1.PREDECESSORS OF LIPOFUSCIN AGE PIGMENT -
PROBABLE ROLE IN BIOLOGICAL PROCESSES
V. T. Vertushkoff
Dnepropetrovsk,
49128, Ukraine
Date
of place: 23.05.2002
Processes of immune response, ageing and
spontaneous carcinogenesis involve polymeric products of unsaturated fatty
acids oxidation.
Age pigment, called lipofuscin, is accumulated
in human and animal organisms, as they grow and advance in age, in normal
amounts. Depending on the animal species and age, as well as conditions
of formation and place of localization, lipopigments are distinguished
by histo-chemical and ultra-structural characteristics, fluorescence and
absorption spectra, solubility in organic solvents. Principal components
of age pigment isolated granules in human and bull cardiac muscle are
lipids (20-50% of dry weight), proteins (30-60%) and unhydrolyzable matter
(9-20%) [1, 2]. Membrane fragments were also found in granules [3]. Column
chromatography data for age pigment lipid fraction has demonstrated that
phospholipids comprise 75% of the latter.
Pigment deposition intensifies under E-avitaminosis,
hypoxia and hyperoxia, atherosclerosis, reflexive actions (for example,
under stimulation of defensive reflex in rats during ingestion), after
intraperitoneal and hypodermic injection of fats and oils, prolonged action
of adrenocorticotropic hormone, estrogen and diethylstilbestrol on animals,
under hypodynamia, action of ionizing radiation, electric irritation,
temperature stress etc., as well as in case of certain human illnesses
characterized by affection of central nervous system.
Pigment accumulation under E-avitaminosis
has been noticed in macrophages and cardiac muscular fibers in rats [4]
and hamsters [5], phagocyte cells of connective tissue, macrophages of
lymph, spleen and liver in monkeys and hamsters. It is assumed that spleen
and liver are supplied with pigment from animal muscles [6]. Acceleration
in lipopigment formation is observed under ingress of foreign substances
into animal organism, in case of feeding with acetanilide, carbon tetrachloride,
under benzene poisoning etc.
Most of the researchers believe that the
lipid pigments result from unsaturated fatty acids autoxidation and subsequent
oxidation products polymerization. Oxidation of unsaturated fatty acids
leads to appearance of hydroperoxides which can cycle with polymers after-forming
[8]. In vivo this process involves proteins. The damage of cell organelle
membranes by lipid peroxidation is assumed the direct reason of lipofuscin
formation [9,10]. The ferruginous compounds, such as hemoglobin, hemin,
cytochrome C and mioglobin are considered to be non-specific catalysts
of the process. As it is assumed, the lipofuscin chemistry does not exclude
the functional role of its predecessors in biological processes [2,11].
The polymeric compounds are the main product
of oil thermal oxidation at temperature 200°-300° C. Maximum polymer
molecular mass resulting upon superheating of corn oil (200°C, 24
hours) exceeds 10.000 dalton [12]. Absorption value in adipose solutions
in chlorophorm at 243-253 nm range is directly related to polymerization
products concentration in fats [13] .
It is revealed [14] that addition of biogenic
amines (catecholamines, serotonin, histamine) to oleic acid which oxidizes
at relatively low temperature (75°C in presence of Fe2+
ions and air oxygen) results in quick accumulation of colored polymeric
products. For instance, in 2-3 minutes after infusing of serotonin (in
75% methyl alcohol) or adrenalin into oleic acid (in a drop of ice-cold
acetic acid) in 2,5•10-3 mol/l concentration, the quantity
of lipopolymers amounted to 1,3-1,5% of the acid original mass. Fractionating
on column with sephadex LH-20 (1 : 1 eluting chlorophorm-methanol mixture)
calibrated by polyethylene glycol displayed that maximum polymer molecular
mass exceeded 6.000 dalton. There results have demonstrated that addition
of biogenic amines to oleic acid is equivalent to rising in temperature
of its oxidation up to 200-400°C (depending on the added amine). Polymers
forming in oleic acid under the action of various amine compounds differ
by absorption and fluorescence spectra [15].
Experiments on mice [16, 17] proved that
polymers extracted from oleic and linoleic acids displayed clearly expressed
physiological effect similar to the effect of biogenic amines, under the
action of which they have formed. Intraperitoneal injection of mice with
saponifiable fraction of polymers formed in linoleic acid under serotonin
action causes the same changes in certain blood properties that serotonin
itself (see the Table). It should be noted that no serotonin contained
in polymer fraction injected to the animals. It was proved by highly sensitive
fluorimetric method [21]. Injecting of mice with saponifiable fraction
of polymers formed in thermally oxidized linoleic acid (165°C, 24
hours) didn't influence the same blood properties. Intraperitoneal injecting
of mice in dose amounting to 250 mg/kg of polymers formed in oleic acid
under adrenaline action caused the state of heavy agitation followed by
convulsions of the whole body. Increase in dose up to 750 mg/kg resulted
in instant death of mice.
Table. Changing of Certain Biochemical Indices
in Rats Blood after Injection of Serotonin (10 mg/kg) and Lipopolymers
(250 mg/kg).
| Conditions of experiment | Time after injection, min | Total blood coagulation time, s, according to [18, 19] | Fibrinolytic activity, %, according to [20] | ||||
|---|---|---|---|---|---|---|---|
| n | M ± m | p | n | M ± m | P | ||
| Initial history | - | 21 | 95 ± 0,04 | - | 21 | 8 ± 3,4 | - |
| Serotonin introduction | 1 5 15 |
21 21 21 |
48 ± 0,03 79 ± 0,04 87 ± 0,07 |
< 0,001 < 0,001 < 0,001 |
9 9 11 |
17 ± 2,0 57 ± 5,8 52 ± 3,0 |
0,02 < 0,001 < 0,001 |
| Initial history | - | 4 | 80,2± 6,7 | - | 4 | 25,2± 7,5 | - |
| Introduction of polymers (linoleic acid + serotonin) | 5 15 |
4 4 |
46,2 ± 8,3 38,0 ± 8,5 |
< 0,05 < 0,01 |
4 4 |
66,0± 8,2 58,5± 6,1 |
0,01 < 0,05 |
| Initial history | - | 4 | 79,0 ± 2,0 | - | 4 | 33,7± 8,3 | - |
| Introduction of polymers (linoleic acid + 165°C, 24 hours) | 5 15 |
4 4 |
71,2 ± 3,1 74,5 ± 8,9 |
> 0,15 > 0,15 |
4 4 |
36,5± 8,4 35,2± 7,2 |
> 0,15 > 0,15 |
V. Vertushkoff, 16,
Kommunarovskaya Str., Flat
332, city
of Dnepropetrovsk, UKRAINE,
49128
It is known [22, 23] that catecholamines,
serotonin and evidently histamine form prostaglandins under interaction
with cyclic endoperoxides of unsaturated fatty acids. Apparently, the monomers
in polymers formed in unsaturated fatty acids under biogenic amines action
represent prostaglandins and prostaglandin-like compounds.
Significant part of lipopolymers injected
to mice gets to spleen. Selective lipopolymers absorption by animal lymph has been shown also [7].
The dynamics of polymers accumulation in spleen
was registered by absorption in lipid solution in chlorophorm at wavelengths
of 245 nm and 400 nm (registration of polymer coloring). The lipids were
extracted from spleen with 2: 1 chlorophorm-methanol mixture [24]. Experimental
group of mice was intraperitoneally injected with polymers formed in oleic
acid under adrenaline action in dose of 150 mg/kg. The control group of
animals was injected with the same dose of polymers formed in thermally
oxidized oleic acid (165°C, 24 hours). It was revealed [15] that absorption
value at 245 nm in the experimental group spleen lipids changed insignificantly
later on, upon reaching its certain maximum. At the same time, having reached
the maximum, the absorption value at 400 nm is sharply reduced (Fig. 1).
That is, considering the absorption at 245 nm, the polymers stay in spleen.
If to be guided by absorption at 400 nm,
there are no polymers observed in the spleen in 1,5 hours after injection.
These results are forcing to suggest, that polymers in animal spleen are
subjected to chemical transformation, causing disappearance of their coloring.
Evidently, depolymerization of polymer structure occurs. After injection
of polymers from thermally oxidized oleic acid to control group mice, dynamics
of absorption in spleen lipids at 400 nm has the same form that at 245 nm
(Fig. 2).
Mice spleen is also featuring de-colorization
of polymers formed in oleic acid under the influence of serotonin, histamine,
as well as such amine compounds as ß-mercapto-ethylamine, aminoethylisothiouronium,
hydroxylamine, mexamine etc. At the same time, coloring of polymers formed
in oleic acid under cystamine influence is preserved in spleen lipids during
observation longer than 3 hours.
Intraperitoneal injection of biogenic amines
in sub-toxic (radiation protective) doses, as well as that of glutathione,
cysteine and amine compounds listed above, to mice and rats also results
in appearance of polymeric products of unsaturated fatty acids oxidation
(Fig. 3) [15].
During five days after total exposure of
mice to 750 r X-ray dose, two clearly marked maximums of lipopigments concentration,
registered by absorption of lipids solution in chloroform at 400 nm, appear
in spleen. The first maximum falls on 6-12 hour period after exposure to
radiation, and the second one, more significant, occurs on the 3-5th days
[14]. Appearance of the first maximum concurs with developing affection
and destruction of the most radiosensitive cells, namely, blood-forming
organs and embryonal tissue. The second rise in lipopigments concentration
correlates with the degenerative changes and mass death of the intestines'
mucous membrane cells (intestinal syndrome). Spleen lipids fractioning on
the column with sephadex LH-20 has shown that maximum molecular mass of
lipopolymers exceeds 4.000 dalton. On the fourth day after radiation treatment
lipopolymers content was equal to 40% of total spleen lipids mass.
As based on the data stated above, it is
supposed that polymer products of unsaturated fatty acids oxidation take
part in the following biological processes.
Immunological reaction. In everyday
life animals and humans are subjected to stress impacts destabilizing their
internal environment. It is assumed that immunological reaction is activated
by any stress factor, initiating defeat and destruction of cells in tissues.
From this point of view, pathogenic bacteria, viruses, fungi and protozoa
should be also regarded as stress agents. Diffusive endocrine system [25,
26] which hormone-producing cells are dissipated in epithelial and connective
tissues of all the organs in fact, responds to changes in the internal environment.
Apparently, hypophysis-adrenal system (hormones of adrenal gland cortex)
takes part in activation of diffusive endocrine system. Responding to tissue
damage, cells of the local diffusive endocrine system evolve excessive amounts
of biogenic amines. Under organism stress reaction condition, high concentrations
of catecholamines, serotonin, histamine act as catalysts of oxidative polymerization
of unsaturated fatty acids included into the structure of plasma membrane
phospholipids. Since lipids in the membrane are associated with proteins,
result of action of massed biogenic amine doses on the cell is formation
of colored lipoproteins of polymeric structure. Generation of lipoprotein
complexes can occur on account of strong chemical bonds between lipopolymers
and proteins, and result from hydrophobic interactions [27, 28]. Part of
polymeric lipoproteins penetrates into cytoplasm, forming, eventually, intracellular
lipofuscin granules. Noticeably larger amount of lipoproteins comes off
plasma membrane of the cells and goes to lymph, lymph nodes, spleen, liver.
In spleen polymeric lipoproteins are absorbed by macrophages where lipopolymers
are subjected to de-structuring with subsequent inactivation of physiologically
active structural units. Macrophages present released protein antigens to
T-lymphocytes thus initiating antigen-specific immune response.
There is no selectivity in the biogenic amines
action on cells. By this reason, macrophages present antigens of normal,
functionally inferior, defective, pathologically altered and other cells
of the own organism. Besides, macrophages present also antigens of pathogenic
bacteria, viruses, fungi and protozoa affecting and damaging tissue cells.
Such antigens are bonded with lipids in the process of their polymerization
running in conformity to free radical mechanism. Apparently, lipoproteins
of polymeric structure can be considered as the natural immune system stimulator
or the mechanism starting the immunological reaction. Per os and intraperitoneal
introduction of lipopolymers extracted from spleen of rats on the 4-th day
after exposure to X-ray radiation in dose of 800r to healthy rats initiates
activation of immunological reaction. Diffusive endocrine system, immune
system, as well as organs of sense and epithelial tissue receptors connected
with peptidergic neurons of the nervous system, are considered to be separate
elements of general system of initial reaction, warning and protection of
the organism [26].
Ageing and spontaneous carcinogenesis.
It is supposed that products of oxidative polymerization of unsaturated
fatty acids getting into post-mitotic cells represent the first and the
main reason of organism ageing. Well-known information found in scientific
literature proves it by implication. Thus, the accumulation of lipopigments
in cells causes pro rata increase of cytoplasm structures damaging, for
instance, the decrease in cytoplasm mass, in number of mitochondria, coarse
endoplasmic retuculum, simplification of Golgi apparatus and vacuoles formation
in cytoplasm [29]. Some human genetic diseases display clearly expressed
examples of connection between the ageing processes and lipofuscinogenesis.
For example, progeria is characterized with significant deposition of age
pigment in cells of numerous organs even in early age. The child of nine
years old resembles an adult of seventy. Such ageing symptoms as atherosclerosis,
gray hair and others take place [30, 31].
Toxic effect of thermally oxidized fats is
pro rata correlated with content of polymer products [32, 33]. Introducing
polymeric fractions extracted from superheated corn oil into ration for
rats in the amount of 2,5 % of diet causes the death of animals within seven
days [39]. Lipopolymers formed in oleic acid under biogenic amine action
suppress the respiration of liver homogenate in pigs [17].
Figure 4 displays the kinetics of peroxide
compounds accumulation in oleic acid, oxidized at 75°C with addition
of vitamin E of carcinogenic poly-cyclic hydrocarbon, 3,4-benzpyrene and
polymers formed in oleic acid under serotonin action. It should be noted
that slowing down of oxidation process by 3,4-benzpyrene and lipopolymers
lasts for indefinitely long period. In accordance with Warburg, carcinogenic
chemical compounds are the agents affecting oxidative phosphorylation. The
highest concentration of carcinogen in mitochondria was found with the help
of fluorescence microscopy, while investigating the distribution of hydrocarbons
in epithelial tissues of mice skin treated with 3,4-benzpyrene.
As it was proposed above, monomers in polymers
formed in unsaturated fatty acids under the influence of biogenic amines
represent prostaglandins and prostaglandin-like compounds. It is well known
that prostaglandins disengage oxidative phosphorylation, considered as the
main element of mechanism of their action on the cell [35]. This data, as
well as anti-oxidative properties of lipopolymers and their ability to suppress
the tissue respiration, allows to state that lipopolymers are also the agents
disturbing oxidative phosphorylation. Mitochondria are the principal source
of intracellular energy. Along with decrease in mitochondria number, cells
of aging organism are featuring mitochondria with symptoms of evident degradation
marked by more frequent detachment of external membrane, reduction of cristae
number, swelling, deposition of pigment, etc. Size of the intra-mitochondrial
pigment granules increases in the ageing process [36]. Most of the data
available clearly proves that considerable activation of energy metabolism
glycolytic phase in the old age occurs predominantly due to glycogenolysis
reactions. In the course of ageing, decrease in the total gaseous metabolism
and activity of oxidative processes takes place [37]. Lipopolymers getting
into cells create energy deficit progressive with the age. As a result,
processes of renewal and restoration of the functional cellular structures
are more and more lagging from processes of their damage, inactivation and
destruction. Lipopigments forming granules in cells are inactive ones, especially
because they are often enclosed in single-layer membrane. However, stress
factor influencing the organism ensure almost continuous getting of new
lipopolymer portions into cells. It has been shown that 7% of the total
lipofuscin amount in human sympathetic vagal ganglia is accumulated in the
first decade of life; accordingly, this figure is equal to 8-14% in the
second decade and 30-33% in the fifth decade [38, 39].
Successive alternation of stress actions
and situations emerging in everyday vital activity and causing destabilization
of the internal environment plays determinative role in lipofuscin formation
in humans and animals. Lipofuscin pigments appear, evidently, under the
influence of any action exceeding the bounds of physiological stimulus.
Speed of lipofuscin accumulation in various species of animals is determined,
on the one hand, by sensitivity of the organism systems, and on the other
hand, by frequency of changes in the medium itself [3]. Ageing of the organism
stipulated by lipopigment getting into cells is the incidental (accompanying)
negative result of activity of the general system of initial reaction, warning
and protection of the organism [26].
Peacock [40, 41] obtained sarcomas in mice
in the place of introduction of superheated cholesterol, its esters and
superheated cotton-seed oil. Lane et al. [42] also observed appearance of
sarcomas in 10% of rats injected with superheated lard or vegetable oil
subcutaneously. Polymerized unsaturated vegetable oils caused appearance
of sarcomas in the place of injections [43].
Many researchers noticed increase in the
number of nuclei in old individuals tissues, as well as increase of polyploid
cells share [37]. This fact proves that correlation between diminished breath
sounds and enhanced glycolysis in cells is as follows. The cells stay in
condition of permanent, continuous stimulation of cell division [44]. However,
high level of differentiation and specialization of cells can impede this
division. Since lipofuscin is formed in dividing cells as well [45], these
cells can also be predecessors of tumor cells.
Fig.1. Absorption dynamics at 245 nm (1) and 400 nm (2) in spleen
lipids solution in chlorophorm after injecting of polymers to mice (oleic
acid + adrenalin). Dotted lines mark absorption values at 245 nm and 400
nm in spleen lipids of intact mice.
Fig.2. Absorption dynamics at 245 nm (1) and 400 nm (2) in spleen
lipids solution in chlorophorm after injecting of polymers to mice (oleic
acid +165°C, 24 hours). Dotted lines mark absorption values
at 245 nm and 400 nm in spleen lipids of intact mice.
Fig.3. Absorption dynamics at 400 nm in lipids solution in chlorophorm
after injecting of adrenalin to mice in dose of 4 mg/kg. 1 - bone marrow,
2 - spleen, 3 - small bowels, 4 - blood. Dotted lines mark absorption
values in lipids of corresponding tissues of intact mice.
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