temperature is lowered, warm-blooded animals increase their metabolism
when exposed to cold temperature. These experiments were also inter-
preted as support for the "wear and tear" theory. However, all of
the experiments on the effects of temperature on life-span can be
interpreted as evidence to support any theory of aging which involves
chemical reactions, as for example, the cross-linking theory, or
even error theories, since the rate of all chemical reactions is

influenced by changes in temperature (168).

Deprivation theories - diffusion. Other nongenetic

 

theories assume that aging is due to inadequate delivery of essential
nutrients and oxygen to the cells of the body. These theories point
to the increasing prevalence of atherosclerosis with advancing age
and the well known effects of vascular degeneration on cellular and
tissue functions for their primary support. However, when the blood
supply is shut off from a tissue by vascular disease, all of the cells
in the area supplied die. This is in sharp contrast to the more or
less chance distribution of cells which drOp out or disappear from

a tissue or organ with advancing age. Furthermore, there is no
evidence for a systematic reduction in oxygen content of the blood
with advancing age. Fasting blood glucose levels (174) and amounts
of other nutrients present in the blood do not fall with advancing
age. It seems plausible that even in advanced old age adequate
supplies of oxygen and nutrients are available to cells and tissues
of the body except under circumstances associated with diseases of

the blood vessels.

24
Carpenter (28), however, has speculated that with advancing age,
changes occur in cell membranes which limit the diffusion of oxygen
and other nutrients from the blood to the inside of the living cell.
However, there is little experimental data which would indicate that
this is the case. It is, therefore, extremely doubtful that senescence

can be ascribed to cellular deprivation of essential materials (168).

Accumulation theories. Cellular aging and senescence

 

have also been ascribed to the accumulation of deleterious substances
in the aging animals. A number of investigators have reported that
highly insoluble lipofuscin particles accumulate in cells of various
tissues, such as neurons and muscle fibers of the heart, with advancing
age (185). It has been argued that the presence of these insoluble
particles interferes with the normal cellular metabolism and ulti-
mately results in cell death. However, at present no evidence of
impaired physiological function associated with the presence of these
age pigments has been provided. The accumulation of these large
molecules has been ascribed by Carpenter (29) to impair diffusing
capacity of cell membranes with advancing age. Since the age pigments
consist of varying prOportions of lipids and protein molecules, it

has been assumed that they represent an accumulation of non-
metabolized molecules. The accumulation theory is a reasonable one

to pursue, but the toxic substances, present in old cells but absent
in young ones, must be isolated and identified. Furthermore, physio-
logical experiments are required to show that the presence of age

pigments impairs the function of the cell (168).

Free radical theory. Harman (74) has pr0posed that

 

aging and cell death stem from the damaging effects of the formation

25

of free radicals. Free radicals are chemical compounds which contain
oxygen in a highly activated state. Hence, they are very unstable
and tend to react quickly with other molecules in their vicinity.

As a result of these reactions enzymes and other proteins may be
altered in structure and function. The rate of formation of free
radicals is accelerated by radiation and inhibited by the presence of
antioxidants. Harman offers as evidence for his theory the increase
in average life-span observed in mice which have been fed antioxi-
dants. Unfortunately, the eXperiments reported by Harman have a

number of limitations so that the theory is still unproved (168).

Cross-linkinggtheories. Many macromolecules of bio-

 

logical importance develop, with the passage of time, cross-linkages
or bonds either between component parts of the same molecule or
between molecules themselves. The formation of these cross-links
alters the physical and chemical properties of the molecules involved
so that they no longer function the same way as before. Since these
cross-links are very stable they are apt to accumulate with the
passage of time. The cross-linking theory first proposed by
Bjorksten (17) holds that the accumulation of these cross-link
molecules is a primary cause of aging.

Most of the experimental evidence supporting this theory has
been derived from studies on extracellular proteins, namely, elastin
and collagen (17,194). It is well established that with aging, cross-
links develop between the two intertwined strands of the collagen
molecule and between adjacent strands. Formation of these cross-
links results in the well known loss of elasticity with advancing age

in many tissues of the body, as for example, the skin and blood

26
vessels. It is also known that the body contains many substances,
primarily aldehydes, which are effective in accelerating the rate
of formation of cross—links. From these observed facts with respect
to extracellular protein, it has been assumed that similar changes
occur in intracellular proteins such as enzymes and also in the DNA
molecule (194). Although cross-linking between different parts of
the DNA molecule has been observed in vitro, no evidence has yet
appeared which indicates these changes also occur in vivo. The
cross-linking theory is, however, a viable one which may well explain
molecular changes which form the basis for known changes in the char—
acteristics of structural proteins at least. The theory leads to
the search for methods whereby cross-links once formed may be broken
down but, perhaps more importantly, to methods which would reduce
their rate of formation in the animal and thus influence the rate
of aging.

Starting with the observations that (I) collagen becomes stiffer
with age and (2) collagen constitutes 25 to 30 percent of the total
body protein and surrounds blood vessels and cells, Kohn (95) has
hypothesized that age changes in collagen may have far reaching
effects on other physiological functions. Although this hypothesis
relates molecular changes in collagen to more general physiological
processes, it is highly speculative and has not been experimentally

tested (168).

Physiological Theories of Aging. Physiological theories of
aging attempt to explain aging and the life-span of various species

of animals either on the basis of a breakdown in the performance of

27
a single organ system or more reasonably in terms of impairments in

physiological control mechanisms.

Single Organ System Hypotheses

 

Cardiovascular system. In the past, physiological

 

theories of aging have focused attention on a single organ system to
explain the total effects of aging. Since failure of the heart and
blood vessels represents a primary cause of death, especially among
the aged, failure of the cardiovascular system has long been regarded
as the primary cause of aging. According to this hypothesis, aging
results primarily from the progressive deterioration of blood vessels
as a result of the develOpment of atherosclerosis. However, athero-
sclerosis is a disease process which occurs in the young and increases
progressively with advancing age. Death from this disease may occur
at any age. Furthermore, aging occurs in many lower forms of animal
species where no blood vessels are present. Hence, the hypothesis
that aging is basically related to reduction in blood supply does

not have general biological significance, even though it is an
important cause of death among humans. Another theory has the
hypothesis that aging is primarily the result of lowered delivery of
oxygen to critical tissues such as the brain. However, experimental
evidence fails to support this hypothesis. The oxygen content of
arterial blood does not fall with advancing age and blood flow to

the brain is only slightly diminished after maturity (94,168).

Thyroid gland. Another hypothesis assumed that aging

 

was due primarily to slowing of metabolic processes at the cellular

level. Since the rate of cellular metabolism is regulated by the

28
thyroid gland, it was assumed that aging was due to the inability
of the thyroid gland to supply adequate amounts of its hormone.
This hypothesis was also supported by some of the superficial simi-
larities between the symptoms of patients with hypothyroidism and
senescent subjects, as for example, loss of hair, slower movements,
lengthened reaction times, drying and wrinkling of the skin, and
the presumed reduction in basal oxygen consumption with advancing
age. However, more detailed studies have shown that the age related
fall in basal oxygen uptake is a reflection of tissue loss with
advancing age and that the oxygen consumption of individual cells does
not change significantly (169). Other experiments have shown that
with the exogenous administration of thyroid stimulating hormones
the thyroid of the older individual responds as well as that of the
young with respect to increasing the output of thyroxin, as evidenced
by increased basal metabolism, heart rate, and uptake of iodine by
the gland (9). Hence, the theory that aging is due to failure of the

thyroid gland can no longer be considered as very probable (168).

Sexyglands. Similarly, failure of the sex glands has

 

been looked upon as a primary cause of aging. In fact, this hypothesis
has led to the administration of sex hormones in an attempt to induce
rejuvenation (119). However, even with a highly purified male sex
hormone, the side effects, as for example, the possibility of
developing prostatic cancer, and the ephemeral results reported have
indicated the futility of this approach to influence overall aging.
Chronic administration of female sex hormones to postmenopausal
women has been urged by some endocrinologists to minimize the overall

effects of aging (69). This recommendation is based on the

29
presumption that aging in women is related to the level of sex hor-
mones present in the body. Although there is evidence that some of
the complications of the menopause can be alleviated by the adminis-
tration of female sex hormones, the possibility of inducing or
activating cancer in the female genital tract by prolonged administra-
tion of the hormone has led most clinicians to utilize the treatment
on the basis of relatively short-term therapy and to avoid chronic
administration (80,161). There is little evidence that aging as a
biological phenomenon can be ascribed to diminished sex hormone

production (168).

Pituitary gland-neurohypophysis. Since the pituitary
gland plays a central role in the modulation of activity of the
adrenal gland, the thyroid, and the gonads, aging has been ascribed
to a failure of this gland to carry out its apprOpriate functions
(163). However, methods for determining blood levels of the regula-
tory hormones produced by the pituitary and the hypothalamus are not
yet available. Since the possibility that the pituitary plays a key
role in aging cannot be ruled out at this time, recent advances in
utilizing radioactive immunological techniques, which are more sensi-
tive than the usual biochemical methods, may resolve this problem in

the near future (168).

Stress theory. The stress theory of aging, develOped
largely by Selye (163) and his collaborators, holds that aging is the
result of the accumulation over time of the effects of living.
According to Selye, any stress which requires adaptation by the
organism leaves a residuum of impairment from which the animal does

not completely recover. These residuals, although they may be quite

30
small with respect to specific events, accumulate over the life-span
so that ultimately the reserve capacities of the animal are exhausted.
The basic assumption of this theory is that there is always residual
damage which persists and accumulates. However, this is not supported

by eXperimental data (168).

Immunological theories. The immune system protects

 

the body not only against invading microorganisms but also against
atypical mutant cells which may form in the body. The immune system
carries out this protective function both by generating antibodies
which react to foreign organisms, proteins, etc., but also by the
formation of special cells which engulf and digest foreign cells and
substances. Aging has a marked impact on the capabilities of the
immune system. The production of antibodies reaches a peak during
adolescence and then declines.

One assumption is that the basic impairment in the immune system
is a loss in the ability to recognize slight deviations in molecular
structure and cellular characteristics so that cells which have under-
gone mutation and would ordinarily be destroyed by the immune system
are no longer recognized and are permitted to grow and develop to
the detriment of the animal. By the same token, impairment of cell
recognition capacity might also result in the development of anti-
bodies which inactivate normal cells in various tissues. It may also
be hypothesized that even though recognition occurs, the immune
system of the senescent animal is unable to produce an adequate
supply of antibodies or phagocytic cells. Experiments have shown
that in senescent animals the supply of stem cells from which the

phagocytic cells are derived, as well as the antibody producing cells,

31
is reduced as a result of a fall in the rate of cell division in the
stem cells (78).

The autoimmune theory of aging (19,196) proposes that aging
results from the deve10pment of antibodies which react even with
normal cells in the body and destroy them. This might occur either
because of failure to recognize normal cells as normal or the
development of errors in the formation of antibodies so that they
now react with normal cells as well as the deviate ones which stimu-
lated the immune cells to form antibodies in the first place. This
hypothesis gains its greatest support from the age—related increase
of autoimmune antibodies found in the blood, and the similarity
between many of the so-called immune diseases (some types of anemia,
rheumatoid arthritis, arteritis, maturity onset diabetes, thyroiditis,
and amyloidosis) and the phenomena of aging.

Although evidence for the key role of the immune system in
aging is still sketchy, the general hypothesis is an attractive one
which can be experimentally tested. It has added merit in that, if
true, it can lead to the development of methods which could poten-

tially influence aging in the human (168).

Aging and Physiological Controls

The overall performance of an animal is closely related to the
effectiveness of a variety of control mechanisms which regulate the
interplay of different organs and tissues (63). Frolkis has advanced
the hypothesis that aging and death of an animal are the result of
failure of adaptive mechanisms. Since adaptation to environmental

stresses involves the appropriate functioning of control mechanisms,

32
the adaptation hypothesis can be considered as a special case of the
broader theory of control mechanism failure.

With advancing age many control mechanisms lose their effective-
ness. Any control mechanism must have a system which will detect
changes for which adjustments must be made. In addition to the
sensing device, there must also be a mechanism which can carry out
the proper adjustment. In the human body, there are many sensing
devices which activate effective processes to carry out readjustments
of body temperature, blood glucose, blood pressure, heart rate, etc.
In developing theories of aging based on control mechanisms, it is
necessary to distinguish between alterations in the sensitivity of
detecting devices and the effectiveness of the organ system or cells
which initiate the changes necessary to maintain stable conditions.

In the mammal the primary control mechanisms Operate either

through the endocrine system or the nervous system (168).

Endocrine Control System. Many metabolic processes are regulated
by hormones which are produced in a gland and are then transported
to the target organs through the blood stream. Although the detailed
mechanisms whereby the hormone produces its effect within target
organ cellsare still unknown, recent studies have shown that tissues
of senescent animals show a reduced binding capacity for many of the
steroid hormones (124) so that the ultimate site of action of a break-
down of the regulatory mechanisms through endocrine control may well
be at the cellular level. There are, however, a number of examples
of the breakdown of endocrine control in terms of overall physiologi—
cal effects. For example, the response: of the kidney to a standard

amount of antidiuretic hormone is significantly less in the aged

33
subject than in the young (124) and also with advancing age, the
rate at which excess sugar is removed from the blood is significantly
reduced (174). The removal of the excess glucose is regulated pri—
marily by the release of insulin from the pancreas. Extensive
experiments have shown that the age deficit is due to the reduced
sensitivity of the beta cells of the pancreas to a slight rise in

blood sugar level (152).

Nervous Control Mechanisms. The nervous system also plays an

 

important role in regulating many physiological activities. Perform-
ances which require coordinated movements mediated through the
nervous system show greater decrements with age than do component
parts of the total performance. For example, the maximum breathing
capacity shows a significantly greater age decrement than does vital
capacity itself (138). The maximum breathing capacity requires the
coordinated movement of the chest cage and the diaphragm for its
performance. In contrast, vital capacity involves only a single
maximum inspiration followed by an expiration, which does not involve
a coordinated rate-adjusted performance.

In mammals, body temperature is normally regulated within fairly
narrow limits. Experimental studies in which subjects of different
ages were exposed to different environmental temperatures have shown
that young subjects can maintain body temperature even when environ-
mental temperatures are either increased or decreased, whereas old
subjects show a significant change in body temperature (96).

The ability to perform muscular work is a function of the inte-
grated activity of the cardiovascular, respiratory, and neuromuscular

systems. Studies have shown that perhaps the greatest decrements

34
shown with age lie in the area of exercise and maximum work per-
formance. Although decrements can be identified within each of the
organ systems involved, they are less than the decrements in total
performance. In both physiological and psychological performances,
the primary finding is that with increasing age the rate of adjustment
or performance diminishes. The slower response observed in many
organ systems in the elderly may well be a reflection of impaired

integrative functions both at the neural and endocrine levels (168).

Summary_of Theories of Aging

 

A brief review of the theories of aging shows that, although
some specific experimental evidence may be found which will support
each of them, it is clear that some are more probable than others.

In some cases, critical tests ofenihypothesis cannot be made at
present because of technological limitations. This is especially
true of the error hypothesis which, although it is still diffuse, is
currently having considerable impact on the direction of research on
aging. Methods which will permit the identification of molecules
which have an error in their assembly will undoubtedly be available
in the near future. When the nature of the error and the point at
which it occurs have been identified, further research can be con-
ducted to develop methods either for reducing the occurrence of error
or for correcting the existing errors.

The cross—linking theory also offers promise for the future.

The first step is to determine whether cross—linking occurs in intra-
cellular proteins as it does in extracellular proteins such as
collagen and connective tissue. It will be possible, with future

research, to identify methods to both reduce the rate of formation

35
of cross-links and even to eliminate them once they are formed.

The autoimmune theory of aging also holds great promise for the
future. Although a great deal of experimental work remains to be
done, the rapid deve10pments in the field provide new methods and
techniques which will make it possible to test many of the specific
aspects, especially at the cellular level.

Although the ultimate cause of aging may lie at the cellular
or even molecular level, current theories in this area fail to take
into account the important consideration of interrelationships
between cells, tissues, and organs. It is in this area that theories
which focus on the role of regulatory and control mechanisms offer
promise for the future. Such theories are currently less elegant
than the cellular and molecular theories but they do address them-
selves to the important problem of explaining aging in the total
animal.

It seems doubtful whether a single theory can adequately explain
all aspects of aging because aging is a highly complex phenomenon
which may require different eXplanatory principles for different

aspects of the process (168).

Effects of Aging in Bodyy§ystems

In Table 4 (adapted from 2), which follows, the first column
is a list of the morphological changes usually attributed to aging
for different body systems. The second column comprises findings
on clinical examination of old people which are usually accepted as
"normal" or inevitable changes imposed by aging. They may induce
the aging individual to seek medical help and appropriate advice but

seldom for investigation or treatment as clinical problems. The

36

TABLE 4.

Effects of Aging In Body Systems'

 

Morphological age changes
in various systems

Related ‘normal‘
age changes

Clinical pathological
trends

 

Skin

Increased pigmentation
epidermis

Atrophy {sweat glands
hair follicles

coHagen

elastin

Thickened blood vessels

Somatic mutations

Diminished subcutaneous

fat

(125,139,146)

Degeneration i

Central nervous system:
special senses

Eye
Loss of orbital fat (139)

Stenosis of lacrimal duct

Lipid deposits in comes (4)
Conjunctivitis sicca

(52)
Shallow anterior chamber
Loss of elasticity and
nuclear sclerosis in lens
Degenerative changes in
muscles of accommodation,
iris. vitreous. retina. and
choroid (51)

Degeneration in cortical
neurones relating to vision
(occipital lobes) and of
intrinsic or extrinsic ocular
muscles

' (Adapted from 2.)

Skin discoloured. thin.
Wrinkled. dry. and fragile.
Tendency to senile
purpura. Campbell de
Morgan spots. Deformed
or atrophic nails. greying
and receSSion of hair

(12. 32. 41.127)

Sunken appearance of eye
Laxity of eyelids
Senile ptosis

Epiphora

Arcus senilis

Reduced tears and dry
cornea

Reduced filtration angle
Presbyopia

Contracted pupils. slowed
reflex

Impaired visual acuity and
tolerance of glare
Reduced fields of vision
Defective color vision
Slowing of dark adaptation
Muscae volentes (objects
floating in field of vision)
Visuo-spatial perception
and discrimination less
accurate

Impaired accommodation
Limitation of upward gaze

Seborrheic keratosis
Abrasions and infections
Pruritis

Heat loss—hypothermia
Onychogryphosis;
Paronychia

Pressme sores
Basal-cell epithelioma
Keratoses—squamous
epithelioma

Entropion

Ectropion

Trichiasis (ingrowing
lashes)

Basal-cell carcinoma of
eyelid

Dacryocystitis
Lacrimal abscess

Necrotizing sclerokeratitis.
corneal ulcers

Glaucoma

Cataract

Retinal detachment
Occlusive vascular disease

Confusional states caused
by sensory deprivation

37

TABLE 4—cont.

 

Morphological age changes
in various systems

Related 'normal‘
age changes

Clinical pathological
trends

 

Ear

Degeneration of organ of
Corti (loss of hair cells)
Loss of neurons in cochlea
(ganglion cells) and tem.
poral cortex

Impaired elasticity affect-
ing vibration of basilar
membrane

Otosclerosis of ossiculuar
chain in middle ear (206)
Excessive wax accumula-
tion

AtrOphy of striae vascularis
(impaired endolymph
production)

Degeneration of hair cells
in semicircular canals (206)

Nose. throat & tongue

Atrophic changes in
mucosae (128)

Neuronal degeneration (taste
buds reduced 64 per cent

by age 75) (7)

Atrophy and loss of
elasticity in laryngeal
muscles and cartilages

Central nervous system:
Brain and spinal cord

Macroscopic changes.-

Meningeal thickening.
cerebral atrophy (brain
weight down 10 per cent
between ages 30 and 70) (25)

Presbyacusis—impaired:

(i) Sensitivity to tone
(high frequency)

(ii) Perception (especi-
ally against background
noise)

(iii) Sound localization

(iv) Cortical sound dis-
crimination

Impaired reflex postural
control

Uncertainty and unrelia-
bility in moving about in
darkness

Impaired sense of taste

and of smell

Diminished responsiveness
of reflex cough and
swallowing

Vocal folds slack. voice
pitch raised; power and
range reduced

Diminished intellectual
responsiveness. perception.
mental agility and
efficiency

Impaired memory and
learning ability

Psychological effects of
deafness (isolation;sus-
picion; depression)

Conductive deafness

Méniére‘s syndrome

Anorexia
Food fads
Malnutrition

Avitaminosis
Risks of gas or food poison-
ing

Reduced Intellectual
reserves predisposing to
acute confusional states
Sustained mental, be-
havioural. and motor
changes of dementia

38

TABLE 4—cont.

 

Morphological age changes
In various systems

Related ‘normal'
age changes

Clinical pathological
trends

 

Cellular changes:

In all cells—deposits of
lipofuscin (‘wear-and-tear
pigment' formed in de-
generating cytoplasm
probably from lysosomes
or mitochondria) (181)

In neurones—(i) loss of
RNA. mitochondria, and
enzymes In cytoplasm;
decline in function; death
of cell (65.110)

(iI) l-Iyallne and
eosinophilic inclusions and
Lewy bodies

(iii) Neurofibrillary
tangles and senile
plaques—different
degenerative changes
occurring with increasing
frequency in people over
60 years of age, but not
directly related with each
other

Corpora amylacea—
occur anywhere in brain
ussbe(62)

Vascular changes:

lntlmal and medial

fibrosis: Siderosis. amyloid.
and hyaline degeneration
Atheroma—increaslng In
extent with age, but
pathogenesis ls multi-
factorial (97.160)

Locomotor system

Muscles—atrophy affecting
both number and size of
fibres conditioned by meta-
bolic disorder and 'func-
tional denervation’.
weakness attributable to
biochemical or hormone
deficiencies

Less resilient. more rigid
In outlook

More self-centered.
withdrawn, and Intro-
verted

Sensorl-motor performance
slower to achieve accuracy
Impaired sensory aware-
ness (pain, touch. heat
and cold. joint-position
sense)

Impaired mechanisms
controlling posture. anti-
gravity support, balance,
and moving equipolse
(nerve conduction

velocity reduced 10 per
cent by age 75)

Loss of muscle bulk
Degenerative joint changes
Decline In physical strength
Limitation of range and
speed of movements
Disability—combined
effects of muscular weak-
ness. joint stiffness. and

Depression

Persecutory symptoms of
paraphrenia

Defective appreciation or
localization of pain
Predispositlon to falls and
Injuries

Transient ischaemic
cerebral episodes
Impending. progressing
and completed strokes

Bunions. subluxatlon of
small joints In hands and
feet

Painful feet (and other
chiropody problems)
Paget’s disease
Muscular wasting.
especially distal

39

TABLE 4—Cont.

 

Morphological age changes
in various systems

Related 'normal'
age changes

Clinical pathological
trends

 

Atrophy {osteoporosis

of bones: osteomalacia
Joints— loss of resilience
and elasticity in ligaments,
cartilage. and periarticular
fissues

Degeneration. erosion.
and calcification in
cartilage and capsule

(14. 72)

Castro-Intestinal
system

impaired central mechan-
isms for sensori-motor
performance:

(i) Less precision in
fine movements and in
rapid alternating move-
ments

(ii) Irregular timing of
action, loss of smooth
flow of one form of action
into another

(iii) Slowing down to
avoid outcome of one
action before planning the
next
Confidence and reliability
of activity reduced
Difficulty with intricate
tasks (especially if com-

plicated by uncompensated

visual defect)

Stooped posture. loss of
height. and other distor-
tions owing to atrophy
and effects of weakness in
skeleton and major

muscle groups responsible

for posture and antigravity
support.

Problems of adaptation to
dentures and altered
alignment of bite

Dental caries; ginglval
recession (33, 81)

Capricious appetite

extremities

Arthritis: ankylosis and
contractures

Fractures. spontaneous and
traumatic

Herniae. extra- and intra-
abdominal

Retained carious stumps
Cysts

Dental sepsis
Temporo-mandibular
anhnfls

Anorexia

Malnutrition

40

TABLE 4—cont.

 

Morphological age changes
in various systems

Related ‘normal'
age changes

Clinical pathological
trends

 

Atrophic changes in jaw.
mucosae. intestinal glands.
and muscularis (111)

No significant age changes
described in liver. and
there are as yet no tests

of function sufficiently re-
fined to detect the impair-
ment sometimes suspected
in older patients

Respiratory system

Coalescence of alveoli
(atrophy and loss of
elasticity in septa)(8)
Sclerosis of bronchi and
supporting tissues
Degeneration of bronchial
epithelium and mucous
glands (79)

thoracic
Osteoporosis vertebrae
(130) rib cage

Reduced elasticity and cal-
cification of costal cartilage
Weakness of intercostal
and accessory muscles of
respiration (92.93. 179)

Asymptomatic alterations
in secretion, motility,

and absorption occur in
‘normal’ aging

Total lung volume un-
changed but vital capacity
is diminished. O2 diffusion
impaired and respiratory
efficiency reduced; as

are sensitivity and effici-
ency of self-cleansing
mechanisms

Kyphosis and increasing
rigidity of chest wall

Functional reserve respira-
tory capacity is therefore
Impaired In old age, but
clinical evidence is minimal
unless evoked by illness.
Compliance changes little
because the rlse to be
expected from diminished
elastic recoil Is offset by
increased lung stiffness
(fibrosis) and loss of flexi-
bility In chest wall

Achlorhydria (incidence
increases over age 60):
related to defective absorp-
tion of iron and vitamins.
and to pernicious anaemia
Dysphagia (pseudo-bulbar
palsy: oesophageal reflux.
pouches. and carcinoma)
Constipation

Peptic ulcer

Diverticulosis

Increased susceptibility to
pneumonia

Pulmonary tuberculosis—
(reactivation of ‘healed‘
tuberculosis)

Carcinoma of bronchus
Pulmonary embolism

Concurrent respiratory
disease and cardiac failure

41

TABLE 4—cont.

 

Morphological age changes

Related 'normal'

Clinical pathological

 

in various systems age changes trends
Cardiovascular
system
Aorta—loss of elasticity Aorta dilated and unfolded Arrythmias

in media and intimal
hyperplasia (68)

Cusps of heart valves de-
generate—less resilient
with nodular sclerosis and
sometimes calcification
which may extend into
interventricular septum

Myocardial changes—
lipofuscin deposits; myo-
cardial fibrosis. and
amyloidosis (182)
Atrophy and fibrosis of
media. and intimal hyper-
plasia in coronary arteries
Brown atrophy only in as-
sociation with debilitating
states—malnutrition,
cancer. pernicious
anaemia, etc. (heart weight
correlates with body weight)
(107)

Atheroma-Incidence in-
creases with age. probably
promoted by hypertension
and cigarettes (178)

(may obstruct venous
return in left side of neck)

Apex beat difficult to locate
if chest rigid or distorted
by kyphoscoliosis
Stiffened valves cause
murmurs—aortic systolic.
mitral regurgitant; not
necessarily significant

No specific age-determined
changes or degeneration

in the heart (i.e. no ‘senlle
heart disease') can be cor-
related convincingly with
impaired cardiac function
in old age but it is accepted
that; (i) Cardiac output
declines owing to reduced
stroke volume. (ii) There-
fore capacity for physical
work Is limited. (iii) A
given amount of exercise
raises heart rate and blood
pressure more in old age
than In youth.

Mental confusion and pro-

found weariness should raise
suspicion of heart disease in

old peOple. They are often
more prominent than an-
ginal pain or even breath-
lessness (because of
restricted activity)

Conduction defects—
bundle branch block prob-
ably indicates significant
heart disease

Aortic stenosis
Pulmonary heart disease
(pulmonary embolism)
Blood-pressure changes—
dlfficult to define hyper-
tension over age 65. SP.
readings alone do not con-
stitute a diagnosis (It
requires evidence of ad-
verse effects on eye. heart.
brain. and kidney). Hypo-
tension often more sinister
in old age.

lschaemic heart disease is
the most common cause of
heart failure In geriatric
medical practice

42

TABLE 4—cont.

 

Morphological age changes
in various systems

Related ‘normal'
age changes

Clinical pathological
trends

 

Benito-urinary system

Thickening of basement
membrane of Bowman’s
capsule and impaired
permeability
Degenerative changes in
tubules
Atrophy and reduced
numbers of nephrons
Vascular changes affect
vessels at all levels from
intimal thickening of the
smallest. to arterlolar hy-
alinization and intimal
hyperplasia in large arteries
Prostatic atrophy—acini
and muscle with focal
areas of hyperplasia
Benign nodular hyper-
plasia present in 75 per
cent of males over 80
years of age
Histological (latent) pros-
tatic carcinoma demonstr-
able In most males aged
over 90 years (clinical
carcinoma very much less)
( 5 . 44,90)

Renal efficiency in waste.
disposal impaired:

(i) The number of
nephrons ls halved in an
average life span

(ii) Renal blood flow is
also halved by age 75.

(iii) Glomerularfiltra-
tion rate and maximum
excretory capacity reduced
by same proportion. The
aging kidney can still
maintain normal homeo-
static mechanisms and
waste disposal within
limits. but it is less
efficient. needs more
time, and its reserves
may be minimal. Therefore
relatively minor degrees
of dehydration.
infection. or Impaired
cardiac output may pre-
cipitate failure

Renal calculi

Renal lnfectlons- pyelone—
phritls. cystitis

Prostatic disease
Gynaecological disorders
Retention

Incontinence

43

TABLE 4—cont.
Other systems

Endocrine

Failure is not a consequence of 'normal' aging but. as in other systems. poverty of
reserve may precipitate evidence of deficiency.

(i) Secretory capacity of the pancreatic (3 cells diminishes, and diabetic
abnormality of glucose tolerance increases with age. The efficiency of insulin in
dealing with excess glucose apparently declines. and aging is the most
conspicuous factor predisposing to clinical diabetes

(ii) Functional thyroid activity decreases with age; B.M.R. and radioactive
Iodine uptake fall. myxoedema is three or four times more common than
thyrotoxicosis in older people.

(iii) Pituitary activity appears to be retained at normal levels with age. but
adrenal activity is impaired.

Homeostasis

Particularly vulnerable in old age to plasma or blood loss. dehydration. potassium
depletion, and metabolic acidosis. At rest the normal old person can maintain a
constant internal environment. but capacity to react to stress. even the demands of
daily living, is markedly lessened owing to two key characteristics of aging:

(i) Poverty of reserve which impairs the ability to restore systemic equilibrium
quickly when it is upset.

(ii) Breakdown in co-ordination because different organs age at different rates.
and functions dependent on the performance of several systems are therefore
impaired.

Central autonomic dysfunction

May contribute to postural hypotension, impaired temperature control and the risk
of hypothermia, loss of appreciation of visceral pain. and defective alimentary
motility.

44

third column shows the more common pathological predispositions of

aging individuals which necessitate medical intervention.

A Model for Studying Aging:
The Red Blood Cell

 

 

The death of a cell may be considered as a progressive phenomenon,
a kind of telescoped death of a higher organism in miniature. The
senescence of a cell may be considered similarly. Investigations
of the mechanism and characteristics of cellular aging represent,
therefore, a larger scope than the aging of a minute part of an
organ of the body. The choice of a model for such a study may be
directed either by consideration of the importance of the cell to be
investigated, or by its relative simplicity in terms of anatomy,
biochemistry, and physiology and, therefore, the probability that
it offers to elucidate some basic problems in the process of cellular
aging. From this point of view, the mammalian red blood cell is a
suitable model for the study of aging at the cellular level (43).
Aging, in a general sense, as exmplified by the various theories of
the mechanism of aging, can be viewed as any change from steady
state. One of the sources of a change in steady state, at the
cellular level, is a change in cell surface-to-volume ratio. The
erythrocyte osmotic fragility test provides a way of evaluating the
surface area/volume (SA/V) relationship of normal and abnormal red
blood cells (201). Therefore, the erythrocyte osmotic fragility
test was utilized to assess changes in human red blood cells with
aging. These changes in surface-to-volume ratio of human red blood
cells, as measured by the osmotic fragility test, would thus reflect

a change from steady state indicative of aging at the cellular level.

45

Osmotic Fragility

 

Changes in the fragility of red cells are closely related to
changes in their osmotic behavior, and although the fragility is
often spoken of as if it were a simple property, it depends in a
complex way on a number of variables. To determine it, red cells
are placed in a series of relatively large volumes of media of
decreasing tonicity separated by intervals of tonicity, and the
tonicity in which there is just commencing hemolysis determines the
fragility. Some investigators prefer to use tonicity in which
there is complete hemolysis, and some plot the whole curve for the
percentage of complete hemolysis as a function of tonicity (149).

The classical model of osmotic swelling and lysis of the red
cell postulates a balloon-like cell in which (1) constant intra-
cellular contents are present with no significant anomalies of
osmotic coefficient, (2) the membrane is virtually inelastic and
offers no significant resistance to deformation from disc to sphere,
(3) the membrane is virtually impermeable except with respect to
water molecules, and (4) the membrane shows minimal resistance to
rupture when the cell reaches the maximal volume which can be
encompassed by its surface area. Each of these postulates can be
regarded as inadequate or to have been incompletely demonstrated,
although the design of most osmotic fragility studies avoids serious
errors due to deviations from these expectations. For example,
cation interchanges occur slowly across the membrane, the intra-
cellular protein is at a concentration in which the osmotic coeffi-
cient may vary significantly with changes in protein concentration
(l,47,l89,205), and in some mammalian cells membrane elasticity may

show significant variations (129). Likewise, in some animal cells

46
cation permeability may show considerable variation with changes in
cell volume (145,164). possibly due to a conformational change in
a membrane receptor site (158), and there is some evidence of a
similar relationship between cell volume and potassium flux in
human red cells (153). The significance of these findings to the
lysis of the cell swollen by a hypotonic environment needs to be
elaborated; aspects of red cell osmotic lysis and departures from
the precise expectations of classical theory have recently been
reviewed by Wessels and his colleagues (207,208,209). It was observed
by Ponder that the red cell does not act as a perfect osmometer even
when the relationship between the volume changes and osmolality
refers only to the volume of the intracellular water space (148).
It was found that there were significant differences in the rela-
tionship of volume to osmolality, depending on whether the cells
were taken from defibrinated blood or from oxalated blood. Another
observation made at this time by Ponder was that the osmolality of
plasma was approximately equal to that of 0.15 M sodium chloride
solution, whereas the red cell had to be suspended in slightly higher
concentrations (1% or 0.172 M chloride) to be isovolumic with cells
in plasma. Other evidence that the osmotic properties of the red
cell are affected by the composition of the suspending medium for
the red cells is given by Ponder (151), who studied the relationship
between red cell volume and tonicity of suspending solutions of
barium, magnesium, strontium and calcium chlorides. The effect on
hemolysis of the addition of low concentrations of calcium and
magnesium salts to hypotonic buffered saline were studied: concen-
trations of calcium chloride as low as 10-4 M significantly diminished

hemolysis of red cells in 145 mOsm saline, whereas magnesium chloride

47
produced no effect below a concentration of 10-2 M. Both the fast
and slow phases of hemolysis were diminished (Figure l). The calcium
treated cells were less osmotically fragile than the red cells
suspended in sodium chloride alone. The osmotic fragility curve
is highly dependent on the specific solution used as the suspending
medium (22).

Clinical Significance of Osmotic
FragilityfiMeasurements

 

 

Increased osmotic fragility, or decreased resistance to hemolysis,
is a feature of spherocytes of all types and may therefore indicate
congenital spherocytosis, idiOpathic acquired hemolytic anemia,
isoimmune hemolytic disease of the newborn infant (more common in
A—B—O incompatibility than in Rh sensitization), and other hemolytic
anemias. Normal osmotic fragility (Figure 2) (122) is found in
symptomatic hemolytic anemia and in paroxysmal nocturnal hemoglobin-
uria. Decreased osmotic fragility is seen in sickle cell anemia and
in hemolytic anemias characterized by abnormally flat target cells,
such as occurs in some of the hemoglobinopathies (Figure 3) (123).
Any history of biOphysical studies of the red cell membrane in
disease states should certainly credit the earliest studies of
mechanical and osmotic fragility. Although these laboratory tests
were not definitively interpretable at the time they were first found
useful for evaluation and diagnosis of clinical disease, their
physiologic significance has now become much more apparent (201).
Stated in its simplest form, the osmotic fragility (O.F.) test pro-
vides a way of evaluating the surface area/volume (SA/V) relationship
of normal and abnormal cells (213). Thus, increased fragility is

generally interpretable as indicative of a decrease in SA/V or

48

 
 
 
    

 

 

SST

FAST-e—o—

__ >NaCl
90(- SLOw-0 O-
FAST.X-X- so ’
Cl * C
80" sr.ow-e-e->~° ° ’0 ’
, f

701.
‘2
a) 60¢-
3
5. 5°’
I 40'I
\° 301-

20'-

’ V
I
IOU-
: l I 1
iso iéo iio I00

OSMOLALITY m mOsm/kg

FIGURE |. EFFECT OF IONIC COMPOSITION OF SUS ‘
PENDING MEDIUM ON OSMOTIC FRAGILITY OF HUMAN
RED BLOOD CELLS. Shown are the fast and slow
phases of hemolysis with NoCI as the Suspending
medium. and the fast and slow phases of hemolysis

with NaCl plus Ca”(l.25mM) as the suspending medium.
CaH decreased the osmotic fragility of the red

blood cells at the same medium osmolality.

HEMOLYSIS

°/o

49

loo-1
90-

eoj
'ro-i

so.
40-

20"
IO‘

 

 

 

FIGURE 2. NORMAL AND ABNORMAL OSMOTIC
FRAGILITY CURVES. A. Increased osmotic

fragility. B. Decreased osmotic fragility (122).

50

HEMOLYSIS

°/o

 

 

 

 

m 32 o'.s bis 0.: o'.e of? 01.? 7's
% NoCl

FIGURE 3. OSMOTIC FRAGILITY IN DISEASE
STATES. A. Hereditary spherocytosis. B.
Thalossemi a major. C . Tho lassemia minor.

0, Hb - E disease. E. l-lb‘ E " thalassemia

(123) .

51
sphering. Conversely, decreased O.F. (increased resistance to lysis
in hypotonic salt solutions) implies either an increase in surface
area or a decrease in cation content or volume in an isotonic medium.
Both hereditary spherocytosis and spherocytosis seen in immune
hemolytic disease are associated with increased O.F., while the
increase in membrane lipid content seen in a variety of disorders
is associated with enhanced osmotic resistance (37). However, in
spite of broadened insights into the pathophysiologic alterations in
red cells implied by altered O.F., it is still essential to recall
the fundamental assumptions upon which the test is based. First, as
a way of estimating red cell surface area, the O.F. test assumes a
normal cell volume in plasma, reflecting primarily a normal total
cation content which should not change in prelytic hypotonic salt
solutions. Inferences regarding decreased surface area from an
increased O.F. must exclude any increase in cell volume. Interpre-
tation of increased surface area based on decreased O.F. must also
be based on normal volumes in plasma and must exclude any excessive
prelytic loss of cation from partially swollen cells, as demonstrated
by Ponder (150) in cells affected by certain detergents and surface
active agents. Finally, interpretation of O.F. as an expression of
SA/V assumes that the intrinsic properties of the membrane have not
changed. In hereditary spherocytosis, however, the membrane itself
becomes very rigid and shows an anomalously low swelling prior to
lysis and shrinkage after lysis (99). Mechanical fragility, on the
other hand, is related also to the shape factor, i.e., decreased
SA/V (sphering) makes the cells less able to tolerate deforming
stresses and, therefore, more mechanically fragile. In addition,

however, mechanical fragility may reflect either a cell with rigid

52

contents, e.g., sickle cell, or cells which have lost their normal
intrinsic membrane deformability, such as metabolically depleted
cells (202). Thus, rather than being a non-specific indicator of
abnormal cells, the fact that mechanical fragility is abnormal in
so many disorders strongly suggests that loss of cellular deforma-
bility for any reason, whether it be shape change or develOpment of
a rigid interior or membrane, is a final common pathway leading to

premature cellular destruction (201).

The Time Course of Osmotic Hemolysis

 

A further dimension to osmotic fragility studies has been the
time course of the hemolytic process. According to Bowdler and Chan
(20), the degree of lysis of erythrocytes in hypotonic solutions is
time—dependent. They showed: (a) Red cells are lost from suspension
in hypotonic electrolyte solutions in two phases. An early rapid
phase, believed to be due to water influx resulting from the higher
chemical potential of water external to the Cell, is followed by a
second, smaller phase of cell loss with much longer half-time.

(b) The second phase is most prominent in the middle of the range

of partial hemolysis and is much less at the extremes of the range
of hemolysis. (c) The second phase is prevented or terminated by
inclusion, or addition, of sucrose to a concentration of 20 mM in
the system, and it is slowed by the presence of 0.005% tannic acid,
without alteration of magnitude of the second phase. (d) The magni-
tude of the second phase is dependent on the dominant cation of the
suspending medium; it becomes progressively greater through the
series Mgz+, Na+, Li+, K+ and Rb+. These findings were interpreted

as being due to increased passive cation permeability in cells

53
undergoing swelling to a volume close to that critical for hemolysis,
with water influx resulting from the unopposed colloid osmotic pres-
sure of the intracellular protein. Wessels and Veerkamp (209) have
similarly shown that osmotic resistance in NaCl solutions is strongly
time dependent. Though the time of lysis depends in some way on the
permeability, it is clear that the rate of hemolysis may also be
affected by the following factors: (a) the surface/volume ratio,
which varies with the shape of red blood cells of various mammalian
species (86,211), (b) differences in membrane elasticity, though not
observed with human erythrocytes (129), (c) the loss of osmotic
material from the cells during the permeation experiments (50,98),
(d) variations in initial intracellular tonicity (141), (e) changes
in membrane prOperties induced by the penetrating substances.
According to Jay and Rowlands (88), when an erythrocyte is placed
in a sufficiently hypotonic environment it swells to a sphere and
hemolyzes, releasing its hemoglobin into the suspending medium
(Figure 4) (30). The cell becomes a ghost cell, having lost all or
most of its hemoglobin. The rate at which the cell volume increases
depends on one or a combination of a driving force, resulting from
an osmotic or a concentration gradient, and the permeability of the
cell membrane to water and to the permeating solute, of which there
is a concentration gradient across the membrane. Jay et a1. (88)
state that hemolysis is a multistage process. The stages are swelling,
popping, reduction in volume accompanied by ion leakage and, finally,
hemoglobin leakage (Figure 5). The classical hemolysis time (TH) is
made up of a swelling time (TSW) and a stress time (TST). Stress
time is not negligible and with the faster permeants it may occupy

more than 75% of the hemolysis time. The stress time can also be

CHANGE IN NENATOCRIT PER CENT

30-

+20.

IOi— NACL, CM

2.0

 

HYPERTONIC HYPOTONIC (

PERCENT

54

ISOTONIC

1.0

   
    

 

“CL. ON PERCENT

 

0.0 0.0 0.4 0.2 ,

 

PWELLI NO NENGJBIS

FIGURE 4. BEHAVIOR OF NORMAL CELL

HYPERTONIC AND HYPOTONIC MEDIA (30).

75

50

IN

PER CENT

HENOLYSIS,

55

To Th

#TSW —’JH—-—Ts' —-—’J

‘—TK —-iI———THb——->I
D

 

 

 

 

. -
‘
I' ‘.
B

i I
I
B

\

   

 

 

c 03 D K
Ac As
Vc Vs VK

FIGURE 5. DIAGRAM OF THE SEQUENCE OF EVENTS N
THE PROCESS OF OSMOTIC HEMOLYSIS. To . initial time
T=O. Th. total hemolysis time; T“, swelling time;
Tst. stress time. TK. potassium leakage time. and Tub,
hemoglobin leakage time. The diameters of the cell

Dc. D, and DK can be measured at the various
times and the corresponding areas and volumes can
be calculated. V0 . the initial cell volume can also
be calculated from D. . the diameter of the cell
at maximum swelling (88).

56
+ . . .
divided into two parts: a K leak time (TK) during which the cell
shrinks and a time (THB) during which hemoglobin is leaving the cell.

The T occupies a substantial part of T from 25 to 65%, and is

HE H'

relatively longer in fast hemolysis.

Red Blood Cell Membrane Components
and Suggested Models

 

 

Biconcavity and deformability are two unique properties of the
human erythrocyte. It is generally accepted that the biochemical
composition of the red cell membrane underlies these biophysical
features (99,201,212). Approximately 44% of the dry weight of the
red cell membrane is lipid, while 49% of the dry weight is protein
and only 8% of the mass is made up of carbohydrate (71). Enzymes
and calcium are also present.

The lipids in erythrocytic membranes are of three types:
neutral lipids, phospholipids, and glycolipids. The neutral lipids
of the red cell membrane consist almost entirely of free, nonesteri-
fied cholesterol (136). Cholesterol accounts for 29% of the dry
weight of membrane lipids (37) and for 43% of the molar concentra-
tion of membrane lipids (172,198). Knowledge about the location of
cholesterol in the erythrocyte membrane is extremely limited. Murphy
(132) observed that cholesterol was concentrated around the periphery
of the biconcave disc. The results of these autoradiographic studies
have been neither confirmed nor denied (27). It has been generally
assumed that on a molecular basis cholesterol is associated with other
lipid molecules. It is not known, however, whether cholesterol is
distributed equally between the outer and inner regions of the lipid
core of the erythrocyte membrane (162,191). Reticulocytes can synthe-

size cholesterol from acetate (10), but mature red cells lose this

57
capacity (116). Red cell membrane cholesterol is in an equilibrium
exchange with the free cholsterol bound to serum lipoproteins (87).
Complete equilibrium between plasma and cell free cholesterol occurred
in approximately eight hours, both in Vitro and in Vivo (131).
Sterols other than cholesterol are capable of exchanging with
cholesterol in the red cell membrane (26). The action of steroid
hormones may be related to their direct incorporation into membranes,
and this may be true for progesterone (46). There are changes in red
cell lipids during storage. Normal red cells stored at refrigerator
temperatures in acid-citrate-dextrose (ACD) undergo a progressive
loss of both cholesterol and phospholipid amounting to approximately
25% in seven weeks (73,193). A decrease in critical hemolytic
volume (an indirect measure of surface area) accompanies this lipid
loss. In contrast to this symmetrical loss of lipid, saline-washed
red cells frozen in saline containing EDTA undergo a progressive,
but selective, loss of glycerophosphatides during storage (199).
This alteration results in a decrease of total lipid phosphorus by
more than 30% after eight weeks of storage. There is little or no
change in the red cell content of sphingomyelin or cholesterol (37).
There are also changes in red cell lipids in vitro. The selective
loss of cholesterol from red cells in incubated serum results from
the esterification of free cholesterol in serum by the serum enzyme
lecithin:cholesterol acyltransferase (LCAT) (87). This loss of
cholesterol from membranes is associated with a pr0portional loss of
surface area, as measured by osmotic fragility (31). When incubated
under conditions leading to metabolic depletion, red cells undergo
a loss of both cholesterol and phospholipid. This change occurs prior

to the onset of measurable hemolysis (36). Similar lipid loss has

58
been observed with red cell ghosts (104). Protein is not lost from
ghosts under these conditions, suggesting that lipid loss occurs, at
least in part, because of an altered membrane affinity for lipid.
The late loss of lipid from intact red cells, however, is accompanied
by a loss of membrane protein. Associated with this lipid loss is
a proportional decresae in membrane surface area, as estimated from
critical hemolytic volume (202). In contrast to the selective loss
of cholesterol, the loss of lipid under these conditions is irrevers-
ible (36,37). There are changes in red cell lipids in disease,
e.g., hereditary spherocytosis (37). After splenectomy in patients
without HS, target cells with increased surface area occur and,
parallel to their appearance, there is an increase in osmotic
resistance. Both evolve to a maximum over several weeks to several
months. The red cell content of lipid is increased 15 to 20% in
patients without HS who have undergone splenectomy, as compared with
normal subjects with intact spleens (35). Comparison of red cells
from patients with and without HS, all of whom have undergone splenec-
tomy, reveals, therefore, a 15 to 20% deficiency of both cholesterol
and phospholipid in the HS cells. However, HS cells after splenectomy
have a lipid content which is, nonetheless, similar to that of red
cells from normal subjects whose spleens are intact. Despite this
similarity, red cells from HS patients without spleens are more
osmotically fragile than red cells from normal subjects with spleens.
This difference indicates that factors in addition to lipid deficiency
per se are responsible for the deficiency of surface area in HS red
cells. Data indicate that an abnormal interaction between ATP,
divalent cations, and the membrane may exist in HS red cells, and

that this abnormality may account for the cells' leakiness to sodium

59
and instability upon ATP depletion (100). Thus, despite considerable
data regarding membrane lipids in HS and the role of lipids in various
membrane phenomena which appear abnormal in HS, the nature of the
membrane defect in this disorder is still unknown. However, the
acquisition of lipid and surface area by HS red cells in vivo appears
to circumvent this defect and to allow a marked prolongation of cell
survival (37). More recently, Shinitzky, Moses and Livne (167) have
reported that erythrocytes affected by HS, obtained from splenec-
tomized patients, showed a varying degree of elevated osmotic fragility.
The increased microviscosity of the membrane lipid core correlated
with the severity of HS. The data supported the proposition that the
defect in HS-affected cells is associated, at least in part, with
alterations in the membrane lipids. Farnsworth et al. (58) have
demonstrated that modifications of the lipid composition of the
cellular and subcellular membranes of rat erythrocytes are associated
with changes in their permeability. Modifications of the lipids are
associated with a significant increase in the osmotic fragility of
the erythrocytes and an indication of a more homogeneous red-cell
population in comparison to the controls. Patients with various
forms of liver disease, including hepatitis, cirrhosis, and obstruc—
tive jaundice, have red cells with an increased content of cholesterol
and phospholipid (34,135). Increases in membrane cholesterol content
correlate with increases in membrane surface area, as measured by
osmotic fragility. Using CrSI, it has been found that normal red
cells progressively acquire surface area and become resistant to
lysis in the circulation of patients with obstructive jaundice over
the course of two to three days, and that osmotically resistant

target cells lose surface area and become osmotically normal over a

60
similar time course in the circulation of normal subjects (34). The
phospholipids of the erythrocyte membrane account for 69% of the dry
weight of membrane lipids and for 54% of the molar concentration of
membrane lipids. The four major classes of phospholipids, arranged
in order of decreasing concentrations, are: phosphatidyl choline
(lecithin), phosphatidyl ethanolamine (cephalin), sphingomyelin,
and phosphatidyl serine (37). Recent studies appear to support the
concept of a nonrandom distribution of phospholipids between the inner
and outer regions of the membrane (91). Lecithin and sphingomyelin
are predominantly located in the outer part of the lipid layer and
phosphatidyl ethanolamine and phosphatidyl serine on the inner part
of the lipid layer (162,191). About 50% of the fatty acids in red
cell membrane phospholipids are saturated and 50% are unsaturated.
Reticulocytes, but not mature red cells, can synthesize fatty acid
de novo (10). Plasma free fatty acid and plasma phospholipids can
exchange with their respective counterparts in red cell membranes
(172). Glycolipids account for a small fraction of the total membrane
lipids of erythrocytes (2% by weight and 3% by molar concentration).
The most abundant erythrocyte glycolipid is globoside (37). Unlike
cholesterol and phospholipids, which are widely distributed in
membranes, globoside seems to be restricted to plasma membranes.
Other red cell membrane glycolipids have antigenic activity corre-
sponding to the Lewis and ABH blood groups (114). It is of interest
that neuraminic acid (sialic acid) is confined to membrane glyco-
proteins and is not a component of membrane glycolipids. Erythrocyte
membrane carbohydrate is found primarily as glyc0protein and glyco-
lipid complexes (135). As a group, the glycoproteins of the

erythrocyte membrane all appear to be integral membrane proteins and

61
are hydrOphobically bound to the lipid bilayer of the membrane (188).
Red cell membrane proteins are not as well characterized as the
lipids, although about half the mass of these membranes is composed
of protein. At least seven to nine major proteins can be defined
by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and
by gel filtration technique (109,147,204). Two membrane proteins
are more or less well characterized. The first is referred to as
spectrin, because of its initial identification in experiments using
red cell ghosts, or as erythrocyte actomyosin because it bears a
strong resemblance to muscleactomyosin.(71,144). Spectrin accounts
for about 20 to 25% of stromal protein and the monomeric form has a
molecular weight of about 240,000 (57,190). Spectrin is soluble in
the presence of chelating agents, contains no lipids or carbohydrate
groups, is rich in glutamic acid, contains no cysteine, and has been
purified to apparent homogeneity as judged by electrophoresis, immuno-
diffusion, and ultracentrifugation (71). It has a tendency to form
insoluble fibers or filaments in the presence of divalent cations,
especially calcium (71). It is believed that Spectrin is located on
the inner surface of the red cell membrane (24). Erythrocyte glyco-
phorin is the other well studied red cell membrane protein. It is
the principal glycoprotein of erythrocyte stroma, accounts for about
10% of total membrane protein, has a molecular weight of about
55,000, and is transmembranous with its carbohydrate moiety located
on the external surface of the red cell (113). About 60% of glyco-
phorin in membrane is sialic acid. This, in turn, accounts for the
characteristic negative charge of the red cell that regulates inter-
action with neighboring cells and the surrounding medium. Moreover,

this protein carries the A, B, M, and N specific blood group antigens

62
as well as receptors for phytohemagglutinin, wheat-germ agglutinin,
and influenza viruses (113). Several enzymes seem to be tightly
bound to the red cell membrane: glyceraldehyde—3-phosphate dehy-
drogenase, aldolase, 3-phosphoglycerate kinase, adenylate kinase,
cyclic AMP-dependent protein kinase, adenosine triphosphatases
(MgZ+-activated ATPase, Ca2+-activated ATPase, [Ca2+ + Mgz+]—activated

+ + +
ATPase, [Na + K + Mg2

J—activated ATPase [195]), and cholinesterase
(11). Evidence strongly indicates that the bulk of the erythrocyte
calcium, and perhaps the whole of it, is present in the membrane
(53,75,195).

Recent evidence suggests that the primary defect in hereditary
spherocytosis is not restricted to the lipids but may also involve
the membrane proteins. Johnsson (89) proposes that the alterations
in red cell lipid components in spherocytes of varying origin may
represent a phenomenon secondary to the abnormal red cell shape
rather than being related to a specific etiological factor. Release
of cholesterol and phospholipid, particularly under conditions of
metabolic depletion (35), increased cation pumping activity, and
increased phospholipid turnover, could all be secondary to altered
lipid-protein interactions (191). Although the exact nature of the
erythrocyte defect has not yet been fully clarified, many lines of
evidence indicate that the primary defect resides in the red cell
membrane protein. These include (1) a partial or complete deficiency
or malfunction of a Spectrin-like membrane protein component of the
erythrocyte (66,105); (2) the presence of a genetically defective
membrane microfilament (83,84,85); (3) reduced levels of membrane
Spectrin phosphorylation probably secondary to a defective membrane

kinase enzyme (70,126); (4) an excess of negative charges of membrane

63
proteins (55); (5) a defect in shape regulation which results from
an alteration of any of the proteins in the shape change complex and/or
accessory regulatory proteins (11,166,214). The net outcome of these
biochemical, physiologic, and structural aberrations results in the
formation of microspherocytes, cells that are rigid, with decreased
surface area to volume ratio, increased mean corpuscular hemoglobin
concentration, increased osmotic fragility, decreased deformability,
and reduced life-span (11,82). The biconcave shape of the red cell
and other properties of it such as its capacity to transform between
the shapes of disc and sphere despite the uniformity of the physical
properties demonstrable over the surface, result at present in the
lack of a completely adequate model. However, there are some early
membrane models: (a) Gorter and Grendel (67) suggested a simple
lipid bilayer. They extracted the erythrocyte with acetone and
measured the monomolecular film formed from the extraction product
with Langmuir's device. They found a total film area of about 200
square microns per cell, about twice their estimate for the surface
area of the erythrocyte. Thus, they concluded that there is just
enough lipid in each cell to cover it twice, and that the lipids are
associated in a bilayer. The fact of the matter is that erythrocytes
do not always behave as if they had a lipid exterior. For example,
their surface tension, which is a measure of the tenacity with which
molecules at the surface of a liquid cling to their own kind, is far
too low. Fats and oils in an aqueous environment have very large
surface tensions because of hydrophobic bonding; lipids extracted from
erythrocytes do not. (b) Later, Danielli and Davson (42) extended
the model to include a coating of protein. The interior, in their

model, was an unspecified "lipoid" region. Danielli suggested that

64
the anomalously low surface tension of erythrocyte lipids is the
result of contamination by protein, which would naturally seek the
surface of a lipid drOplet and thereby change its character. The
hydrophilic behavior of intact cells could also be explained by this
assumption, which led Danielli and Davson to propose the first
complete membrane model in 1935. Danielli envisioned a membrane
with a lipoid center, coated on either side with protein. The
phospholipids in his model are oriented in two monomolecular layers,
with their hydrophobic tails toward the inside of the structure and
their hydrophilic phosphates on the surface, contacting the layers
of protein. The fundamentals of this structure are still accepted
today, although there have been numerous proposals for modification.
(c) The unit membrane, first proposed by Robertson (157) in the
1950's, explained the trilaminate appearance of many membranes in
the electron microscope by including an extended layer of protein on
either side of the bilayer. In the middle 1950's Robertson found a
way to verify the basic features of the Danielli model with the
electron microscope. He devised staining techniques that resolve
most membranes into two distinguishable lines on micrographs,
whereas both electron and light microscopists had hitherto seen only
single lines. It was later demonstrated by others that partial
extraction of membrane lipids with acetone leaves the double lines
intact, suggesting that the stain was revealing protein layers on
the two surfaces of the membranes. Robertson's membrane model has
rather thin protein layers on the two surfaces, more consistent with
an extended B-structure than with the compact globular proteins
suggested by Danielli. Furthermore, Robertson's model is not neces-

sarily symmetrical--whereas the internal surface in the model is

65
coated with protein, the outer surface could be either a mucoprotein
or a mucopolysaccharide. These features, along with the single
lipid bilayer leaflet 40 to 65 A thick, is known as the unit membrane
model, a name that implies a homogeneity in structure. There are
some modern membrane models. The size and solubility properties of
membrane proteins suggest that they are not confined to the surface
but are interspersed among the lipids: (a) The protein crystal model
is a regular arrangement of protein and bilayer. A number of workers
have published electron micrographs in which the membranes, prepared
by freeze-etching, seem to be composed of regular, repeating units,
or subunits. This concept represents a considerable departure from
a continuous lipid layer. David E. Green (192) would discard both
the Danielli and the unit membrane hypotheses for an alternate model
for the protein-lipid interactions in membranes called the protein
crystal model. The presence of protein within membranes, rather than
merely on their surface, not only explains the hydrophobic interac-
tion between protein and lipid, but also helps to explain certain
aspects of membrane permeability. In addition, it provides enough
room to allow the proteins to assume a globular form like that of
most enzymes, which frequently have diameters of 40 A or more.
Various physical measurements carried out on membrane proteins
suggest that they are indeed globular, and, therefore, inconsistent
with the space allotted them in the unit membrane. (b) A more
irregular structure is known as the fluid mosaic model. This fluid
mosaic model introduced by Singer (176) features the same intimate
contact between lipid and protein, but a less rigid arrangement and
stoichiometry. Rather than being discretely arranged, proteins

float freely in a phospholipid bilayer (54). Blank (18) presents

66
even another model. The measured two dimensional yields of protein
monolayers are high and parallel the rigidity of erythrocyte membranes
with regard to the dependence on divalent cations in the aqueous
phase. Although the surface properties of membrane proteins have
not been measured, it is likely that this protein layer would also
be far from fluid, and could be the basis for the mechanical stability
of the membrane. The rheological properties of protein monolayers
suggest that the membrane proteins normally present could account
for the mechanical stability of the membrane and for some of the
observed rheological prOperties of erythrocyte membranes. However,
a membrane having this structure would also be thicker and con-
siderably less fluid, at least in parts, than has been suggested in
recent membrane models. Skalak (177) reports that the mechanical
behavior of the red cell membrane is consistent with the fluid
mosaic model of the structure of the membrane. Electron microsc0pe
studies on red cell membrane have provided some ultrastructural
information on membrane structure. It has been reported that
examining the red cell ghost membranes by electron microscopy and
metal shadowing revealed that the membrane consists of short cylinders
(or plaques) with heights of approximately 30 A and diameters of
100 to 500 A. Cylindrical particles approximately 100 A in diameter
and normal to cell surface have been demonStrated in the membrane
interior by the use of freeze-cleaving technique. A model of the
red cell membrane composed of cylindrical apoprotein molecules bound
at their central belts by lipids has also been suggested (203).
However, the information based on metal-shadowed membranes loses
much of its appeal when the artifacts and limitations of the technique

are considered. Many problems also remain in transferring the

67
information from freeze—cleavage replicas to a detailed model of the
architecture of the erythrocyte membrane. It is not known whether
the intramembranal particles represent intact proteins or only
hydrophobic regions of proteins. Nor is it possible to assess the
extent of heterogeneity of the particles in the A fracture face and
their physical and chemical relationship to the particles in the B

fracture face (188).

RBC Shape:Deformability:Surface ArealYolume Relationship

In the biconcave disc shape, it appears likely that the shape is
a net result of the equilibrium between surface tension, ratio of
cell radius to membrane thickness, pressure differential across the
membrane, and extensional stiffness (177) of the membrane, assuming
the erythrocyte membrane is in fact elastic. Rakow et al. concluded
that the elasticity of the red cell membrane is attributable to one
or more of the membrane proteins (155). Evidence has accumulated
that indicates that the conformation of Spectrin (and probably actin)
determines red cell shape and deformability (85). Shrinkage of
human red cell ghosts is controlled by membrane "contractile" pro-
teins possessing Ca++-ATPase activity (144) and phosphorylation-
dependent shape changes in erythrocytes are related to the association
of Spectrin per se (190). Mohandas et al. (126) have demonstrated
that red cells can be shown to sphere suddenly when heated to a
specific temperature, between 48 and 50°C. This is the temperature
at which spectrin, and only spectrin, denatures or melts. It has
also been demonstrated that the distortion of the red blood cell
cytoskeleton induces a redistribution of integral membrane proteins

which modifies the surface properties of the cell (115). Sheetz

68
postulates that red cell membrane shape change is dependent on a
protein shape change complex and/or accessory regulatory proteins
(166). Lin has reported that divalent cation, such as calcium,
causes fusion of lipids in the membrane with resultant shape and
stability changes (106). Sheetz states that there are two different
categories of shape change, one physical and one enzymatic, each
having a different molecular basis (165). Investigators (60,108,143)
have reported that cross-linking of membrane proteins is related to
shape and deformability changes in the red blood cell and that this
cross-linking has an enzymatic basis (173). Beck (13) reports that
cell shape changes result from monolayer condensation and/or expan-
sion of plasma membranes when clustering of intrinsic proteins occurs.
Shape, intrinsic membrane deformability, and the fluidity of cell
contents, the major determinants of erythrocyte deformability, are
interdependent. The extreme flexibility of the cell as a function
of its biconcave shape is dependent on the intrinsic deformability
of the membrane and fluidity of the intracellular hemoglobin solu-
tion. Fluidity of contents in a shear field requires a deformable
membrane which transmits force to the cell interior. Shape may be
a function of cell content (e.g., sickle hemoglobin at low oxygen
tension) and also of the membrane (e.g., spherical shape at low
intracellular ATP concentration) (202). Shape is an important
parameter, for a sphere is recognized to be a geometric form of
considerable rigidity (101). ATP is widely recognized as the source
of high energy phosphate requisite to the maintenance of normal
intracellular cation composition in the erythrocyte by means of an
ATPase-related active cation transport mechanism. ATP is also

essential for the preservation of the normal biconcave disc shape

69
(134). Reduction of the intracellular ATP results in the classical
disc-to-sphere shape transformation (149). It has been demonstrated
that the post-transfusion survival of erythrocytes (133) is directly
dependent on erythrocyte ATP levels, an observation which clearly
relates cell survival to its metabolic integrity. More recently, it
has been observed that ATP and Ca++ were linked to contractility of
erythrocyte ghosts, a finding which suggested that ATP-related shape
change is a function of ATP-Ca++-erythrocyte membrane interaction
(144). Studies have been interpreted to indicate that a sensitive
ATP-Ca++-Mg++ intracellular relationship may regulate the physical
state of hemoglobin and non-hemoglobin protein at the inner membrane
surface. Ca++ accumulation favors a protein gel formation at the
inner cell surface, whereas ATP and Mg++ move the equilibrium toward
the sol state. Gel formation would result in increase of membrane
thickness, and gel-induced stiffening of the membrane could increase
the viscosity (49,195). Another Ca++-modu1ated system in the
erythrocyte membrane might be a Ca++-dependent contractile protein
whose presence in the membrane is strongly suggested by: the ATP-
dependent shape change (134), the ATP-Ca++-linked contraction of
ghost membranes (144), the demonstration of actomyosin-like protein
(112), and the evidence shown (159) for Ca++-dependent ATPase activity
of certain erythrocyte fibrillar proteins. Ca++ may induce membrane
rigidity and shape change by producing a denser thickened gel struc-
ture at the inner membrane surface and by stimulation of the membrane
protein to a more dense, less compliant contracted state. Most
mechanisms causing decreased erythrocyte deformability have their
effect either by reducing membrane flexibility, by reducing fluidity

of cell contents, and/or by causing increased sphericity, i.e.,

70
reduction of the membrane surface area-to-volume relationship (101).
Mechanisms altering the erythrocyte surface area-to-volume relation-
ship are (a) Heinz body formation, (b) fragmentation, (c) partial
phagocytosis, and (d) aging. Heinz body formation in disorders of
unstable hemoglobin, defects of the pentose phosphate pathway, or
disorders of unbalanced globin synthesis causes increased viscosity
of the cell contents; because of membrane attachment of Heinz bodies,
the overlying membrane is made rigid. If the entire cell is not
eliminated, the splenic pitting mechanism is thought to tear the
rigid Heinz body containing portions from such cells, with a rela-
tively excessive loss of surface area as compared to volume. The
surviving cell may be spherical and hence more rigid than before
passage through the splenic cord—sinus circulation (39). Erythro-
cyte fragmentation also results in increasing sphericity causing
sufficient cell rigidity to make the cell susceptible to eventual
mechanical trapping and destruction by the spleen. Partial phago-
cytosis of erythrocytes is a special case of fragmentation, and the
result is increasing sphericity (200). Apparently, phagocytic
fragmentation occurs when sensitized cells contact phagocytes, and/or
the release of erythrocyte contents as a result of mechanical injury
stimulates phagocytosis. In addition to fragmentation as a mechanism
for the termination of erythrocyte life-span, many investigators
have inquired whether decreasing metabolic capacity resulting from
a reduced amount of enzymes or substrates could prejudice survival
(156). Studies indicate that significant differences exist in ATP
concentration between the youngest and oldest erythrocytes separated
by centrifugation. These data and the preceding discussion of ATP

as a determinant of erythrocyte deformability, strongly suggest that

71
eventual erythrocyte destruction may be a consequence of age-related
decrease in ATP/Ca++ ratio due both to ATP depletion and calcium
accumulation (101). Recent analysis of various models of membrane
structure by Bull and Brailsford (27) suggests that a protein chain
meshwork, interacting to provide a skeletal network, would provide
mechanical properties corresponding to those observed in the erythro-
cyte. Skalak et al. (177) and Evans (56) have developed general
two-dimensional elastic material models to describe the continuum
mechanical behavior. Shape changes of the erythrocyte are signifi-
cant in their potential adverse modification of the rheologic prOper-
ties of the erythrocyte, and thus the survival of cells, as well as
the potential compromise of exchange processes at the capillary level.
An understanding of the mechanisms of shape change has importance
for the insights into structural organization and dynamics of the
membrane protein and lipid components and environmental factors,
which affect membrane properties and integrity. However, there is
no satisfactory model to account for the biconcave shape of the red
cell and its capacity to transform between the shapes of disc to
sphere despite the uniformity of the physical properties demonstrable

over the surface (56,64,102).

MATERIALS AND METHODS

Approximately ten milliliters of blood were drawn by venipunc-
ture from each of 44 subjects and from a control subject on the
day of testing. Thirty of these subjects were male blood donors
obtained from the American Red Cross and ranging in age from 18 to
59 years. The remaining 14 subjects (seven male and seven female)
were from the Lansing and Ann Arbor, Michigan, Senior Citizens'
Centers and ranged in age from 60 to 82 years. Heparinization was
achieved by drawing the blood into Vacutainer tubes containing pre-
measured amounts of sodium heparin (143 IU). Heparin was the anti-
coagulant of choice because its effect on erythrocyte osmotic
response is most similar to that of defibrination (23). Packed
cell volumes were determined using the microhematocrit method.
Hemoglobin (cyanmethemoglobin method) and red blood cell (RBC) counts
were determined using the Coulter Counter, Model ZF.a From these
values, red cell indices (MCV, MCH, MCHC) were calculated. Only
hematologically normal subjects (MCV = 76-96 cuu; MCH = 27-32 pug;
MCHC = 32—36% [175]) were included in the study. Each blood sample

was centrifuged at 2,500 rpm for 10 minutes in an International

Centrifuge (IEC) Universal Model UV,b the plasma was removed and

 

a . .
Coulter Electronics, Inc., Hialeah, FL.

b . . .
International Equipment Co., Needham Heights, MA.

72

73
the volume measured. A volume of each subject's red blood cells was
added to its respective plasma specimen to yield a packed cell
volume (PCV) of approximately nine percent. (This PCV value insured
that the optical density of the experimental supernatants would be
within the range of the spectrophotometer used.) The RBC osmotic
fragility test was then performed on each of the PCV-adjusted cell/
plasma suspensions by the following procedure. Four 16 x 100 mm
glass culture tubes were prepared for each of the following osmolali-
ties (mOsm/kg) of NaCl: 300, 130, 125, 120, 115, 110, 105 and 0
(distilled deionized water). The osmolality of each solution was
determined to within 1 1.0 mOsm/kg by the use of an Advanced Instru-
ments Model 3L Laboratory Osomometer.C The pH of each solution was
adjusted to 7.40 with NaOH using a Beckman Model SS-2 pH Meter.d
Reagents were analytical grade and distilled deionized water was
used for all solutions. A Fisher Model 241 Automatic Pipettere was
used to simultaneously expel 50 microliters of the previously pre-
pared RBC/plasma suspension and 5 milliliters of the appropriate NaCl
solution into an appropriately labeled test tube. Each test tube
was incubated for seven minutes at room temperature (22°C) and then
the hypotonic solution was returned to isotonicity with 200 micro-
liters of 4 M NaCl added with an Eppendorf pipette. The quadrupli-
cate samples were then centrifuged at 2,500 rpm for 10 minutes and
the supernatants were transferred to a duplicate set of test tubes.

The Optical density of each supernatant was analyzed by using a

 

cAdvanced Instruments, Inc., Newton Heighlands, MA.
d
Beckman Instruments, Inc., Fullerton, CA.

eFisher Scientific Co., Pittsburgh, PA.

74
Gilford Model 240 Spectrophotometerf at a wavelength of 414 nm. The
percentage of cells hemolyzed was determined as a proportion of the

total cells in the suspension as follows:

O.D. of supernatant solution
O.D. of RBC in distilled water (100% hemolysis)

The O.D. of the supernatant from RBC prepared in the same manner but
suspended in isotonic saline was subtracted from each O.D. value
obtained; this correction allowed for the small number of cells
traumatically lost by mechanical damage during the procedures.

Analysis of the osmotic fragility data was performed using a
Wang 'NXHB programmable calculator, for which the regression analysis
was adapted (48). Curves of fractional hemolysis (H) versus osmo-
lality of the test medium were linearized using the transform

1-H . .
h = In ———. RegreSSIOn lines were then calculated for curves of

H
h versus osmolality. From these results, the osmolality at which
50% of the erythrocytes in the suSpension hemolyzed (H50) was
determined using the equation

_ _ y-intercept
50 slope of regression line

Correlation coefficients were then calculated for H50 versus

age and slope of the regression line versus age.

 

f . . .
Gilford Instrument Laboratories, Inc., Oberlin, OH.

 

RESULTS

Statistically significant positive correlation was demonstrated

between osmotic fragility and age in healthy subjects between the

ages of 18 and 82 years (r = +0.35, p<0.05). Fifty percent hemolysis

(H50) occurred at osmolalities varying from 116 mOsm/kg to 136 mOsm/kg
(Figure 6 and Table 5).

Red cell variability, as expressed by the lepes of the osmotic

fragility curves, increased significantly with age (r

_ +0.50, p<0.01)
(Figure 7 and Table 5).

75

76

.muOOnosm

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coaosaom oceamm mo >uwacaoamo ocuv 0mm cow3uon mangOHumHmm .omm momuo> 0mm .b whomeh

77

 

 

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o:
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:8.
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>AHmOAmOHoumEa: mo mom one A>ueaflomflam> HHOO can we o>flumoeccev O>HDO >ueawmmum OHuOEmO one mo

macaw ozu camBuoo mflnmcoHumHom .amm msmao> o>azu wuwaemcum OADOEmO mo omoam .h ousmflm

t'8

79

.s. U18:

m¢<u> 2. U o (

00 OF 00 On 0' On ON
- b b n b p E e

 

IN.
U>¢D
>P-J.O(¢k WkozflO
k0 UtOi—fl
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80

TABLE 5.

 

COMPARISON OF AGE
WITH H50 AND SLOPE

OF OSMOTIC FRAGILITY CURVE

 

V P

SLOPE vs. AGE ‘O.5O p< 0.0l

DISCUSSION

In 1943, Newman and Gitlow (137) found that in Old age the
osmotic fragility of erythrocytes showed hardly any change; however,
a mixture of ammonium oxalate and potassium oxalate was used as the
anticoagulant and only heparinized and defibrinated blood are suitable
for osmotic fragility studies (213). In 1948, Olbrich (140) succeeded
with the aid of a more refined technique in demonstrating a slight
increase in fragility (15). In 1974, Detraglia et al. found dif-
ferences in osmotic fragility of erythrocytes between an elderly and
a young population. Age-related changes in erythrocyte osmotic
fragility were identified in the present study. These experiments
expanded the earlier work of Detraglia and this study refines osmotic
fragility measurements by the following methods: (1) sample varia—
bility was reduced by automatic pipetting; (2) pH-adjusted but
unbuffered saline solutions diminished buffer-induced aberration;

(3) the apparently sigmoid osmotic fragility curves were treated as
linear transforms to permit statistical treatment of results.

Since the dimensions of the cells with respect to volume and
hemoglobin concentration were unaffected by age, the increased
osmotic fragility could arise from (a) a reduction in mean cell
surface area, resulting in partial sphering, or (b) diminished
capacity of the membrane to withstand tensile stress. Classic

osmotic fragility theory denied the latter; however, the work of

81

82
Bowdler and Chan (20) showed prelytic changes to occur in the
membrane attributable to tensile stress less than that required for
lysis. A recent study supports the former. Hegner et al. (77)
report a decrease in erythrocyte membrane phospholipid content in
an older donor group compared with a younger donor group. This
reported decrease in membrane lipid would lead to a decrease in the
surface area/volume and would be consistent with increased osmotic
fragility (201). Both the decrease in membrane lipid and the
increase in osmotic fragility reflect a change from steady state
indicative of aging at the cellular level.

Red cell variability, as expressed by the slopes of the osmotic
fragility curves, is increased in red cells in Older subjects; thus,
the number of distinct types of erythrocytes in a given pOpulation
from an aging person has a greater variety of types of cells (45,135).
It is well known that the immune system undergoes drastic deteriora-
tive changes with age (187). The spleen, like most lymphoid tissues,
becomes smaller with age (15,21,59) concomitant with vascular archi-
tectural changes (6,187) manifest in a resultant decline in efficiency.
Conceivably the filtration function of the spleen becomes less
efficient. If this is the case, the spleen may not trap and remove
red blood cells as efficiently and may allow more cells that are
spheres (reflecting loss of membrane) to enter the general circula-
tion. This decline in the efficiency of the spleen with age, as
evidenced by a decrease in filtration ability and vascular archi-
tectural changes, would lend credence to the wear and tear theory of
aging. The results of this study could also be interpreted as
support for the immunologic theory of aging, in that one assumption

of this theory seems to be supported. The assumption is that the

83

basic impairment in the immune system is a loss in the ability
(e.g., of the spleen) to recognize slight deviations in molecular
structure and cellular characteristics (reflected by increased
osmotic fragility) so that cells which have undergone change
(e.g., spherocytes) and would ordinarily be destroyed by the immune
system are permitted to remain to the detriment of the animal.
Indeed, these rigid spheres may give rise to the initial insult to
vascular walls and this insult may lead to the formation of atheroma-
tous plaques and subsequently to atherosclerosis. If this is the
case, it would be support for the cardiovascular theory of aging.

Bjorksten (17) prOposes that the accumulation of cross-link
molecules is a primary cause of aging. Recent Observations on the
self-association of spectrin (190) and cross-linking of membrane
proteins in the red blood cell tend to suggest that the human red
blood cell membrane may serve as a model for the investigation of
the cross-linking theory of aging, and indeed may prove to be a

very suitable model of aging at the cellular level (43).

LIST OF REFERENCES

 

10.

ll.

LI ST OF REFERENCES

Adair, G. S.: The thermodynamic analysis of the observed
osmotic pressures of protein salts in solutions of finite
concentrations. Proc. Roy. Soc., Series A, 126, (1929):

16-24. 1

Adams, G.: Essentials of Geriatric Medicine. Oxford Univer-
sity Press, Oxford, 1977.

Adelman, R. C.: Age—dependent control of enzyme adaptation.
Adv. Gerontol. Res., 4, (1972): 1-23.

 

Alsbirk, P. H.: Corneal thickness. 1. Age variation, sex
difference and oculometric correlations. Acta Ophthalmologica,
56, (1978): 95-104.

Alwall, N.: Ageing kidney. Lancet, II, (1978): 635.

Andrew, W.: Age changes in the vascular architecture and cell
content in the spleen of 100 Wistar Institute rats including
comparisons with human material. Am. J. Anat., 79, (1946):
1-73.

Arey, L. B., M. J. Tremain, and F. L. Monzingo: The numerical
and topographical relation Of taste buds to human circum-
vallate papillae throughout the life-span. Anat. Rec., 64,
(1935): 9-25.

Auerbach, O., L. Garfinkel, and E. C. Hammond: Relation of
smoking and age to findings in lung parenchyma: A microscopic
study. Chest, 65, (1974): 29-35.

Baker, S. P., G. W. Gaffney, N. W. Shock, and M. Landowne:
Physiological responses of five middle-aged and elderly men to
repeated administration of thyroid stimulating hormone (thyro-
trOpin; TSH). J. Gerontol., 14, (1959): 37-47.

Ballas, S. K., and E. R. Burka: Pathways of de novo phospho-
lipid synthesis in reticulocytes. Biochim. Biophys. Acta, 337,
(1974): 239-247.

Ballas, S. K.: Disorders of the red cell membrane: A reclassi-
fication of hemolytic anemias. Am. J. Med. Sci., 276, (1978):
4-22.

84

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

85

Bean, W. 8.: Nail growth: Twenty-five years' Observation.
Arch. Intern. Med., 122, (1968): 359-361.

Beck, J. S.: Echinocyte formation: A test case for mechanisms
of cell shape changes. J. Theor. Biol., 71, (1978): 515-524.

Bennett, G. A., H. Waine, and W. Bauer: Changes in the Knee
Joint at Various Ages. With Particular Reference to the Nature
and Development of Degenerative Joint Disease. The Commonwealth
Fund, New York, 1942.

Betke, K.: Ageing changes in blood cells. In Structural
Aspects of Ageing, ed. G. H. Bourne, Hafner Publishing Co.,
Inc., New York, 1961, p. 229-245.

Bierman, E.L.: Fat metabolism, atherosclerosis and aging in
man: A review. Mech. Aging Dev., 2, (1973): 315-332.

Bjorksten, J.: The crosslinkage theory of aging. J. Am.
Geriat. Soc., 16, (1968): 408-427.

Blank, M.: Membrane proteins and membrane rheology. Bio-
rheology, 128, (1975): 89.

Blumenthal, H. T., and A. W. Berns: Autoimmunity and aging.
In Advances inicerontological Research, Vol. 1. Academic
Press, New York, 1964, p. 289-342.

Bowdler, A. J., and T. K. Chan: The time course of red cell
lysis in hypotonic electrolyte solutions. J. Physiol., 201,
(1969): 437-452.

Bowdler, A. J.: Blood and reticuloendothelial diseases. 2.
The spleen. Medicine, 31, (1977): 1745-1758.

Bowdler, A. J.: Membrane stability and the osmotic response
of red cells. Department of Health, Education, and Welfare
Public Health Service Grant Application, September 1, 1975.

Bowdler, N.: The effect of calcium on the osmotic fragility
of the human red cell. Unpublished data.

Bretscher, M. 5.: Human erythrocyte membranes: Specific
labelling of surface proteins. J. Mol. Biol., 58, (1971b):
775-781.

Brody, H.: Aging of the vertebrate brain. In Development and
Aging in the Nervous System, ed. M. Rockstein and M. L. Sussman,
Academic Press, New York, 1973, p. 121-133.

Bruckdorfer, K. R., J. M. Graham, and C. Green: The incorpora-
tion of steroid molecules into lecithin sols, B-lipoproteins
and cellular membranes. Eur. J. Biochem., 4, (1968): 512-518.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

86

Bull, B. S., and J. D. Brailsford: The biconcavity of the red
cell: An analysis of several hypotheses. Blood, 41, (1973):
833-844.

Carpenter, D. G., and J. A. Loynd: An integrated theory of
aging. J. Am. Geriat. Soc., 16, (1968): 1307-1322.

Carpenter, D. G.: Diffusion theory of aging. J. Gerontol.,

Castle, W. B.: In Pathologic Physiology: .Mechanisms of
Disease, 3rd ed., ed. W. A. Sodeman, Saunders, Philadelphia,
1961, p. 899.

Cohen, G., and P. Hochstein: Generation of hydrogen peroxide
in erythrocytes by hemolytic agents. Biochemistry, 3, (1964):
895-900.

Colomb, D.: Stellate spontaneous pseudoscars. Arch. Dermatol.,
105, (1972): 551-554.

Comfort, A.: The Biology of Senescence. Rinehart, New York,
1956.

Cooper, R. A., and J. H. Jandl: Bile salts and cholesterol in
the pathogenesis of target cells in obstructive jaundice.
J. Clin. Invest., 47, (1968): 809-822.

Cooper, R. A., and J. H. Jandl: The role of membrane lipids
in the survival of red cells in hereditary spherocytosis. J.
Clin. Invest., 48, (1969): 736-744.

Cooper, R. A., and J. H. Jandl: The selective and conjoint
loss of red cell lipids. J. Clin. Invest., 48, (1969): 906-
914.

C00per, R. A.: Lipids of human red cell membrane: Normal
composition and variability in disease. Seminars in Hematology,
7, (1970): 296-322.

Courtney, R.: Age changes in the periodontium and oral mucous
membranes. J. Mich. Dental Assoc., 54, (1972): 335-342.

Crosby, W. H.: Normal functions of the spleen relative to red
blood cells: A review. Blood, 14, (1959): 399-408.

Curtis, H. J.: Biological Mechanisms of Aging. Thomas,
Springfield, Illinois, 1966.

Damon, A.: Predicting age from body measurements and observa-
tions. Aging and Human Development, 3, (1972): 169-173.

 

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

87

Danielli, J. F.: The theory of penetration of a thin membrane.
In The Permeability of Natural Membranes, ed. H. Davson and J.

F. Danielli, Cambridge University Press, London, 1943, p. 341—

352.

Danon, D.: Biophysical aspects of red cell aging. In The
Physiology and Pathology of Human Aging, ed. R. Goldman and
M. Rockstein, Academic Press, New York, 1975.

Darmady, E. M., J. Offer, and M. A. Woodhouse: The parameters
of the aging kidney. J. Pathol., 109, (1973): 195-207.

Detraglia, M., F. B. Cook, M. Stasiw, and L. C. Cerny:
Erythrocyte fragility in aging. Biochim. Biophys. Acta, 345,
(1974): 213-219.

De Venuto, F.: Osmotic fragility and spontaneous lysis of
human red cells preserved with addition of progesterone. Proc.
Soc. Exp. Biol. Med., 128, (1968): 997-1000.

Dick, D. A.: Cell Water. Butterworths, Inc., Washington, D.C.,
1966.

Documenta Geigy - SCIENTIFIC TABLES, ed. K. Diem and C. Lentner,
Ciba-Geigy Limited, Basle, Switzerland, 1970.

Dintenfass, L.: Molecular and rheological considerations of
the red cell membrane in view of the internal fluidity of the
red cell. Acta Haematol., 32, (1964): 299-313.

Donlon, J. A., and A. Rothstein: The cation permeability of
erythrocytes in low ionic strength media of various tonicities.
J. Membrane Biol., I, (1969): 37-52.

Duane, A.: Accommodation. Arch. Ophthalmol., 5, (1931): 1-14.

Duke-Elder, S., and A. G. Leigh: System of Ophthalmology,
Vol. VIII. Diseases of the Outer Eye, Part 2. C. V. Mosby,
St. Louis, 1965.

Dunn, M. J.: Red cell calcium and magnesium: Effects upon
sodium and potassium transport and cellular morphology.
Biochim. Biophys. Acta, 352, (1974): 97-116.

Dyson, R. D.: Cell Biology - A Molecular Approach. Allyn and
Bacon, Inc., Boston, 1974, p. 286-293.

Engelhardt, R.: Impaired reassemblance of red blood cell
membrane components in hereditary spherocytosis. In Membranes
and Disease, ed. L. Bolis, J. F. Hoffman and A. Leaf, Raven
Press, New York, 1976, p. 75-80.

Evans, E.A.: A new material concept for the red cell membrane.
Biophys. J., 13, (1973): 926-939.

 

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

88

Fairbanks, G., T. L. Steck, and D. F. H. Wallach: Electro—
phoretic analysis of the major polypeptides of the human
erythrocyte membrane. Biochemistry, 10, (1971): 2606-2617.

Farnsworth, P., D. Danon, and A. Gellhorn: The effect of fat-
free diet on the rat erythrocyte membrane. Brit. J. Haematol.,
2, (1965): 200-205.

Finch, C. E., and L. Hayflick: Handbook of the Biology of
Aging. Van Nostrand Reinhold Co., New York, 1977.

Fischer, T. M., C. W. M. Haest, M. St6hr, D. Kamp, and B.
Deuticke: Selective alteration of erythrocyte deformability

by SH-reagents. Evidence for an involvement of spectrin in
membrane shear elasticity. Biochim. Biophys. Acta, 510, (1978):
270-282.

Franck, J., and R. Platzman: Physical principles underlying
photochemical, radiation-chemical and radiobiological reactions.
Radiat. Biol., 1, Part I, (1954): 191-253.

Franklin, E. C., and D. Zucker-Franklin: Current concepts of
amyloid. Adv. Immunol., 15, (1972): 249-304.

Frolkis, V. V.: Neuro-humoral regulations in the aging
organism. J. Gerontol., 21, (1966): 161-167.

Fung, Y. C., and P. Tong: Theory of the sphering of red blood
cells. BiOphys. J., 8, (1968): 175-198.

Gaubatz, J., N. Prashad, and R. G. Cutler: Ribosomal RNA gene
dosage as a function of tissue and age for mouse and human.
Biochim. BiOphys. Acta, 418, (1976): 358-375.

Gomperts, E. D., J. Metz, and S. S. Zail: A red cell membrane
protein abnormality in hereditary spherocytosis. Brit. J.
Haematol., 23, (1972): 363-370.

Gorter, E., and F. Grendel: Bimolecular layers of lipids on
chromocytes of blood. J. Exp. Med., 41, (1925): 439-443.

Gozna, E. R., A. E. Marble, A. Shaw, and J. G. Holland: Age—
related changes in the mechanics of the aorta and pulmonary
artery of man. J. Appl. Physiol., 36, (1974): 407-411.

Greenblatt, R. B.: Estrogen therapy for postmenopausal females.
New Eng. J. Med., 272, (1965): 305-308.

Greenquist, A. C., and S. B. Shohet: Defective protein phos-
phorylation in membranes of hereditary spherocytosis erythrocytes.
FEBS Lett., 48, (1974): 134-135.

Guidotti, G.: The composition of biological membranes. Arch.
Intern. Med., 129, (1972): 194-201.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

89

Gutman, E., and V. Hanzlikova: Basic mechanisms of aging in
the neuromuscular system. Mech. Age. Dev., 1, (1972): 327-349.

Haradin, A. R., R. I. Weed, and C. F. Reed: Changes in physical
properties of stored erythrocytes: Relationship to survival
in vivo. Transfusion, 9, (1969): 229-237.

Harman, D.: Free radical theory of aging: Effect of free
radical reaction inhibitors on the mortality rate of male
LAFl mice. J. Gerontol., 23, (1968): 476-482.

Harrison, D. G., and C. Long: The calcium content of human
erythrocytes. J. Physiol., 199, (1968): 367-381.

Hayflick, L.: The limited in vitro lifetime of human diploid
cell strains. Exp. Cell Res., 37, (1965): 614-636.

Hegner, D., D. Platt, H. Heckers, U. Schloeder, and V.
Bruninger: Age-dependent physicochemical and biochemical
studies of human red cell membranes. Mech. Age. Dev., 10,
(1979): 117-130.

Heidrick, M. L., and T. Makinodan: Nature of cellular deficien-
cies in age-related decline of the immune system. Gerontologia,
18, (1972): 305-320.

Hernandez, J. A., A. B. Anderson, W. L. Holmes, N. Morrone, and
A. G. Foraker: The bronchial glands in aging. J. Am. Geriat.

Hershel, J., et al.: Replacement estrogens and endometrial
cancer. New Eng. J. Med., 300, (1979): 218-222.

Jackson, D., and P. R. J. Burch: Dental caries as a degenera-
tive disease. Gerontologia, 15, (1969): 203-216.

Jacob, H.: Dysfunctions of the red cell membrane. In The Red
Blood Cell, 2nd Ed., Vol. 1, ed. Douglas Mac N. Surgenor,
Academic Press, New York, 1974, p. 270-292.

Jacob, H. S., A. Ruby, E. S. Overland, et al.: Abnormal
membrane protein of red blood cells in hereditary spherocy-
tosis. J. Clin. Invest., 50, (1971): 1800-1805.

Jacob, H., T. Amsden, and J. White: Membrane microfilaments
of erythrocytes: Alteration in intact cells reproduces the

hereditary spherocytosis syndrome. Proc. Natl. Acad. Sci.,

69, (1972): 471-474.

Jacob, H.: Tigentening red-cell membranes. New Eng. J. Med.,
294, (1976): 1234-1235.

Jacobs, M. H., and S. A. Corson: The influence of minute
traces of c0pper on certain hemolytic processes. Biol. Bull.,
67, (1934): 325-326.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

90

Jain, M. K.: Role of cholesterol in biomembranes and related
systems. In Current Topics in Membranes and Transport, Vol. 6,
ed. F. Bronner and A. Kleinzeller, Academic Press, New York,
1975, p. 1-57.

Jay, A. W. L., and S. Rowlands: The stages of osmotic haemoly-
sis. J. Physiol., 252, (1975): 817-832.

Johnsson, R.: Red cell membrane proteins and lipids in sphero-
cytosis. Scand. J. Haematol., 20, (1978): 341-350.

Kalant, N., S. Satomi, R. White, and E. Tel: Changes in renal
glomerular basement membrane with age and nephritis. Canad.
J. Biochem., 55, (1977): 1197-1206.

Keith, A. D., and W. Snipes: Dynamic aspects of biological
membranes. In Horizons in Biochemistry and Biophysics, Vol. 4,
ed. E. Quagliariello, F. Palmeri, and T. P. Singer, Addison-
Wesley Pub. Co., Inc., Advanced Book Program, Reading, Mass.,
1977, p. 31-62.

Kent, S.: The aging lung. Part 1. Loss of elasticity.
Geriatrics, 33, (1978): 124-132.

Kent, S.: The aging lung. Part 2. Decline of pulmonary
function. Geriatrics, 33, (1978): 100-111.

Kety, S. S.: Human cerebral blood flow and oxygen consumption
as related to aging. J. Chronic Diseases, 3, (1956): 478-486.

Kohn, R. R.: Principles of Mammalian Aging. Prentice-Hall,
Englewood Cliffs, New Jersey, 1971.

Krag, C. L., and Kounty, W. B.: Stability of body function in
the aged. II. Effect of exposure of the body to heat. J.
Gerontol., 7, (1952): 61-70.

Kramsch, D. M., and W. Hollander: Interaction of serum and
arterial lipoproteins with elastin of the arterial intima and
its role in the lipid accumulation in atherosclerotic plaques.
J. Clin. Invest., 52, (1973): 236-247.

LaCelle, P., and A. Rothstein: The passive permeability of
the red blood cell to cations. J. Gen. Physiol., 50, (1966):
171-188.

LaCelle, P. L.: Alteration of membrane deformability in
hemolytic anemias. Seminars in Hematology, 7, (1970): 355-371.

LaCelle, P. L., and R. I. Weed: Reversibility of abnormal
deformability and permeability of the hereditary spherocyte.
Blood, 34, (1969): 858.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

91

LaCelle, P- L., and R. I. Weed: The contribution of normal and
pathologic erythrocytes to blood rheology. Prog. Hematol., 7,
(1971): 1-31.

LaCelle, P. L., R. I. Weed, and P. A. Santillo: Pathophysio-
logic significance of abnormalities of red cell shape. In
Membranes and Disease, ed. L. Bolis, J.F. Hoffman, and A. Leaf,
Raven Press, New York, 1976, p. 1-17.

Lamb, M. J.: Biology of Aging. A Halsted Press Book. John
Wiley and Sons, New York-Toronto, 1977, p. 2.

Langley, G. R., and M. Axell: Changes in erythrocyte membrane
and autohaemolysis during in vitro incubation. Brit. J.
Haematol., 14, (1968): 593—603.

Limber, G. K., R. F. Davis, and S. Bakerman: Acrylamide gel
electrophoresis of the human erythrocyte membrane. Blood,
36, (1970): 111-118.

Lin, G. S. B., and R. I. Macey: Shape and stability changes in
human erythrocyte membranes induced by metal cations. Biochim.
BiOphys. Acta, 512, (1978): 270-283.

Lindner, J., and G. Johannes: Contribution on the ageing of
arterial connective tissue. In Connective Tissue and Aging,
ed. H. G. Vogel, Excerpta Medica, Amsterdam, 1973, p. 68-78.

Lux, S. E., and K. M. John: Diminished spectrin extraction
from ATP-depleted human erythrocytes. Evidence relating
spectrin to changes in erythrocyte shape and deformability.
J. Clin. Invest., 61, (1978): 815-827.

Maddy, A. H.: Erythrocyte membrane proteins. Seminars in
Hematology, 7, (1970): 275-295.

Mann, 0. N. A., and P. O. Yates: Lipoprotein pigments -
Their relationship to ageing in the human nervous system. I.
The lipofuscin content of nerve cells. Brain, 97, (1974):
481-488.

Manson, J. D., and R. B. Lucas: A microradiographic study of
age changes in the human mandible. Arch. Oral Biol., 7,
(1962): 761-769.

Marchesi, V. T., and E. Steers: Selective solubilization of
a protein component of the red cell membrane. Science, 159,
(1968): 203-204.

Marchesi, V. T., T. w. Tillack, R. L. Jackson, et al.: Chemical
characterization and surface orientation of the major glyco-
protein of the human erythrocyte membrane. Proc. Natl. Acad.
Sci., 69, (1972): 1445-1449.

92

114. Marcus, D. M., and L. E. Cass: Glycosphingolipids with Lewis
blood group activity: Uptake by human erythrocytes. Science,
164, (1969): 553-555.

115. Marikovsky, Y., J. K. Khodadad, and R. S. Weinstein: Influence
of red cell shape on surface charge topography. Exp. Cell
Res., 116, (1978): 191-197.

116. Marks, P. A., A. Gellhorn, and C. Kidson: Lipid synthesis in
human leukocytes, platelets and erythrocytes. J. Biol. Chem.,
235, (1960): 2576—2583.

117. Maynard Smith, J.: Review lectures on senescence. I. The
causes of ageing. Proc. Roy. Soc. (Biol. Sci.), 157, (1962):
115-127.

118. Maynard Smith, J.: Theories of aging. In prics in the
Biology of Aging, ed. P. L. Krohn, Interscience Publishers,

119. McGrady, P. M., Jr.: The Youth Doctors. Coward-McCann, New
York, 1968.

120. Medawar, P. B.: An unsolved problem of biology. Lewis,
London, 1951. (An inaugural lecture delivered at University
College, London.)

121. Medvedev, Z. A.: The nucleic acids in development and aging.
In Advances in Gerontological Research, ed. B. L. Strehler,
Academic Press, New York, Vol. 1, p. 181-206.

122. Miale, J. B.: Laboratory Medicine Hematology. The C. V. Mosby
Co., St. Louis, 1977.

123. Ibid., p. 680.

124. Miller, J. H., and N. W. Shock: Age differences in the renal
tubular response to antidiuretic hormone. J. Gerontol., 8,
(1953): 446-450.

125. Miyahara, T., S. Shiozawa, and A. Murai: Effect of age on
amino acid composition of human skin collagen. J. Gerontol.,
33, (1978): 498-503.

126. Mohandas, A. C., S. B. Shohet, and A. C. Greenquist: Effects
of heat and metabolic depletion on erythrocyte deformability,
spectrin extractability and phosphorylation. In The Red Cell.
Progress in Clinical and Biological Research, Vol. 21, ed. G. J.
Brewer, Alan R. Liss, Inc., New York, 1977, p. 453-477.

127. Montagna, W., and P. F. Parakkal: The Structure and Function
of Skin, 3rd Ed. Academic Press, New York, 1974.

128. Montgomery, P. W.: A study of exfoliative cytology of normal
human oral mucosa. J. Dental Res., 30, (1951): 12-18.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

93

Moore, T. J.: Glycerol permeability of human fetal and adult
erythrocytes and of a model membrane. J. Lipid Res., 9, (1968):
642-646.

Morgan, D. B., and H. F. Newton-Jones: Bone loss and senescence.
Gerontologia, 15, (1969): 140-154.

Murphy, J. R.: Erythrocyte metabolism. IV. Equilibration of
cholesterol-4-C14 between erythrocytes and variously treated
sera. J. Lab. Clin. Med., 60, (1962): 571-578.

Murphy, J. R.: Erythrocyte metabolism. VI. Cell shape and
the location of cholesterol in the erythrocyte membrane. J.
Lab. Clin. Med., 65, (1965): 756-774.

Nakao, K., T. Wada, and T. Kamiyama: A direct relationship
between adenosine triphosphate-level and in vivo viability of
erythrocytes. Nature, 194, (1962): 877-878.

Nakao, M., T. Nakao, and S. Yamazoe: Adenosine triphosphate
and maintenance of shape of the human red cells. Nature, 187,
(1960): 945-946.

Neerhout, R. C.: Disorders of the red cell membrane: A review
of biochemical and physiologic alterations of erythrocyte
membranes which may lead to morphologic changes and shortened
cell survival. Clin. Pediat., 7, (1968): 451-464.

Nelson, G. J.: Composition of neutral lipids from erythrocytes
of common mammals. J. Lipid Res., 8, (1967): 374-379.

Newman, B.. and Gitlow, S.: Blood studies in the aged - The
erythrocyte in the aged male and female. Am. J. Med. Sci.,
205, (1943): 677-687.

Norris, A. H., N. W. Shock, M. Landowne, and J. A. Falzone, Jr.:
Pulmonary function studies: Age differences in lung volumes
and bellows function. J. Gerontol., 11, (1956): 379-387.

Olansky, 8.: Background paper: Medical Research on Aging,
White House Conference on Aging, Washington, D.C., 1961.

Olbrich, 0.: Blood changes in the aged. Part III. Edinb.
Med. J., N053, 55, (1948): 100-115.

Olmstead, E. G.: Mammalian Cell water. Physiologic and
Clinical Aspects. Lea and Febiger, Philadelphia, 1966, p.
55-68.

Orgel, L.E.: The maintenance of the accuracy of protein synthe-
sis and its relevance to ageing. Proc. Natl. Acad. Sci., 49,
(1963): 517-521.

Palek, J., P. A. Liu, and S. C. Liu: Polymerisation of red
cell membrane protein contributes to spheroechinocyte shape
irreversibility. Nature, 274, (1978): 505-507.

94

144. Palek, J., W. A. Curby, and F. J. Lionetti: Relation of
Ca++-activated ATPase to Ca++-linked shrinkage of human red
cell ghosts. Am. J. Physiol., 220, (1971): 1028-1032.

145. Parker, J. C., and J. F. Hoffman: Interdependence of cation
permeability, cell volume and metabolism in dog red cells.
Fed. Proc., 24, (1965): 589.

146. Partridge, S. M.: Biological role of cutaneous elastin. In
Advances in Biology of Skin, Vol. 10, ed. W. Montagna, J. P.
Bentley, and R. L. Dobson, Meredith Corporation, New York,
1970, p. 69-87.

147. Pennell, R. B.: Composition of normal human red cells. In
The Red Blood Cell, 2nd Ed., ed. Douglas Mac N. Surgenor,
Vol. 1, Academic Press, New York, 1974, p. 93-146.

148. Ponder, E., and E. J. Robinson: The measurement of red cell
volume. V. The behaviour of cells from oxalated and from
defibrinated blood in hypotonic plasma and saline. J. Physiol.,
83, (1934): 34-48.

149. Ponder, E.: Hemolysis and Related Phenomena. Grune and
Stratton, New York and London, 1948.

150. Ponder, E.: The prolytic loss of K+ from human red cells.
J. Gen. Physiol., 30, (1947): 235-246.

151. Ponder, E.: Volume changes, ion exchanges and fragilities of
human red cells in solutions of the chlorides of the alkaline
earths. J. Gen. Physiol., 36, (1953): 767-775.

152. Pozefsky, T., J. L. Colker, H. M. Langs, and R. Andres: The
cortisone-glucose tolerance test: The influence of age on
performance. Ann. Internal Med., 63, (1965): 988-997.

153. Poznansky, M., and A. K. Solomon: Effect of cell volume on
potassium transport in human red cells. Biochim. BiOphys.

154. Price, G. B., and T. Makinodan: Aging: Alteration of DNA-
protein information. Gerontologia, 19, (1973): 58-70.

155. Rakow, A. L., and R. M. Hochmuth: Thermal transition in the
human erythrocyte membrane: Effect of elasticity. Bio-
rheology, 12, (1975): 1-3.

156. Ramot, B., F. Brok, and L. Ben-Bassat: Alterations in the
metabolism of human erythrocytes with aging. Plenary Session
Papers: XII, Cong. Int. Hematol., New York, 1968.

157. Robertson, J. D.: The ultrastructure of cell membranes and
their derivatives. Biochem. Soc. Symp., 16, (1959): 3-43.

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

95

Romualdez, A., R. I., Sha'afi, Y. Lange, and A. K. Solomon:
Cation transport in dog red cells. J. Gen. Physiol., 60,
(1972): 46-57.

Rosenthal, A. S., E. M. Kregenow, and H. L. Moses: Some char-
acteristics of a Ca2+-dependent ATPase activity associated
with a group of erythrocyte membrane proteins which form
fibrils. Biochim. Biophys. Acta, 196, (1970): 254-262.

Rosing, D. R., D. R. Redwood, P. Brakman, T. Astrup, and S. E.
Epstein: Impairment of the diurnal fibrinolytic response in
man: Effects of aging, type IV hyperlipoproteinemia, and
coronary artery disease. Circulation Res., 32, (1973): 752-
758.

Ryan, K. J., and D. C. Gibson, eds.: Menopause and Aging.
U. 8. Government Printing Office, DHEW Publ. No. [NIH],
(1973): 73-319.

Sackmann, E., O. Albrecht, W. Hartmann, and H. J. Galla:

Domain formation in lipid bilayers and biological membranes.

In Structural and Kinetic Approach to Plasma Membrane Functions,
ed. C. Nicolau and A. Paraf, Springer-Verlag, New York, 1977,

p. 28-44.

 

Selye, H.: The future for aging research. In Perspectives
in Experimental<3erontologyr ed. N. W. Shock, Charles C.
Thomas, Springfield, Illinois, 1966, p. 375-385.

Sha'afi, R. I., and J. J. Hajjar: Sodium movement in high
sodium feline red cells. J. Gen. Physiol., 57, (1971): 684-
696 O

Sheetz, M. P.: Cation effects on cell shape. In Cell Shape
and Surface Architecture. Progress in Clinical and Biological
Research, Vol. 17. Alan R. Liss, Inc., New York, 1977, p. 559-
567.

Sheetz, M. P.,.D. Sawyer, and S. Jackowski: ATP-dependent red
cell membrane shape change - A molecular explanation. In The
Red Cell. Progress in Clinical and Biological Research, Vol.
21, ed. G. J. Brewer, Alan R. Liss, Inc., New Y ork, 1978,

p. 431-451.

Shinitzky, M., S. Moses, and A. Livne: Elevated microviscosity
in membranes of erythrocytes affected by hereditary spherocy-
tosis. Brit. J. Haematol., 31, (1975): 117-123.

Shock, N. W.: Biological theories of aging. In Handbook of
the Psychology of Aging, ed. J. E. Birren and K. W. Schoie,
Van Nostrand Reinhold Co., New York, 1977, p. 103-115.

Shock, N. W.: Metabolism and age. J. Chronic Diseases, 2,
(1955): 687-703.

170.

171.

172.

173.

174.

175.

176.

177.

178.

179.

180.

181.

182.

183.

96

Shock, N.W.: Physiological aspects of aging in man. Ann.
Rev. Physiol., 23, (1961): 97-122.

Shock, N. W.: Physiological theories of aging. In Theoretical
Aspects of Aging, ed. M. Rockstein, Academic Press, New York,
1974, p. 119-136.

Shohet, S. B.: Mechanisms of red cell membrane lipid renewal.
In Membranes and Disease, ed. L. Bolis, J. F. Hoffman, and
A. Leaf, Raven Press, New York, 1976, p. 61-74.

Siefring, G. E., A. B. Apostal, P. T. Velasco, and L. Lorand:
Enzymatic basis for the Ca2+-induced cross-linking of membrane
proteins in intact human erythrocytes. Biochemistry, 17,
(1978): 2598-2604.

Silverstone, F. A., M. Brandfonbrener, N. W. Shock, and M. J.
Yiengst: Age differences in the intravenous glucose tolerance
tests and the response to insulin. J. Clin. Invest., 36,
(1957): 504-514.

Simmons, A.: Technical Hematology. J. B. Lippincott Company,
Philadelphia, 1976, p. 459.

Singer, S. J., and G. L. Nicolson: The fluid mosaic model of
the structure of cell membranes. Science, 175, (1972): 720-731.

Skalak, R., A. Tozeren, R. P. Zarda, and S. Chien: Strain
energy function of red blood cell membranes. Biophys. J., 13,
(1973): 245—263.

Solomon, R. D.: Biology and pathogenesis of vascular disease.
Advan. Geront. Res., 2, (1967): 285-354.

Stockwell, R. A.: The cell density of human articular and
costal cartilage. J. Anat., 101, (1967): 753-763.

Strehler, B.: Immortality (in Essays of Finalists in 3rd
Westinghouse Science Talent Search). Science Service,
Washington, D.C., 1943, p. 71-73.

Strehler, B. L., and C. H. Burrows: Senescence. Cell biologi-
cal aspects of aging. In Cell Differentiation, ed. 0. A.
Schjeide and J. deVallis, Van Nostrand Reinhold, New York,
1970, p. 266-283.

Strehler, B. L., D. Mark, A. S. Mildvan, and M. Gee: Rate
and magnitude of age pigment accumulation in the human myocar-
dium. J. Gerontol., 14, (1959): 430-439.

Strehler, B. L.: Dynamic theories of aging. In Aging: Some
Social and Biological Aspects, ed. N. W. Shock, Publ. No. 65,
Am. Assoc. Adv. Sci., Washington, D.C., 1960, p. 273-303.

184.

185.

186.

187.

188.

189.

190.

191.

192.

193.

194.

195.

196 O

197.

97

Strehler, B. L., ed.: The Biology of Aging. Publ. No. 6,
Am. Inst. Biol. Sci., Washington, D.C., 1960.

Strehler, B. L.: On the histochemistry and ultrastructure of
age pigment. In Advances in<§erontological Research, Vol. 1,
ed. B. L. Strehler, Academic Press, New York, 1964, p. 343-384.

Strehler, B. L.: Origin and comparison of the effects of time
and high-energy radiations on living systems. Q. Rev. Biol.,
34, (1959): 117-142.

Strehler, B. L.: Time, Cells and Aging, 2nd Ed. Academic
Press, New York, 1977.

Tanner, M. J.: Erythrocyte glyc0proteins. In Current Topics
in Membranes and Transport, Vol. 11. Academic Press, New York,
1978, p. 279-325.

Triantaphyllopoulos, D. C., and E. H. Krikke: Effect of
plasma proteins on the fragility of erythrocytes. Canad. J.
Physiol. Pharmacol., 44, (1966): 401-406.

Ungewickell, E., and W. Gratzer: Self-association of human
spectrin. Eur. J. Biochem., 88, (1978): 379-385.

Van Deenen, L. L. M., and J. De Gier: Lipids of the red cell
membrane. In The Red Blood Cell, 2nd Ed., ed. Douglas Mac N.
Surgenor, Vol. 1, Academic Press, New York and London, 1974,
p. 147-211.

Vanderkooi, G., and D. E. Green: Biological membrane structure.
I. The protein crystal model for membranes. Proc. Natl.
Acad. Sci., 66, (1970): 615-621.

Van Gastel, C., D. Van Den Berg, J. De Gier, and L. L. M.

Van Deenen: Some lipid characteristics of normal red blood
cells of different age. Brit. J. Haematol., II, (1965): 193-
199.

Verzér, F.: The aging of collagen. Sci. Am., 208, (1963):
104-114.

Vincenzi, F. F., and T. R. Hinds: Plasma membrane calcium
transport and membrane-bound enzymes. In The Enzymes of
Biological Membranes,Vol. 3, ed. A. Martonosi, Plenum Press,
New York, 1976, p. 261-281.

Walford, R. L.: The Immunologic Theory of'Aging. Williams and
Wilkins, Baltimore, 1969.

Watson, J. D.: The Double Helix. New American Library, New
York, 1969.

198.

199.

200.

201.

202.

203.

204.

205.

206.

207.

208.

209.

210.

98

Ways, P., and D. J. Hanahan: Characterization and quantifica-
tion of red cell lipids in normal man. J. Lipid Res., 5,
(1964): 318-328.

Ways, P.: Degradation of glycerophosphatides during storage
of saline-washed, saline suspended red cells at -20°C. J.
Lipid Res., 8, (1967): 518-521.

Weed, R. I., and C. F. Reed: Membrane alterations leading to
red cell destruction. Am. J. Med., 41, (1966): 681-698.

Weed, R. I.: Disorders of red cell membrane: History and
perspectives. Seminars in Hematology, 7, (1970): 249-258.

Weed, R. I., P. L. LaCelle, and E. W. Merrill: Metabolic
dependence of red cell deformability. J. Clin. Invest., 48,
(1969): 795-809.

Weinstein, R. S., and N. S. McNutt: Ultrastructure of red
cell membranes. Seminars in Hematology, 7, (1970): 259-274.

Weinstein, R. S., J. K. Khodadad, and T. L. Steck: Ultrastruc-
tural characterization of proteins at the natural surfaces of
the red cell membrane. In The Red Cell. Progress in Clinical
and Biological Research, Vol. 21, ed. G. J. Brewer, Alan R.
Liss, Inc., New York, 1978, p. 413-427.

Weisberg, H. F.: Osmotic pressure of the serum proteins.
Ann. Clin. Lab. Sci., 8, (1978): 155-164.

Weiss, A. D.: Auditory perception in relation to age. In
Human Aging, ed. J. Birren, 0.8. Government Printing Office,
Washington, D.C., 1963, p. 111-140.

Wessels, J. M. C., and J. H. Veerkamp: Some aspects of the
osmotic lysis of erythrocytes. II. Differences in osmotic
behaviour of erythrocytes after treatment with electrolyte
and non-electrolyte solutions. Biochim. BiOphys. Acta, 291,
(1973): 178-189.

Wessels, J. M. C., and J. H. Veerkamp: Some aspects of the
osmotic lysis of erythrocytes. III. Comparison of glycerol
permeability and lipid composition of red blood cell membranes
from eight mammalian Species. Biochim. Biophys. Acta, 291,
(1973): 190-196.

Wessels, J. M. C., D. T. F. Pals, and J. H. Veerkamp: Some
aspects of the osmotic lysis of erythrocytes. I. A reexamina-
tion of the osmotic lysis method. Biochim. Biophys. Acta, 291,
(1973): 165-177.

Wheeler, K. T., and J. T. Lett: On the possibility that DNA
repair is related to age in non-dividing cells. Proc. Natl.
Acad. Sci., 71, (1974): 1862-1865.

211.

212.

213.

214.

99

Whittam, R.: Transport and Diffusion in Red Blood Cells.
Arnold Ltd., London, 1964, p. 2.

Williams, A. R.: Viscoelasticity of the human erythrocyte
membrane. Biorheology, 10, (1973): 313-319.

Williams, W. J.: Hematology. McGraw-Hill Book Co., New York,
1977.

Zail, S. S.: The erythrocyte membrane abnormality of heredi-
tary spherocytosis. (Annotation). Brit. J. Haematol., 37,
(1977): 305-310.

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