X-RAY DIFFRACTION STUDIES OF HUMANAORTIC ELASTIN RESIDUES

Gerald Weissmann, Sigmund Weissmann

J Clin Invest. 1960;39(11):1657-1666. https://doi.org/10.1172/JCI104189.

Research Article

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X-RAY DIFFRACTION STUDIES OF HUMANAORTIC ELASTINRESIDUES*

By GERALDWEISSMANNt AND SIGMUNDWEISSMANN

(Fronm the Department of Medicine, New York University School of Medicine, the Third(N.Y.U.) Medical Division, Bellevue Hospital, New York, N.Y., and the Materials

Research Laboratory, College of Engineering, Rutgers University,New Brunswick, N.J.)

(Submitted for publication June 1, 1960; accepted July 8, 1960)

Abnormal calcification is readily identified bothgrossly and microscopically in the atheroscleroticplaques of aging human aortic intima (1). Con-currently, and frequently independently, consider-able amounts of calcium are deposited in the me-dia. This deposition need not occur in regions ofprior lipid deposition (2); indeed, even in the ab-sence of atherosclerosis, the calcium content ofaorta has been found to increase with age (3).

Such calcific deposits, mainly from athero-sclerotic plaques, have been studied by Carlstrom,Engfeldt, Engstrom and Ringertz (4), who ten-tatively identified the crystallites as hydroxyapatite(Ca10(PO4)6(OH2) . Their studies have indi-cated that the X-ray diffraction powder pattern ofgrossly visible plaques is essentially similar to thatgiven by bone. Further studies have indicatedthat the dry mass of human arterial wall is great-est in the media, at a region where elastin is thepredominant fiber (5). It appeared possible,therefore, that the hydroxyapatite crystallites mightbe associated with aortic elastin residue (2).

Elastin is generally separated from the otherproteins of aorta by virtue of its marked insolubilityin dilute alkali (6), formic acid (7) or hot water(8). Each of these methods has been used toseparate elastin residues of relatively similar prop-erties and amino acid composition, although therelationship these products bear to the native pro-tein is by no means clear (7). Such productshave been studied by chemical analysis (2) andelectron microscopy (9), recently aided by the

* This work was aided by grants from the UnitedStates Public Health Service (M-2022) and from theArmed Forces Epidemiological Board (DA-49-007-MD-590).

t Present address: Strangeways Research Laboratory,Cambridge, England. Part of this work was done whilethe author was a Fellow of the Arthritis and Rheuma-tism Foundation (1958-1959).

solubilization of elastin by a relatively specificpancreatic enzyme, elastase (10). The currentstudy was undertaken to demonstrate the presenceof crystallites in human aortic elastin residues(AER) by means of X-ray diffraction analysis.Elastin was prepared from aortas obtained at ne-cropsy from individuals aged 8 to 86. In orderto demonstrate the possible association of hydroxy-apatite with elastin rather than with other con-nective tissue elements, a form of extraction wasemployed which would separate elastin on the basisof its insolubility in water at autoclave tempera-tures.

MATERIALS ANDMETHODS

Preparation of aortic elastin residues.' Human aortaswere obtained within 24 hours after death from theOffice of the Chief Medical Examiner, New York City.Only samples free of moderate to marked intimal plaqueformation were employed. The intima-media portionwas stripped from the adventitia as in the preparations ofKirk and Dyrbye (11) at 40 C. Immediately thereafter,this portion (from aortic root to bifurcation of iliac ves-sels) was diced into 0.5 cm" segments, washed 3 timeswith ice cold physiologic saline (0.9 per cent) by cen-trifugation in the cold, blotted dry until no further mois-ture could be observed on Whatman no. 1 filter paper,and weighed. This was designated the wet weight.Next, these wet aortic slices (6.0 g) were placed in ab-solute ethanol (250 ml) and homogenized in the baffledflask (500 ml) of a VirTis 45 homogenizer for 10 min-utes at top speed (rated at 45,000 rpm) employing thetwin-blade attachment. An ice bath kept the tempera-ture below 10° C. The disintegrated aorta and ethanolwere centrifuged at 2,000 rpm for 20 minutes, and theresidue washed 3 times each with absolute ethanol andethyl ether, then dried in vacuo over CaCl2 to a constantweight designated as the dry weight. (During this pro-

1 The term "aortic elastin residue" is employed todistinguish the final product from that obtained by Lans-ing (2). In the present study, sections of intima as wellas of media have been employed to include crystallitesassociated with the elastic membranes of aortic intimain the material finally examined by X-ray analysis.

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GERALDWEISSMANNAND SIGMUNDWEISSMANN

WHOLE, DRIED AORTIC POWDER

Homogenized 30 min in H20

Centrifuged

PPT.

FRACTION A(Acid mucopolysaccharide,water soluble proteins)

Supernatant

FRACTIONC(Collagen as gelatin,neutral polysaccharides)

Supernatant

PPT. with 80% ethanol

FRACTION ES(Soluble proteinderived from elastin)

RESIDUE 1

Heated in autoclaveat 20 pounds, 3 hours,centrifuged

PPT.

FRACTION E, AORTIC ELASTIN RESIDUE*Treated with elastase,

dialyzed, centrifuged

PPT.

FRACTION R(Protein-free

hydroxyapatite)

* Used in most X-ray diffraction analyses.

FIG. 1. OUTLINE OF PROCEDUREUSED IN PREPARING AORTIC ELASTIN RESIDUES (AER, SEE TEXT).

cedure, designed to dry and defat the specimens, a yellowcolor was imparted to the ethanol during homogeniza-tion, and to the first two ethanol washes.) Designatedas dried aortic powder, this product, stored at room tem-perature, was used for further fractionation as late as

one year after preparation. To determine whether dif-ferences in the nature of aortic elastin residues would ap-

pear between dried and fresh aortas, in several early ex-

periments the wet aortic slices (6.0 g) were placed di-rectly in the homogenizer with distilled water (280 ml).They were then immediately carried through the frac-tionation procedure described below.

Further fractionation2 of powdered aorta was car-

ried out after portions had been allowed to stand withdistilled water (2.0 g per 80 ml) at 40 C for 16 hoursto rehydrate (Figure 1). Dried, rehydrated aorticpowder (2.0 g) was added to distilled water (280 ml)in the baffled flask (500 ml) of the VirTis 45 homoge-nizer, which was run at top speed for 30 minutes in a

room at 40 C. Constant replenishment of an ice bathkept the temperature of the homogenate below 100 C.This procedure yielded an opaque mixture to whichethanol (560 ml) was added. All subsequent procedures

2 For sake of clarity, the fractions containing the finalproduct (AER) have been italicized.

were performed at room temperature (23-25° C). Fol-lowing centrifugation at 2,000 rpm for 45 minutes, thesupernatant solution was filtered through glass wool, andpotassium acetate (8.5 g) was added to precipitatepolyanionic material (12). The precipitate was washed3 times each with absolute ethanol and ethyl ether anddried in vacuo over CaCI2. Judged by its metachromasiawith toluidine blue, and precipitability by polyvalent co-

balt salts (13), this product was presumed to containwater soluble acid mucopolysaccharide-protein complexes.Chemical analysis of 10 samples of this fraction (Frac-tion A, Figure 1) gave values, as per cent, within theranges: hexosamine 4.5 + 0.5, nitrogen 11.2 0.8, hy-droxyproline 0.0.

The residue (Residue 1, Figure 1) was washed 3 timeseach with ethanol and ether and dried as above. Whenthe weights of Fraction A and the elastin-containingResidue 1 were added, the yield was found to be 87 to96 per cent. The proportion of the two fractions variedsomewhat with age.

The dried residue was placed in distilled water (20 mlper g) and heated in an autoclave at 20 pounds for 3hours, maintaining a temperature of approximately 3000 C.Two vol of water were added to the resultant gray sus-pension which was then centrifuged at 2,000 rpm for 40minutes. The supernatant solution was filtered, evapo-

Supernatant

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X-RAY DIFFRACTION STUDYOF AORTIC ELASTIN

rated over steam to one-fifth the original volume, pre-cipitated with two vol of ethanol, and dried and weighedin the above manner. Several samples of this material,designated Fraction C (Figure 1) yielded upon chemi-cal analysis the following, as per cent: hexosamine 2.4 +

0.6, nitrogen 13.1 + 0.8, hydroxyproline 6.9 + 0.5. Basedon the high hydroxyproline and hexosamine concentra-tions, this fraction probably contained collagen that hadbeen converted to gelatin, and neutral polysaccharides.The residue was washed 3 times each with alcohol andether, and dried as before. This product, designatedFraction E (Figure 1) represented AER. Fractions Cand E were obtained in yields of 78 to 92 per cent fromResidue 1. AER appeared as a fine white or beigepowder which could conveniently be used for X-ray dif-fraction analysis. The hydroxyproline content of severalsamples was 1.3 + 0.3 per cent. While not strictly com-parable with elastin prepared by alkali or formic acidtreatment, this product most closely resembled the ma-terial studied by Partridge, Davis and Adair (8).

Treatment with elastase. It has been shown that treat-ment of elastin with elastase results in the formation ofsoluble proteins (14). To demonstrate the intimate as-sociation of hydroxyapatite crystallites with elastin, sam-ples of AERwere treated with this enzyme. Were suchassociation to exist, the finding of a hydroxyapatite X-raydiffraction pattern would be expected in the ethanol-pre-cipitated, nondialyzable supernatant solution of elastase-treated aortic residue. In such an experiment, two sam-ples (100 mg) of aortic elastin residue (A, 55-39) wereincubated in a 15 ml Erlenmeyer flask with commerciallyobtained, crystalline elastase 3 (20 mg) previously dis-solved in 5.0 ml of barbital buffer, pH 8.6, 0.1 M. Twosimilar samples without enzyme added to buffer servedas controls. After incubation for 18 hours at 370 C ina constant-temperature water bath, the contents of thefour incubation vessels were transferred quantitatively todialysis bags (Visking, /Y8 inch) with 5 ml of additionalbuffer. The contents of the bags were dialyzed againstdistilled water (6 L) with constant stirring for 24 hoursat 40 C. Thereafter, the contents were transferred tocentrifuge tubes (25 ml), made up to 15 ml with distilledwater, and spun at 3,000 rpm for 45 minutes. All resi-dues were washed 3 times each with alcohol and etherand dried as before. All supernatant solutions represent-ing that protein which had been made soluble, but non-dialyzable, by such treatment had ethanol added to 80per cent vol/vol. After addition of 1 per cent KAc, theywere allowed to stand for 3 hours at 40 C. The fine pre-cipitates, which formed only in the tubes containing elas-tase-treated material, were washed and dried as above.It was possible that incomplete centrifugation might leavecrystallites in the nondialyzable supernatant solutions ofelastase-treated AER. The appearance of crystals in theethanol-precipitated supernatant solutions, as disclosed byX-ray diffraction analysis, would in such case not be dueto the specific action of elastase, but to a nonspecificprecipitation of crystallites by any protein in the presence

3 Nutritional Biochemicals, Cleveland, Ohio.

of ethanol. To exclude this possibility, 1.0 ml of a 1 percent human serum albumin (American Red Cross) wasadded to the dialyzed supernatant solution of similar non-elastase-treated control samples. Following the additionof albumin, alcohol was added with KAc as above, andthe precipitate which formed immediately was washedand dried as before. All samples were subjected to X-raydiffraction analysis by the powder method.

X-ray diffraction. X-ray diffraction studies were per-formed in a Debye-Scherrer powder camera with a 57.3mmradius. The powdered residue was placed in 0.2 mmLindermann glass capillary tubes and exposed for 20hours. Copper radiation (CuKa = 1.54 A), with a nickelfilter, and Kodak no-screen X-ray film were used. Thepatterns were analyzed by measuring the diffractionangles e corresponding to various interatomic spacings ofthe crystalline sample. The interatomic spacings d werecomputed from the Bragg equation

nX= 2 d sin 0

where n is a whole number indicating the order of re-flections and X is the wavelength of the X-rays expressedin A units. The intensities of the diffraction lines werevisually estimated and the crystalline diffraction patternwas identified with the aid of the X-ray Powder DataFile (formerly known as the A.S.T.M. Card IndexSystem).

It should be emphasized that a typical, sharp dif-fraction pattern is obtained by the powder method if thecrystallites of the sample are not too small (above 1,000A) and exhibit a regular three-dimensional periodicity oftheir atomic structure. Proteins, because of their largeand irregular periodicity, and amorphous materials, be-cause of the absence of three-dimensional periodicity, donot give rise to conventional powder diffraction patterns.These materials exhibit only diffuse halos near the pri-mary beam.

Analyses. Nitrogen was determined by the Kjeldahlmethod, hexosamine by the method of Schloss (15). Hy-droxyproline was determined by the method of Troll andCannon (16). Elemental analyses for calcium and car-bon were performed by Dr. Carl Tiedcke of Teaneck, N. J.

RESULTS

Analytic and X-ray studies of whole aortic resi-dues. The analytic and gravimetric data obtainedfrom nine samples of AER are listed in Table I.The last two columns indicate a visual estimate ofthe relative amounts of protein and crystallinematter observed by X-ray diffraction analysis.Dry weight of the samples was relatively con-stant, averaging 24 per cent of the wet weight, anddid not vary appreciably with age. There was atrend toward a weight increase in AER with ad-vancing age. It is apparent that while the nitrogencontent decreased with advancing age, the per-

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GERALDWEISSMANNAND SIGMUNDWEISSMANN

FIG. 2. X-RAY DIFFRACTION POWDERPATTERNS OF HUMANAORTIC ELASTIN RESIDUES. (CuKa radiation, filter, 20hours' exposure.) 1, Age 8 years. 2, Age 30. 3, Age 37. 4, Age 41 [arrows indicate the faint crystalline patternof hydroxyapatite (Ca,0(P04),(0H)2]. 5, Age 80, 24 hour exposure. 6, Age 86, prepared from fresh aorta (seetext). 7, Identical with 6, but with superimposed single crystal patterns which could not be identified as hydroxy-apatite. 8, Age 86, prepared from dried aortic powder (see text).

centage of calcium rose markedly. Similarly, thecarbon content varied inversely with age.

AER obtained from individuals aged 8, 28, 30and 37 years did not exhibit crystalline diffractionpatterns but showed strong, diffuse haloes charac-teristic of a high protein content (Figure 2, nos.1-3). This finding was in good agreement withthe high nitrogen and carbon content of these sam-ples. The first crystalline pattern was given bythe elastin residue extracted from the aorta of a41 year old woman (Figure 2, no. 4). Despitethe strong, diffuse scattering associated with thehigh protein content of the specimen, a diffractionpattern emerged which could be identified as hy-

droxyapatite-like. (The techniques employed arenot capable of distinguishing unequivocally be-tween hydroxyapatite and closely related apatitesand their solid solutions. For instance, fluorapa-tite and tribasic calcium phosphate give closelysimilar patterns, but the former may be excludedfrom human material, and patterns given by tri-basic calcium phosphate agree less well with theobserved data in Table II. For these reasons,the observed pattern will be referred to as hy-droxyapatite.)

In elastin residues prepared from the aortasof individuals aged 63, 80 and 86, the diffuse pro-tein scattering was definitely decreased. Con-

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X-RAY DIFFRACTION STUDYOF AORTIC ELASTIN

7.

FIG. 2.-Continued

TABLE I

Analytic and gravimetric data for nine isolated human aortic residues *

Analytic values of elastinX-ray diffraction pattern

Whole Elastin Hex-Sample Age Sex aorta residue Nitrogen Calcium Carbon osamine Protein Crystal

%dry weight % % % %55-15 8 M 25.0 54.0 16.3 0.8 49.6 ++++ 055-38 28 M 25.5 48.3 16.8 0.1 49.6 ++++ 055-20 30 M 24.1 49.4 15.8 2.8 47.5 0.2 ++++ 055-65 37 F 24.2 49.0 15.4 0.1 ++++ 055-45 41 F 20.0 53.0 14.0 17.3 48.7 0.1 +++ +55-46 63 M 23.5 62.9 13.9 10.5 41.5 0.6 + + + +55-32 80 F 28.1 59.0 8.4 31.4 25.5 + +++

A1 55-39 86 F 21.0 62.9 9.3 28.2 29.1 0.4 + +++A2 55-39t 86 F 21.0 68.4 10.8 22.4 32.3 0.6 + ++++ -

* The last two columns refer to the relative strength of protein and hydroxyapatite patterns as seen in X-ray diffrac-tion patterns. These are graded from 0 (absent) to ++++ (marked) and were estimated visually (see Figures 2 and3).

t Prepared directly from sample 55-39 without prior drying.$ Single crystal patterns superimposed upon diffraction lines.

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GERALDWEISSMANNAND SIGMUNDWEISSMANN

TABLE II

d-Spacings* calculated from X-ray diffraction analysis (Powder method) of isolatedhuman aortic elastin residues

Hydroxy- Human aortic elastin residuesapatitet

(Hexagonal Ao55-39 A155-39 Ai55-39ao =9.42 Fraction Fraction Fraction

hkl co =6.88) A255-39 AM5-39 Ai55-52 55-45 Ai55-39 D R ES

100 8.17 VW 7.09 8.38 VW 8.14 VW 8.17 VW 8.28 8.22 VW101 5.26 VW 5.18 VW 5.18 W 5.14 M 5.12 VW 5.08 5.09 VW200 4.07 VW 4.14 VW 3.99 VW111 3.88 VW 3.93 VW 3.85 VW201 3.51 VW 3.57 S 3.54 S002 3.44 W 3.46 S 3.42 S 3.42 S 3.44 S 3.42 S 3.46 M 3.42 S102 3.17 VW 3.16 W 3.17 W 3.17 S 3.16 VW 3.15 VW 3.14 VW 3.18 W210 3.08VW 3.07 VW 3.11 VW 3.09 VW211 2.81 VS 2.84 W 2.85 M 2.86 VS 2.84 VS 2.84 M 2.84 S 2.84 M 2.87 S112 2.78 S 2.79 VS 2.78 VS 2.79 VS 2.79 VS 2.80 VS 2.79 VS300 2.72 M 2.71 W 2.71 S 2.74 S 2.70 S 2.73 S 2.71 M202 2.63 VW 2.63 VW 2.63 VW 2.63 VW 2.63 VW 2.64 VW301 2.53 VW 2.49 VW 2.53 VW 2.53 M 2.57 VW 2.57 VW 2.59 VW 2.58 VW 2.58 VW310 2.26 W 2.25 VW 2.25 VW 2.24 VW 2.27 W 2.24 W 2.27 VW311 2.15 VW 2.13 VW 2.15 VW 2.15 VW 2.16 VW 2.14 W 2.16 VW113 2.07 VW 2.06 VW 2.07 VW 2.06 W 2.07 VW303 2.00 VW 2.01 VW 2.00 VW 2.00 VW 2.00 VW222 1.94 W 1.95 W 1.94 W 1.98 W 1.94 W 1.94 M 1.95 W 1.94 VW312 1.89 VW 1.91 VW 1.91 VW 1.91 VW 1.89 VW213 1.84 W 1.85 W 1.84 W 1.88 VW 1.84 W 1.82 M 1.84 VW411,004 1.72 W 1.72 WV 1.72 W 1.72 W 1.71 W 1.70 W 1.72 VW313 1.61 VW 1.65 VW 1.65 VW 1.60 1.60 VW 1.61 VW501 1.59 VW 1.56 VW 1.58 W331 1.53 VW 1.54 VW 1.53 W 1.54 W 1.54 VW 1.54 W

* I ntensities estimated visually: very strong (VS) = 100, strong (S) = 70-90, medium (M) = 50-70, weak (W)= 30-50, very weak (VW) = <20.

t X-Ray Power Data File Card no. 9-342 (prepared by P. De Wolff), Techn. Phys. Dientst., Delft, Holland, 1959.

comitantly, the diffraction pattern of hydroxyapa-tite became more conspicuous (Figure 2, nos. 5-8).

The d spacings and corresponding intensities ofall samples analyzed are given in Table II. Theseare compared with a standard diffraction pat-tern of hydroxyapatite from the X-ray powderdata file. It will be seen that virtually all linesare in close agreement with those of the stand-ard pattern. The slight deviations in d spacingsobserved in some of the samples may indicate thepresence of solid solutions of hydroxyapatite aspostulated by Posner and Stephenson (17).

One preparation derived from an 86 year oldwoman, (Figure 2, no. 7) exhibited, superimposedupon the powder pattern of apatite, diffractionpatterns characteristic of single crystals. The co-incidence of the single crystal diffraction spotswith lines not belonging to the hydroxyapatitepowder pattern indicated that they were of differ-ent, not yet identified, origin. All samples thatexhibited the diffraction pattern of hydroxyapa-tite had a considerable calcium content. Since, as

indicated above, X-ray diffraction analysis canonly indicate the mineral content present in crys-talline form, it was not unexpected that no directcorrelation could be observed between the total cal-cium content determined chemically and the in-tensity of the diffraction lines (compare Tables Iand II).

Treatment of human aortic elastin residue withelastase. Treatment of AERwith elastase yieldedtwo nondialyzable fractions. Fraction R was afinely powdered, white precipitate with approxi-mately twice the calcium and one-third the ni-trogen (in per cent) of the original sample (TableIII). X-ray diffraction analysis of this fractionexhibited the highly resolved diffraction patternshown in Figure 3, no. 10, which was virtuallyfree of the diffuse scattering associated with thepresence of protein.

Fraction ES was formed by ethanol precipita-tion of the nondialyzable supernatant solution re-sulting from elastase treatment of AER. Thismaterial was flocculent and finally fibrous, but en-

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X-RAY DIFFRACTION STUDYOF AORTIC ELASTIN

tirely white, whereas the original residue wasslightly beige. When subjected to X-ray dif-fraction analysis, in addition to the expected pro-tein scattering, Fraction ES showed a patternreadily identified as hydroxyapatite (Figure 3, no.11).. In order to demonstrate that the hydroxy-apatite crystallites in Fraction ES were bound toprotein, it was necessary to show that they wouldnot remain in the supernatant fraction if they wereunassociated with nondialyzable material. Sepa-ration might have occurred if larger crystalliteswere to precipitate during centrifugation, leavingthe finer crystallites to float in the supernatant.Consequently, a diffraction pattern was obtainedzwithout rotating the specimen. It may be seen

in Figure 3, no. 1 la, that the lines are just as con-tinuous as in the rotated specimen. This indicatedthat the crystallite size was of the order of mag-nitude of 0.5 to 5 ju. Larger crystallites wouldhave given distinct spots of single crystals. Frac-tion R also yielded a diffraction pattern with con-tinuous lines when the specimen was examinedwithout rotation. This would imply that super-natant and precipitate were not separated on thebasis of crystallite size.

Control samples carried through this procedure,save for the substitution of inert buffer for elastase,yielded only one fraction (Fraction D, Tables IIand III). This fraction was essentially identicalwith that of the starting material by chemical and

10

FIG. 3. X-RAY DIFFRACTION POWDERPATTERNS OF HUMANAORTIC RESIDUES. (CuKa radiation, nickel filter, 20 hrs.

exposure.) 9, Elastin residue (A, 55-39) incubated in barbital buffer and dialyzed (Fraction D, Figure 1). 10,Insoluble precipitate of another sample of A, 55-39 after elastase digestion and subsequent dialysis. Note the disap-pearance of the diffuse diffraction halo associated with protein (Fraction R, Figure 1). 11, Ethanol-precipitated,nondialyzable supernatant of sample A, 55-39 after elastase digestion. Note the characteristic hydroxyapatite pattern,indicated by arrows, and the prominent diffraction halo associated with protein (Fraction ES, Figure 1). 11a, Iden-tical sample as 3. Pattern obtained without rotation of specimen. The continuous lines indicate that the crystallitesize of the sample is between 0.5 and 5 A.

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GERALDWEISSMANNAND SIGMUNDWEISSMANN

TABLE III

Treatment of aortic elastin residue with elastase (sample A155-39) *

X-ray diffraction patternYield

Sample Symbol elastin Calcium Nitrogen Carbon Protein Crystal

mg/lOO mg % % %Original elastin A155-39 28.2 9.3 29.1 + +++Dialyzed elastin D 89.8 24.5 10.2 31.0 + +++Elastase-treated,

nondialyzable R 23.1 53.9 3.1 9.8 0 ++++residue

Elastase-treated,nondialyzable ES 5.3 ++++ +supernatant

* After dialysis of the digestion products, centrifugation yielded the fractions R and ES; Fraction ES was reprecipi-tated with 80%vol/vol ethanol from the supernatant of such a dialyzed digestion mixture (Figure 3). X-ray diffractionintensities are as graded in Table I.

X-ray diffraction analysis (Figure 3, no. 9). Thesupernatant of this fraction did not yield a precipi-tate upon the addition of alcohol. When, however,albumin was first added to the supernatant of asimilar control preparation, a precipitate readilyformed upon the addition of ethanol to 80 per centvol/vol. This precipitate did not show a crystal-line pattern, but merely displayed the expected,diffuse protein scattering.

Samples of reprecipitated gelatin formed fromthe collagen of aorta at autoclave temperature(Fraction C) were also subject to X-ray diffrac-tion analysis, as were samples of acid mucopoly-saccharide-protein complexes (Fraction A) whichwere extracted by the initial homogenization incold water. No trace of a crystalline pattern wasobtained from these fractions, consequently anyclose structural bond (not disrupted by the iso-lation procedure) between hydroxyapatite andmaterials in these fractions appeared to have beenexcluded.

DISCUSSION

These 'studies confirm the presence of a hy-droxyapatite as the principal crystallite of humanaorta. In addition, they provide evidence thatthis mineral may be closely associated with iso-lated elastin, rather than with collagen, polysac-charide or lipid. The AER, isolated by the pro-cedure detailed above, showed an increase of cal-cium and a decrease of nitrogen (expressed as percent) with increasing age. These findings are inagreement with those of Lansing, Alex and Rosen-thal (3), although the yields and percentages ofcalcium differ from those reported by them. This

difference is undoubtedly due to the inclusion inour specimens of intimal calcific deposits. X-raydiffraction analysis of AERhas shown a decreaseof protein content, coincident with an increase ofcrystalline hydroxyapatite with advancing age.These changes are probably related to the chemicalmodifications described above.

The intimate structural association of hydroxy-apatite with elastin has been established by thefollowing evidence.

1. Isolated AERgave the characteristic X-raydiffraction pattern of hydroxyapatite, while neithercollagen prepared as gelatin, nor water-solublepolysaccharide fractions yielded crystalline pat-terns.

2. Elastase treatment of AER liberated into thesupernatant solution of the digestion mixtures anondialyzable protein, which, upon ethanol pre-cipitation, showed hydroxyapatite structure onX-ray diffraction analysis.

3. Powder diagrams of samples not rotated inthe camera served to exclude the possibility thatthe crystallites floating in the supernatant solutionwere separated from those in the precipitate on thebasis of size alone. Since insoluble hydroxyapatitecould not be found in the aqueous supernatant ofsuspensions of elastin not treated with elastase,intimate bonds between elastin and these smallhydroxyapatite crystallites were presumed to ex-ist. Probably these are strong molecular forcesbetween protein and crystal, since they surviveautoclave conditions.

4. The coincidental precipitation of small hy-droxyapatite crystals floating in suspensions of

1664

X-RAY DIFFRACTION STUDYOF AORTIC ELASTIN

dialyzed elastin by another soluble protein suchas albumin has not been observed.

Postulation of elastin-mineral association wouldnot exclude a much weaker, yet physiologicallysignificant association of hydroxyapatite with col-lagen or lipid which would be disrupted by an ex-traction procedure involving fat solvents and au-toclave temperatures. Indeed, were extractionto break such forces, insoluble hydroxyapatitecrystallites would be found in the elastin fractionsas finally isolated.

Increased crystallinity of aortic elastin with ag-ing may be sufficient to account for the embrittle-ment thought to be responsible for hemodynamicchanges in the aging aorta, whose elastic proper-ties have been attributed to macromolecular ag-gregation (18). Crystallization in polymeric sub-stances usually diminishes their elastic properties.4Examples of this phenomenon are found in nat-ural and synthetic rubbers, as well as in syntheticfibers. Crystallization of such polymers was foundto be associated with an increase of stiffness. Atenfold increase in Young's modulus during crys-tallization was shown in results obtained on un-vulcanized rubber as well as on Neoprene (19).Impregnation of fibrous proteins by many smallcrystallites would thus increase brittleness by theformation of anchor-points for elastin fibers, witha concomitant decrease of their rubber-like mo-bility. Amorphous deposits do not show theseeffects, whereas crystallites by means of secondaryvalence forces (e.g., hydrogen bonds), can estab-lish firm linkages with fibers and thus establishanchor-points (19). In view of these functionalconsiderations, studies of crystallinity may be morefruitful in the study of aging tissues than simplechemical analysis which does not distinguish be-tween crystalline and amorphous deposits of min-eral.

Further quantitative studies of the crystal-fiberrelationship in blood vessel wall seem appropriate.Since the number and size of crystallites are ofvital importance in determining strength andelastic properties of analogous polymer systems,techniques such as low-angle X-ray scattering

4 While this study has been concerned with the deposi-tion and/or formation of mineral crystal upon a polymer,polymers themselves may exhibit crystallinity under ap-propriate conditions. The resultant loss of elastic proper-ties is essentially similar.

and Fourier analysis of line profiles (20) shouldgive valuable quantitative information in this re-gard. Such studies, using the elastin preparationsdescribed above, are now in progress.

SUMMARY

1. Elastin residues from human aortas have beenprepared on the basis of their insolubility in waterat autoclave temperature.

2. The crystallites associated with such residuesbecame prominent in aortas from older individualsand were identified as hydroxyapatite by X-raydiffraction analysis.

3. In aortas obtained from young subjects, onlya diffuse X-ray scattering characteristic of proteinwas observed; this scattering decreased with ad-vancing age.

4. These X-ray findings paralleled a decrease innitrogen and an increase in the calcium contentof isolated aortic elastin residues with advancingage.

5. The intimate structural association of hy-droxyapatite crystallites with elastin in aortic tis-sue has been demonstrated by means of elastase.Treatment of elastin residues with this enzymecaused the liberation of a protein into the non-dialyzable supernatant solutions of the digestionmixture, which upon ethanol precipitation gavethe typical diffraction pattern of hydroxyapatite.

6. Neither collagen nor polysaccharide fractionsobtained from human aortas exhibited crystallitepatterns.

ACKNOWLEDGMENT

The authors gratefully acknowledge the advice of Dr.Maxwell Schubert, in whose laboratory most of the ini-tial experiments were performed, and who suggested themethod of fractionation. We should also like to thankDr. Lewis Thomas for encouragement and support, andDrs. Isidore Fankuchen and Ludwig Eichna for helpfulsuggestions.

REFERENCES

1. Duff, G. L., and McMillan, G. C. Pathology ofatherosclerosis. Amer. J. Med. 1951, 11, 92.

2. Lansing, A. I. Chemical Morphology of ElasticFibers in Trans., Second Josiah Macy Jr. Founda-tion Conference on Connective Tissues. New York,1951, p. 45 ff.

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