37?
/ '/-
THE EFFECTS OF EDTA CHELATION THERAPY ON PLAQUE
CALCIUM AND MINERAL METABOLISM IN ATHEROSCLEROTIC RABBITS
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
Doctor of Philosophy
By
Foster M. Walker, M.S.
Denton, Texas
May, 1980
Walker, Foster M., The Effects of EDTA Chelation Therapy
on Plaque Calcium and Mineral Metabolism in Atherosclerotic
Rabbits. Doctor of Philosophy (Biology) , May, 1980,
97 pp., 3 tables, 24 illustrations, bibliography, 44
titles.
New Zealand albino rabbits exhibited calcified aortic
plaques and maximum average serum cholesterol levels of 1200 mg
percent after twenty-three weeks on an atherogenic diet (250
to 500 mg percent cholesterol in ten percent corn oil; 200,000
I.U. vitamin D3 per month). One month following termination of
the atherogenic diet, rabbits were treated with disodium
edetate (Na2EDTA, 50 mg/kg body weight) via the marginal
ear vein, on alternating days for a total of twenty infusions
each. Aortae were examined for tissue calcium both quantitatively
(direct microcomplexometric analysis) and histologically six
weeks after completion of EDTA chelation therapy.
Rabbits infused with EDTA exhibited significantly less
(p. 05) aortic calcium (300 microequivalents/gram of tissue),
than those infused with normal saline (635 microequivalents/
gram of tissue), and those which were not infused (778 micro
equivalents/gram of tissue). Medially calcified plaques
were seen in non-infused animals, while none were noted in
those which were infused with EDTA.
Quantitative analysis of calcium, magnesium, zinc, and
cadmium in twenty-four-hour urine samples from the rabbits
on both infusion and non-infusion days was done utilizing
atomic absorption spectroscopy. The excretion levels of
calcium and zinc were significantly greater (p .05) on
infusion days in EDTA-treated rabbits compared to non-EDTA
treated rabbits, while that of magnesium was greater (p .05)
on non-infusion days. Cadmium excretion remained unchanged.
These results indicate that disodium EDTA chelation
therapy can affect the removal of plaque calcium from the
aortae of atherosclerotic rabbits, possibly contributing to
the breakdown of atheromatous lesions. In addition, the
excretion patterns of zinc and magnesium must be considered
in a mineral replacement therapy to accompany chelation
therapy.
TABLE OF CONTENTS
LIST OF TABLES .....................
LIST OF ILLUSTRATIONS . . . . . . . . .
Page . .. . . . i
.. . . .II
Chapter
I. INTRODUCTION . . . . . . . . .
II. LITERATURE REVIEW .0-0-0 - - -
.1
.3
Chemistry of EDTA Clinical Effects Effects on Lipid Metabolism Effects on Trace Minerals Toxicological Effects Mode of Action
III. METHODS AND MATERIALS.-.-.-.-.-.-.-.-...-.-.-.25
Cholesterol Determinations Infusion Procedures Histology Tissue Calcium Determinations Urine Mineral Determinations
IV. RESULTS.....-......-.-.-.-.-.
Serum Cholesterol Histology and Tissue Calcium Mineral Excretion in Urine
V. DISCUSSION . - - - * - - - - - -
.
&.- - . . 39
89
Summary
BIBLIOGRAPHY...-.-.-.-.-.-.-...-.......-.-. - & -.-.-... 92
LIST OF TABLES
Table Page
I. Formation Constants of Various Metal-EDTA Complexes ....................... 15
II. Aortic Tissue Calcium Levels . . . . . . . . 48
III. Urinary Mineral Excretion .............. 62
LIST OF ILLUSTRATIONS
Figure
1.
Page
6Structure of Ethylene Diamine-tetraacetic acid
2. Structure of EDTA-chelated Calcium . . . . .
3. Mean Serum Cholesterol Levels in Control andExperimental Animals
4. Aorta of Rabbit on Atherosclerotic
5. Aorta of Rabbit on Atheroscletotic
6. Aorta of Group A Rabbit. . . . .
7. Aorta of Group A Rabbit . . . . .
8. Aorta of Group B Rabbit.0.0.0.0.0
9. Aorta of Group B Rabbit ......
0. Aorta of Group C Rabbit . . . . .
1. Aorta of Group C Rabbit . . . . .
2. Aorta of Group D Rabbit . . . . .
3. Aorta of Group D Rabbit . . . . .
4. Aorta of Group E Rabbit........
5. Aorta of Group E Rabbit . 0.0 .
6. Aorta of Group F Rabbit . . . . .
7. Aorta of Group F Rabbit . . . . .
8
.... . ..* . .41
Diet. ... ... 44
Diet . ..... 44
46
46
50
. .0 .0 .0 . .0 . 0. . 50
52
52
. . . . . . . 0. 0. 55
. .0 .0 .0 .0 . . . . 55
. . . . .. ..57
57
59
. .0 . . . 0. .59
18. Urinary Calcium and Magnesium Excretion in Group B Rabbits .................. ..........
19. Urinary Zinc and Cadmium Excretion in Group B Rabbits . . . . . a. 0. 0. 0. 0. 0 . . . . .
20. Urinary Calcium and Magnesium Excretion in Group C Rabbits............ ........
ii1
1
I
1
I
1
1
1
1
64
66
68
. . . .
Figure Page
21. Urinary Zinc and Cadmium Excretion in Group C Rabbits ..................... . ........ 70
22. Urinary Calcium and Magnesium Excretion in Group D Rabbits ...................... ....... 73
23. Urinary Zinc and Cadmium Excretion in Group D Rabbits...............................75
24. Urinary Calcium and Magnesium Excretion in Group D Rabbits .................. . ........... 77
25. Urinary Zinc and Cadmium Excretion in Group D II Rabbits........................... ........ 79
26. Urinary Calcium and Magnesium Excretion in Group E Rabbits ..*................. .......... 82
27. Urinary Zinc and Cadmium Excretion in Group E Rabbits ..*............................ 84
iii
ABBREVIATIONS
EDTA
Na2EDTA
MgNa2 EDTA
AAS
CaNa 2EDTA
DTPA
CDTA
HEEDTA-N
BAL
EGTA
CALCEIN
TCA
I.V.
Ethylenediaminetetraacetic Acid
Disodium Ethylenediaminetetraacetate
Magnesium Disodium Ethylenediaminetetraacetate
Atomic Absorption Spectrophotometer
Calcium Disodium Ethylenediaminetetraacetate
Diethylenetriamine pentaacetate
Cyclohexane Trans 1,2-Diaminetetraacetate
(2-Hydroxyethyl) Ethylenediaminetetraacetate
2,3 Dimercaptopropanol
Ethylene Glycol Bis-( -Aminoethyl Ether) -N,N'tetraacetic acid
Fluoresceinbismethyleneiminodiacetic Acid
Trichloroacetic Acid
Intravenous
LV
CHAPTER I
INTRODUCTION
Atherosclerosis is recognized as the most prevalent
chronic disease in the United States (Keys, 1970; Kannels,
1971; Holding, 1972). Currently, there is no therapeutic
procedure for the treatment of the non-regressive athero
sclerotic lesion, other than invasive by-pass surgery. As
a result, considerable interest has been developed in the
use of chelation therapy using synthetic chelating agents
such as ethylenediaminetetraacetic acid (EDTA) for the
treatment of atherosclerosis to determine if it is effective
in the dissolution of the atherosclerotic plaques (Seven, 1960;
Lamar, 1968; Klevay, 1975).
The intravenous infusion of EDTA theoretically removes
calcium deposits by chelating and subsequently excreting
unbound calcium ions via the urine. This action may cause
modifications of the metabolic functions of calcium and
other minerals. Specifically, a rapid turnover of calcium
may be produced through the stimulation of the parathyroid
glands (Perry, 1969; Meltzer, 1961; Boyle, 1963), which may
therefore contribute to the breakdown of atherosclerotic
lesions by the removal of metastatic calcium from the plaques
(Lamar, 1968).
2
Hass (1966) has found that albino rabbits developed
calcified atherosclerotic-like plaques similar to those
found in human arteries, when placed on a cholesterol in
corn oil diet and injected with Vitamin D3 (cholecalciferol)
at monthly intervals, for a total of approximately four
months. The research described here seeks to detail the
effects of chelating agents on the metastatic calcium deposits
of atherosclerotic lesions and, subsequently, on mineral
metabolism in New Zealand albino rabbits. Therefore, this
study wasdesigned to show that plaque calcium is removed
from the artery by chelation therapy, and as a result of this
decalcification, plaque size is reduced, thereby returning
the blood flow in the affected vessel to a normal level. In
addition, if the above can be done without deleterious side
effects, the accomplishment of which is dependent upon
development of an effective mineral replacement protocol, then
this study could assume significance in atherosclerosis research.
CHAPTER II
LITERATURE REVIEW
Chemistry of EDTA
Ethylenediaminetetraacetic acid (Ethylenebis iminodi
acetic acid) (EDTA) belongs to a class of molecules known
as chelates, which are employed to remove a metal ion from
the influence of its immediate environment. The metal chelate
compounds are defined as metal complexes in which two or
more of the donor groups coordinated to the metal are bound
by chemical linkage of some sort (Martel, 1960). These mole
cules are polydentate ligands, the structure of which provides
several sites, each able to associate with a central metal
ion (Wong, 1975). As a result, the ligand forms a molecular
sheath around the ion, and the complexing capacity of the metal
is chemically saturated. The latter effect, in particular,
prevents any further interaction of metal ion with anything
else, as long as the complex remains intact. It was noted
by Martel (1960) that for alkaline earth ions, the stability
of the metal chelate is due almost entirely to the entropy
change which occurs upon complex formation, and that the
result is a "cage" for the metal, with little covalent bond
formation. Figure 1 illustrates the generalized structure
of a molecule of EDTA, and Figure 2 demonstrates the structure
of a divalent cation-EDTA complex.
3
4
Clinical Effects
The earliest reported research activity in the use of
the salts of ethylenediamine tetraacetic acid (EDTA) for the
removal of metastatic calcium deposits was in 1946 at the
University of Zurich, and in 1947 and 1948, at the University
of Bern- (Huff, 1974).
In 1950, Popovici succeeded in the experimental control
of serum calcium levels in rabbits by administration of EDTA
(Endrate), in which a transient decrease in hypercalcemia and
a significant increase in calciuria was obtained. Proescher
(1951) found that disodium EDTA (Na2EDTA) completely inhibited
the coagulation of human and animal blood in vitro, due to the
binding of serum calcium ion; and Grant (1952) utilized the
same binding properties in employing EDTA solutions as collyrium
for calcium removal and clearing of post-keratitis corneal
opacities.
An early study on human calcium metabolism by Spencer
(1952), indicated that slow infusion of Na 2EDTA causes no
permanent decrease in serum calcium levels despite the pro
duction of excess calciuria.
The first reported use of intravenous administration of
Na 2EDTA to treat hypercalcemic patients with bone destructive
diseases (Holland, 1953) met with limited success due to
excessively rapid infusion. However, Spencer (1956) recommended
the desirability of the chelation of excess ionized calcium
in the circulation and the excretion of calcium as the chelate
FIGURE 1. - Structure of Ethylene Diaminetetraacetic Acid
6
HO
C
CH
o HO
CH7 2
N-CH--CH-N
CH CH 2 21
C
HO 0
C
0 HO
(Martel, 1960)
FIGURE 2. - Structure of EDTA-Chelated Calcium
8
Co
ca CH 2
CH/ 'co 2 CH S/ /2
2CCH
(Martel, 1960)
9
in the place of free calcium, in order to decrease hyper
calcemic toxicity and to prevent the development of nephro
calcinosis. Clark (1955) also obtained successful treatment
of nephrocalcinosis with EDTA chelation therapy.
EDTA has been recognized as an effective therapeutic
agent which may be used when a rapid reversal of digitalis
intoxication is desired. This action reduces the amount of
calcium available to the actomyosin complex of the heart
muscle, thereby decreasing the effects produced by digitalis.
Jick (1959) was able to decrease or abolish cardiac arrhythmias
of conduction disturbances in patients believed to be intoxi
cated with digitalis. Gubner (1957) also found that EDTA
reduced premature ventricular contractions, terminated digi
talis-induced ventricular tachycardia, improved atrioventricular
conduction in heart block, and increased the rate of the idio
ventricular pacemaker in complete heart block. The chelation
of blood calcium apparently induced a rapid re-entry of
potassium ions into the cells, which promoted a quicker
restoration of sinus rhythm than that produced by the intra
venous infusion of potassium ions.
Na2 EDTA infusion has been effective in treating angina
pectoris. Meltzer (1960) treated ten male patients, each
receiving between 57 and 163.5 grams of Na 2 EDTA over a period
of two to three months. Three months after completion of
the treatments, nine of the ten had significant reduction in
the number and severity of anginal attacks, five of nine
10
electrocardiograms showed improvement, and.no significant
toxicity was encountered.
Na2EDTA infusion also has been regarded as an excellent
method of reducing extensive calcinosis associated with
advanced scleroderma. Klein (1955) and Muller (1959)
obtained definite clinical improvement by intravenous
administration of the chelate, with accompanying dissolution
of large quantities of pathologic calcification. Neldner
(1962) found similar results, but a follow-up study revealed
that although many patients manifested improvement immediately
after chelation, this improvement was temporary in most
instances.
One of the most promising areas for employment of chelating
properties of EDTA has been in the treatment of occlusive
vascular disease. Bolick (1961) demonstrated the effective
ness of EDTA for the removal of calcium from internal
atheromatous plaques in vitro, in which total calcium content
of coronary vessels and calcium removal rates were determined.
The results indicated that coronary atheromatous calcification
can be as extensive as that in aortic atheromas, and that
calcification may occur in two different forms. One form
exhibited characteristic hematoxylin-ringed lacunas when
calcium was removed. These atheromas were slowly extracted
by EDTA, and were believed to contain calcium in discrete
granules. The other form of calcification showed no lacunas,
appeared to be more diffusely infiltrated by calcium, and
11
were rapidly extracted by EDTA. In vivo studies by Koen
(1963) examined the effects of subcutaneous injections
of MgNa2 EDTA on rabbits maintained on a cholesterol-oil
enriched diet. It was found that the intimal surface of
the aortas showed amelioration of atheromata and a marked
decrease in phospholipid. It appeared that chelation therapy
produced a more rapid destruction of phospholipids, a slower
synthesis of phospholipids, and an increased turnover of
phosphate. In human clinical studies, Clark (1960) administered
EDTA intravenously to patients with advanced atherosclerotic
disease. Three to five grams of Na2 EDTA were given, for a
total of 90 to 150 grams, over several weeks. Overall relief
from manifestations of the disease were reported to be superior
to that obtained with other methods, such as by-pass surgery,
excision or stripping of the artery, or various types of heart
stimulates. The best results were found in patients with
intermittent claudication and in occlusive vascular disease
affecting the brain. Good results were also obtained in a
study on fifty patients with various forms of occlusive athero
sclerosis, who were treated with Na 2EDTA for periods of three
months to three years (Lamar, 1968). Improvement in clinical
symptoms found in these patients was interpreted as arising
from the breakdown of atherosclerotic plaques as the EDTA
chelated and removed metastatic calcium from the lesions,
thus widening the contracted arterial lumen, increasing the
blood carrying capacity of the vessels, and subsequently
12
correcting the metabolic failures caused by tissue ischemia.
More recent studies reported in Russian (Nikitina, 1972) and
Czechoslovakian (Brucknerova, 1972) literature indicated
beneficial effects with EDTA on cerebral, coronary, and peri
pheral circulation, and concluded that chelation is the treatment
of choice for vascular disease producing claudication.
A corollary effect of chelation therapy has been the
reduced requirement for insulin that was noted in diabetic
patients (Meltzer, 1962). Pento (1974) reported on investi
gations concerning the insulin secretion in rats and rabbits,
the results demonstrated the importance of precise calcium
homeostasis for normal insulin responsiveness. The author
postulated that insulin release involves stimulus-secretion
coupling, where calcium uptake and its subsequent interaction
with microtubular protein triggers emiocytosis of beta-cell
secretory granules.
Effects on Lipid Metabolism
The effects on different methods of administration of
EDTA salts on cholesterol metabolism in animals has been
thoroughly investigated. Uhl (1953) reported that subcutaneous
administration of Na2 EDTA augmented the dietary-induced
hypercholesteremia in a fashion similar to that induced by
its oral administration, and that both oral and parenteral
Na 2 EDTA protected against deposition of dietary-derived
cholesterol in rabbit liver. There was further indication
13
that ingested Na2EDTA augmented considerably the otherwise
slight hypercholesterolemia which occurs with excess
cholesterol feeding, but the effect was not observed with
parenteral administration. Koen (1963) noted that serum
cholesterol values were higher in EDTA-treated animals,
and this result was attributed to the lipotropic action of
the chelate. It appears that if EDTA is given to animals
on a high cholesterol diet, the drug protects the liver from
fat infiltration or acts as a clearing factor if given after
fat infiltration has occurred.
In studies concerned with the effects of EDTA on lipid
metabolism in humans, Perry (1955, 1960) noted a reduction
of abnormally high serum cholesterol in atherosclerotic and
hypertensive patients following parenteral administration
of EDTA. The infusion of Na2 EDTA in patients with elevated
plasma levels of total esterified fatty acids and trigly
cerides produced a reduction in lipid levels in most cases
(Olwin, 1968). However, these reductions proved to be
transitory, because there was a return toward pre-therapy
levels by the end of four weeks following the last infusion.
A satisfactory explanation for the plasma lipid-lowering
mechanism of EDTA has not been offered, although the possible
relation of the alteration in lipid metabolism to the removal
of essential elements such as calcium, magnesium, zinc, and
copper by the chelate seems plausible. Klevay (1975) found
a thirty-six percent decrease in serum cholesterol after
14
the I.V. administration of Ca Na2 EDTA to rats, along with
a marked zincuria. This action probably resulted from an
increased loss of zinc compared to that of copper, which
decreased the zinc-to-copper ratio, which in turn had been
found to antagonize hypercholesterolemia.
Effects on Trace Minerals
Many metal chelates have apparent great stability
in vitro. In the body, however, many factors may influence
the stability of the metal chelates.
The relative stability of a metal chelate is reflected
by its stability constant, K, (Table I). In vivo, this
constant cannot be used with any definite assurance to
predict reactions because of intricate physiological compe
tetions. In addition, pH and the tendency of the metal to
form insoluble hydroxides, the distribution and metabolism
of the chelate, and the competition of physiological complexing
radicals for the metal ions, all influence stability of
chelates in the body (Johnson, 1960).
As indicated above, the effects of chelation on trace
minerals and the resultant influence on metabolism and
other functions could be significant. Daily injections of
one to twelve grams of Ca Na2 EDTA, given intravenously to
twenty-two human patients, produced markedly increased
concentrations of urinary zinc, and to a lesser extent,
iron and manganese were similarly altered (Perry, 1957).
15
TABLE I
FORMATION CONSTANTS OF VARIOUS METAL-EDTA COMPLEXES
K
Mg 4.9 X 108
Ca 4.0 X 1010
Fe 1.6 X 1014
Co 1.3 X 1016
Zn 1.6 X 1016
Cu 2.5 X 1018
Ni 3.2 X 10 1 8
(Wong, 1975)
16
But the day-to-day variations in urinary concentrations of
other trace elements such as molybdenum, lead, tin, nickel,
titanium, vanadium, chromium, cadmium and silver, did not
present discernable trends.
The infusion of Na2 EDTA has been shown to produce a
diuresis of copper, zinc, or both, in patients with sclero
derma (Rukavina, 1957) or porphyria (Peters, 1957; Price,
1959). EDTA chelation has also increased urinary manganese
excretion (Koen, 1963), the metal being mobilized from de
posits such as bone and nervous tissue to soft tissue, and
then to the blood. Koen also found that the copper content
of livers and aortae in atherosclerotic rabbits treated with
Na2 EDTA showed a significant decrease. The effects of
Na2 EDTA on urinary phosphorus excretion was found to fluctu
ate, with no discernable pattern (Spencer, 1960) .
Administration of EDTA to normocalcemic and hypercalcemic
patients produced increased urinary calcium excretion, but
serum calcium levels were decreased temporarily only in the
normocalcemic patients (Spencer, 1960). Perry (1960) gave
Ca Na2 EDTA parenterally to five hospitalized patients,
which produced markedly augmented zincuria, and excretion
increased more than tenfold less than twenty-four hours
after the infusions. Smaller increases in cadmium, lead,
manganese, and vanadium excretion were also observed.
Kaman (1975) found increased excretion of calcium and zinc,
and decreased urinary magnesium after twenty Na2 EDTA infusions
in an atherosclerotic patient.
17
Schroeder (1974) obtained a reversal of cadmium-induced
hypertension by either feeding zinc or the administration of
a cadmium-binding chelate. Schroeder in turn related deaths
from hypertension more with renal cadmium or to a higher
ratio of cadmium to zinc. The mechanism of this action was
not determined, but it possibly may involve displacement of
cadmium from tissues by zinc in excess. In renal function
investigations using dogs, it was determined that when plasma
calcium ion levels were decreased by EDTA, vasodilations were
mostly observed in the efferent arterioles of the kidneys;
and that sensitivity of the efferent arterioles to angio
tensin was greater than that of the afferent arterioles
(Kover, 1976). It is conceivable that with diminished
calcium ion concentration, renin secretion, angiotensin
production, and sensitivity to angiotensin of vascular
smooth muscle cells are all affected.
Pearl (1977) found that lowering of extracellular fluid
calcium concentration increased renin release, and that
the inhibition of renin release produced by angiotensin was
calcium-dependent. Therefore, if EDTA caused a marked
calcium gradient from the interior to the exterior of the
cell, intracellular calcium stores would be released. As
a consequence, smooth muscle cell contraction would increase
producing an inhibition of renin release by the juxtaglomer
ular cells. As the efflux of calcium increased, there would
be a subsequent decrease in intracellular calcium, smooth
muscle would relax, and renin release would be increased.
18
Toxicological Effects
The use of I.V. EDTA to dissolve plaque calcium, during
early stages of development, was hindered by overdoses and
the resultant kidney (Meltzer, 1961), spleen and liver damage
and even death (Holland, 1953; Dudley, 1955).
Nephrotoxicity has been considered as potentially the
most dangerous side effect. The nature of EDTA nephrotoxicity
is not known, but there is a definite association of vacuolar
changes in the tubular epithelium of the kidneys with a large
dose of the chelate. Schwartz (1966) found intstudies of
lysosomal enzymes after doses of 1.0 to 2.5 grams of Ca Na2
EDTA over twenty-four hours, that certain biochemical changes
place, and increased with greater doses. Data implies there
was a relationship between morphologic (vacuolization) and
enzymatic changes, the mechanism of which is unknown.
However, various in vitro studies have reported safe
levels of the chelate which produced no renal damage.
Proescher (1951) found that rabbits could tolerate as much
as 80 mg of EDTA per kg without toxic symptoms. Foreman
(1953) concluded that a dosage level of 50 mg per kg per
day would be safe for humans, and Meltzer (1961) reported
that three grams of Na 2EDTA per dose to be without danger
of nephrotoxicity. In toxicological studies with rats,
Craven (1975) found that the ED-50 for the first appearance
of renal histological damage in animals treated daily for
sixteen days with EDTA, was 203 mg per kg per day. No renal
19
lesions developed in animals given 62.5 mg per kg per day
for sixteen days.
Hypocalcemia can occur if the chelating agent is admini-4
stered too rapidly, but Seven (1960) indicated administration
of the disodium salt at a rate no more rapid than 15 mg per
minute produced no symptoms.
Several comments have appeared concerning the production
of skin and mucous membrane lesions by the administration of
Na2EDTA for prolonged periods (Perry, 1955, 1957; Clark, 1955).
At times, the lesions seemed similar to those produced by
avitaminosis B6 , and in two cases (Perry, 1957) lesions
appeared even though the patients were on supposedly adequate
doses of vitamins. However, these lesions tended to clear
rapidly upon cessation of the therapy alone, almost always
within six days. There is evidence that under certain
conditions, Na2 EDTA may have anti-vitamin B6 activity or under
other conditions it may have pyridoxine-like activity (Price,
1961). The mechanism by which Na 2EDTA may produce a pyri
doxine-like effect on tryptophan metabolism is not clear.
It is likely that the chelate produces this action as a result
of some effect on the trace metals. The administration of
Na2 EDTA could then show pyridoxine-like or anti-vitamin B6
activity depending upon whether or not it was correcting or
creating an imbalance.
A drop in bloodpressure in hypertensive experimental
animals has been reported after infusion with Na2EDTA
20
(Schroeder, 1955), and is possibly linked to a vasodepressor
activity for the metal-binding compound. Perry (1972) used
the chelating properties of Ca Na2 EDTA to test the possibility
that metal-binding was involved in hypertension, by infusing
hypertensive animals. The results indicated that blood pressure
could be lowered to normal by the chelator, but not below.
Accordingly, Seven (1960) reported that a significant depressor
response in normotensive patients who were treated with
Na 2EDTA was not observed.
Mode of Action
The proposed action of EDTA in chelation therapy for
cardiovascular disease is its affect on the metabolic
functions of calcium, zinc, and possibly other trace minerals
(Kitchell, 1961). First, chelators affect the removal of
calcium, most probably from sites where it could be deposited
in association with lipid in early atheromatous lesions.
Also, the parathyroid glands are stimulated by a lowered
circulating calcium, which causes a more rapid turnover of
calcium. Finally, it is possible that the altered availability
of a metallic ion, perhaps an alkaline earth or transition
metal, might affect an enzymatic process or even a physical
property, such as membrane permeability, with the resultant
metabolic effects. Wilder (1962) found that EDTA mobilized
calcium from in vitro atherosclerotic vessels when perfused
with a five percent solution, and the quantity of calcium
21
released was proportional to the degree of atherosclerosis
present. He believed that a possible action of EDTA is an
interaction with polyvalent cations to form soluble non
ionic complexes, which prevents the component cation from
participating in its normal metabolic process.
Meltzer (1961), in analyzing the possible toxicity of
EDTA, arrived at a basic concept for use of the chelator.
EDTA removes calcium from serum which is promptly replaced
from the calcium reserve, which in turn is restored by
loosely bound calcium from metastatic sites, thus producing
the beneficial effects. A similar hypothesis was expressed
by Boyle (1963), in work with arthritic patients. He theo
rized that lowering of the calcium ion in the extracellular
fluid, if continued long enough, could result in the mobili
zation of aberrant deposits of the metal in disease. Also,
intermittent decrease of serum calcium ions might stimulate
parathyroid activity, and parathyroid hormone has sobulizing
effect on both apatite and bone matrix.
Lamar (1966) considered that the effect of EDTA is pro
duced only upon metastatic calcium and not upon normal
tissue calcium. He reported that it has been shown consis
tently by the lack of the development of osteoporosis or
of increased dental caries, and by the increase of roent
genographic bone density which has been observed in cases
of EDTA-treated osteoporotic patients, normal calcium was
not deleteriously affected.
22
Wartman (1967) in studies using Mg EDTA, described
the physiological effect As being indirect, mediated by
a decrease in ionized calcium levels in the blood, which
stimulates production of parathyroid hormone. The hormone
in turn mobilizes bone mineral and solubilizes organic matrix,
releasing hydroxyproline and hexosamine into the plasma. It
is therefore reasonable to assume that the chelator can
cause changes in the metabolism of connective tissue com
ponents of atherosclerotic tissue such as aorta. Further
indications that the action is hormonal, are that both elastin
and apatite are removed simultaneously and that the percentage
of calcium in elastin is not changed in Mg EDTA-treated
animals.
Lamar (1975) theorized that the calcium complexed in
the EDTA molecule comes from the aberrant or metastatic
calcium deposits. He suggested that dental and bone calcium
is too tightly bound to the organic protein framework of
the tissues to be mobilized in much more than the normal
constant physiological amounts of daily turnover, unless the
framework has been damaged by trauma or disease.
The pharmacologic aspects of EDTA chelation indicate
that ninety-five percent of the chelate appears in the urine
after twenty-four hours, and less than 0.5 percent remains
in the body after forty-eight hours (Craven, 1975). The
compound is not metabolized and is neither reabsorbed nor
secreted in any segment of the tubules, so that alteration
23
in urine flow rate and or in pH do not affect overall excretion
rate. Smith (1977) found an increased rate of destruction
of lipoprotein produced by EDTA in samples of aortic intima.
The greatest effect was found in the amorphous atheroma lipid
fraction of fibrous and gelatinous plaques, where the rate
of destruction was increased six-fold by EDTA. The plausible
explanation for the action was by enzyme degradation, either
as a result of direct action, or by an alteration of the
interaction between lipoproteins and components of extra
cellular matrix, so that the lipoprotein becomes more
available for destruction.
In summary, a review of the literature indicates that
the use of EDTA for the removal of calcium has been proven
to be effective in the treatment of calcinosis, scleroderma,
in problems of arrhythmia resulting from digitalis toxicity
or from coronary artery sclerosis, and has been disappointing
in the dissolution of renal calculi. EDTA seems to be more
suited for use in man than other chelators, such as diethylene
triaminepentaacetate (DTPA), cyclohexane trans 1,2-diamine
tetraacetate (CDTA), (2-hydroxyethyl) ethylenediaminetetra
acetate (HEEDTA-N), 2,3, dimercaptopropanol (BAL). A more
recently developed chelator, ethylene glycol Bis-(B-amino
ethyl ether) -N,N' -tetraacetic acid (EGTA) shows considerable
promise due to a greater specificity for calcium than do
other chelators. However, few studies employing EGTA have
been reported to date.
24
The maximum effectiveness of EDTA therefore may prove to
be in the treatment of calcific atherosclerosis, and in
other metastatic calcifications of soft tissues, when applied
under conditions of dosage, concentration, and rate to produce
no deleterious side effects.
The research described here endeavored to determine
the effectivenss of Na2 EDTA chelation therapy on the regression
of the calcified plaques associated with atherosclerosis in
New Zealand albino rabbits. It should be recognized that there
is a difference between rabbit and human plaque formation:
calcification in the rabbit aorta is localized in the media,
while that in the human artery is found intimally. Further
mire, the rabbit serum cholesterol levels recorded in this
study (1200 mg %) exceeded even the highest levels found in
the most serious human disease states (1000 mg %); and the
very low density lipoprotein fraction is the principle choles
terol carrier in the rabbit, while the human counterpart is
the low density lipoprotein fraction. (Hass, 1966) However,
the calcium in the rabbit artery, due to its location, may
present a greater challenge to chelation therapy than would
the intimal calcification of the human artery. In addition,
the excretion levels of several important metallic divalent
cations was monitored in order to provide data for the
development of mineral replacement protocols to be used in
conjunction with the therapy.
CHAPTER III
METHODS AND MATERIALS
Thirty-six New Zealand albino (Oryctolagus cunniculus)
rabbits (Pel-Freez, Rogers, Arkansas) 3-4 kg each, were
individually caged and maintained on a diet of Purina Rabbit
Chow (Ralston Purina Co., St. Louis, Mo.). These animals
were placed on a therapeutic dose of tetracycline (Tetrachel
S, Rachelle Laboratories), 20 mg/day/rabbit, for a period of
two weeks as a precaution against infections (Bailey, 1977).
Twenty-four of the rabbits were placed on a cholesterol
diet, which was prepared by adding a sufficient amount of
a 10 percent solution of cholesterol in corn oil (Mazola)
to the Rabbit Chow to give concentrations of 250 -. to 500 mg
cholesterol per 100 gm diet. Additionally, 105 International
Units of Vitamin D3 (cholecalciferol, Sigma Chemical Co.,
St. Louis, Mo.) suspended in corn oil (Mazola) were given
subcutaneously to these rabbits at three-day intervals
every four weeks during the diet, for a total dosage of
106 International Units each. Hass (1966) has shown that
this regimen consistently has produced a severe chronic
calcified medial degeneration of the arterial system. The
remaining twelve rabbits were maintained on an untreated
Rabbit Chow diet to serve as controls.
25
26
Cholesterol-fed animals remained on this atherogenic
diet for a total of twenty-three weeks, then were placed
on untreated Rabbit Chow before chelation therapy (infusion)
was begun. Four rabbits from this group remained on the
atherogenic diet throughout the chelation process (Group D
below). One month after completion of the cholesterol diet,
the rabbits were divided into the following seven groups
for infusion of Na 2EDTA: A - control diet, no infusion;
B - control diet, EDTA infusion; C - control diet, saline
infusion; D1 - cholesterol diet, EDTA infusion; D - choles
terol/control diet, saline infusion; E - cholesterol/control
diet, saline infusion; F - cholesterol/control diet, no
infusion.
Cholesterol Determinations
Total serum cholesterol levels were monitored every
three-to-four weeks during cholesterol feeding, and at the
beginning, midpoint, and completion of chelation treatment.
Blood was drawn via the middle ear vein, using a 10 ml
disposable syringe, while the animals were placed under
Innovar-vet (Droperidol, Cutter Laboratories) anesthesia
(1.0 ml/7.9 kg), and the blood was then placed in a plain
glass vacutainer (-Sherwood Medical Industries, Deland, Florida)
for centrifugation.
Cholesterol determinations were performed using ferric
acetate-uranium acetate and sulfuric acid-ferrous sulfate
reagents (Parekh, 1970). (These reagents function as
27
precipitants to extract the cholesterol and also to provide
color development). The author reported percent recoveries
of cholesterol in the range of 98.5 - 100.7, and reproduc
ibility of the method was the best of those presented to date
in the literature, Specifically, the ferric acetate-uranium
acetate in the acetic acid performs as a precipitant of
proteins as well an an extractant of cholesterol from serum.
It not only separates proteins from the serum but success
fully overcomes interference by other chromogens, notably
the lipids and bilirubin. Additionally, the reagent composed
of ferrous sulfate in sulfuric acid confers unusual stability
of milieu to the iron-cholesterol reaction.
Apparatus
A Coleman Jr. II Spectrophotometer (13 mm light path
and a 20nM band pass) , acid resistant dispensor, glass tube
cuvetts with polyethylene caps (Ames Co.,, Elkhart, Ind.),
unscratched and calibrated, and reaction tubes, 16 X 125 mm,
with polyethylene caps (Ames) was used for the cholesterol
analyses.
Preparation of Reagents
All chemicals used were of analytical grades. Ferric
acetate-uranium acetate (Fe (C2 H3 02 ) -UO 2 (C2H30 2 )2) reagent
was prepared by converting 0.5 grams FeCl3 - 6 H20 (Fisher
Scientific Co., Fair Lawn, N.J.) to Fe(OH)3 by adding 3.0 ml
of concentrated ammonium hydroxide (Fisher). The precipitate
was washed, filtered, rewashed, and dissolved in glacial
28
acetic acid (Mallinckrodt Chemical Works, St. Louis, Mo.)
to a one-liter volume. Therafter, 0.1 gm of powdered
uranium acetate (UO2 (C2 H3 02 )2 * 2 H20) was added and the
contents were shaken before and after standing overnight.
Sulfuric acid-ferrous sulfate (H2So 4 -FeSO4 ) reagent
was prepared by dissolving 0.1 gm of anhydrous ferrous
sulfate (Fisher Scientific Co., Fair Lawn, N.J.) in 100 ml
of sulfuric acid (Fisher), and the contents mixed by
whirling. After cooling, concentrated sulfuric acid was
added up to a volume of one liter.
Procedure
Fifty microliters of sera and of cholesterol standard
(Ames Co., Elkhart, Ind.) were added to reaction tubes,
then 10 ml of ferric acetate-uranium acetate reagent, and
each tube was mixed by inversion and by vortex. The tubes
were allowed to stand for five minutes and then were
centrifuged for five minutes at 2,100 X G. Three ml
aliquots of the supernatant were transferred to the cuvetts,
2.0 ml of sulfuric acid-ferrous sulfate reagent added, the
cuvetts were capped with polyethylene caps and swung at 1800
angle ten times for mixing. The samples were read against
a blank of 3.0 ml of acetate reagent and 2.0 ml of sulfate
reagent, at 560 nM after twenty minutes.
29
Infusion Procedures
The rabbits were infused with either disodium EDTA or
normal saline.
N-a 2 EDTA Solution
Preparation of Na2EDTA solution was accomplished by first
adding 100 gm of Na2 EDTA to a two liter beaker, along with
approximately 600 ml of distilled water, and the mixture
stirred until a uniform suspension was attained. Fifty ml
of a solution containing twenty-one gm per 100 ml sodium
hydroxide (Fisher Scientific Co., Fair Lawn, N.J.) was
added and the mixture was heated and stirred to ensure
complete dissolution. After cooling, the pH was adjusted
to 7.25, by the addition of small volumes of hydrogen
chloride or sodium hydroxide. The solution was then filtered
into a dne-liter volumetric flask, brought up to volume
with distilled water, and mixed by inversion. One hundred
ml of this solution was added to normal saline to produce
a total volume of one liter. Sterilization of the EDTA
solution and the normal saline was accomplished by autoclaving
at 1200C for twenty minutes (Clark, 1955).
Infusion was performed via the marginal ear veins, alter
nating ears, utilizing a thirty ml plastic syringe and a Harvard
apparatus syringe pump. The dosage was fifty mg per kg body
weight, at a rate not greater than fifteen mg per minute (Seven,
1960). Non-EDTA treated rabbits were infused with normal saline
30
in the same manner, using equivalent volumes as determined
from the EDTA dosage levels. Innovar-Vet, as described
previously, was used as anesthesia. Each rabbit was in
fused a total of twenty times, on alternating days.
Histology
Histological studies of the rabbit aortas were done to
ascertain the presence of atherosclerotic calcification and
lipid accumulation.
Procedure
Dahl's Method (Luna, 1968) utilizing alizarin red S and
light green, SF yellow, SH, was employed to detect the presence
of aortic calcium. Calcium salts, with the exception of calcium
oxalate, are stained intense reddish-orange, with a pale green
background counterstain, using this method.
After the rabbits were sacrificed via sodium pentobar
bital, thoracic aortas (from the heart to diaphragm) were
excised, cleaned of excess connective tissue, washed in cold
saline, and divided into two equal longitudinal sections.
One section was placed in Carnoy's fixative (ten percent
glacial acetic acid, thirty percent chloroform, sixty percent
absolute ethanol), and the other section was utilized
immediately for direct microcomplexometric determination of
tissue calcium. Ten mm sections of the aortic arches selected
for histological study were washed in distilled water for six
to eight hours. The tissue then was dehydrated through
31
a series of alcohol (50, 70, 95, 100 percent) and toluene
rinses, and subsequently embedded in paraplast for sectioning.
The aortae were cut into ten micron cross sections utilizing
a microtome (American Optical, Buffalo, N.Y.) and the sections
were placed on glass slides. These sections next were re
hydrated through a series of xylenes and alcohol (100 and 95
percent), stained with alizarin red S and light green, de
hydrated with graded alcohols (70, 95, 100 percent), 50/50
absolute ethanol/xylenes and xylenes, then fixed with permount.
Tissue Calcium Determinations
The quantitation of tissue calcium levels in the rabbit
aortae samples was accomplished using the direct microcomplexo
metric analysis of Mori (1959). This method employs complexo
metric titration of calcium with the disodium salt of 1,2
diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), in
the presence of fluoresceinbismethylene-iminodiacetic acid
(calcein) indicator.
Reagent Preparation
All chemicals used were of analytical grade. The indicator
solution was prepared by adding four mg of calcein (Sigma
Chemical Co., St. Louis, Mo.) to 100 ml of 0.25 N potassium
hydroxide (Fisher Scientific Co., Fair Lawn, N.J.) and kept
refrigerated to maintain stability. CDTA (Sigma) solution
was prepared by dissolving forty-five gm in one liter of
distilled water. This stock solution was diluted to 100 times
to prepare the working solution.
32
Stock standard calcium solution was prepared by first
drying calcium carbonate (Fisher) in an oven overnight at
1100C, then by adding 0.001 gm to a 100 ml volumetric flask
containing twenty-five ml of distilled water. Five ml of
1.0 N hydrochloric acid (Fisher) was added to the mixture,
and complete dissolution and evolution of CO2 was accomplished
by heating to approximately 606C. After cooling, the volume
was brought up to the mark with distilled water. One ml of
this stock standard solution corresponded to twenty micro
equivalents of calcium. The working standard calcium solution
was prepared by exactly diluting 1:20 with distilled water,
and 1.0 ml of this working standard solution represented
one microequivalent of calcium.
Standardization of the CDTA solution was done by pipeting
2.0 ml of the working standard calcium solution into a small
beaker, to which 5.0 ml of 3.0 N KOH and several drops of
calcein solution were added. The mixture was then well
shaken, and titrated with the working CDTA solution until the
color changed from yellowish green (with fluorescence) to
pink (without fluorescence). This titrated volume of CDTA
solution then represented two microequivalents of calcium.
Procedure for Determination of Tissue Calcium
The remaining longitudinal sections of the freshly excised
thoracic aortas (described above), after being blotted on
filter paper and cut into four to five small pieces, were
33
placed into pre-weighed small glass beakers which contained
5.0 ml of 10 percent tricholoroacetic acid (Fisher Scientific
Co., Fair Lawn, N.J.). The beakers were weighed and the
contents of each were placed into a twenty five ml capacity
micro-stainless steel blender (Waring Products, New Hartford,
Conn.), and 5.0 ml of the 10 percent TCA were added. The
contents were homogenized for approximately six minutes.
The final volume of the homogenate was adjusted to 15 ml
with the 10 percent TCA, then was centrifuged at 1,500 XG
for 15 minutes, and the clear supernatant used for
calcium analysis. Two ml of this supernatant were added to
a small beaker which contained 8 ml of 3.0 N KOH, several
drops of calcein were added and the mixture was titrated with
the standardized CDTA solution. Blank titrations were done
by replacing the supernatant with 2.0 ml of 10 percent TCA.
Mineral Determinations in Urine
Atomic absorption spectroscopy was chosen as the method
to quantitate the amounts of minerals chelated by the Na2 EDTA
and subsequently excreted by the kidneys into the urine.
The excretion levels of the divalent cations of calcium,
magnesium, zinc, and cadmium were selected for study due
to their possible metabolic interrelationships, effects
on the atherogenic process, and in the case of cadmium, effects
on blood pressure.
34
Apparatus
A Perkin-Elmer Atomic Absorption Spectrophotometer,
Model 370 (Perkin-Elmer Corporation, Norwalk, Conn.) was
used for the analyses. Air/acetylene fuel and hollow cathode
lamps (combination calcium, magnesium, zinc) and cadmium
were utilized. The Model 370 instrument incorporates a single
grating and reads outwavelength directly in nanometers,
with normal slit setting optimized for flame sampling for
operation with the standard burner/nebulizer system. General
analytical procedures as presented in Perkin-Elmer Instruction
Manual (September, 1976) and by Christian (1970) were utilized.
Preparation of Reagents
Lanthanum chloride, 1.0 M stock solution was prepared
by dissolving 162.92 gm of lanthanum oxide (Fisher Scientific
Co., Fair Lawn, N.J.) with approximately 320 ml of concentrated
hydrochloric acid (Fisher) and diluted to one liter with
deionized (10 megohm) water. Calcium chloride solution was
prepared by drying calcium chloride solution powder (Fisher)
overnight at 1100 C., then by dissolving 100.009 gm in a mini
mal amount of concentrated hydrochloric acid, and diluting
to one liter with deionized water. This produced a 100-mM
stock solution.
Magnesium chloride solution was prepared by dissolving
2.432 gm of magnesium metal (Fisher) in a minimal amount of
concentrated hydrochloric acid and diluting to one liter
35
with deionized water. Cadmium solution was prepared by
dissolving 1.000 gm of cadmium metal (Fisher) in a minimal
amount of concentrated hydrochloric acid and diluting to
one liter with deionized water.
All deionized water used to prepare standard solutions
and urine samples was at least ten-megohm purity, obtained
from a millipore water purifying system (Model 1200 System,
Millipore Corp., Bedford, Mass.). Calibration of the atomic
absorption spectrophotometer was accomplished using diluted
standards prepared from the stock solutions.
Procedure
Twenty-four hour urine samples were collected on both
infusion and non-infusion days. Twelve of the rabbits
undergoing chelation treatment were placed in individual
metabolism cages (Hoeltge Co., Cincinnati, Ohio) and the urine
was collected in 500 ml polyethylene containers (Nalgene,
Co., Boston, Mass.). Twenty-four-hour urine samples pre
viously had been taken in the same manner as above from
twelve control rabbits on three consecutive days. After
collection, the urine was well mixed by vigorous shaking,
and the volume was measured to the nearest millimeter.
The volume to be preserved was then placed into 25 ml
polyethylene bottles (Nalgene), which had been triple-washed
in deionized water and previously charged with a lentil
sized crystal of thymol (Grun-Baum, 1970). The thymol has
36
the effect of keeping the urine bacteriostatic. These storage
bottles were stoppered with polypropylene caps (Nalgene),
marked with indelible ink showing the code number of the sub
ject, date, and actual start and end time of collection.
The urine samples then were placed in a storage freezer which
was maintained at -10 0C, until mineral urinalysis was per
formed. The samples were prepared for analysis by slow
thawing at room temperature. Ten ml of urine from each
rabbit for each day were pooled, and 25 ml of the mixture
was wet-digested with 1:5 sulfuric acid : nitric acid
(Fisher), according to the method of Christian (1969).
Next, the pooled samples were diluted with ten megohm deion
ized water except that lanthanum chloride stock solution was
used to dilute samples to be run for calcium analysis, in
order to avoid interference in the flame. Dilutions were
accomplished using volumetric pipettes and flasks in order
to ensure accuracy. The dilution levels of the four elements
to be analyzed were dissimilar due to the different urine
concentrations and linear working ranges of each. The
following urine dilutions were utilized to ensure the
mineral concentrations met the above criteria: calcium,
1:1,000; magnesium, 1:2,000; zinc, 1:5; cadmium, undiluted.
The atomic absorption spectrophotometer was calibrated
in the direct-reading concentration mode, by first setting
zero using a blank solution, then utilizing a standard
solution to set the scale at the maximum level of the linear
37
working range for the element to be analyzed (Perkin Elmer,
Corp., 1976). Intermediate concentrations which fell be
tween zero and the maximum were utilized to check the
linearity of instrument. After the instrument had been
adjusted for optimum performance and calibrated with the
standard solutions, the urine samples were analyzed. The
meter was read directly as concentration due to the linear
response of the standard solutions. Thus, by including
the dilution factor, calculation of the element concentrations
in the undiluted urine samples was a simple matter.
Statistical Methods
The statistical method employed to examine all quantitative
results in this study was a test of significance for sample
means. This method was chosen because of relatively small
sample sizes (N - 10), and,because the universe standard
deviations were not known, as a result sample standard
deviations were used as estimates. Therefore, the theoretical
sampling distribution of difference was assumed to be a t
distribution with a mean equal to zero and a standard deviation
that is the estimated standard error of the difference. All
significant differences were recorded at the (p<.05) level.
Serum Calcium
Analyses of the levels of serum calcium before and
after EDTA infusion will not be included in the present
investigation because pilot studies by the author utilizing
38
flame photometric methods, as well as reports in the literature
(Perry, 1957; Spencer, 1960), found no significant changes
in serum calcium levels produced by EDTA infusion.
CHAPTER IV
RESULTS
Serum Cholesterol
Hass (1966) has shown that cholesterol feeding in
conjunction with Vitamin D 3 injections would produce
severe chronic calcified medial degeneration in the arteries
of rabbits after a period of three to four months, and that
atheromatous lesions developed when maximum serum cholesterol
levels reached 750 mg percent or more. The twenty-four
rabbits which were maintained on the cholesterol diet had a
mean cholesterol level of 923 mg percent and an average
standard deviation of 283 mg percent, over a period of
twenty weeks. The average maximum reached 1,206 mg percent
with a standard deviation of 275 mg percent (Figure 3).
The control group averaged 68.0 mg percent serum cholesterol
with a mean standard deviation of 17.2 mg percent. The
average body weight increased from 3.2 to 3.5 kg over the
same time period. The animals ate normally and there were
no significant differences in weight gain between experi
mental and control groups. No grossly visible behavioral
differences nor pathological changes in the animals were
noted.
39
FIGURE 3. - Mean Serum Cholesterol Levels in Control and Experimental Rabbits
41 -J
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42
Rabbits were sacrificed at periodic intervals to
ascertain the efficacy of the atherogenic diet. After
twenty-one weeks on the diet, two rabbits were selected
at random and sacrificed via pentobarbital sodium injection.
The aortae were excised and examined histologically to
determine the presence of atheromatous plaques. Extensive
calcification in both aortae was observed with Dahl's method
of calcium staining as well as extensive lipid deposition.
(Figures 4 and 5). These findings resulted in a decision
to remove most of the animals from the cholesterol diet,
and after an interval of four weeks, to start Na2 EDTA/saline
infusion. After infusion was completed, all groups were
placed on Purina Rabbit Chow diet and held for six weeks,
at which time all were sacrificed via pentobarbital sodium,
and aortae were excised for histological examinations and
tissue calcium determinations.
Histology and Tissue Calcium
Groups A, B, and C: Control Diet
Each of these groups was maintained on a normal Rabbit
Chow diet. Group A consisted of three animals which were not
subjected to infusion, while Groups B and C were infused with
EDTA and saline, respectively. Six weeks after infusion, his
tological examination of the aortae revealed no lipid localization
in the intima nor atheromatous plaque with calcification
(Figures 6 and 7). The average aortic tissue calcium level
FIGURE 4. - Aorta of Rabbit on Atherosclerotic Diet
FIGURE 5. - Aorta of Rabbit on Atherosclerotic Diet
t
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FIGURE 6. - Aorta of Group A Rabbit
FIGURE 7. - Aorta of Group A Rabbit
46
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47
(Table II) was significantly greater (p .05) than found in
those controls which were infused with Na2 EDTA solution,
(Group B below).
Group B was comprised of four rabbits which were in
fused with Na2 EDTA solution. The aortae showed no calcified
atheromatous plaque (Figures 8 and 9), nor any evidence of
lipid deposition upon histological examination. When aortic
tissue calcium was quantified (Table II), significantly
less (p .05) was found than in those control animals which
were infused with saline (Group C) and in those which were not
infused (Group A). Group C was composed of four rabbits
which were infused with normal saline solution. Histological
examination of the aortae from this group revealed an absence
of calcified plaques and lipid deposition (Figures 10 and 11).
The average aortic tissue calcium level (Table II) was sig
nificantly greater (p .05) than that found in animals which
were infused with Na2 EDTA solution (Group B) but not signifi
cantly greater (p .05) than those which were not infused.
Groups D, E, and F: Cholesterol Diet
Each of these groups was maintained on the atherogenic
diet until four weeks before infusion was started, except
subgroup DI which remained on the cholesterol diet
throughout infusion. Group D was made up of eight rabbits
all of which were infused with Na2 EDTA. However, this group
was divided in half, one subgroup (DI) remained on the diet
48
TABLE II
AORTIC TISSUE CALCIUM LEVELS
Group Nuber Diet Infusion CAlcim-N Animals Clim
(microequivalents/gm)
A 3 Control None 291 6
B 4 Control EDTA 198* 8
C 4 Control Saline 334 8
D 4 Cholesterol EDTA 232* 8
D 4 Cholesterol/ EDTA 351* 8 Control
E 4 Cholesterol/ Saline 635 8 Control
F 3 Cholesterol/ None 777 6 Control
* Significant difference by t test (p. 05)
FIGURE 8. - Aorta of Group B Rabbit
FIGURE 9. - Aorta of Group B Rabbit
50
7')
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FIGURE 10. - Aorta of Group C Rabbit
FIGURE 11. - Aorta of Group C Rabbit
52
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53
throughout infusion, the other subgroup (DII), was placed
on normal Rabbit Chow during infusion. Histological
examination of the aortae of this group showed no medial
calcification, but areas of lipid accumulation as evidenced
in Figures 12 and 13. The average aortic tissue calcium
(Table II) found in this group was significantly less (p .05)
than that found in experimental animals which were infused
with saline (Group E) or not infused (Group F). Analysis
of aortic tissue calcium within Group D revealed that those
rabbits which remained on the cholesterol diet during infusion,
(subgroup DI) had significantly less (p .05) calcium than
those which were removed from the atherogenic diet before in
fusion (subgroup D11 ). This suggests an influence on the
chelation process by excess dietary cholesterol.
Group E was composed of four rabbits which were infused
with normal saline. Considerable calcified plaque development
and lipid deposition were shown by histological examination
of the aortae (Figures 14 and 15). Aortic tissue calcium
levels (Table II) were significantly greater (p. 05) than
calcium levels found in those experimental animals which were
infused with Na 2 EDTA (Group D), but not greater (p .05) than
the calcium found in the aortic tissue of experimental rabbits
which were not infused (Group F).
Group F consisted of three rabbits which were not
infused. Histological examination of the aortae from the
animals in this group showed extensive plaque calcium
and lipid accumulation (Figures 16 and 17). The average
FIGURE 12. - Aorta of Group D Rabbit
FIGURE 13. - Aorta of Group D Rabbit
55
FIGURE 14. - Aorta of Group E Rabbit
FIGURE 15. - Aorta of Group E Rabbit
57
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FIGURE 16. - Aorta of Group F Rabbit
FIGURE 17. - Aorta of Group F Rabbit
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60
aortic tissue calcium (Table II) found in this group was
significantly greater (p<.05) than that found in experi
mental rabbits which were infused with Na2 EDTA (Group D).
There was no significant difference (p<.05) in aortic
tissue calcium levels from those experimental animals
which were infused with normal saline (Group E).
Mineral Excretion in Urine
Only twelve individual metabolism cages were aVailable
for the collection of twenty-four-hour urine samples.
Therefore, urine collection was done on one-half (twelve) of
the total number of rabbits undergoing infusion for five
infusion days and five non-infusion days, then from the
other half for the same time period. Consequently, twenty
four-hour-urine samples were collected for a total of ten
infusion days and ten non-infusion days for each animal. In
addition, twenty-four hour urines were collected from twelve
randomly selected rabbits for three consecutive days prior
to the start of the atherogenic diet in order to determine
baseline excretion levels (Group G).
Groups B and C: Control Diet
These groups were maintained on a normal Rabbit Chow
diet previous to and during chelation therapy. Group B
was infused twenty times with Na2EDTA solution, and Group C
twenty times with normal saline. Twenty-four hour urines
were collected from each group on ten infusion days and ten
61
non-infusion days for mineral analyses. Mean excretion levels
for calcium, magnesium, zinc and cadmium for each group
are shown in Table III. Those control animals infused with
Na2 EDTA(Group B) showed significantly greater (p<.05)
excretion of calcium, magnesium and zinc than controls
infused with normal saline (Group C), on both infusion and
non-infusion days. Group B zinc excretion exhibited a
consistent pattern of higher levels on infusion days compared
to non-infusion days (Figure 19). This characteristic was
not found amoung Group C animals (Figure 21). Group B
but not Group C, showed significantly greater (p<.05)
excretion of calcium, magnesium, and zinc than did control
rabbits which were not infused (Group G). No significant
(p .05) difference in cadmium excretion was noted among
the control groups.
Groups D and E: Cholesterol Diet
These groups were maintained on a cholesterol diet until
one month before infusion was started, except subgroup D1 ,
which remained on the atherogenic diet during the chelation
treatment. The rabbits in Group D were infused twenty
times with Na2 EDTA: and those in Group E twenty times with
normal saline. Twenty-four hour urines were collected from
each group on ten infusion days and ten non-infusion days
for mineral analyses. Table III shows the average excretion
levels of the four minerals analyzed for each group. There
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H
H H HH H H
H N H(N H NH (N H N
62
0 L()
(N
LC)
N
LO 0 m
0
z
C
0 Z
H
0
0
C
-P
0 u
4
0
4
4-' 0 C
H
0
C) 4-' U)
0
z 0 H
rz
H
H
H H H
w
F:4
z
0 U) PQ
0
0
Cl)
C) H
0
to
0 0 4-4
H
m 0
4-) Z -HC) -Hr- tor
4-- :5
cC ~ H
4 4-4
(1
04
0 n
0
C)
4
to
C)
4-' 44)
to -)
5-4 C
-4 -H rd
to
C0 0 -H
4-4 o H i I\ S4-I ~ o 0
HZE H(N
FIGURE 18. - Urinary Calcium and Magnesium Excretion in Group B Rabbits
()
z4
(I0 0 ZZ
LL.
Z ... z 0l |
I I
0 LO)
64
z
.2
H
H
E
2
H
2
H
H
0* 6 ('J
0Ik 1 qft.
FIGURE 19. - Urinary Zinc and Cadmium Excretion in Group B Rabbits
66
z
z
>- 0
N 0 LLH
zz
-10
H H zz
LLU
z
H
z
'H z
H
z
H
c~~iW SS
FIGURE 20. - Urinary Calcium and Magnesium Excretion in Group C Rabbits
0
2 DD SLU.
oz <
G
>0
>0
)
)
>0 ~il trW
0 09 rO)
0N LO 0
-lvf/
68
z
z
H
H
C') 7
EU 0 a
6
E H
7
H
7
H
7
H
0I I
FIGURE 21. - Urinary Zinc and Cadmium Excretion in Group C Rabbits
70
zf 0
2Z
Q 0
:D 0 U-0 Z
Z
z H 0 z
C
z
HE
1-4
z
H
H
z
I I I I I I Laz '' I ) 0 0C504
p~'.LC) ~ 4. ()% OD t--- r6 -I
71
was no significant difference (p<.05) in mineral excretion
levels between subgroups DI and DII, except that a signifi
cantly higher (p<.05) zinc excretion by the rabbits in sub
group D I was noted on infusion days only.
Both subgroups DI and D excreted zinc at higher levels
(p<.05) on infusion days than did cholesterol-fed rabbits
which were infused with saline (Group E). On non-infusion
days, subgroup DI exhibited higher (p<.05) excretion of
zinc and magnesium than did Group E. Group D showed no
significant difference (p<.05) from control rabbits which
were infused with Na2 EDTA (Group B),, in any of the mineral
excretion levels, except that Group B zinc excretion was
greater than that of subgroup DI on infusion days.
All of the animals on the atherogenic diet which were
infused with Na2 EDTA .(Group D),, showed significantly higher
(p<.05) excretion of calcium and zinc than did untreated
controls (Group G). No significant differences (p<.05)
were found in cadmium excretion between Groups D and E,
nor between these two groups and the controls.
A significant (p<.05) increase in zinc excretion on
infusion days compared to non-infusion days in Group D was
noted (Figures 23 and 25). However, this result was not
found in any of the other mineral excretion levels (Figures
22 and 24).
FIGURE 22. - Urinary Calcium and Magnesium Excretion in Group D Rabbits
:D 0 Lf)
02 4Z
LA. 2
z d OO
-U
-
9 0e.
73
I I
2:
H
2:
H
2:
H ()
z'0
E H;
2:
H
2
H
2:
H
2:
H
2
H0 t
0 re)
0. C'4
%4140
-1 N1
FIGURE 23. - Urinary Zinc and Cadmium Excretion in Group DI Rabbits
zz 4-
IIz
0 o 0 4D U 6 6 6
75
(
(
(
z
1z
(
(
(
(
()
z
'U
hi
z
z
H
z
H
z
0A."i.
FIGURE 24. - Urinary Calcium and Magnesium Excretion in Group D1I Rabbits
77
U3 -4 2. Q
- 0 2 :III>e
z 0u Z LL
0 z z
z z
3 2'
H H
}-1 1'
z 71
2:
I I I I 1 0 0 0. 0. .q
D 641
~m's
FIGURE 25. - Urinary Zinc and Cadmium Excretion
in Group D I Rabbits
79
u)
o z
ZH
-4 _
N 0
IIIIt, Z
80
Group E was composed of cholesterol-fed rabbits which
were maintained on a control diet during normal saline in
fusion. Calcium excretion in this group was significantly
greater (p< .05) than that of saline-infused controls (Group
C) on both infusion and non-infusion days, and in non-infused
controls (Group G) on non-infusion days only. Zinc excretion
in Group E was greater (p <.05) than in Group G on infusion
days, and Group E magnesium excretion was greater (p <.05)
on non-infusion days than that found in Group G. No signifi
cant differences (p. < 05) in any excretion levels between
infusion and non-infusion days were noted in this group
(Figures 26 and 27). Cadmium excretion exhibited no signifi
cant (p<.05) differences between Group E and any other group.
FIGURE 26. - Urinary Calcium and Magnesium Excretion in Group E Rabbits
82
Ld 0
z 0 L
>0
L)
0* 0*I0
Q I
LLZ Z
z
z
.c
HE z-
H
H
H
H
LO0 0" 0
, I W/t
FIGURE 27. - Urinary Zinc and Cadmium Excretion in Group E Rabbits
:>- z
-< 0 0
GOMM
z z
_ z
0. 06,
0
84
z H:I(
(
(
(
(
(
(
(
)
)
)
F
z
z(1)
H 0
z c
E H
H
z
H
z
H
z
H
z
0 (cr
0 LO)
0 ro'
)I 1 1 I -4--
0 (NJ
0
-1 Vq /b 11
6
CHAPTER V
DISCUSSION
A group of twenty-four New Zealand albino rabbits,
following a twenty-three week atherogenic diet, were treated
with either intravenous infusions of disodium ethynelediamine
tetraacetic acid (Na2EDTA) or normal saline.
There can be little doubt that Na2EDTA produced a marked
decrease in the plaques of induced atherosclerosis in these
rabbits. Histological evidence indicated that calcified
plaques were present in the animals which were fed the
atherogenic diet and subsequently infused with saline or
were not infused, but were absent from those which were infused
with Na2 EDTA. This effect was manifest whether the animals
were cholesterol-fed during Na 2EDTA administration or main
tained on control diet. Concurrently, aortic tissue calcium,
when quantitatively analyzed, was shown to be significantly
reduced (p .05) in both control and atherosclerotic animals
which were infused with Na2 EDTA when compared to non-EDTA
infused animals.
The precise anti-atherosclerotic mechanism is not known,
but because calcium deposits in plaques are usually con
sidered to be an end stage of atheroma formation, and
85
86
pronounced calcifications are strongly associated with
stenosis of the involved segments and ischemic myocardial
lesions (Meyer, 1977), it would seem plausible that removal
of calcium could cause the plaque to become amorphous and
either absorbed or dissolved by the blood. In this regard,
Smith (1977) found that EDTA increased the rate of destruction
of lipid material in samples of intima. The greatest effect
was found in the amorphous atheroma lipid fraction of fibrous
and gelatinous plaques, where the rate of destruction was
increased six-fold by EDTA.
An alternative explanation might be that EDTA alters
the interaction between lipids and components of the extra
cellular matrix, so that the lipid material becomes more
available for destruction. In this regard, it is possible
that the altered availability of a metallic ion might affect
an enzymatic process or even a physical property, such as
membrane permeability, with the resultant metabolic effects.
The removal of calcium from metastatic tissue deposits by
Na2 EDTA should be reflected by increased urinary calcium,
since the action of the chelator is to bind serum calcium
into a stable, soluble, nonionized complex which is relatively
rapidly excreted in the urine (Soffer, 1961). Accordingly,
increased urinary calcium was found in those rabbits on
the control diet and infused with Na2 EDTA compared to those
which were infused with saline or were not infused. However,
among those rabbits which were fed the cholesterol diet, no
87
significant increase in urinary calcium was noted in the
Na2EDTA treated animals when compared to those infused with
normal saline, even though aortae from the latter group
contained significantly greater (p<.05) tissue calcium.
Furthermore, both of these groups excreted significantly
greater (p<.05) urinary calcium than either of the control
diet groups which were infused with saline or were not
infused.
The increased calcium excretion by the cholesterol-fed,
saline-infused animals is perplexing. However, this un
expected result might be explained by a decreased activity
and reduced size of the parathyroid glands, which could have
been produced as a consequence of hypervitaminosis D (Guyton,
1971), while the animals in Group E were on the atherogenic
diet. This reduction in parathyroid activity could account
for the significantly greater calcium excretion by these
animals compared to saline-infused controls which were never
subjected to Vitamin D3 injections (Group C), or to those
baseline controls which were not infused (Group G).
A second possible explanation for the increased calcium
excretion by the cholesterol-fed, saline infused animals
could be a partial obstruction of the bile ducts by
cholesterol crystal precipitation, which would tend to pro
duce an accumulation of bile (including calcium ions) in
the blood. This condition in turn could produce an increased
excretion of the ions into the urine in order to maintain
serum calcium balance.
88
Conversely, there was an increase in calcium excretion
by the rabbits which were injected with Vitamin D3 and infused
with Na2 EDTA. This group (D) also exhibited significantly
less (p<.05) aortic tissue calcium than did those animals
which were Vitamin D3 -injected and saline-infused. These
results might be explained by the compensatory mechanism
of the maintenance of calcium homeostasis caused by para
thyroid stimulation due to Na2 EDTA interference (Huff, 1974).
This mechanism would cause the release of the more labile
calcium sources, much coming from metastatic pathologic
deposits, which ultimately would be excreted in the urine.
It is probable that this mechanism would not be activated
in the non-EDTA-treated animals.
The action of saline infusion to produce the increased
calcium excretion in Group E seems unlikely, because saline
infusion in the control diet group (C) did not result in
significantly greater (p<.05) urinary calcium than in non
infused baseline controls (Group G); and calcium excretion
in the EDTA infused control group (B) was significantly
greater (p<.05) than in the saline-infused controls.
Zinc was increased to large quantities in the urine on
infusion days, which is in agreement with previous studies
which showed as much as a ten-fold increase in zincuria
following EDTA treatment in man (Perry, 1951, 1960). Due
to the metabolic importance of zinc, this result becomes
particularly important in consideration of replacement therapy
89
to supplement EDTA chelation. Specifically, zinc has been
reported to be involved in DNA and RNA synthesis, protein
and carbohydrate metabolism, reproduction, bone growth,
learning, as well as many enzymes. In addition, cadmium
and zinc are intimately related; if cadmium were to replace
zinc in the renal tissue, or if the cadmium-to-zinc ratio
were increased due to a loss of zinc, hypertension could be
induced (Schroeder, 1974). This is turn may antagonize the
effects of chelation therapy on the regression of athero
sclerosis.
There was not a clear-cut increase in the excretion of
magnesium on infusion . days. It appeared, to the contrary,
that magnesium was conserved to compensate for the loss of
calcium, as observed by Koen (1963), and that magnesium
excretion was increased on non-infusion days. Again, this
observation becomes particularly important in mineral
replacement therapy to supplement the EDTA chelation when
it is recognized that magnesium is important as a catalyst
for many intracellular enzyme reactions, particularly those
relating to carbohydrate metabolism. In addition, low
magnesium concentration can cause greatly increased irrita
bility of the nervous system, peripheral vasodilatation,
cardiac arrhythmias (Guyton, 1971); and the ion may be
replaced by beryllium in certain magnesium-dependent enzymes,
resulting in inactivation (Schroeder, 1960).
90
In future studies, the effects of EDTA chelation on
other trace elements should be examined. The quantitation
of normal levels of metals considered to be essential for
mammals as cofactors for certain metalloenzymes - manganese,
cobalt, copper and molybdenum - in both serum and urine
should be done. Additionally, the effects of EDTA chelation
on enzymes such as carbonic anhydrase, lactate dehydrogenase,
carboxypeptidase, uricase, and possibly phosphodiesterase
could be determined. A study to examine the changes in
parathyroid hormone before, during, and for an extended
period following Na2 EDTA chelation would be helpful in
determining the most probable effect of the chelate on
calcium metabolism. Evaluation of a more specific calcium
chelator, such as EGTA, relative to the effects on athero4
sclerotic plaques should be considered.
In summary, New Zealand albino rabbits exhibited calcified
aortic plaques and a maximum average serum cholesterol level
of 1,200 mg percent after twenty-three weeks on an atherogenic
diet of cholesterol and vitamin D3 . The rabbits were infused
with Na2 EDTA or normal saline, beginning one month after com
pletion of the diet, for a total of twenty infusions each,
on alternating days. Histological and histochemical examination
of the aortae revealed, qualitatively and quantitatively,
that rabbits treated with Na 2EDTA had significantly less
(p .05) aortic calcium than those infused with normal saline
or those which were not infused. Twenty-four hour urine
91
samples of those infused with Na2 EDTA and normal saline
were analyzed via atomic absorption spectroscopy for
calcium, magnesium, zinc and cadmium. It was found that
excretion of calcium was increased on infusion days by
Na 2 EDTA treatment, which, along with the histological and
histochemical results, indicates that Na 2 EDTA was effective
in the removal of the aortic metastatic calcium. Similarly,
zinc excretion was increased on infusion days, and returned
to normal levels on non-infusion days. However, the excretion
pattern of magnesium in general exhibited an increase on non
infusion days, with a return to normal on infusion days.
The excretion patterns of zinc and magnesium must be included
in the consideration of mineral replacment therapy to
accompany EDTA treatment.
These results suggest that Na2EDTA, a non-specific
cation chelator, affects the removal of plaque calcium
from arterial walls, thereby contributing to the regression
of atherosclerosis.
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