STUDIES ON HYALURONIC ACID AND RELATED SUBSTANCES

I. PREPARATION OF HYALURONIC ACID AND DERIVATIVES FROM HUMAN UMBILICAL CORD

BY ROGER W. JEANLOZ AND ENRICQ FORCHIELLI*

(From the Worcester Foundation for Experimental Biology, Shrewsbury, and the Department of Physiology, Tufts College Medical School, Boston)

(Received for publication, December 9,1949)

In studying the chemical structure of hyaluronic acid it has been found necessary to modify existing methods of isolation and determine the de- gree of purity of the compounds obtained by analysis, electrometric titra- tion, and viscosity determinations.

Two procedures based on the work of Meyer and Palmer (l), Robert- son, Ropes, and Bauer (2), and Hadidian and Pirie (3) have, been studied; In Method A, the human umbilical cords are digested with pepsin and trypsin, and the crude hyalnronate is precipitated with ethanol and.then further purified from the remaining proteins by Sevag’s procedure. : :In Method B, the cords are, extracted with a saline solution and the hyal- uronate is precipitated with ammonium -sulfate and pyridine and then further purified by Sevag’s procedure.

By Method B small quantities :of a highly pure product, free from traces of enzyme, are obtained withzease and rapidity. With Method :A all the available hyaluronic acid is extracted, and it is more suitable for handling large quantities of starting material, but yields a hyaluronate contaminated with a polysaccharide sulfate. This polysaccharide may be removed with pyridine and ammonium sulfate precipitation.

Both viscous-free acid and the sodium salt were prepared and a study was made of various derivatives; the products were obtained by treatment with diazomethane, with acetic anhydride and pyridine, and with tri- phenylchloromethane and pyridine.

The viscosity of these various derivatives was determined at various concentrations, and the action of heat on pure, highly viscous hyaluronate was studied.

EXPERIMENTAL

Method A. Pieparation by Enzymatic-Digestion

1. Digestion of Proteins-3000 gm. of human umbilical cord, correspond- ing to 410 gm. of dried, crude material, free from blood and placental

* This investigation was aided by a grant from G. D. Searle and Company.

495

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496 HYALURONIC ACID. I

tissue and stored under acetone for 2 to 10 weeks, were cut into 2 cm. segments and washed .with’ 12 liters of fresh acetone. The washing wa$ repeated twice with 12 liters of distilled water, each with a 2 to 4 hours soaking period, and completed with a third portion of distilled water and soaking for 18 to 24 hours. The cords were passed through a power- driven meat grinder and diluted with an equal volume of distilled water; the pH was adjusted to 2.0 by the addition of 300 ml. of 2 N HCl. 9 gm. of pepsin (Difoo, 1: 10,000) were added and the mixture, protected by a layer of toluene, was incubated for 24 hours at 37”. The pH was checked and adjusted at frequent intervals during the incubation. After 24 hours, the pH was raised to 7.4 with 90 ml. of 10 N NaOH, 16 gm. of trypsin (Difco, 1:250) were added, and the mixture was again incubated at 37” for 24 hours. After the digestion was completed, the toluene was sepa- rated and the residual mixture centrifuged for 30 minutes.’ The pre- cipitate was discarded, the solution cooled at 5’ was acidified to pH 2.0 with 5 N HCI, 2 volumes of 95 per cent ethanol were added to the super- natant liquid,2 and the mixture was centrifuged. The precipitate was suspended in ab’out 1000 ml. of distilled water, placed in cellophane mem- branes, and dialyzed against running tap water for 24 hours.

2. Elimhaation of Reskhial Proteins by Sevag’s Procedure-A mixture of 3.3 liters of chloroform and 1.7 liters of amyl alcohol and 1 liter of a water solution containing 300 gm. of sodium acetate and 160 gm. of glacial acetic <acid was added to the dialyzed solution (volume, 5 liters). This mixture was shaken vigorously in a mechanical shaker for 10 ‘minutes and then centrifuged for 30 minutes. The supernatant aqueous phase

1 In a similar experiment, starting with i200 gm. of human cord, centrifugation was substituted by filtration on a Biichner funnel, with Whatman No. 4 filter paper prepared with a filter aid (Celite). The hyaluronic acid solution obtained was subjected only two or three times to the Sevag procedure and dialyzed ai in previous experiments. It was then precipitated with 2 volumes of 95 per cent ethanol, washed twice with 95 per cent alcohol, and-thrice with ether and dried at room temperature. The last traces of ether were removed by drying in a vacuum desiccator (Fraction II). The yield was 12.5 gm.

In another similar experiment, starting with 700 gm. of cord, the centrifugation was carried out at 20,000.r.p.m. in a Sharples centrifuge. The hyaluronate (Fraction II&) was precipit,ated with alcohol, washed with alcohol, and ether-dried as above to yield 7.0 gm. of hyaluronate. The analysis of Fractions II and III and the vis- cosity determinat,ions before precipitation indicate that a portion of the hyaluronic acid is lost during the isolation. In one instance the most, viscous fractions remained on the filter, and, in the other instance, the most viscous element was retained in the rotor of the Sharples centrifuge along with the proteins. Therefore, the proportion of polysaccharide sulfate is increased through loss of hyaluronic acid, as is also shown by the higher sulfur content and lower viscosity.

*At this point the mixture was preferably deaerated by evacuation, since this facilitated the settling of the precipitate,

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1%. 11’. JEANLOZ AND E: FORCRIELLI 497

containing the hyalu,ronic acid m siphoned and .treated repeatedly with the same mixture of chloroform and amyl alcohol until no more precipi- tate appeared at the interface. 2 volumes of 95 per cent alcohol were added to the aqueous phase containing the hyaluronic acid (volume, 6 liters). The precipitate was centrifuged for 10 to 15 minutes, dissolved in distilled water, dialyzed against running tap .water for 24 hours, and then against several changes of distilled water for 24 hours. The 4 liters of solution containing 29 gm. of hyaluronate (Fraction &.J3 were saturated with chloroform after dialysis and stored at 5”. It contains i,n general about 20 per cent of polysaccharide sulfate and can be purified by the application of the second method (B4), affording a yield of 5.6 per cent of pure hyaluronate based on the weight of dried cords.

Method B. Preparation by Precipitation with Ammonium Sulfate and Pyridine

1. Saline &rtructi~n--Human umbilical cords (1400 gm. or 190 gm. of dry material) were collected, washed, and prepared the same way as in Method Al. The material was ground and suspended in 4 liters of 0.1 M

sodium chloride solution saturated with chloroform. This extraction was allowed to proceed in the cold for 24 hours with occasional stirring. The mixture was placed in a cloth bag and the fluid was expressed. After a second extraction of the residue in the same way, the combined extracts were acidified at 5” to pH 1.5 to 2.0 with 180 ml. of 5 N HCl and centri- fuged. The precipitates were added to the residue and set aside for enzymatic digestion (Method B4).

2. Precipitation with Ammonium Sulfate-To the solution were added, with vigorous stirring, 2700 gm. of ammonium sulfate and the mixture was kept for precipitation at about 5”, which was complete after several hours. After centrifugation, the solution was treated according to Method B3 and the precipitates were dissolved in water and dialyzed against running tap water for 24 hours and then against distilled water for 24 hours. This solution was treated according to the Sevag technique (Method A2) and afforded 3 gm. of pure hyaluronate (Fraction IV,) in solution.

3. Precipitation with Pyridine-To the ammonium sulfate solution from above were added 500 ml. of pyridine, with vigorous stirring, and the mixture was allowed to stand for about 4 hours. To complete the pre- cipitation 2500 gm. of ammonium sulfate were added slowly, with vigor- ous stirring, and the mixture was refrigerated for several hours. The

3 Fractions are identified by roman numerals and their form or derivatives by a letter (e.g., Fraction I in solution Is, lyophilized IL; the free acid prepared from Fraction. I,. IA., etc.).

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498 HYALUi%ONIC ACID. I

bulk of the preci@tate rose to the interface, some-precipitate adhering to the sides of the vessel. The’subjacent liquid was siphoned, as completely as possible, and the precipitate, along with the pyridine, was centrifuged for 30 minutes to 1 hour. The pyridine was siphoned and the pellicles formed at the interface were washed with 95 per cent alcohol and centri- fuged. The pellicles were cut into small pieces, suspended in distilled water, and dialyzed against tap water for 24 hours and then against dis- tilled water for 24 hours. The material was further purified by the Sevag procedure as described in Method A2. After final dialysis, the solution (500 ml.) contained 1 gm. of hyaluronate (Fraction Vs).

An additional 1200 gm. of ammonium sulfate were added to the am- monium sulfate-pyridine solution and the mixture was treated as de- scribed in the preceding fractionation. This makes certain that no hyaluronic acid remains in the ammonium sulfate liquor. If a precipitate was present, the material was treated as in the previous extraction.

4. Extraction and PuriJication of Re&hal Hyaluronic Acid-The com- bined cord residues were purified by enzymatic digestion as described in Method Al. The final solution (975 ml., Fraction VIs) obtained con- tained 7 gm. of impure hyaluronate, contaminated with about 30 per cent of polysaccharide sulfate and 5 to 10 per cent of proteins. This solution of hyaluronate was purified by a new precipitation with ammo- nium sulfate in the presence of pyridine as follows.

500 gm. of ammonium sulfate and 50 ml. of pyridine were added with vigorous agitation. The mixture was allowed to stand for 1 t.o 2 hours in the cold and then centrifuged for 1 hour. The pellicle formed was washed with alcohol, centrifuged, cut up, suspended in water, and dia- lyzed for 24 hours against running tap water. ,This hyaluronic acid solu- tion was treated a second time with ammonium sulfate and pyridine to insure complete removal of the sulfur-containing compound, then com- pletely dialyzed; it contained 4 gm. of hyaluronate (Fraction VII*). The combined yield of all the fractions was 8 gm. or 4.2 per cent, based on the weight of the dried cords.

The two ammonium sulfate-pyridine liquors were dialyzed in a similar manner and the dialyzed solution was concentrated by lyophilization, yielding 2.5 gm. of residue.

Analysis-Nitrogen (Kjeldahl) 2.90, acetyl 7.4, sulfur 3.8

The study of this sulfur-containing polysaccharide which was found as the contaminant of hyaluronic acid isolated from human umbilical cord will be described in a forthcoming publication.

AnaZy&-The analyses were performed either on hyaluronie acid in solution (exact concentration established by drying an aliquot to constant

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R. W. JEABLOZ. AND E.--F#XXHIELLI 499

weight), on lyophilized, or alcohol-precipitated and ether-dried samples. The analyses obtained from these three types of samples were all identical.

The absence of glycogen in each fraction was demonstrated by the absence of a color reaction with iodine.

Water Content-It is very difficult to remove moisture completely from dried hyaluronic acid, as was previously observed by Meyer and Palmer (1) and as is the case with numerous other polysaccharides. Our samples have been dried to constant weight in vacua at 115O in the presence of phosphoric anhydride for a minimum period of 15 hours. The samples were weighed in closed vessels.

Acetyl-The determinations were made after acid digestion, according to Hadidian and Pirie (3), with the apparatus described by Markham (4).

TABLE I

Analysis of Various Hyaluronate Fractions

I Nitrogen I Ash as Preparation No. I-

Kjeldahl

)W cent

Sodium hyaluronate 3.49 (theoretical)

I 3.40 II 3.74

III 4.27 IV 3.64 V 3.45

VI 4.15 VII , 3.64

-

-~ DlUllas so2

9.3 cent ger cent

3.49 19.70

3.04 18.2 2.63 21.0 2.85 18.2 2.36 14.6 2.28 15.4 2.94 ‘17.5 2.94

-

-

-

Na

per cent

5.73

6.5 5.3 4.15 4.65 2.4

-

- -

-

Acetyl

per cent

10.73

9.6

9.5 10.2 10.4 9.6

10.0

-

-

per cent

0.0

1.2 1.5 2.0 0.10 0.14 1.67 0.16

Nitrogen-Determinations were made according to the Kjeldahl and Dumas methods. The former method produced consistent results and the latter, as is the case with many other natural products, gave results that were too low and inconsistent.

Suljur-The determination was carried out by the combustion procedure of Pregl, as described by Niederl and Niederl (5). The residue from the combustion, due to the presence of sodium, was included in the sample to be precipitated.

Metals-The metals present were converted to the sulfate by incinera- tion after the addition of sulfuric acid. The salts were then dissolved in a large volume of water and sodium was determined by a flame photome- ter (Table I).

4 Nitrogen (Dumas method), sulfur, and methoxyl were determined by the Ana- lytical Division of G. D. Searleand Company, and by F, Weiser, Base& Switzerland.

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500 HYALURONIC ACID. I

Derivatives.

Free Acid and Sodium Salt-To test the hypothesis that anhydride linkages exist between the chains of high viscosity hyaluronic acid (6), the free acid was prepared from the hyaluronate and its neutral equivalent determined by electrometric titration according to Unruh et al. (7). At the same time the viscosity was compared with that of the starting ma- terial. The free acid has previously been prepared by precipitation with glacial acetic acid (1). The partial degradation which always adcompa- nied this step vitiated any viscosity comparison with the starting ma- terial. We have obviated this difliculty in the preparation of the free

PH

6

3 0 2 4 6 8 IO 12

ML. Na OH 0.0165 N

FIG. 1. Electrometric titration of 53.0 mg. of Hyaluronic Acid IV* with 0.0165 N sodium hydroxide in 1 N sodium bromide solution.

acid by passing a solution of hyaluronate through a column of synthetic ion exchange resin, a method which -has been applied to other water- soluble polyuronides (8).

Procedure-Through a column of Dowex 505 20 cm. high and 3 cm. in diameter, previously washed with 200 ml. of 2 N sulfuric acid, and then 2000 ml. of distilled water, 120 ml. (containing 0.40 gm.) of hyaluronate solution (Fraction IVa) were passed under a slight pressure of nitrogen. The column was washed with 60 ml. of distilled water; further washing was not done in order to avoid dilution. The percolate (200 ml.) con- tained 0.290 gm. of free Hyaluronic Acid IV*. To 40.0 ml. of this solu-

6 This product was kindly furnished by The Dow Chemical Company, Midland, Michigan.

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R. W. JEANLQZ AND E. FORCHIELLI 501

tion were added 20 ml. of 2 N sodium bromide solution plus 40 ml. ‘of distilled water and the free acid was titrated with 0.0165 N sodium hy4 droxide solution with glass and calomel electrodes in the Beckman model G pH meter. The results are shown in Fig. 1.

After the titration, the solution containing the sodium salt and a slight excess of NaOH was dialyzed in the cold for 2 days against re- peated changes of distilled water and then lyophilized to residual Sodium Hyaluronate IVN. Hyaluronic Acid IA Was prepared in the same manner starting from Hyaluronate Is.

Analysis- Hyaluronic Acid. Calculatedi Ash 0, neutral equivalent 379.3

‘I “ IA. Found. ““1.6, “ ‘* 382

“ ” Iv& “ “ 1.3, “ ‘( 395

SodiumHyaluronate IVn. Calculated. N 3.49, acetyl 10.73, ash 17.70, Na 5.79 Found. “ 3.52,* “ 9.10, “ 16.6, “ 5.1

* Kjeldahl.

Methyl Ester of Hyaluronic ‘Acid-50 ml. of solution containing 75 mg. of free Hyaluronic Acid IV, (pH 3.4) were shaken in a separatory funnel with three 10 ml. portions of 2 per cent diazomethane in ether until there was no further evolution.of gas. The ether layer was dis- carded and the aqueous solution, now at pH 6.8, dialyzed against dis- tilled water for 24 hours in the cold (5”). Part of the solution of Methyl Hyaluronate was lyophilized for analysis and the remainder used for a viscosity determination.

Analysis-Methoxyl. Calculated, 7.9; found, 5.35, 5.2g6

Acetyl Hyaluronate-In order to estimate the molecular weight by os- motic pressure measurements in chloroform or benzyl alcohol solution and gain some idea of the shape of the molecules by viscosity determinations, an acetyl derivative was prepared. To avoid any degradation the acety- lation was carried out in a medium of anhydrous pyridine and acetic an- hydride, after the hyaluronic acid had been swollen in formamide, according to the method described by Hadidian and Pirie (9).

2 gm. of lyophilized Hyaluronate I, were .dried for 2 hours at 120” in vacua in the presence of phosphoric anhydride, then dispersed in 80 ml. of formamide.’ 80 ml. of anhydrous pyridine were added and, after

6 The analysis refers to the repeating period, i.e., 1 molecule of N-acetylglucos- amine and 1 molecule of glucuronic acid less 2 molecules of HzO.

7 Contrary to the observations of Hadidian and Pirie (9), the dispersion in form- amide is not dependent on the temperature at which it occurs but rather on the physical state of hyaluronic acid. A- product lyophilieed in a very dilute solution and with a very porous structure will become dispersed very readily within a few minutes at room temperature, no particles remaining visible, whereas products

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502 HYALURONIC ACID. ‘I

being thoroughly mixed with 80 ml. of acetic anhydride, the solution was allowed to stand at 25” for 40 hours. The resulting deep brown solution was then poured on 300 gm. of ice and immediately dialyzed, first against running tap water until the acetic acid and pyridine were completely removed, then against distilled water for 24 hours. After centrifugation for 30 minutes, 0.17 gm. of insoluble Acetate A was recovered. The solution was lyophilized to yield 1.5 gm. of ,pale yellow soluble Acetate A.

The acetylation was also carried out for 1 hour at 80”. The products obtained, Acetates B, are dark green in color. After lyophilization, the solubility of these acetates is greatly diminished.

Analysis-Di-0-acetyl hyaluronate. Calculated. Acetyl 26.69, Ash 14.63 Tri-0-acetyl “ ‘I ‘I 32.66, “ 13.47 Acetate A, soluble. Found. “ 29.6, “ 11.4

‘I “ insoluble. “ “ 30.0, “ 10.9 I‘ B, soluble. ‘I I‘ 26.4, “ 15.6 “ “ insoluble. “ ‘I 29.9, ‘r 11.2

The products correspond to mixtures of di- and triacetates. Soluble Acetate A (0.27 gm.), finely suspended in 1000 ml. of distilled water, was passed through a column of Dowex 50. The suspension was lyophilized and the residue, which contained only 0.5 per cent ash, was resuspended in 100 ml. of water. This suspension was then agitated in a separatory funnel with 50 ml. of a 2 per cent ether solution of diazomethane, added in three portions. The acetylhyaluronic acid dissolved completely in the water. The ether layer was discarded and the last traces of ether were removed from the aqueous layer by evacuation. The solution was lyo- philized and yielded 0.270 gm. of residual product.

Analysis-Methoxyl. Calculated, 5.95; found, 0.65, 0.57

This product was dispersed in 20 ml. of pyridine, and 10 ml. of acetic anhydride were added. After 2 days of contact at room temperature, the suspension was heated for 2 hours at 100” with moisture protection, cooled, and poured on to 100 gm. of ice. A resulting slight precipitate which formed was centrifuged; the centrifugate was dialyzed .against running tap water, then against distilled water until all the pyridine and acetic acid were completely removed, and the dialyzed solution was finally lyophilized.

Analysis-Acetvl. Found, 28.6

It was not possible, therefore, to increase the proportion of acetyl groups by acetylation in the presence of pyridine. The same experiment

dried in the form of films remain unchanged in contact with formamide even after several days at a slightly elevated temperature (about 50”).

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R. W. JEANLOZ AND E. FBRCFIIELLI 503

was performed on the soluble Acetate B with the same result. The products were insoluble in organic solvents, such as benzyl alcohol, chlo- roparaffins, acetone, and pyridine, and thus it was not possible to make physicochemical measurements.

Trityl Hyaluronate-This compound was prepared by the method of Low and White (lo), in an attempt to determine whether position 6 of the glucosamine moiety was free or glucosidically linked.

500 mg. of lyophilized Hyaluronate IL, dried in the same manner as the material which was used for acetylation, were introduced in a tube with 10 ml. of anhydrous pyridine and 2 gm. of triphenylchloromethane. The tube was sealed and shaken for 20 hours at 55”, followed by 2 hours at 100”. Only a small part dispersed and seemingly entered into the reaction. The contents of the tube were cooled and then poured into 20 ml. of anhydrous acetone. This mixture was filtered and the precipitate washed thoroughly with acetone and dried by exposure to air; it was then left in contact with distilled water in the cold for 1 week. The insoluble residue was filtered, carefully washed with water, and the washings were collected and lyophilized. The residual gray powder, which weighed 0.130 gm. after desiccation over calcium chloride, contained 0.53 trityl groups per repeating unit period.8

The lyophilized aqueous solution yielded 0.390 gm. of hyaluronic acid in which no trityl groups could be found.

0.5 gm. of dried, lyophilized Hyaluronate IL was dispersed in 30 ml. of formamide, to which 25 ml. of pyridine and 2 gm. of triphenylchloro- methane were added. The mixture was kept at 50” for 24 hours, pro- tected from moisture, and, after cooling, poured into 200 ml. of acetone and treated as above. 50 mg. of insoluble product, containing 0.95 trityl group per repeating unit, were isolated. No trityl groups could be de- tected in the hyaluronic acid obtained from the aqueous solution.

V&o&y-The viscosity was determined with an Ostwald viscosimeter having a capillary approximately 9 cm. long and a flow time of 30 to 35 seconds for distilled water. A total volume of 4.0 ml. was used for each determination which was run at 25” in 0.05 M sodium chloride solution buffered at pH 7.0 with phosphate (11, 3).

In order to obtain data on the shape of the molecule and on the factors which affect viscosity, the measurements were made with varying con- centrations, and curves of the intrinsic viscosity (ratio of specific viscosity to concentration) as a function of, concentration were established.

8 The trityl content was determined by hydrolysis in hot, concentrated sulfuric acid followed by dilution with water, filtration, washing with water, and drying; the insoluble triphenylcarbinol, dried to constant weight, was identified by its melting point.

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504 HYALURONIC ACID. I

Viscosity of Various Fractions-The various fractions obtained -(Frac- tions I, IV, V, VII) by the procedures described showed practically the same high values for viscosity when determinations were made before lyophilization or precipitation and drying (Fig. 2). When the viscosity is low, this is due to the presence of more or less large quantities of poly- saccharide sulfates, as shown by sulfur analysis, as the viscosity of chon- droitinsulfuric acid solution is very low (12) in comparison with that of hyaluronic acid. The viscosities of hyaluronic acid isolated by the vari- ous methods are all much greater than those reported by Blix and Snell-

I I I

0.05 0.10 0.15

GRAMS IN 100 ML. SOLUTIOI’J

FIG. 2. Intrinsic viscosities of solutions of hyaluronate fractions before lyophili- zation or precipitation (Curves Is and 111s to VIIs). Intrinsic viscosity of pleural fluid calculated from the data of Meyer and Chaffee (13) (Curve VIII).

man (12) for their product, which indicates a molecular weight or weight of the micelle of aggregation many times greater than that found by these authors. The relatively low viscosity of their products may be due to incomplete extraction, as in the case of Fraction III described above.

An attempt to purify Fraction IV further through fractionation by centrifugation at 20,000 r.p.m. for 3 hours gave no conclusive results.

The various fractions represent the total hyaluronic acid contained in umbilical cords, Their relative viscosity is very high compared to the other hyaluronic acid described.s (The relative viscosity of Fraction V at

0 See summary in Hndidian and Pirie (3) and Meyer (6).

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R. W. JEANLOZ AND E. FORCHIELLX 505

a concentration of 0.3 gm. per 100 ml. = 485; and at 0.1 gm. per 100 ml. = 10.) Pleural fluid has an intrinsic viscosity, calculated with the data of Meyer and Chaffee (13) for an infinite dilution, approximately the same as that of the hyaluronic acid described in this paper, but the increase as a function of concentration is much greater (Curve VIII, Fig. 2). This indicates that if the viscosity of the fluid is indeed due to hyaluronic acid the aggregation of the latter is much greater in its native state and is partially destroyed in the course of isolation.

Ogston and Stanier (14) recently obtained from synovial fluid a com- plex of hyaluronic acid and protein which possessed a relative viscosity of 39 at the concentration of 0.1 gm. per 100 ml. and was homogeneous in the ultracentrifuge. It is therefore probable that the proteins are re- sponsible for the high viscosity of fluid containing hyaluronic acid in its native state and that any purification to obtain the polysaccharide pro- tein-free should cause degradation of the aggregate of particles.

InJEuence of Time and Temperature-Numerous observations on the stability of hyaluronic acid in the presence of oxygen and oxidation-reduc- tion systems have been published (15). In this paper, only the influence of time and temperature on viscosity has been studied (Fig. 3).

Precipitation with 95 per cent ethanol of Fraction Vs followed by ether washing and finally drying at room temperature does not affect the vis- cosity (Curve V1, Fig. 3). The viscosity is reduced by lyophilization and more so when the solution is in a more dilute state (Curves VZ and Vd.

When hyaluronate is lyophilized or obtained by alcohol precipitation and dried to constant weight in a vacuum at loo”, a readily soluble prod- uct is obtained. Its viscosity is greatly reduced (Curve Vd, Fig. 3), es- pecially at high concentration, indicating a “denaturation” of the micelle in which the long filaments become shorter and more compact. The viscosity at infinite dilution is always relatively high, which is possibly due to the interparticle interaction. The “denaturation” is not reversible, for a product left in solution in the presence of salt for 15 days at about 5” did not regain its viscosity (Curve V,).

Since dried products heated in the presence of air or oxygen-free nitro- gen were equally degraded, it appears that oxygen is not the determining factor under these conditions.

A sterile, dialyzed solution retained the same viscosity after being stored for 6 months at 5”, but its viscosity was diminished in the presence of the buffer used for the viscosity measurement (Curve VS, Fig. 3).

The degradation depends primarily on the temperature (Curves Vr and VS, Fig. 3). The viscosity decreases slowly as the temperature is raised to 90’ and above this threshold value it decreases very rapidly

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506 HYALURONIC ACID. I

(Fig. 4). The rate of degradation is initially constant but decreases after 2 hours (Fig. 5).

No evolution of carbon dioxide occurs during this loss in viscosity and acetyl and nitrogen analysis and periodate consumptionlo before and after do not differ.

0.05 0.10 0.15

GRAMS IN 100 ML. SOLUTION

FIG. 3. Influence of precipitation, lyophilisation, and heat on the intrinsic vis- cosity. Hyaluronate Vs precipitated with ethanol and dried with ether (Curve Vi). Hyaluronate Vs lyophilized from a solution with a concentration of 0.4 gm. in 100 ml. (Curve V,). Hyaluronate Vs lyophilized at a concentration of 0.12 gm. in 100 ml. of solution (Curve V,). Lyophilized hyaluronate dried at 100” for 24 hours under a high vacuum (Curve V,). Dried hyaluronate after standing 15 days in buffered 0.05 M sodium chloride at 5” (Curve VS). Hyaluronate Vs after standing 30 days in buffered 0.05 M sodium chloride solution at 5” (Curve Ve). Hyaluronate Vs heated at 60” for 1 hour in the presence of nitrogen (Curve VT). Hyaluronate Vs heated at 100’ for 1 hour in the presence of nitrogen (Curve VS).

InJluence of Substitution of Reactive Groups-The viscosity of the free Hyaluronic Acid IV, (Curve IVr, Fig. 6) obtained from Fraction IV,, in the presence of the previously used buffer, is identical with that of the starting material (Curve IVS). However, the viscosity of the sodium salt is slightly lower, which is probably due to the slight excess of alkali required to insure complete conversion to the salt and also to the various subsequent treatments (Curve IV$ .

lo Jeanloe, R, W., and Forchielli, E., unpublished work.

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R. W. JEANLOZ AND E. FORCHIELLI 507

I I I I

50 70 90 110 30 60 90 120

TEMPERATURE TIME IN MINUTES FIG. 4 FIQ. 5

FIQ. 4. Influence of temperature on the relative viscosity. Hyaluronate VIIs in water heated at various temperatures for 1 hour at a concentration of 0.09 gm. in 100 ml. of solution.

FIQ. 5. Influence of time and temperature on the relative viscosity. Hyaluronate VIIs in water heated at 100’ for variable periods at a concentration of 0.09 gm. in 100 ml. solution.

GRAMS IN 100 ML. SOLUTION

FIQ. 6. Influence of substitution of the reactive groups on the intrinsic viscosity. Hyaluronic Acid IV, (Curve IVr) obtained from Hyaluronate IVs. Methyl Hy- aluronate IVs (Curve IV2). Water solution of 11.3 mg. of Methyl Hyaluronate IVs in 10 ml. mixed with 2.5 ml. of 0.016 N sodium hydroxide left standing 3 hour at 25” and then acidified with 1 ml. ofO.l N acetic acid (Curve IVS). Sodium Hy- aluronate IVN obtained by neutralization of Hyaluronic Acid IV, (Curve IV,). Acetate of Hyaluronate I (Curve IV&).

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508 HYALURONIC ACID. I

Esteritication of the carboxyl group causes degradation of the micellar structure (Curve IV,, Fig. 6); the viscosity is not regained by treating with alkali but, on the contrary, it is considerably lowered (Curve IVI). The same two-step decrease in viscosity is observed when the hydroxyl groups are acetylated (Curve IVE) and the acetyl groups subsequently removed under mildly alkaline conditions (7 intrinsic 4.0 for a concentra- tion of 0.15 gm. per 100 ml.).

DISCUSSION

The above data show that it is possible to extract hyaluronic acid com- pletely from human umbilical cord and to obtain it in a state of purity of about 95 per cent as indicated by the sulfur analysis and the electro- metric titration. The best method for obtaining large quantities of such a product is a combination of degradation of t,he proteins by enzymatic digestion (Method A), followed by fractionation with ammonium sulfate in the presence of pyridine (Met.hod B4). The yield, based on dried cord, can be as much as 5 to 6 per cent.

To obtain an enzyme-free product, which is desirable for the assay of hyaluronidase, it is necessary to extract with an aqueous salt solution (Methods Bl and B2) but large quantities of hyaluronic acid would still remain in the cords, being mechanically retained by the proteins.

The elimination of the sulfate-containing polysaccharide is important, for it is present in relatively large proportion (about 20 per cent) and, in the case of the hyaluronidase assay, it has been shown that chondroitin- sulfuric acid and heparin can function as inhibitors (16). This purifica- tion step seems to have been generally ignored, for in most of the preparations described in the literature a high sulfate content is indi- cated (3, 18).” In contrast to those of Hadidian and Pirie (3), all the fractions obtained by precipitation with ammonium sulfate with or with- out the presence of pyridine have always contained less than 0.2 per cent of sulfur, corresponding to less than 4 per cent of chondroitinsulfuric acid. Further, all the fractions after enzymatic digestion had a high acetyl content, the low content of Fraction I being explained by the relatively strong contamination with polysaccharide sulfate. The differ- ences in the salt values depend on the process of purification: in the first method, the pH is at times slightly higher than 7, which causes complete neutralization of the hyaluronic acid, whereas in the second method the working pH is always below 7.‘” Also in contrast to the observation by

1x One single method for purification has been described by Meyer et al. (17). lx Fractions II, III, and VI contain a low proportion of proteins; these impure

products (with large proportions of polysaccharide sulfate) were submitted only two or three times to the Sevag procedure.

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R. W. JEANLOZ AND E. FORCHIELLJ 509

Lundquist (19), the viscosities of the fractions obtained through ensy- matic digestions were never unstable.

Hyaluronic acid prepared by precipitation in the presence of acid is always contaminated by salt and the neutral equivalents observed for such preparations are always greater than 500 (3, 2, 20). Passage of the sodium salt through a column of acid ion exchange resin readily converts it to the free acid with a minimum of ash content. A sample of hyalu- ronic acid, so prepared, which had a very high viscosity and yielded a sodium salt of the expected composition, was found to have a neutral equivalent of 395, close to the theoretical value of 379. Therefore, it is necessary to reject Meyer’s hypothesis, according to which the high vis- cosity is due to anhydride linkages between the chains (6). If the small discrepancy of 5 per cent between the actual results and those calculated should indicate the proportion of carboxyl groups chemically combined, it more reasonably may be attributed to ester linkage with hydroxyl groups of another chain.

From the viscosity measurements it follows that the molecular chains of hyaluronic acid are joined in the native state in micelles of a molecular weight of several million. The depolymerization of these large aggregates occurs at the time of extraction regardless of the precautions taken. The most depolymerized being the most dispersible, a rapid and incomplete extraction yields products of relatively low viscosity. If, however, the extraction is pushed to completion, as in the instance of enzymatic di- gestion of proteins followed by fractionation of the residual polysac- charides, the purification involves depolymerization, as is shown in Fig. 2, and the product, free of its slightly viscous impurities, has a lower viscosity (Curve VIIs) than the crude initial product (Curve VI,). Therefore it seems impossible to obtain in the pure state products pos- sessing a viscosity equal to that of the pleural fluid described by Meyer and Chaffee (13).

The irreversible loss of viscosity due to high temperature or esterifica- tion is compatible with the hypothesis of Ogston and Stanier (14) that the particles of hyaluronic acid are interconnected in a micelle with a loose, sponge-like structure. The sudden fall of the viscosity at a de- termined temperature should be due to the breaking of the interparticle links and is similar to the breakdown of the starch micelle by heat (21). The breakdown at room temperature under the action of diazomethane is evidence of the rBle of the carboxylic groups in these interconnections between particles.

Because of the slight solubility of hyaluronic acid in non-aqueous media it is very difficult to esterify hydroxyl groups and thereby obtain acetyl derivatives soluble in non-polar solvents. This difficulty is shared by other

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510 EIYALURONIC ACID. I

polysaccharides containing amino sugars. All measurements of molecular weight, or of the dimensions of the molecular aggregate, can therefore be only an approximation of the average among the various aggregates of the molecules of hyaluronic acid.

The salt content of the acetates is in general below the theoretical value. It is possible that under the action’of acetic anhydride an esterification between the chains takes place with formation of tridimensional polymers aggregated in micelles, which would explain the great drop in solubility. As in the case of the free hyaluronic acid esterification degrades the mi- celle and permits solution. It seems, however, that the greater part of the carboxylic groups cannot be reached. Either they may be engaged in ester linkage or they may be protected by the rest of the molecule. The difficulty in obtaining a more complete acetylation could also arise from these same causes.

The formation of a trityl derivative indicates that the hydroxyl group in position 6 of the glucosamine moiety is not involved in the linkage with glucuronic acid. However, the poor yield from tritylation, which is due to the general difficulties of esterification, does not allow definite conclusions to be drawn.

SUMMARY

A combination of methods for the preparation of hyaluronic acid based on enzymatic digestions and precipitations by ammonium sulfate in the presence of pyridine is described. Complete isolation of hyaluronic acid from human umbilical cord in the form of the sodium salt, possessing a very high viscosity, and in a high state of purity is effected. The free acid was obtained by passage of the sodium salt through an acid ion ex- change column and its acid equivalent was found to be 395 (theoretical, 379). Starting from this acid, the sodium salt and the methyl hyalu- ronate were prepared. Acetylation experiments in pyridine solution yielded products containing only two to three acetyl groups per repeating unit. The formation of a derivative containing one trityl group seems to indicate that position 6 of the glucosamine moiety is not linked.

The viscosity measurements confirmed the view that hyaluronic acid consists of aggregates of filaments of high molecular weight. These measurements also showed that the aggregates rapidly depolymerized above 90” or when subjected to conditions aimed at esterifying the free carboxyl or hydroxyl groups.

BIBLIOGRAPHY

1. Meyer, K., and Palmer, J. W., J. Biol. Chem., 114, 689 (1936). 2. Robertson, W. van B., Ropes, M. W., and Bauer, W., J. Biol. Chem., 133, 261

(1940).

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R. W. JEANLOZ AND E. FORCHIELLI 611

3. Hadidian, Z., and Pirie, N. W., Biochem. J., 42, 260 (1948). 4. Markham, R., Biochem. J., 36, 790 (1942). 5. Niederl, J. B., and Niederl, V., Micromethods of quantitative organic analysis,

New York, 2nd edition, 160, 191 (1942). 6. Meyer, K., J. Biol. Chem., 176, 993 (1948). 7. Unruh, C. C., McGee, P. A., Fowler, W. F., Jr., and Kenyon, W. O., J. Am.

Chem. Sot., 69, 349 (1947). 8. Green, J. W., and Pigman, W. W., Abstracks, American Chemical Society, 113th

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10. Low, W., and White, E. V., J. Am. Chem. Sot., 65,243O (1943). Il. Madinaveitia, J., and Quibell, T. H. H., Biochem. J., 34, 625 (1940). 12. Blix, G., and Snellman, O., Ark. Kemi, Mineral. o. Geol., 19 A, No, 32 (1945). 13. Meyer, K., and Chaffee, E., J. Biol. Chem., 133,83 (1940). 14. Ogston, A. G., and Stanier, J. E., Biochem. J., 46,364 (1950). 15. Robertson, W. van B., Ropes, M. W., and Bauer, W., Biochem. J., 36,903 (1941).

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16. McLean, D., J. Path. and Bact., 64, 284 (1942). 17. Meyer, K., Smyth, E. M., and Dawson, M. H., J. Biol. Chem., 128,319 (1939). 18. Byers, S. O., Tytell, A. A., and Logan, M. A., Arch. Biochem., 22, 66 (1949). 19. Lundquist, F., Acta physiol. &and., 17, 44 (1949). 20. Meyer, K., and Palmer, J. W., J. Biol. Chem., 107, 629 (1934). 21. Meyer, K. H., and Bernfeld, P., Helv. chim. acta, 23,890 (1940).

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Roger W. Jeanloz and Enrico ForchielliUMBILICAL CORD

AND DERIVATIVES FROM HUMAN PREPARATION OF HYALURONIC ACID

RELATED SUBSTANCES: I. STUDIES ON HYALURONIC ACID AND

1950, 186:495-511.J. Biol. Chem. 

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