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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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

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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

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7')

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FIGURE 10. - Aorta of Group C Rabbit

FIGURE 11. - Aorta of Group C Rabbit

52

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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

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FIGURE 17. - Aorta of Group F Rabbit

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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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