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Permeability of hyaluronic acid solutions.

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The Effects of Matrix Concentration, Calcium, and pH
The permeability of hyaluronic acid solutions
was measured in a chamber closed at each end by semipermeable membranes. permeability was strongly concentration dependent below 1.0 gm% hyaluronic acid.
Small changes in the ionic environment of 1.0 gm%
hyaluronic acid solutions also altered permeability. The
permeability of such solutions decreased by a factor of
4.7 in the presence of as little a s 5 mM calcium ion and
showed optimum permeability at pH 7.0. The pH optimum was not noted in hyaluronic acid preparations
known to contain only small amounts of protein.
Hyaluronic acid is found in connective tissue
throughout the body. In the early 1950s, Day (1,2) demonstrated the important role of hyaluronic acid in determining the rate of solvent flow through the extracellular
space of connective tissue. Subsequently, only the study
of Ogston and Sherman in 1961 (3) has provided some
insight into the mechanisms of bulk solvent flow
through a hyaluronate matrix. In the current study we
used a simple apparatus to directly measure the flow of
solvent through solutions of hyaluronic acid under vari-
From the Departments of Medicine and Bacteriology (Immunology), University of North Carolina, School of Medicine,
Chapel Hill, North Carolina.
Supported by Research Grant AM-I9992 from the National
Institutes of Health.
Marcus E. Carr, Jr., PhD, MD: Post-Doctoral Trainee; Nortin M. Hadler, MI): Associate Professor of Medicine and Bacteriologv
(Immunology) and recipient of an Established Investigatorship from
the American ileart Association.
Address reprint requests to Dr. Hadler, Dept. of Medicine,
938 Faculty Laboratory Office Building 231H, University of North
Carolina, Chapel Hill, NC 27514.
Submitted for publication January 28, 1980; accepted in revised form July 30, 1980.
Arthritis and Rheumatism, Vol. 23, No. 12 (December 1980)
ous conditions of pH, ionic strength, and calcium concentration.
The flow of a solution through a well defined
volume of sample material is governed by Darcy’s equation. This is an empirical relationship based on a classic
experiment of flow through filter beds (4).
D = Q v v:/Ftp
D is the permeability or Darcy constant of the medium.
All of the other parameters-viscosity of the permeating
solution (v), length of the sample column (q),cross sectional area (F) in the direction of flow (Q/t), and the
pressure applied ( p e a r e known or are easily measured.
Hyaluronic acid from human umbilical cord (H-175 I,
Grade 1) was purchased as a potassium salt from Sigma
Chemical Company (St. Louis, MO). It was dissolved by placing the hyaluronic acid in test tubes, layering the solvent on
top, and allowing the tube to stand in a water bath at 37°C for
24 hours. This was sufficient to allow all bubbles to rise to the
top. The tubes were then placed in a refrigerator at 2°C. Just
prior to use, the tubes were removed from the refrigerator and
brought to 35°C by heating in a water bath for 30 minutes.
In addition to the Sigma samples, two other umbilical
cord hyaluronic acid preparations were examined. Hyaluronic
acid from human umbilical cords (batch #7017) was obtained
from Miles Laboratories (Goodwood, South Africa). The
sample was received as a powder of a potassium salt, and was
stated to contain 1.4% protein and to have an intrinsic viscosity of 69 at 25°C. A third sample in the form of Sodium hyaluronate was purchased from the laboratory of Professor E. A.
Balazs. The preparation had a stated specific viscosity of 2700
centipoise and was reported to contain 0.9% protein.
Ionic strength of all solutions was set by adding NaCl
from a stock I.OM solution. All solutions contained 0. IOM sodium chloride unless otherwise indicated. Phosphate buffers
were prepared as 0.50M stock solutions and the pH was set by
the addition of 8.OM NaOH or concentrated H,PO,. The buffer was always diluted 1 to 10 in all hyaluronic acid solutions.
The pH of all hyaluronic acid solutions was 7.0 unless otherwise indicated. All chemicals were of reagent grade and all solutions were prepared in doubly distilled and deionized water.
Perfusion measurements were made by using the apparatus illustrated in Figure 1. The perfusing fluid was forced
through the hyaluronic acid matrix by a pressure head generated by a nitrogen gas tank. The gauges on the tank were used
as a first approximation of the applied pressure, but precise
pressure measurements were made with a mercury rnanometer. Centimeters of mercury were converted to dynes/crnz by
multiplying by the factor 1.33322 X 104. Flow was measured
by observing the movement of the meniscus in a pipette which
served as a flowmeter. The attachment of graph paper to the
shaft of the pipette allowed for h e r gradations of rneasurement. By switching between 1.0 and 0.1 ml pipettes, we were
able to measure both fast and slow flow. All experiments were
performed at ambient temperature. In all cases, the perfusing
fluid was identical in composition to the hyaluronic solution
being perfused minus the hyaluronic acid. This apparatus is
very similar to the one described previously (5).
The hyaluronic acid solution was contained within a
plexiglass chamber (Figure 2) closed at both ends by membranes. The chamber itself was threaded at both ends in order
to accept the female portion of a Millipore filter holder (cat.
#SXO001300). The Unipore membranes were purchased from
Biorad Laboratories and had a pore size of 1000 Angstroms.
The membranes were supported by grids which were cut from
the male end of a Millipore filter holder. Both grids and rnembranes were sealed into place by silicon rings. The inside diameter of the chamber was 0.476 crn and the length 1.0 cm.
The chambers were filled with hyaluronic acid solutions by
two techniques: for the more dilute solutions, pasteur pipettes
were used; for the more concentrated solutions, the gel-like
flow in
silicon ring
flow out
support grid
.silicon ring
perfusion chamber
silicon ring
Figure 2. Chamber used to contain hyaluronic acid solutions during
perfusion measurements.
material was spooned into the chamber. In all cases, loading
was accomplished by removing one end of the chamber.
The membranes themselves offer some resistance to
flow. Blank measurements were made prior to each hyaluronic acid solution measurement with the chamber filled with
water and at the same pressure as was used to measure flow
through the hyaluronic acid solutions. The inverse of the flow
value for the filters plus water was subtracted from the inverse
of the flow value for the filters plus hyaluronic acid.
Ultraviolet (UV) spectra of the three hyaluronic solutions were obtained by utilizing a Cary 118 spectrophotometer. The solutions were scanned from 300 to 260 nm in 1.0 cm
pathlength quartz cells. The hyaluronic acid samples were all
prepared in 0.1M KCl and were unbuffered. A solution of Nacetylglucosamine and glucuronic acid in O. 1M KCl was scanned as a control. A solution of 0.1M KCl served as a blank for
all scans.
Figure 1. Schematic diagram of the apparatus used to measure the
flow through hyaluronic acid solutions. 1, Nitrogen tank used to apply
pressure head. 2, Mercury manometer: a, mercury pot; b, vertical mercury column; e, meter stick; d, spill pot. 3, Three-way valve to control
pressure head. 4, Connection to allow loading of perfusion fluid. 5,
Chamber containing test sample. 6, Three-way stopcock to allow
switching between flowmeters. 7, e, 1.0 ml pipette for fast flow; f, 0.10
ml pipette for slow flow.
The ability of the described instrument to measure accurately the very low flows observed in concentrated hyaluronic acid is illustrated in Figure 3. A major
point of concern was whether the hyaluronic acid would
be forced through the solvent and onto the exiting
membrane. If this occurred, one would observe nonlinear flow; the flow would gradually slow with time.
The fact that the volume flow is linear with time therefore indicates that coating of the membrane is not occurring during the period of measurement. A second
method used to check for membrane coating was to reverse the direction of solvent flow after a given period of
flow in one direction. The results obtained with reversed
flow were essentially the same as the original results.
10” 2.0t-
[HA] in gm0lo
Figure 3. Volume flow of water through hyaluronic acid (Sigma preparation) solutions versus time in seconds. The concentrations of
hyaluronic acid are indicated at the right in gm%. All solutions contained 0.10M sodium chloride and were buffered at pH 7.0 with
0.05M sodium phosphate.
Permeability displays a large concentration
dependence below 1.5 gm% (Figure 4). The effect of
Ca++ ion concentration on the permeability of 1%
hyaluronic acid solutions (Sigma preparation) is depicted in Figure 5. Even solutions with concentrations
of Ca++as low as 5 mM are nearly five times less permeable than those with no added Ca++.The effect of pH
on the permeability of 1% hyaluronic acid solutions
(Sigma preparations) is depicted in Figure 6. At neutral
pH there is an increase in permeability which falls off at
either higher or lower pH.
The results of UV scans of the three hyaluronic
acid preparations are presented in Figure 7. As can be
seen, the Sigma hyaluronic acid Preparation has an absorbance at 280 nm that is 2.2 times that of the Miles
product. The control solution of nearly equimolar
amounts of N-acetylglucosamine and glucuronic acid
has a negligible absorbance between 300 and 260 nm.
Figure 4. The effect of the concentration of hyaluronic acid (HA) on
the permeability or Darcy constant (D) of the solution. These solutions are all buffered at pH 7.0 in 0.05 sodium phosphate, 0.10 sodium
chloride. If the sodium chloride is omitted from the 1.0% solution, D
measures 49.6 X lo-”. The study illustrated employed the Sigma
preparation of hyaluronic acid.
6.3 X
ml/second through a 1.0 gm% solution as
compared to 5.4 X
for a 0.84 gm solution reported
by Ogston (3).
The rather marked effects of small changes in the
ionic environment of the hyaluronic acid matrix on the
permeability of the solution have not been detailed previously, but they were not totally unexpected in view of
the reported effects of such changes on the physical
properties and conformation of hyaluronic acid. The
structure of hyaluronic acid as revealed by x-ray diffraction is a left hand helix. The aggregation of these
The permeability of a hyaluronate matrix to a
simple solvent at neutral pH reported here is in agreement with that reported previously. We found a flow of
[Ca++]in mM
Figure 5. The effect of calcium ions on the permeability of 1.0 gm%
hyaluronic acid solutions (Sigma preparation). All solutions were buffered at pH 7.0 with 0.05M phosphate.
chains in the putty-like matrices subjected to x-ray diffraction is dependent on pH and counter ions. Sodium
ions tend to stabilize the hyaluronic acid matrix by
forming intermolecular salt bridges (6,7). Under certain
conditions the chains are arranged anti-parallel to one
another (8,9) and can be further stabilized through calcium bridges (9).
The rheologic properties of solutions of hyaluronic acid are also sensitive to ionic strength and pH
(10). Light- scattering (11,12) and optical rotary dispersion (ORD) studies (13) have provided additional
evidence of effect of ionic strength and pH on the conformation of hyaluronate in solution. Analysis by I3C
nuclear magnetic resonance (NMR) has demonstrated
the importance of calcium in determining the order
present in a thick matrix of hyaluronic acid (14).
The permeability pH optimum of 7.0 (Figure 6)
cannot be explained by any known property of the hyaluronate itself in the matrix (15). A contaminant, most
likely a protein, probably serves as a modulator of matrix permeability. In 'recent experiments we have demonstrated that the Miles preparation, which appears to
have the least protein content (Figure 7), lacks the pH
optimum of the Sigma preparation. Future studies will
aim at the identification and isolation of the constituent
present in some hyaluronic acid preparations that imparts the pH sensitivity to matrix permeability. The current study illustrates the importance of comparing the
physical properties of preparations of matrix of varying
3 0 0 nm
Figure 7. Ultraviolet (UV) spectra of three different preparations of
hyaluronic acid. All solutions contained 0. IOM potassium chloride.
HAS = Sigma preparation (4.08 mg/ml); HAB = Balazs preparation
(4.58 mg/ml); HAM = Miles preparation (4.70 mg/ml); NAG + GLU
= N-acetyl glucosamine (5.0 mg/ml) + glucuronic acid (4.89 mg/ml).
All measurements were made at 22.1'C. The UV absorption at this
range of wavelength reflects contamination of the hyaluronic acid
preparation particularly by proteins.
degrees of purity. We may at times purify away the
most interesting properties of the matrix.
The sensitivity of hyaluronate matrix permeability to slight alterations in solvent suggests a
scheme by which a cell communicates with and thereby
alters its extracellular environment (16). The cell may
control its juxtaposed extracellular matrix by secreting
various ions that act on proteins which in turn modulate
changes in the matrix. Given this model, the matrix no
longer appears to be a space filling substance, but rather
takes on the role of a dynamic extension of the cell that
is directly under cellular control.
"5.5 6
Figure 6. The effect of pH on the permeability (D X lo-") of 1.0 gm%
hyaluronic acid solutions (Sigma preparation). All solutions contain
0.10Msodium chloride. The Miles preparation has no demonstrable
pH sensitivity of permeability.
1. Day TD: Connective tissue permeability and the mode of
action of hyaluronidase. Nature 166:785-786, 1950
2. Day TD: The permeability of interstitial connective tissue
and the nature of the interfibrillary substance. J Physiol
17:l-8, 1952
3. Ogston AG, Sherman TF: Effects of hyaluronic acid upon
diffusion of solutes and flow of solvent. J Physiol 156:6774, 1961
4. Darcy H: Les Fontaines Publiques de la Ville de Dijon.
Pans, 1856
5. Carr ME, Shen LL, Hermans J: Mass-length ratio of fi-
brin fibers from gel permeation and light scattering. Biopolymers 16:l-15, 1977
Guss JM, Hukins DWL, Smith PJC, Winter WT, Arnott
S, Moorhouse R, Rees DA: Hyaluronic acid molecular
conformation and interactions in two sodium salts. J Mol
Biol 95:359-384, 1975
Winter WT, Smith PJC, Arnott S: Hyaluronic acid: structure of a fully extended 3-fold helical sodium salt and
comparison with the less extended 4-fold helical forms. J
Mol Biol 99:219-235, 1975
Sheehan JK, Gardner KH, Atkins EDT: Hyaluronic acid:
a double helical structure in the presence of potassium at
low pH and found also with the cations ammonium, rubidium and caesium. J Mol Biol 117:113-135, 1977
Winter WT, Arnott S: Hyaluronic acid: the role of divalent cations in conformation and packing. J Mol Biol
117:761-784, 1977
Gibbs DA, Merrill EW, Smith KA, Balazs EA: Rheology
of hyaluronic acid. Biopolymers 6:777-79 1, 1968
11. Hallett FR, Gray AL: Quasi-elastic light scattering studies
of hyaluronic acid solutions. Biochim Biophys Acta
343:648-655, 1974
12. Mathews MB, Decker L: Conformation of hyaluronate in
neutral and alkaline solutions. Biochim Biophys Acta
498:259-263, 1977
13. Chakrabarti B: Effect of counterions on the conformation
of hyaluronic acid. Arch Biochem Biophys 180:146-150,
14. Napier MA, Hadler NM: Effect of calcium on structure
and function of a hyaluronic acid matrix: carbon-13 nuclear magnetic resonance analysis and the diffusional behavior of small solutes. Proc Natl Acad Sci USA 75:22612265, 1978
15. Hadler NM, Napier MA: Structure of hyaluronic acid in
synovial fluids and its influence on the movement of solutes. Semin Arthritis Rheum 7:141-152, 1977
16. Hadler NM: The biology of the extracellular space. Clinics Rheum Dis (in press)
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acid, hyaluronic, solutions, permeability
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