BIOCHEMICAL APPROACHES TO AGING: Norton Rothstein
CONFOUNDING VARIABLES: MAMMALIAN SPECIES
1. Short-lived strains might not
manifest age related conditions.
2. Strain differences produce possibly
different aging characteristics.
3. Diet and other conditions can
cause variation in life span even of the same strain of rats.
4. Rats should be raised pathogen
free (barrier-reared). Such animals tend to be longer lived,
thus implicating infectious agents in shortening life span.
5. Some strains of rats with age
tend to be lean while others become obese.
6. Environmental factors such as crowding affect longevity.
CONFOUNDING VARIABLES: CELLS IN CULTURE
1. While cells may be derived
from almost any species and any tissue type, and may be stored
for long periods in liquid nitrogen; there is an unproven relationship between
cells in culture and
aging in vivo.
2. The number of population
doublings drops significantly (40 to 28 using skin for 15 versus
75-year olds, though less if diabetics are removed) its relevance to aging
is not clear, for if an
aged subject undergoes even 70% of their original potential for doubling,
there does not appear
to be a serious danger of running out of cells.
3. Because SV-40 virus can
transform even late-passage (where there is a stead decline in the
number of cells doubling) into an "immortal" line, the relation between
cell aging and aging is
even further obscured.
loose specialize cells and are repopulated by stem cells regardless of the tissue of origin. This
it hard to correlate such in vitro cells, with in
As cells divide and redivide there occurs a selection in which specialized cells tend to die out, and the
culture adapts to the medium.
can be significant variation in cells from biopsy to biopsy, even in the same subject.
7. Cells are being stressed in vitro to highly unnatural conditions thus possibly confounding a relationship
to aging in vivo.
8. It has been repeatedly shown that the life-span of cells in vitro are determined by the cumulative number
of population doublings and not
the calendar time in culture-counter
to in vivo.
cultures, even from cloning experiments are heterogeneous; i.e., some are dividing rapidly, some
and some not dividing.
No one has yet succeeded in making non-dividing cells divide normally.
11. It has been found in human skin fibroblast that the cells centrally located existed at a much lower
generation level than those
at the circumference, which divided more frequently. This might help explain
the differences in pro-life active
capacity found in sub cultured ceils.
12. Serum used in culture medium, trypsin used to free cells from culture vessels and other conditions might
affect culture growth.
Cortisone and hydrocortisone extended life span of human cells in culture. Early
passage cells where most
responsive, yielding a 30-40% extension. It was inhibitory, or less effective on older cells, presumable because such
cells process decreased (about 40%) sites.
14. Only 2%of cells from old
donors could produce colonies of 256 cells or more, whereas
the majority of cells from young donors could do so.
PROPERTIES OF SKIN FRBROBLAST CULTURES FROM YOUNG & OLD
Onset of senescent phase (PD)a 35.2 ± 2.1 22.5
In vitro life-span (PD)
44.6 ± 2.5 33.6 2.1
Cell population replication rate, hrs
20.8 +_ .8 24.3 ± .9
percentage replicating cells 87.1 ± 1.6
79.6 ± 2.5
Cell number at confluencyb 7.31 ± .42
5.06 ± .52
Percent of cells able to form 69.0
48.0 ± 4.4
colony of 16 cells
Sister chromatid exchanges/cell 67.9
± 1.6 56.1 ± 1.4
b. ability to
maintain population, 10 cells/cm
weeks after plating at low cell densities
14. In vitro cells do not as in vivo
cells do have increase in content and size of tRNA.
15. Similarly for residual bodies
there is no increase in aged donors.
16. Unfortunately when preparing in vivo cells they are generally passage
a few times before
use, thus making the difference between the two obscure.
17. Confluence is used to determine Phase III. A cell population is harvested when a monolayer
covers a vessel (confluency), after about 45 doublings (this period termed
phase II) the cells will
not reach confluence, their number decreasing with each passage, Phase III.
18. A number of biochemical changes
have been observed, much more needs to be done. No distinction was made as to
whether the change occur in Phase II or Phase III cells, and some studies do not differentiate age of donor.
19. The relationship between
life span and potential for population doublings has not yielded
any conclusive results from the few studies that have been done.
20. Experiments attempting
to show weither the control of the potential for doubling resides in
the cytoplasm or nuclei have proven inconclusive. For example, such factor for the WI-38 cells
appears either to lie in either the cytoplasm or in activators of the
nucleus. These where
determined in transformed cell experiment in which parts of Phase II and
Phase III cells where
21. With tissue transplants,
the age of the donor does not seem to be important, but rather the age
of the recipient: none did well with old recipients. Grafts have survived
nearly 2 times the life span
of the animals, and others have obtained in longer lasting results. Affects of the procedure
infusions of blood vessels and connective tissue confound conclusions.
22. Red blood cells, particularly human erythrocytes, since they cannot
components for they are without nucleus, must survive on its original complement
of structural and function elements. The subsequent deterioration of enzymes
and membranes during its life span
of 120-130 days can be regarded as a model for deteriorative processes in
aging. The lack of
and spec deteriorative mechanisms are present confound conclusions.
23. It is difficult to resolve if related changes observed in experiments
are reflective of their
significance in humans, plus the extremely subtle nature of the changes
make progress difficult.
1. The generation of free radicals
occurs in biological systems and can be brought about by
ionizing radiation, UV radiation or by certain enzymatic and nonenzymatic reactions.
2. Biological systems are protected
from these effects b; a number of radical scavenging
molecules and by enzyme systems designed to remove superoxide radicals or
which result from free radical reactions.
3. Skin spots are one sign of the
incomplete protection these systems afford.
4. Large doses of dietary antioxidants
can function to reduce the amount of free radicals,
although extension of life span is not affected — at least in mammals.
5. Much more basic information remains
to be obtained before on can begin to put even a tentative picture as to what effect lipid metabolism has on aging and vice
6. In addition to free radicals,
superoxide radicals are generated by a number of processes both
biological and chemical, including auto oxidation of hydroquinone, leukoflavins,
catecholamines, thiols, reduced dyes, tetra-hydropteridines, and ferredoxins,
and as for
enzymes they include xanthine oxidase, aldehyde oxidase, NADH and NADP oxidase,
flavoprotein dehydrogenases, etc.
7. Lipid peroxidation results in
the formation of
malonaldehyde and thence age pigment.
8. Free radicals have
the potential to initiate in addition to malonaldehyde formation, addition,
scissions cross-linking, and aromatic hydroxylations, and these reactions
could result in
mutagenic and carcinogenic effects, transcription errors, DNA damage, and protein inactivation.
Though there is little unequivocal evidence to show that these reactions
are major events in the
aging process, the idea of a small but steady burden on the tissues cannot be
9. Hydroxyl radical is considered
to be responsible for 90% of the damage to DNA caused by
ionizing radiation and that H20? causes strand breakage.
10. Age pigments, generally
known as LIPOFUSCIN are believed to be derived from subcellular organelles as a result of cross-linking of membranes--the
cross-linking is thought to be derived
from peroxidation of lipids. Since
the cross-linked products are not readily susceptible to the
action of proteolytic enzymes, they accumulate with time.
11. The amount accumulated can in terms of intracellular volume be quite
6 to 50% in very old animals depending on tissue.
1. Feeding mice a variety of antioxidants has caused an improvement in mean
life span, which
appears to be species-related rather than general.
2. What role antioxidants play in
lengthening life is yet to be determined.
3. Vitamin E reduce the lipid
peroxidation, and unsaturated fatty acids increased it (and
therefore pigment: format ion).
4. Studies with vitamin E suggest
that age pigment accumulation is neither life threatening or
even seriously debilitating.
5. Vitamin C has no effect on brain
6. Little work on the direct effects
of vitamin C on aging has been done.
7. Centrophenoxine has been shown
to reduce age pigments.
8. Age pigment represents a
mechanical accumulation of indigestible material, a result,
(and possible burden) rather than a cause of aging.
1. For men serum level increased
from 177mg/dl to 248, from 17 to 55 years, then leveled
off; rapid rise occurring between 20 & 39.
2. For women A similar pattern
with a substantial jump in concentration between
49 and 59, with a higher level (285 vs. 259) in the 60 to 69 year age group,
3. Results with rats varied greatly for cholesterol: increasingly dependent
on strain tested,
and even the very laboratory.
4. Food restriction sharply reduced
and delayed the degree o of increase.
1. Though there are age related
changes in membranes (mitochondrial function, enzyme
activity, lipid composition of mitochondrial and microsomal membranes) no
if they initiate and develop the process of aging or are simply a result of the
2. If age related change occurs to membrane, their nature varies with type
3. Effects of diet can bring about
as great a change in membrane composition as aging.
4. Age-related changes might
change the susceptibility of membranes to fragmentation
and this would yield dissimilar preparations for analysis, or even different lipid
5. Membrane turnover does not appear
to change with age.
There are disturbing contradictions and
lack of consistency in results, from
author to author.
MITOCHONDRIA, MICROSOMES, & LYSOSOMES
1. Certain enzyme levels in
microsomes show age-related changes, but it is not known
if they result from membrane changes, altered enzymes, or a reduction in
the number of enzyme
2. Results are influenced by
differences in sex, strain, and maintenance conditions, and tissue
3. Finally there is no direct link
that has been established between enzyme changes and aging.
4. Enzyme changes could be secondary to changes related to gene expression,
mitochondrial and microsomal metabolism in old organisms should point the
these primary facets of aging.
5. For mitochondria there is a reduction
in the number
and volume density with age. There is also a fraction of mitochondria with increased fragility in old preparations.