Lipoprotein (a) is a surrogate for ascorbate
Matthias Rath and Linus Pauling
PROCCEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA 1990, 87, 6204-6207
Introduction
Lipoproteins consist of particles, each of which is a globule of lipid
molecules, surrounding by an apoprotein shell. Lipoprotein(a) [Lp(a)]
was discovered by Blumberg et al. (1) and by Berg (2). It shares with
low-density lipoprotein (LDL) the lipid and apoprotein composition Ñ mainly
apoprotein B-100, apo B, consisting of a polypeptide chain of 4,536 amino-acid
residues. The unique feature of Lp(a) is an additional glycoprotein,
designated apoprotein(a), apo(a), which is linked to apo B by disulfide
groups.
The cDNA sequence of apo(a) (3) shows a striking homology to plasminogen,
with multiple repeats of kringle 4, one kringle 5, and a protease domain.
The isoforms of apo(a) vary in the range of 300 to 800 kDa and differ
mainly in their genetically determined number of kringle 4 structures
(3). Apo(a) has no plasmin-like protease activity (4), but a serine protease
activity has been demonstrated recently (5). Like plasminogen, Lp(a)
has been shown to bind to lysine-sepharose, immobilized fibrin and fibrinogen
(6) and the plasminogen receptor on endothelial cells (7-9). This binding
is inhibited by e -aminocaproic acid, certain other amines, and plasminogen.
Lp(a) and Ascorbate in Different Species
Lp(a) has been detected in the plasma of humans other primates (10)
and the European hedgehog (Erinaceus europeus) (11). The presence of
apo(a) in some other species can not be excluded since no comprehensive
immunological or genetic screening has been reported yet. Most mammals
synthesize ascorbate, usually in the range of 30 to 300 mg per day per
kg of body weight. A few species, including humans, other primates, the
guinea pig, and the Indian fruit-eating bat, have lost the ability to
synthesize ascorbate.
We observed that Lp(a) is found primarily in the plasma of those species
that are unable to synthesize ascorbate. Vice versa, most mammals having
an endogenous ascorbate supply lack detectable Lp(a) in their plasma.
It was the recognition of the correlation in mammal species of the two
events, the loss of the ability to synthesize ascorbate and the detection
of apo(a) and Lp(a) in the plasma of these species, that caused us to
formulate the hypothesis that apo(a) serves as a surrogate for ascorbate.
We have not found any earlier description of this hypothesis in the scientific
literature.
The loss of the ability to synthesize ascorbate is the result of a mutation
of the gene encoding for L-gulono-g -lactone oxidase (GLO), which catalyzes
the conversion of gulonolactone to ascorbate (12). In the case of the
primates this mutation happened about 40 million years ago (13). Since
ascorbate has numerous important metabolic functions and since the dietary
ascorbate uptake was on average less than 10% of the amount synthesized
by comparable animal species, this loss placed a great stress on the
primates. This deficiency may have led to evolutionary effective mutations
to reduce this stress and in particular to acquire the ability to synthesize
apoprotein(a). This could have happened, in part, through the modification
of another kringle-containing protein, such as plasminogen.
There is, however, another possibility. Other animals might be found
in the future with functional genes for both apo(a) and GLO. In this
case, it would be more likely that plasma ascorbate levels play a regulatory
role in apo(a) synthesis.
Our hypothesis led to the prediction that the guinea pig, unable to
synthesize ascorbate, would be found to produce detectable amounts of
Lp(a). In fact, we were able to demonstrate apo(a) immunoreactivity in
the blood of guinea pigs by SDS-PAGE and subsequent immunoblotting (unpublished
observation). In another experiment the European hedgehog, known to have
Lp(a) in its blood was studied for its ability to synthesize ascorbate.
One of our colleagues, Dr. Constance Tsao, has shown that the hedgehog
liver has not lost its ability to synthesize ascorbate (personal communication).
This indicates that the genes for both apo(a) and GLO are present in
the same animal. This observation supports the hypothesis of a regulatory
role of ascorbate in the synthesis of apo(a).
Since the ability to synthesize apo(a) has survived millions of years
in evolution, this protein must have one or more valuable functions.
Some of these functions are discussed in the following sections.
Ascorbate and Lp(a) Strengthen the Extracellular MATRIX and Promote
the Healing of Wounds
Ascorbate is essential for the protection of the extracellular matrix
system. This is in part due to the increased rate of synthesis of collagen
when the ascorbate level is high. Ascorbate is required (one ascorbate
molecule per hydroxyl group) for many enzyme-catalyzed hydroxylation
reactions, including the conversion of procollagen to collagen by the
conversion of lysine and proline to hydroxylysine and hydroxyproline.
One of the manifestations of scurvy resulting from the extreme depletion
of ascorbate is capillary fragility, followed by massive hemorrhages
throughout the tissues (13). In such conditions inhibition of fibrinolysis
would be advantageous. Because of its unique properties, Lp(a) is an
ideal molecule to meet this requirement. Apo(a), because of its homology
to plasminogen, would target the Lp(a) particle to sites of increased
vascular permeability. In this situation, the ability of Lp(a) to competitively
inhibit the binding of plasminogen to fibrin and the plasminogen receptor
would be beneficial. In fact, Lp(a) has been shown to have antifibrinolytic
properties (14).
It seems reasonable to us to propose that one way in which Lp(a) serves
as a surrogate for ascorbate is the strengthening of the extracellular
matrix in the blood vessels and other organs, particularly with low ascorbate
concentrations. In fact, apo(a) has been detected in non-lesioned areas
of arterial wall from children (15). Lp(a) in the arterial wall would
strengthen the arteries, but atherosclerosis would occur, as described
later, if this function were to operate to too great an extent.
Another way in which Lp(a) functions as a surrogate for ascorbate is
in accelerating the healing of wounds. Both high plasma ascorbate (16)
and high plasma Lp(a) (17) have been reported to accelerate the process
of wound healing. A possible mechanism for Lp(a) is its binding to fibrin
and other extracellular matrix components (5,18), thereby compensating
for a decreased rate in collagen formation, particularly when the ascorbate
concentration is low.
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Ascorbate, Uric Acid, and Lp(a) as Antioxidants
During evolution of primates a major factor in lengthening their life-span
may have been improved protective mechanisms against damage by oxygen
radicals. Free-radical-mediated lipid peroxidation seems to be critically
involved in cardiovascular disease and in cancer, rheumatoid arthritis,
and other pathological and degenerative processes, including aging. Ascorbate
has been shown to completely protect plasma lipids against detectable
peroxidative damage induced by aqueous peroxyl radicals, with other antoxidants
(a -tocopherol, b -carotene, bilirubin, proteinthiols) being less effective
(19).
Other investigators have reported similar results for the guinea pig
(20). The primates after having lost the ability to synthesize ascorbate
may well have been under evolutionary pressure to develop other antioxidative
mechanisms. Uric acid has been reported to be a moderately effective
antioxidant and the much increased level of urate in primates, in comparison
with other animals, has been described as a response to the low level
of ascorbate (21).
We now suggest that apoprotein(a), with over 100 disulfide groups per
molecule, is also effective as an antioxidant, acting also in this way
as a surrogate for ascorbate. It would be especially effective in preventing
peroxidation of lipids in the Lp(a) particle because of its presence
in the shell surrounding the lipid sphere, where it could destroy the
peroxyl radicals before they reach the lipids. Some of the internal disulfide
groups in the kringles of apoprotein(a) might be reduced by ascorbate
to thiol groups.
Another possibility is the formation of disulfide radicals by adding
or subtracting an electron, the latter giving a product analogous to
the superoxide radical. Immunoblots of homogenized arterial wall taken
at autopsy support this hypothesis (22). Twenty-four hours after death
the apo(a) extracted was not degraded and had the same molecular size
as the apo(a) in the pre-mortem blood. In contrast, apo B is known to
be partially degraded under these conditions.
Lp(a), Ascorbic Acid, and Cardiovascular Disease Ascorbic acid levels
were found to be decreased in the plasma and leukocytes of coronary heart
disease (CHD) patients (23). Furthermore, the concentrations of ascorbate
in atherosclerotic lesions of human arterial wall are considerably lower
than in the areas without lesions (24). In contrast, plasma Lp(a) levels
were found to be elevated in CHD patients and patients with other forms
of atherosclerosis (25,26).
There is a thousand-fold range of Lp(a) blood concentrations in human
beings determined largely by heredity (27, 28) and to some extent by
environmental factors, especially nutrition (29). Lp(a) above 30 mg/dl
doubles the risk of CHD, and if in addition LDL is elevated the risk
is increased by a factor of 5 (30). There is no correlation between Lp(a)
levels and cholesterol plasma levels, and in normolipemic CHD patients
the only risk factor for CHD is found to be elevated Lp(a) (22). This
observation indicates that Lp(a) can cause atherosclerosis without hyperlipidemia.
The importance of Lp(a) in human atherosclerosis has been revealed by
a quantitative study of the amount of this lipoprotein in the wall of
the ascending aorta of coronary bypass patients (22). Lp(a) deposition
in the arterial wall was found to correlate with the extent of plaque
development in both the human aorta and the coronary arteries (15). Furthermore,
a selective accumulation of Lp(a) over LDL was established in both human
arteries (22) and occluded coronary bypass vein grafts (31).
As discussed above, the development and retention of Lp(a) in evolution
strongly support a beneficial role of Lp(a). The great range of concentrations
of Lp(a) found in human plasma suggests that the control mechanisms for
apo(a) synthesis at the optimum level have not yet been developed. In
addition, the atherogenicity of Lp(a) seems to be closely related to
the ascorbate concentrations in plasma and tissue. We suggest that ascorbate
deficiency increases plasma Lp(a).
It is also known that ascorbate deficiency affects the integrity of
the endothelial cell lining (32), thus promoting the infiltration of
Lp(a) and other lipoproteins. On the basis of these considerations, we
postulate that ascorbate can reduce or prevent the development of atherosclerosis
by lowering plasma Lp(a), decreasing lipoprotein infiltration into the
arterial wall, and preventing lipid peroxidation.
Ascorbate could prevent the atherogenicity of Lp(a) also in another
way. Since the binding of Lp(a) to fibrin involves lysyl groups, we suggest
that, because of its involvement in hydroxylation reactions, ascorbate
could convert these groups to hydroxylysyl groups and thus contribute
to preventing the attachment of Lp(a). The binding of Lp(a) might also
be affected by chemical modification of the lysine-binding site of the
Lp(a) particle itself and ascorbate could interfere with this modification.
The Guinea Pig as an Animal Model for Atherosclerosis
It is known that the formation of atherosclerotic plaques can be induced
in the rabbit and other animals by feeding a high-cholesterol diet. This
can also be done with the guinea pig. However, the guinea pig is in a
remarkable way different. It has been reported that atherosclerotic deposits
in the arteries of the guinea pig were formed on an ascorbate deficient
diet without additional cholesterol (33). We have verified that these
deposits are not formed by guinea pigs given higher doses of ascorbate
but are formed by the animals on an ascorbate deficient diet without
the administration of large amounts of cholesterol (unpublished experiments).
Histological examinations showed that the atherosclerotic process in
the guinea pig resembles that in humans. Dissociation of the endothelial
cells with parietal adhesion of coagulated lipemic plasma has been observed
(34). Since we have been able to detect Lp(a) in the plasma of the guinea
pig, we predict that Lp(a) will be found to be deposited in the arterial
wall of hypoascorbemic guinea pigs and to contribute to plaque formation.
Because of its similarity to man with respect to ascorbate and Lp(a)
metabolism the guinea pig should be an ideal animal model for atherosclerosis
research (16).
Lp(a) and Ascorbic Acid in Cancer and Diabetes Mellitus
Similar to the inverse correlation of Lp(a) and ascorbic acid in atherosclerosis,
a high incidence of cancer is associated with low levels of ascorbate
(35) and also high levels of Lp(a) (36). Similar observations were made
in diabetes mellitus for ascorbate (37) and Lp(a) (38). Despite different
causes, the progression of these diseasesis dependent on the integrity
and stability of the tissue, particularly the extracellular matrix (39).
Ascorbate depletion in these pathological states will cause Lp(a) to increase
and make up the deficiency at the sites of the disease progression. We
therefore predict that Lp(a) will be found in the vicinity of cancer processes.
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Roles of Lp(a) and Ascorbate
A striking relationship of Lp(a) and ascorbate is that species that
have lost the ability to synthesize ascorbate have detectable amounts
of Lp(a) in their plasma. Plasma Lp(a) and ascorbate levels are inversely
correlated in wound healing, atherosclerosis, cancer, diabetes, and other
pathological conditions.
Additional evidence for the Lp(a)-ascorbate connection comes from another
observation. In patients with trauma and after myocardial infarction,
the Lp(a) plasma levels were found to increase gradually, following acute
phase proteins such as C-reactive protein, haptoglobin, and others with
a relatively late maximum at 2 weeks (17).
Inversely, plasma ascorbate levels were found to decrease for approximately
2 weeks after myocardial infarction (23). The fall in ascorbate may be
explained by mobilization of ascorbate at the site of the lesion, through
migration of leucocytes.
Brown and Goldstein have suggested that Lp(a) might play a role in wound
healing (40). We now suggest a broader role of Lp(a) in tissue maintenance
and repair. In brief, we propose that Lp(a) is a late member in the chain
of responses to cellular damage. Its role under physiological and pathophysiological
conditions would be the attempt to reconstitute cellular and extracellular
integrity. The fact that animals with plasma Lp(a) levels below the detection
level do not suffer disadvantages strongly suggests that in its physiological
role Lp(a) can be replaced. We therefore propose that not only is Lp(a)
a surrogate for ascorbate, but also ascorbate is a surrogate for Lp(a).
Conclusion
We have marshaled the evidence that high levels of Lp(a) and low levels
of ascorbate are associated with an increased incidence in mortality
from cardiovascular disease, cancer, and other diseases. We suggest that
Lp(a) levels may be decreased by ascorbate. There is epidemiological
evidence (41) that dietary ascorbate supplementation is equally effective
in reducing the mortality rate for heart disease, cancer, diabetes, and
other diseases in the elderly. Moreover, preliminary studies have shown
that the process of atherosclerosis in both guinea pigs (42) and humans
(43) can be reversed by adequate amounts of ascorbate.
We have thus described a metabolic regulatory mechanism that seems to
have significant implications for the most frequently occurring diseases
in the industrialized countries. The application of this mechanism will
significantly expand the scope of conventional therapy. It may lead the
way to new approaches in the effective prevention and treatment of cardiovascular
and other diseases.
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