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Tna JOURNAL OP INVES!FIGATTWE DEBMATOLOOT

Copyright 1956 by The Williams & Wilkins Co.Vol. 47, No. 3

Printed in U.S.A.

EFFECT OF ETHYLENEDIAMINETETRAACETIC ACID (EDTA)AND TETRAHYDROXYQTJINONE ON SCLERODERMATOUS

SKIN

HIsToLoGIc AND CHEMICAL STUDIES*

M. K. KEECH, M.D.,t D. S. McCANN, PH.D., A. J. BOYLE, MD., Pir.D4AND H. PINKUS, MD.

The considerable literature dealing withmany aspects of progressive systemic sclerosis(acroscierosis) reveals no etiologic factor as thecause of this disease. Rodnan (1) recently re-viewed the clinical, serological and immunohisto-chemical evidence suggesting that it is "an ab-normal perhaps heritably determined, immuneresponse." Rukavina et at. (2) gave a compre-hensive discussion on the chemical reasons forthe possibility that scleroderma, as well as otherchronic diseases, may represent an aberrationin trace or abnormal metal actions; either atrace metal deficiency, abnormal metal excess,metalloensyme competition by an extraneousmetal or the dependence of many metallo-enzyme systems upon chelation.

A derangement in calcium and/or magne-sium metabolism would appear to be a likelypossibility as the cause of scleroderma since ithas been shown that the ubiquitous sulfatedmucopolysaccharides (found essentially in car-tilage, tendon, heart valves, skin and the wallsof large blood vessels) serve as cation exchangeresins in a complex physiological system (3). Itwas demonstrated that 1 meq of cation (cal-cium, sodium, barium) was bound to 1 g of drycartilage (sulfate content 1 mecljg) and thatthere was an excellent correlation between cat-ion binding capacity and sulfate content (4).The concepts that the extracellular fluid andground substance together form one physico-chemical system (5) and the possibility that

Supported by U. S. Public Health ServiceTraining Grants TI AM 5141 and AM 07194,U. S. Public Health Service Research Grant AM05776 and the Michigan Chapter Arthritis Founda-tion.

The Paul B. Elder Company, Bryan, Ohio sup-plied a generous amount of tetrahydroxyquinone.

Reprint requests to: M. K. Keech, M.D.,Anderson Clinic, South 25th and Army-NavyDrive, Arlington, Virginia 22206.

Received for publication December 1, 1965.* From the Departments of Medicine,t Chemis-

try and Dermatology, Wayne State Universityand Detroit General Hospital, Detroit, Michigan.

235

connective tissue which does not calcify mayaccumulate calcium (6) are suggested in viewof the fact that the connective tissues of thebody contain calcium in both ionic and boundforms in excess of that present in the blood (7).

Salts of ethylenedianiinetetraacetic acid(EDTA) have long been used in clinical medi-cine, as for example in the dissolution ofurinary calculi (8, 9), to dissolve metastaticcalcium deposits in the kidney (10), in the sepa-ration, concentration and transfusion of plate-lets (11), blood preservation (12), lead poison-ing (13, 14), hemachromatosis (15, 16, 17, 18),acute ferrous sulphate poisoning (19, 20, 21),porphyria (22), plutonium poisoning (23),corneal opacities (24), lime burns for the eye(25), hypertension (26, 27), as a means oflowering blood cholesterol (27, 28, 29), inangina pectoris (10, 30) and in calcinosisuniversalis (31, 32, 33). EDTA combines withmetal ions to form cyclic complexes which arewater-soluble and virtually undissociated (34).When the sodium salt (Na,EDTA) is rapidlyadministered the serum calcium is lowered andan excess of urinary calcium is excreted. ThusSpencer et at. (35) found that in 3 normalindividuals the actual urinary calcium excre-tion corresponded to 73—84 per cent of thetheoretical calcium binding power of the in-jected EDTA dose. (1 g NaEDTA complexes108 mg calcium at a pH above 6.5).

In 1955 Klein and Harris (36) described asevere ease of calciuic scleroderma that re-sponded to intravenous Na2EDTA with x-rayevidence of diminution of articular and cu-taneous metastatic calcific deposits, and in-creased mobility of affected joints. A post-treatment skin biopsy specimen was reportedas indicating some clinical improvement, al-though the illustrations are not convincing(Table I). Stimulated by this, others treatednon-calcific cases with varying results (2, 37,38). After a three week course (total 45 g of

236 THE JOTJBNAL OF INVESTIGATIVE DERMATOLOGY

intravenous Na,EDTA) three patients im-proved, but only one post-treatment skin biopsyspecimen showed some return to normal (2).Nine of 23 cases improved after a three weekcourse of the same salt, but three sets of pre-and post-treatment skin biopsy specimensshowed no significant change (37) (Table I).The three patients described by Johnson in1960 (38) were presumably those reported bythe same group in 1957 (2).

Winder and Curtis (39) treated 19 patientswith acroscler3sis with 45-135 g Na,EDTAover 6 weeks although one had 180 gms in 8weeks. Seven were thought to be improved,four possibly improved and eight were un-changed. No skin biopsy studies were done.Birk and Rupe (40) treated thirteen cases withthe same salt with encouraging results, butagain no post-treatment skin biopsies wereperformed. Most authors agreed that resultswere better in the acrosclerotic type of selero-derma (38, 39, 40, 41) but emphasized thatthe ultimate usefulness of the drug had stillto be evaluated (37, 39). This group of opti-mistic reports were brought into perspectiveby a follow-up study by Neidner et al. (42)of 60 patients two to four years after theircourse of chelation therapy. These patients in-cluded those described by Muller at at. (37)where it had been noted that the drug was

not curative, that the response of a given indi-vidual was unpredictable, and why improve-ment only occurred in some eases and not inothers remained unexplained. Neidner at at.(42) suggested that the immediate subjectiveand objective (temporary) improvement couldhave been related to bed-rest plus physicaltherapy. The analysis revealed that repeatedcourses of Na2EDTA did not increase thechances of improvement, and that there was nosignificant difference in the clinical course oftreated and untreated cases.

The present paper forms part of a largerreview of 27 patients with progressive systemicsclerosis (PSS) seen in the same clinic overthe past four years. The clinical, pathologicaland EKG aspects were presented and a schemeoutlining the pathological sequence of eventswas postulated (43). In view of the fact thatprevious reports dealt only with short coursesof EDTA usually totalling 45—60 g and lastingonly a few weeks with little or no histologicchange in the skin, we thought it worth whileto examine specimens of skin, both histologi-cally and chemically, from four patients afterprolonged courses of intravenous Na2EDTAtotalling 132—212 g and given over a period offifteen months. In addition, two patients weregiven oral tetrahydroxyquinone (THQ) for thesame period of time. Kelly and Pinkus (44)

TABLE IEffect of Na5EDTA on skin biopsies reported in literature

Author Ref.no. Type of case °' of

casesTotal

EDTA(s)Length oftreatment

Post-treatment skinbiopsy resuits

Klein and Harris (1955)* 36 Calcific 1 60 8 weeks Improved

Rukavina et al. (1957) 2 Acroselerosis,non-calcific

3 45 8 weeks 2 unchanged1 minor improve-

ment

Muller et al. (1959) 37 Aeroselerosis,non-ealcific

3 45 3 weeks No significantchange

Johnson (1960)t 38 Acrosclerosis,non-calcific

3 45 3 weeks 1 minor improve-ment

Present Authors — Acrosclerosis,non-calcific

4 132—212 15 months 3 improved (Fig-ures 1—3)

* Theonly report with photomicrographs.t Presumably the same cases as Rukavina at at. (1957).

SCLERODERMA AND EDTA 237

reported clinical and histological regression ofkeloids with this compound. The effect wasthought to be possibly via an enzyme systemor by a chelating mechanism. Eight untreatedcases of scieroderma served as histologiccontrols, five as chemical controls and normaland involved skin from the same patient werecompared. In addition, normal skin from elevenindividuals without scieroderma were analyzedfor calcium and magnesium.

MATERIALS AND METHODS

General details are furnished in Tables II—IV.1. The four patients treated with Na5EDTA had

severe, non-calcifie acroselerosis, and each hadRaynaud's phenomenon, mask-like facies, wide-spread skin involvement, esophageal hypomotilityand pulmonary changes with alveolar-capillaryblock. In addition H. W. had a thickened perio-dontal membrane, sclerodactyly, resorption of un-gual tufts, gangrene of two digits necessitatingamputation, joint involvement and progressiveesophageal changes in spite of treatment withEDTA. The EKG showed marked LAD, PAC's,

occasional PVC's and RV outflow tract hyper-trophy.

2. The two patients treated with oral hydroxy-quinone (THQ) also had non-calcific acroscierosiswith Rayiiaud's phenomenon and moderate skininvolvement. L. S. had esophageal hypomotility,resorption of ungual tufts and typical changes inthe lung parenchyma. Both had pulmonary func-tion tests at the lower limits of normal.

3. The eight untreated histologic controls wereclassic cases of PSS, several very severe; six hadesophageal involvement, and three expired.

4. Three of the five untreated cases of scle-roderma that served as chemical controls were in-cluded in the group of histologic controls. The re-maining two were also classic cases of PSS.Involved and uninvolved skin from the same pa-tients were analyzed (Table III).

5. Normal forearm skin from eleven individualsof the same age range but without sclerodermawere chemically analyzed for comparison (TableIV).

Therapy: Administration and Dosage1 (a). Di.todium EDTA (Endrate: Abbott

Laboratories) was administered intravenously inintermittent 5 day courses over 15 months on an

Histologic analyses of skin biopsies from cases of progressive systemic sclerosis before and after treatment

Cases Biopsy site Total medication Skin histology

J. B., NM 58, No. 388056 (ondigoxin and reserpine)

H. W., NF 32, No. 160588

J. G., NM 44, No. 82964

E. C., NF 43, No. 133043 (ondigoxin)

Left suprascapular region

Flexor surface, forearm

Dorsal aspect, middle pha-lanx, left 3rd digit


PT120 EDTA190 EDTA

PT132 EDTA

PT150 EDTA212 EDTA

PT100 EDTA180 EDTA

TypicalImproved(Fig. 1)



Typical

No change

L. S., NF 41, No. 57048

H. S., NP 55, No. 91884

Right calf


PT21.6 THQ40.5 THQ

PT40.5 THQ

Typical

No change

TypicalNo change

* Race, sex, age and hospital number. The skin was involved at all biopsy sites.f Na2EDTA: 0.5 gm/30 lbs body weight in intermittent five day courses over 15 months. Tetrahy-

droxyquinone (THQ): one 30mg tablet three times daily continuously. PT = pre-treatment.

TABLE II

238 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE IIIChemical analyses of skin biopsy specimens from involved and uninvolved skin

from cases of progressive systemic sclerosis

CaseBiopsy Site pg Ca/mg skin pg Mg/mg skin

Involved Uninvolved Involved Uninvolved Involved Uninv01ved

After EDTA treatment

J. B., NM 58, No. 388056

H. W., NF 32, No. 160588

J. G., NM 44, No. 82964

E. C., NF 43, No. 133043

Left suprascapu-lar region

Flexor surface,forearm

Dorsal aspect,middle pha-lanx, left 3rddigit


Right thigh

Left thigh(slightly in-volved)

Right thigh

Right thigh

.14

.15

.12

.27

.35

.15

.26

.14

.10

.15

.15

.17

.31

.14

.13

.12

After THQ treatment

L. S., NF 41, No. 57048

H. S., NF 55, No. 91884

Right leg

Flexor surface,forearm(slightly in-volved)


None taken

.19

.35

.26

—

.20

.17

.21

—

Untreated cases of scieroderma

M. C., NF 36, No. 343157A. M., NF 44, No. 90093E. M., NM 55, No. 2947

R. C., NM 56, No. 471253

P. M., NP 65, No. 129347

Right upper armRight wristMiddle phalanx,

right 3rd digitFlexor surface,

left forearmFlexor surface,

right forearm

Left thighLeft thighLeft thigh

None taken

Right thigh

.24

.29

.20

.30

.23

.16

.28

.21

—

.26

.16

.13

.13

.15

.24

.13

.21

.17

—

.17

* Race, sex, age and hospital number. Biopsies of involved skin in the first six cases were taken ad-jacent to their previous biopsies.

outpatient basis. One half a gram per 30 lbs bodyweight was dissolved in 500 cc of 5 per cent glu-cose in water and infused over one hour. The totaldose ranged from 132—212 g depending on bodysize.

,Side effects: These were minimal and did notnecessitate discontinuation of therapy. A sensationof localized burning at the infusion site sometimesoccurred, and two patients had gastric irritation,controlled by antacids. The patient H. W. is de-scribed below.

(b). The patient H. W. (for clinical details seeabove) had intermittent courses of magnesium-EDTA (Abbott Laboratories) over 15 months

from January, 1962 to March, 1963, with a totaldose of 152.5 g without any side effects. However,therapy was discontinued because there was severeinfection and gangrene of the fingers; and thepossible effect of this salt on infection was notknown. Amputation of two digits was performedin July, 1963. Na2EDTA was started in February,1964 and continued until May, 1965 with a totaldose of 132 g during which time serial esopha-grams demonstrated further dilatation of theesophagus. As noted above, the EKG was abnor-mal, probably due to pulmonary and/or cardiacinvolvement. On one occasion, near the beginningof disodium salt therapy, the patient had mild


hypocalcemia with cramps in the calves, a positiveChvostek's sign and cardiac arrhythmia. Thesesigns and symptoms were quickly relieved by in-travenous calcium. Throughout this second coursethe patient experienced attacks of dizziness andpalpitations, but an EKG taken during an attackonly showed sinus tachycardia.

2. Thirty mg of tetrahydroxyquinone (THQ)was taken three times a day continously for 15months. There were no side effects.

Skin biopsy specimens were taken from thesame involved sites before, during and at the endof treatment. The histologic specimens were em-bedded in paraffin, cut and stained with hema-toxylin and eosin, orcein-Giemsa, Mallory andVon Kossa's method for calcium. All the sectionswere stained at one time in order to eliminatevariation in stain.

The samples for chemical onelysis were storedfresh frozen and were likewise analyzed at onetime. They were lyophylized, digested with aperchloric-nitric mixture, the salts then taken upin water and spectrographed for calcium andmagnesium using lithium as an internal standard.

RESULTS (TABLES is—v)

General. The four patients treated withNa2EDTA cooperated well and there was sub-jective and objective softening of the skin.However, there was no evidence of improve-ment in the viscera as judged by esophagramsand x-rays of the lung parenchyma. In fact H.W. developed gangrene of two digits while onMgEDTA, necessitating amputation, and pro-gressive esophageal involvement while onNa2EDTA.

One patient (H. S.) on THQ did not exhibitany significant change. The other (L. S.) whohad more cutaneous involvement, noticed somesoftening of the skin, but this was not asmarked as in those patients receivingNa2EDTA.

Histology (Table II)Sections of all the untreated cases of sclero-

derma showed severe changes consisting ofbroadening of the collagen bundles, leading tomore or less complete obliteration of inter-fascicular spaces. The papillary layer was simi-larly involved and the vaseularity of thepapillae was reduced even where the pattern ofepidermal ridges was preserved, as it was in themajority of eases.

The pre-treatment specimens of the experi-mental group showed similar changes, and nosignificant differences were observed aftertreatment with THQ and in one of the eases

E. J., NF 33,No. 331951

P. D., NF 34,No. 334791

B. C., NF 38,No. 124412

V. P., NF 45,No. 325386

H. B., NM 65,No. 461774

F. F., WM 71,No. 367694

All normal skin from the volar aspect of theforearm or the thigh.

treated with EDTA (E. C.). In the other threeEDTA eases, the following changes werefound: J. B.: pre-treatment sections of skinfrom the suprascapular region showed the epi-dermis was only 3—4 cell layers thick, heavilypigmented and covered by a dense horny layer.Epidermal ridges were largely absent. The con-nective tissue of the dermis was very dense,and the normal difference between the papil-lary and reticular layers was almost lost.Fibrocytes and capillary blood vessels weresparse, the vessels appeared rigid and therewas a considerable number of pigmentedmacrophages in the upper dermis (Fig. JA).

TABLE IVChemical analyses of eleven normal skin

biopsy specimens

Case

B. B., NF 29,No. 265007

K. C., NF 32,

Biopsy site

Right forearm

Left thigh

Left forearm

Left forearm

Left forearm

Left forearm

Right forearm

Left forearm

Left forearm

Left forearm

Left forearm

pgCa 5gMgmgskio mgskin

.20 .12

.38 .31

.25 .12

.32 .23

.25 .19

.37 .15

.25 .21

.43 .23

.31 .15

.32 21

.36 .16

A. C., NF 54,No. 357464

A. M., NM 58,No. 177068

A. 1%., WM 59,No. 367670


FIG. 1. Sections of suprascapular skin of J.B., H and E, X 170. A. before treatment, B.after 190 gm EDTA. The large triangular or stellate cells in the upper dermis in A. arepigmented macrophages.

FIG. 2. Sections of skin from the flexor surface of the forearm of 11W., 11 aad E, X 170.A. before treatment, B. after 132 gm EDTA.

Sections taken after 190 g of EDTA showedno great change in the lower part of theepidermis, but the horny layer was moreloosely woven and was similar to normal skin.The dermal collagen formed thinner and morewavy bundles separated by interfascicularspaces. The blood vessels and fixed tissue cellsappeared more nearly normal, and the numberof pigmented macrophages was much reduced(Fig. 1B).

11. W.: The epidermis of the flexor sur-face of the forearm was of average thicknessbefore and after treatment, although the hornylayer was somewhat more dense in the pre-treatment specimen. The dermal collagen wasfar less sclerotic before treatment than in J. B.,but the normal structural difference betweenpapillary and reticular layers was obliterated.Fibrocytes were scarce and the small bloodvessels were rigidly open (Fig. 2A). Sections

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taken after 132 g EIDTA showed improve-ment in the condition of fibers, cells and bloodvessels (Fig. 2B).

J. G.: Biopsy specimens were taken fromthe dorsal aspect of the middle phalanx of theleft third finger. Before treatment the skin wasso sclerotic that difficulty was experienced incutting the sections (Fig. 3A). The epidermiswas not much thinner than normal, but thedermal collagen was so dense that all struc-tural details were lost. Fibroblasts and capil-laries were scarce. After 150 g of EIDTA thecollagen bundles of the retieular layer werewell defined though still thick (Fig. 3B), thepapillary collagen remained dense, but thecapillaries were fairly normal. The epidermishad the average configuration for this part ofthe body and was covered by extremely thick,dense keratin. Two months after completion ofthe course of 212 g of the drug there was addi-tional improvement (Fig. 3C) especially in theepidermis and papillary connective tissue. Themalphighian and granular layers of the epi-dermis were thicker, the horny layer wasthinner and less dense. The dense eollagenouszone at the lower border of the epidermis was

thinner, and the population of cells in thedermis had increased. In spite of matched stath-ing, the dermal collagen appeared lighter thanin Fig. 3B, presumably because the bundleswere thinner.

The Von Kossa stains on all the sectionswere negative, whether they were untreated,pre-treatment or post-treatment specimens. Itshould be noted that the effect of MgEDTA onH. W. was not evaluated, since the remedy wasgiven before the main research program wasbegun. She received 152 g over 15 months, thecourse ending one year prior to the pre-treat-ment biopsy for the course of Na2EDTA whichis recorded.

Chemistry

Calcium and magnesium determinations weremade on skin biopsy specimens from 11 nor-mal donors as well as from the uninvolvedand involved sites from 11 patients sufferingfrom scleroderma. The latter group comprised4 patients treated with EDTA, two treatedwith THQ and the remainder untreated. Nor-mal calcium values (Table IV) were .29 tgper mg dry weight with a standard deviation

FIG 3. Sections of dorsal skin of the finger of J.G., H and E, X 153. A. before treat-ment, B. after 150 gm EDTA, C. two months after completion of the full course of 212 gm.The poor condition of the section shown in A. is due to the extreme sclerosis which madecutting difficult.

/ a

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•4


of .07 while the seleroderma average (TableIII) ran .23 .07 pg/mg dry skin. The differ-ence is not significant statistically. However,closer inspection shows that the specimens fromthe involved sites of the three patients whoresponded histologically to EDTA treatment allshow calcium levels at the lower end of theoverall range (Table V). This was not truefor E. C., the patient who also failed to showhistological improvement. The magnesium val-ues for normals were .19 .05; for the groupwith scleroderma .17 .05 cg/mg dry tissue.Literature averages for the two metals quotedby Rothman (45) are 27—46 mg per 100 g dryskin for calcium and 18—30 mg per 100 g dryskin for magnesium. The same author pointsout that the calcium content of skin increaseswith increasing age. Analysis of the 11 controlspecimens show a slight trend in this direction.The same correlation is somewhat more markedin the twenty seleroderma samples. It was notpossible to establish any correlation of mag-nesium values in skin with age.

DISCUSSION

Histologic findings. Three of the four pa-tients treated with EDTA showed a returntoward normal in their skin biopsy specimens,

while there was no significant difference in thetwo treated with THQ. These results are farbetter than those recorded in the literature(Table I). This is probably related to themuch greater dose and far longer time of ad-ministration (15 months as against 3—8 weeks).All the EDTA eases had definite softening ofthe skin clinically, as did one of the patients onTHQ who had a similar though milder response.The decreased vaseularity and loss of inter-faseieular spaces in the eorium of the pretreat-ment specimens is felt clinically as stiff, hardskin; while the increased vaseularity, returnof fixed tissue cells and the loosening of thecollagen bundles in the dermis as well as thehorny layer of the epidermis is reflected asclinical softening. The reappearance of the(normal) interfascieular spaces denotes a newinflux of ground substance which allows a morenormal passage of nutrient and waste ma-terials to and from the cells. As these biopsyspecimens were non-calcifie (Von Kossa nega-tive and a normal calcium content) the ob-served softening could not have been due toloss of calcium, and suggests that EDTA actson the connective tissue itself, probably viathe parathyroids as discussed below.

Biopsy site. The biopsy specimens were

TABLE VSummary of chemical analyses

case no. of cases Age Range(yr)

No. ofbiopsies

pg Ca/mg skinAverage and range

pg Mg/mg skinAverage and range

Normal controls 11 29—71 11 .29 (.20—43) .19 (.12—31)

SelerodermaUntreated

Involved skinUrnnvolved skin

After EDTAInvolved skinUninvolved skin

After THQInvolved skinUninvolved skin

5

4

2

36—65

32—58

41—55

54*

44t

21*

.25 (.20—30)

.23 (.16—28)

.17 (.12—27)

.25 (.14—35)

.27 (.19—35)

.26

.16 (.13—24)

.17 (.13—21)

.14 (.10—17)

.18 (.12—.31)

.19 (.17—20)

* One patient in each group had no biopsy of uninvolved skin.t One patient had no uninvolved skin and biopsy from a lesser involved area gave similar values to

the severely involved area.Rothman45 qnotes average figures from the literature for human skin: calcium 27—46 mg/100 g dry

tissue and magnesium 18—30 mg/100 g dry tissne.


taken from the same skin areas at intervals ofabout 8 months. Therefore, the second andthird specimens were near a previous area ofhealing, but this transient increase in localmetabolic activity could not have been thecause of the changes observed as only threeof the six cases that underwent serial studydemonstrated any alteration.

Relation to calcium and magnesium

The normal skin calcium and magnesiumlevels obtained throughout this study confirmRothman's refutation of the older belief thatcalcium is retained by patients with sclero-derma (45). He found normal values in theuninvolved skin of one patient with sciero-derma and a normal calcium balance and nor-mal excretion of excess calcium administeredby mouth or by injection. "Thus the reasonwhy scieroderma is so often associated withcalcinosis remains obscure (and) requires fur-ther study." He also points out that "nothingis known about the distribution, linkages, andphysiological role of calcium in the corium ex-cept that it represents a vital constituent ofthe cementing substance of the endothelialcells.... There is some indirect evidence thatcalcium may be bound to connective tissuefibers in a non-ionized form." The only strikingresponse to EDTA by a scieroderma patientreported in the literature was the calcific caseof Klein and Harris (36) (Table I). It isnoteworthy, therefore, that three of the EDTAtreated non-calcific cases discussed in this re-port responded both clinically and histologi-cally. It may be relevant that E. C. was takingdigoxin for congestive heart failure, but J. B.(who responded histologically) was also on thisdrug, in addition to reserpine, for hypertensiveheart failure. The possible influence of thesemedications, as well as differences in individualresponse on the outcome of EDTA therapy,requires further study.

The fact that the involved, but not the un-involved, sites of the EDTA treated patientswho responded histologically to EDTA showedrelatively low calcium values suggests that theaction of EDTA is on the abnormal non-calcific collagen bundles.

Boyle and co-workers (46) discuss the possi-ble mechanisms by which EDTA affects theconnective tissue in a wide range of circula-tory and scierosing diseases. They point out

that Na,EDTA initially lowers the ionic cal-cium in the blood, so that which is not carriedto bone is replenished from tissue sources andcalcium turnover is increased, aided by theknown, concurrent alteration in cell membranepermeability. This, they suggest, stimulates aparathyroid response which is largely responsi-ble for many effects for example, the differ-ence in time manifestation of atherosclerosis inthe sexes owing to the cyclical stimulation ofparathyroid activity in the female. They alsomention the effect of NaEDTA on trace metalswith possible secondary enhancement or inhibi-tion of enzyme systems, though such an argu-ment is far less tenable in view of the fact thatadministration of CaEDTA does not show theeffects obtained with the sodium or magnesiumsalts.

Trace metals and enzyme systems

The progressive sclerosis throughout the bodyin scieroderma points to a possible defect inproteolysis. Three well-characterized proteolyticenzymes have been demonstrated in the skin(47): two dermo-proteases and a peptidase(48, 49, 50, 51). These are believed to playsignificant roles in trauma, vesiculation andinflammation of the skin. Cathepsins (47) areubiquitous components of animal tissues espe-cially kidney, spleen, liver and lung, and areinvolved in autolysis of dead tissues. Theyare also present in lysosomes and may play animportant role in many diseases.

Although a mammalian collagenase has longbeen suspected (52) especially in relation tothe involuting post-partum uterus, only re-cently has real evidence of its existence beenforthcoming. buck and Patel (53) found thatthe alkaline extract of porcine pancreas wascollagenolytic, and that it was not inhibitedby EDTA, soybean trypsin inhibitor or mer-curobenzoate, and was not enhanced by theaddition of CaCl2 thus ruling out contamina-tion with bacterial collagenase, the presence ofa "collagen mucoproteinase", a denatured col-lagen or a non-specific cathepsin as possiblecauses of the collagen breakdown.

Yet another possible mechanism of actionstems from a series of papers by the samegroup (54, 55, 56, 57, 58). They found a loss ofinsoluble collagen from the uninjured skin ofanimals away from the area of trauma. Thiswas termed the "distant dermal collagen re-


sponse" and was found to occur following localcroton oil injury; administration of cortisol, orstress in the form of sham adrenalectomy ordaily intraperitoneal injections of large amountsof isotonic saline. It could be mediated bylocal tissue enzyme release as suggested by theirmost recent paper (59) where extracts of in-tact and necrotic rat skin were shown to exhibitcollagenolytic activity that was riot inhibitedby EDTA, soybean trypsin inhibitor or amercuric salt. Collagenolytic activity has alsobeen demonstrated in bone cells (60), thisactivity being entirely confined to the intra-cellular large granule fraction that sedimentsbetween 700 and 15,000 g (61).

it is conceivable that the stress of intrave-nous infusions of Na,EDTA could have playeda part in the loss of dermal collagen demon-strated histologically, either by (1) stimulationof parathormone, (2) direct activation of localautologous proteolytic enzymes (dermopro-teases; cathepsins), (3) stimulation of endoge-nous pancreatic collagenase (presuming humanand porcine pancreas possess the same en-zymes) or (4) stimulation of endogenous corti-sone which is known to depress fiber forma-tion. The first is the most likely, since theparathyroids are known to respond to loweringof the serum ionic calcium and parathor-mone in turn is known to cause collagen break-down (62). Although the mechanism of thisturnover has not yet been established it isquite possible that a collagenase intermediaryis involved. In fact, the addition of parathy-roid extract enhances the release of hydroxy-proline from rat bone in tissue culture by acollagenolytic process (63).

The second and third suggestions remain asintriguing possibilities with far-reaching impli-cations. For example, if the pancreas secretescollagenase, this would be continuous through-out life but would fluctuate according to re-quirements, e.g. increasing post-partum andpossibly slowly decreasing throughout life, thusallowing sclerosis to occur in sites such as bloodvessels. It is interesting that the administrationof porcine pancreatic collagenase to a patientwith severe PSS produced a definite thoughtemporary response, as evidenced by a decreasein skin content of hydroxyproline and an in-creased urinary excretion of hydroxyproline(64).

The last suggestion seems quite unlikely asthe stress would appear too slight to producesufficient cortisone; nor has intravenous ad-ministration of either the sodium or magnesiumsalts of EDTA resulted in an elevated urinaryoutput of 17 hydroxy corticoids (65). Finally,in animal experimentation, "control" groups re-ceived as many injections of physiological salineas the "treated" groups received of EDTA. Themarked differences obtained between two suchgroups (66) must then be due to somethingother than the "stress of the injection".

Our results suggest a long chain of effectsending in enzymatic tissue breakdown. To ourknowledge this is the first study of its kind,and is worthy of further investigation of alarger number of patients, preferably not oncardiac drugs. In addition, this approach maybe developed to elucidate the ill-understood,altered enzyme systems in disease (for exam-ple, the collagenolytic activity of normal andpathologic human skin could be studied beforeand after various treatments or modes ofstress).

SU1%MARY

The skin from four patients with progres-sive systemic sclerosis were examined beforeand after a prolonged course of intravenousNa2EDTA totalling 132—212 gm over fifteenmonths. Two patients were similarly studiedfollowing 40 g of tetrahydroxyquinone (THQ)by mouth, and all were compared with cases ofuntreated scieroderma as well as with normalcontrols. Three of four patients treated withEDTA showed histological improvementwhereas the remainder were unchanged.

The calcium and magnesium content of bothinvolved and uninvolved skin from treated anduntreated cases of scieroderma were within therange of normal controls similarly studied, andwere also within the ranges quoted in theliterature (45). However, the involved skinsamples from the three histologically-respon-sive patients exhibited calcium levels that werebelow the range of the normal controls.

The literature on the rationale and thera-peutic use of EDTA is reviewed, and the possi-ble mechanisms underlying the results are dis-cussed with special reference to endogenousproteolytic enzymes. It is suggested that thisapproach could be modified to study alteredenzyme systems in human disease.

SOLERODERMA AND EDTA 245

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