Cardiovascular studies on copper-deficient swine

EXPERIMENTAL

AND

MOLECULAR

Cardiovascular

3, lo-18 (1964)

PATHOLOGY

Studies

on Copper-Deficient

HI. Properties of isolated Aortic DAVID A. KIMBALL, Department

of

Pathology,

Elastin’

F. COULSON, AND WILLIAM

WALTER University

Swine

of

Received

Utah

College

February

of Medicine,

H. CARNES Salt

Lake

City,

Utah

5, 1963

Spontaneous aortic rupture has been reported in copper-deficient swine (Carnes et aE., 1961; Shields et al., 1962). The most conspicuoushistological lesions in segments not involved by grossrupture were focal breaks in the medial elastic laminae and an accumulation of amorphous material with some staining characteristics of elastin between the laminae. Mechanical tests of the aortas showed a marked reduction in tensile strength (Shields et al., 1962) and other alterations of mechanical properties (Coulson and Carnes, 1962) that indicated a defect in the elastin as well as the collagen or in their mutual insertion. A preliminary report of altered properties of the elastic tissue has been made (Kimball et al., 1962). A further analysis of the isolated elastin is reported here. The isolation or aortic elastin has been carried out by the use of concentrated formic acid according to Hass (1942). This is basedupon the observation of Florence and Loiseleur (1934) that formic acid has a selective solvating action upon various proteins. Ayer et al., (1958) have shown that a formic acid extracted residue of aorta is free of other tissue elements and that its elastic recoil is unimpaired. MATERIALS

AND METHODS

Purebred Yorkshire swine were obtained from a local breeder. In the first experiment the animals were from two separate litters born 1 day apart, 8 animals from a litter of 13, and 7 from a litter of 12. The animals were delivered to the laboratory at 4 and 5 days after birth and were placed on a copper-deficient milk diet (Shields et al., 1962). All animals were supplemented with 30 mg of iron per kilogram of body weight per day. A supplement of 0.5 mg of copper per kilogram of body weight per day as sulfate was added to the milk of six animals at 16 and 17 days of age. Nine animals received no other supplement. All animals were continued on the diet until they died or were sacrificed by exsanguination under sodium pentobarbital anesthesia.All deaths occurred between 71 and 102 days of age. In the secondexperiment 18 York-white pigs were placed on the basal diet of milk supplementedwith iron at 4-6 days of age. Five of these received no other supplement. Six received a supplement of copper as in the first experiment. Seven received the copper supplement and additional minerals and vitamins. The supplements are given in Table I. 1 This work was supported by Public Health Service Grant H5609(Cl) from the National Heart Institute, and by Research Training Grant 2G-310(Cl) of the Division of General Medical Sciences. 10

AORTIC

ELASTIN

IN

COPPER-DEFICIENT

TABLE SUPPLEMENTS

ADDED

TO BASAL

I DIET

OF EVAPORATED Dietary

X0.

Group I II III

Control Copper deficient Copper deficient supplemented

Pigs

Iron (30 m&kg/day)

11

SWINE

MILK Supplements

Copper (OS mp/kg/day)

Other minerals and vitamin9

12

+

14

+

-

+

+

7

+

a The mineral supplement consisted of manganese chloride, aluminum sulfate, sodium fluoride, potassium iodide, zinc sulfate, cobalt nitrate, and nickel acetate. Vitamins added were thiamin, riboflavin, nicotinic acid, pyridoxine, pantothenic acid, inositol, para-aminobenzoic acid, biotin, pteroylglutamic acid, coba!amine, choline, ascorbic acid, and vitamins .4, D, E, and K.

At autopsy, transverse rings of aorta 4 mm wide and free from obvious defects or branches were cut by razor blades from the descending thoracic aorta. These were cleansed of blood and surrounding tissue and rinsed in saline. The rings were desiccated over P-Or, at a reduced pressure for at least 4 days and stored in the desiccator until treatment with formic acid. Each ring was weighed dry and then placed in a sealed bottle containing concentrated formic acid (88%)) in a water bath at 45°C for specified intervals of time. One ml of formic acid was used for every 5 mg of dry tissue. Following extraction, the rings were washed in running water for 24 hours. The width and thickness of each ring was measured with a calibrated stereomicroscope. They were then stretched as described below and load-extension curves were plotted. The mechanical device for stretching has been described (Coulson and Carnes, 1962). It contains a motor regulated by a control which can be operated at different relative speeds from 1 to 10. The motor drives a lead screw which has a platform supporting the lower hook. For every revolution of the lead screw the lower hook is moved a distance of 1 mm. This change in distance is measured by a lo-turn potentiometer, which is connected to the lead screw through a series of pulleys and a plastic belt, and is recorded on the S axis of a Moseley X-Y recorder (Model 3s). The motor can be reversed, bringing the hooks closer together. The upper hook is suspended in a fixed position from a Statham transducer. The tension produced in the transducer is recorded on the Y axis of the Moseley recorder. Each aortic ring was suspended between the two hooks in a normal saline bath. The starting length, Lo, of each ring was measured as the distance between the supporting edges of the hooks when the stretched loop exerted a force of 1 gm. The motor was adjusted to move the lower hook at a rate of 3 mm per minute. The distance moved and the tension produced were recorded until the rings ruptured. Following rupture, the rings were again desiccated over PZO;, at a reduced pressure for at least 4 days and weighed, so that a comparison of weights before and after formic acid extraction could be made. RESULTS In a preliminary experiment, the effect of formic acid on the weight and tensile strength of normal swine aorta was determined. Loops of aorta, obtained from mature

12

DAVID

A. KIMBALL,

WALTER

F. COULSON,

AND

WILLIAM

H.

CARNES

hogs at the slaughter house, were cut from a 15cm segment of the descending thoracic aorta. Thirty-one loops were desiccated, weighed, and treated with formic acid at 45°C for periods of 6, 24, 48, 96, 192, and 360 hours. Figure 1 shows the reduction in weight after formic acid extraction for varying periods of time. The weight of the residue dropped rapidly during the first 6 hours, then reached a 100 4

90aog 70E d 6o ; B 50-

-+Y

4022 L z 30-

"1

20 #.",J , I 6 12 24 40 96 192 HOURS IN FORMIC ACID AT 45' C

o-,,

FIG. I. Decrease determination.

in

aortic

residue

after

formic

acid

360

extraction.

Each

point

represents

one

plateau which decreased only slightly between 12 and 96 hours. Finally, the entire residue went into solution by 360 hours. Figure 2 shows the tensile strength of the residue after varying periods of extraction. This also dropped rapidly during the first 6 hours, when it reached a plateau at about 10 kg per square centimeter and then

48

96

HOURS

IN FORMIC

ACID AT 45'

C

strength

during

formic

extraction.

0 6 12 24

FIG. 2. Decrease one determination.

in aortic

tensile

azid

192

Each

point

represents

AORTIC

ELASTIN

IN

COPPER-DEFICIENT

13

SWINE

slowly decreased after 96 hours. Both the tensile strength and the weight remained relatively constant during the period between 24 and 96 hours. Therefore, 36 hours was selected as the optimum time for extraction in the remaining experiments. During formic acid extraction it was observed that the aortic rings became gelatinous and swollen to about twice normal size. They returned to their former shape upon washing with tap water. This phenomenon was reported by Hass (1942). After removal from the formic acid, gross differences could be observed between the elastic residues of the copper-deficient and control pigs. The former were limp, their sides were collapsed and adherent to each other, and gross linear defects were seen separating the walls into layers. The loops of elastin residue from the controls maintained their oval shape and had a homogeneous appearance without gross defects. Both control and copper-deficient residues were white and shiny. The copperdeficient loops, before extraction, were thicker than the controls (Coulson and Carnes, 1962). After extraction they were approximately the same thickness as the controls. A comparison of dry weights before and after extraction is shown in Table II. The percentage of elastin residue after formic acid extraction in the conTABLE PERCENTAGE

01’ INSOLUBLE

ELASTIS

RESIDUE

II AFTER

FORMIC

.%CID EXTRACTION

Residue in percentage of original weight

NO.

Group I II III

Control Copper Copper

Pigs

44 -C 5.7 24 * 4.3 20 k 1.1

12

deficient deficient

The aortic segments standard deviation.

14 7

supplemented were

extracted

for

36 hours

at 45°C.

Values

given

are

the mean

and

one

trol was almost twice that in the copper-deficient aortas. The copper-deficient supplemented group fell within the same range as the deficient group without the supplement. These results indicate a greater proportion of formic acid soluble material in the deficient aortic loops. It became important, therefore, to learn whether the aortic elastin of the copperdeficient animals was reduced in amount or whether it had a lower resistance to formic acid. This could not be learned without determining elastin content by another independent method. However, it was possible to show that the curve of weight loss of the deficient aortas was like that of the controls. Therefore, the percentage of the residue after 36 hours extraction gives a valid comparison of the relative amounts of insoluble residue. The aortic rings from 6 control and 5 deficient pigs were washed, dried, and weighed after successive intervals of 36, 72, and 108 hours extraction. The percentages of residue at each time are shown in Table III. It is evident that the loss of weight in the first 36 hours accounts for the difference between the copper-deficient and control aortas. The rates of loss between 36 and 108 hours are alike. These rates are slow but they are somewhat faster than that of the older pigs (Fig. 1). During stretching there were qualitative differences between the groups in the manner of rupture. The control loops broke sharply and cleanly with no visible defect before the break. The deficient loops frayed progressively. The inner layers

14

DAVID

A. KIMBALL,

WALTER

F. COULSON,

AND

TABLE III Loss OF AORTA IN FORMIC

RATE OF WEIGHT

Residue

in percentage

I Control (6 pigs)

Time in formic acid (hours)

WILLIAM

H. CARNES

ACID

of original

weight

II Copper deficient (5 pigs)

Ratio II/I

36

46.7

(42.6-52.0)

23.9

(19.5-27.8)

0.51

72

39.9

(35.3-44.8)

21.0

(17.0-25.6)

0.52

10s

32.2

(29.6-34.4)

17.6

(14.2-21.4)

0.54

Values

given

are the means

with

ranges

in parentheses.

ruptured first, the defects progressing peripherally. This phenomenon produced a marked aberration in the load-extension graphs, quite like those observed by Coulson and Carnes (1962) with whole fresh copper-deficient pig aorta. A comparison of typical control and copper-deficient breaking curves is shown in Fig. 3. The curves too ----

75 i

" w z 2

CONTROL Cu DEFICENT

n ,s' I

/'

50I'

I #'

/'

25 -

0

I 5 EXTENSION

FIG. 3. Force developed during extension These are superimposed tracings of typical

I IO (mm)

of aortic curves.

I II I I I I 1 I I I I I

I 15

loops

I 20

at constant

rate

to the breaking

point.

for the controls rose sharply with a steeper slope than those of the deficients. They reached a higher maximum force and then fell sharply. The copper-deficient loadextension curves were not as steep: they reached a lower tension and then continued to stretch as the different layers broke at different elongations, giving a ragged appearance to the second part of the curve. A composite graph of many of these curves can be seen in Fig. 4. Table IV compares the tensile strengths of the control and the copper-deficient aortic residues. The tensile strength was calculated from the load-extension curves as F,,,/2A, where F,,, is the force in grams at the yield point and A is the area calculated as a rectangle from the thickness and width of the cross section at the narrowest point of the ring. An average was taken of 2 or 3 loops from each animal. The tensile strength of the control loops was about 3 times that of the deficient loops. There was no significant difference between the extensibilities of control and copperdeficient loops (Table IV). The isolated aortic elastic tissue was not solid but consisted of multiple fenestrated membranes. Inasmuch as there was a large difference between the groups in the percentage of elastin residue, the question arose whether a difference in the density

AORTIC

ELASTIN

IN

0

COPPER-DEFICIENT

5

IO EXTENSION

15

SWINE

I5 i mm1

20

FK. 4. Force-extension curves of elastin residue. The curves have been superimposed the tangents pass through the origin (see Fig. 6). The straight fall to the baseline after has been omitted for clarity. Each curve represents a sample from a different pig. TABLE MECHANICAL

Group Control Copper Values

deficient given

are the means

IV

PROPERTIES

OF AORTIC

Tensile strength (kg/cm”)

X0. pigs

ELGTIE

Extensibility ($5 original length)

Elastic modulus (pm/cm’)

6

1.86

k 0.85

131 k

21.6

1405

9

0.57

*

120

17.1

648

and one standard

so that breaking

0.15

5

C 434 k

158

deviation.

of the porous membranes might account for an apparent difference strength. No way was found to accurately measure the cross sectional solid elastin. However, the concentration of elastin was calculated from of the hydrated specimen and the dry weight of the residue. Figure 5

in tensile area of the the volume shows that

5.0 40-

;

3.0

g I '; El 5

20-

w .i 5 I-

OB-

IO-

0.6 04I

0.1

FIG. 5. Relation of tensile strength culated as dry weight of the residue tensile strength is given in the text.

02 03 0.405 CONCENTRATION OF ELASTIN I pmlcm3)

to concentration per unit volume

IO

of elastin residues. Concentration is calof the hydrated specimen. Calculation of

DAVIDA.KIMBALL., WALTER

16

F. COULSON,AND

WILLIAM

H. C.~RNES

the tensile strength was related to the concentration of elastin in the specimen but that this did not account entirely for the difference between the groups. Comparing specimensof similar elastin concentration, the tensile strength of the controls was about twice that of the deficients. An elastic modulus was calculated for each curve using the formula (hF/2A)/ (ALILl). A tangent was drawn to the straightest portion of each curve (Fig. 6).

EXTENSION (mm)FIG.

6.

Determination

of elastic

modulus.

The

calculations

are explained

in the text.

The slope of this line was measured as the change in force, AF, compared to the change in length, AL. L1 is the length of the loop at the point where the tangent crossesthe base line. The cross sectional area described earlier is ‘4. This modulus is proportional to Young’s modulus.” The elastic modulus of the controls was more than twice that of the deficients (Table IV). DISCUSSION It is clear that copper deficiency in growing swine results in a deficiency of the aortic elastin. There is a lower than normal amount of insoluble elastin that may reflect an impairment of its synthesis or a modification of its properties. The excess of a buffer-soluble protein in these aortas with the proline:hydroxyproline ratio of 8.5: 1 (Weissman et al., 1961) suggests that a fraction of the elastin is soluble. It is not clear whether this is a soluble precursor of normal elastin or a product of the lysis of insoluble elastin. The reduced tensile strength and lowered coefficient of elasticity of the insoluble elastin residue might also be explained by postulating the synthesis of an abnormal elastin or the action of an elastolytic agent upon the normal product. A choice between these hypothesesmust await other evidence. The abnormality of elastin undoubtedly accounts, in part at least, for the altera3 Young’s modulus (I’), is characteristic of elastic materials defined by the equation F = I’. (AL/L,) -A, where F is the force in dynes, AL is the extension beyond the unstretched length, Lo, in centimeters, and .4 is the cross sectional area in square centimeters. The elastic modulus (M) used here is related to Young’s modulus as L, is related to Lo, disregarding the difference in units. The modulus, M, was preferred because the intersection of the tangent with the baseline could be determined with greater accuracy than could the unstretched length.

AORTIC

ELASTIN

IN

COPPER-DEFICIENT

SWINE

17

tion of mechanical properties of the copper-deficient swine aorta. The lowering of the elastic modulus of the first phase of the load-extension curve of whole aorta (Coulson and Carnes, 1962) closely approximates that of the isolated elastin. The mechanical properties of elastin have been compared to those of rubber with a high extensibility and a low tensile strength. The mechanical behavior of isolated elastin virtually obeys Hook’s law, stress being directly proportional to strain. Estimated values of Young’s modulus for various elastin-containing tissues vary widely but they are far lower than the values for relatively pure collagen (Rigby et al., 1959). However, the second phase of the load-extension curve of whole aorta is also markedly altered. This phase has a modulus far exceeding that of elastin and it has generally been considered a property of the collagenous component (Burton, 1954). It appears necessary, therefore, to postulate a deficiency of collagen also, unless one can attribute the second phase of the curve to another component. Attention is directed to the properties of the collagen in experiments now under way in this laboratory. The relationship of the chief structual components of the aorta under these experimental conditions may be summarized in the following manner: The elastic tissue of aorta forms fenestrated parallel lamellae (Hass, 1942). These are bound together by muscle cells (Keech, 1960; Pease and Paule, 1960: Karrer, 1961). The collagen fibers form loosely arranged bundles that lie parallel and close to the elastic lamellae and may pass through the fenestrations. Direct connections between the collagen and elastin are apparently limited (Karrer, 1961). The smooth muscle is believed to contribute very little to the resistance to stretch even when contracted (Langren, 1952; Alexander. 1954: Burton. 1954). The biphasic load-extension curve of whole aorta has been interpreted to signify that the collagen is slack and does not contribute to the force until the vessel has been extended considerably. The brunt of the force is then borne by the collagen until its maximum extensibility is reached. \Vhen the collagen breaks, a load is placed upon a segment of elastin locally which far exceeds its tensile strength. This causes a sudden break before the maximum extensibility of the whole elastin ring has been exceeded. It is postulated that both collagen and elastin contribute to the maintenance tension of blood vessels but that stability of tension is dependent upon elastic tissue (Burton, 1954). The law of Laplace states that tangential tension is equal to the product of the contained pressure and the radius. It follows that strain in the aorta would be greatest in the widest portion. A defect of elastin would be expected to have its most serious consequences in the ascending aorta. This is the site where rupture invariably occurred in the copper-deficient swine. SUMM.4RY

Elastin was isolated from the aortas of copper-deficient swine by extraction with formic acid. The residue was compared to that isolated from the aortas of controls given copper. The percentage of insoluble elastin residue in the copper-deficient animals was reduced to half that of the control animals. The tensile strength of the deficient elastin was about one-third that of the control. Force-extension curves were markedly altered in the deficients, showing an irregularity in the breaking pattern. The elastic modulus of the deficients was about half that of the controls. These changes in properties of elastin may be correlated with the spontaneous aortic rupture reported in copper-deficient swine.

18

DAVID

A. KIMBALL,

WALTER

F. COULSON,

AND

WILLIAM

H.

CARNES

REFERENCES R. S. (1954). Influence of vasoconstrictor drugs on the distensibility of the splanchnic venous system, analyzed on the basis of an aortic model. C&c. Res. 2, 140-147. AYER, J. P., HASS, G. M., and PHILPOTT, D. E. (1958). Aortic elastic tissue: Isolation with use of formic acid and discussion of some of its properties. A.M.A. Arch. Pathol. 65, 519-544. BURTON, A. C. (1954). Relation of structure to function of the tissues of the wall of blood vessels. Physiol. Rev. 24, 619-642. CARNES, W. H., SHIELDS, G. S., CARTWRIGHT, G. E., and WINTROBE, M. M. (1961). Vascular lesions in copper-deficient swine. Federation Proc. 20, 118. CLARK, J. H. (1933). Elasticity of veins. Am. J. Physiol. 105, 418-427. COULSON, W. F., and CARNES, W. H. (1962). Cardiovascular studies on copper-deficient swine. II. Mechanical properties of the aorta. Lab. Invest. 11, 1316-1321. FLORENCE, G., and LOISELEUR, J. (1934). Contribution g l’etude des substances collagknes: I. PrCparation et propriCt& de differents collagknes. Bull. Sot. Chim. Biol. 16, 52-63. HASS, G. M. (1942). Elastic tissue. I. Description of a method for the isolation of elastic tissue. A.M.A. Arch. Pathol. 24, 807-819. KARRER, H. E. (1961). An electron microscope study of the aorta in young and aging mice. J. Ultrastrut. Res. 5, 1-27. KEECH, M. K. (1960). Electron microscope study of the normal rat aorta. J. Biophys. Biochem. Cytol. 7, 533-538. KIMBALL, D. A., COULSON, W. F., and CARNES, W. H. (1962). Some properties of aortic elastin of copper-deficient swine. Federation Proc. 21, 121. KR~\FKA, J., JR. (1937). The mechanical factors in arteriosclerosis. A.M.A. Arch. Pathol. 23, ALE&UNDER,

l-19.

LANGREN, S. (1952). Baroreceptor activity in the carotid sinus nerve and the distensibility of the sinus wall. Acta Physiol. Scandinav. 26, 3.5-56. PEASE, D. C., and PAULE, W. J. (1960). Electron microscopy of elastic arteries; the thoracic aorta of the rat. J. Ultrastruct. Res. 3, 469-483. REUTERWALL, 0. P. (1921). Die elasticitit der Gef%sw%nde und die Methoden ihrer nHheren Priifung. Acta Medica Sand. suppl. 2, l-175. RIGBY, B. J., HIRAI, N., SPIKES, J. D., and EYRING, H. (1959). Mechanical properties of rat tail tendon. J. Gen. Physiol. 43, 265-283. SHIELDS, G. S., COULSON, W. F., KIMBALL, D. A., CARNES, W. H., CARTWRIGHT, G. E., and WINTROBE, M. M. (1962). Studies on copper metabolism. XxX11. Cardiovascular lesions in copper-deficient swine. Am. J. Pathol. 41, 603-621. WEISSMAN, N., SHIELDS, G. S., and CARNES, W. H. (1961). Connective tissue changes in aortas from pigs on a copper-deficient diet. Federation Proc. 20, 160.