Hyaluronic acid (HA) is a high molecular weight non-sulfated
glycosaminoglycan composed of repeating disaccharide unit of
D-glucuronic acid (Glc UA) and A-acettyl-D-glucosamine (NAG). The HA is
recognized as a major participant in important biological processes as
cell motility, proliferation, differentiation and migration. The HA
degrading enzymes, the HAases are found in various human organs and body
fluids and in the external secretions of various other organisms. (2-5)
There are three groups of HAases according to their HA degradation
mechanisms namely, (1) hyaluronate 4glycanohydrolase
(hyaluronoglucosaminidase: EC 18.104.22.168), (2) hyaluronate
3-glycanohydrolase (hyaluronoglucuronidase: EC 22.214.171.124), and (3)
hyaluronate lyase (EC 126.96.36.199). (6) The HAases present in various
mammalian tissues belong to the first group of enzymes and are of
particular clinical interest as they have been demonstrated to be
involved in the pathophysiology of many human disorders. (2-11) Due to
their physiological importance, a rapid and sensitive method to measure
HAase activity has become increasingly necessary. A variety of assay
methods have been used to measure HAase activity, i.e., turbidometric,
viscometric, and colorimetric methods, and newer methods such as
spectrophotometric, fluorogenic, radiometric, agarose plate based,
ELISA-like, HPLC, zymography, capillary electrophoresis, FACE- based and
ECL-assisted assays. (2,12-14)
Despite several quantitative assay methods available, the
Morgan-Elson colorimetric method modified by Reissig et al (1) is
considered to be the best and widely used method to determine the HAase
activity in clinical samples including serum, plasma, saliva, urine and
other body fluids. In the Morgan-Elson reaction the NAG in the reducing
end is successfully transformed into a chromogen under alkaline
condition at 100[degrees] C and subsequently by the action of p-dimethyl
amino benzaldehyde (p-DMAB) in glacial acetic acid and hydrochloric acid
mixture (9:1; v/v) to give a reddish purple colored product that can be
detected at 585 nm. However, this method is limited by the interference
of variation in the pH of the sample; presence of other sugars, amino
acids and [Mg.sup.+2] and also the final color development often require
90 minutes. Reissig et al (1) improved this method by substituting a
concentrated PTB reagent (0.8 M, pH 9.1; 50 [micro]l/ assay volume of
300 [micro]l, to a final concentration of 0.13 M PTB in the reaction
mixture) for the original carbonate buffer. As a result of modification,
the final color development required just 20 minutes and about 2 folds
increase in the color yield. In spite of the significant improvement,
the estimation of HAase activity in clinical samples such as serum,
plasma and other body fluids was not satisfactory as there was a massive
interference by turbidity upon increasing the protein concentration
above 400 [micro]g in the reaction mixture. While, less than 400
[micro]g of protein in the reaction mixture did not cause any turbidity.
Hence, removal of turbidity in cases when excess of protein used was
only through centrifugation at a high centrifugal force of about 18,000x
g. Although, the adoption of the curvilinear interpolation by Asteriou
et al. (15) overcomes the turbidity interference, this approach was not
suitable especially for the assays of HAases performed at acidic pH,
since the resulting turbidity colored reaction mixture was often
unstably suspended. Hence, balancing the protein content of the assay in
order to get the measurable activity and at the same time preventing the
turbidity formation is a critical practical difficulty. Therefore, it is
highly unlikely to pick up the HAase activity can be picked up in cases
of lesser protein levels (< 400 [micro]g protein) or results in
turbidity in cases of higher protein levels (> 400 [micro]g protein)
in specimens turbidity would cause anomalous results. However, use of
larger concentrations of the clinical sample (protein content > 400
[micro]g) is quite essential to get the measurable activity. Therefore,
considering these limitations, we attempted to re-investigate the
Reissig et al. (1) method by using an optimal concentration of PTB
reagent (2 M, pH 9.1; 50 [micro]l/ assay volume of 300 [micro]l, to a
final concentration of 0.33 M PTB in the reaction mixture) to
drastically reduce turbidity.
MATERIALS AND METHODS
Healthy donors (20-25 yrs age group), osteoarthritic and diabetic
patients were recruited with informed consent according to the
Declaration of Helsinki and as per University of Mysore institutional
review board protocols to donate up to 5 ml of whole blood. Hyaluronic
acid (HA) and A-acetyl glucosamine were purchased from Sigma Chemicals
Co. St Louis, USA. All other chemicals used were of analytical grade.
Determination of hyaluronidase activity
Hyaluronidase activity was determined according to the native
method of Reissig et al (1955) and reinvestigated Reissig et al method.
Reissig et al method: Enzyme was incubated with HA (50 [micro]g) in
a final reaction volume of 300 [micro]l of 0.1 M sodium formate buffer
(pH 3.8) containing 300 mM NaCl and incubated for 150 min at 37[degrees]
C. The reaction was stopped by adding potassium tetraborate, (0.8 M, pH
9.1, 50 [micro]l/assay volume of 300 [micro]l to a final concentration
of 0.13M). The reaction mixture was kept in boiling water bath for
exactly 3 min. The coloring reagent p-dimethyl amino benzaldehyde
(pDMAB) in glacial acetic acid and hydrochloric acid (1.5 ml, 9:1, v/v)
was added to give a reddish purple colored product that can be detected
at 585 nm. The change in absorbance was monitored at 585 nm. Activity
was expressed as u moles of N-acetyl glucosamine released/min/mg
In reinvestigated method: To stop the reaction, 2 M potassium
tetraborate (pH 9.1; 50 [micro]l/ assay volume of 300 [micro]l, to a
final concentration of 0.33 M PTB in the reaction mixture) was used
instead of 0.8 M.
The primary endpoint (OD at 585 nm; NAG estimation), was measured
using both native and reinvestigated methods at different protein levels
0, 500, 1000, 1500 and 2000 [micro]g. Repeated-measures analyses for OD
was done with a means model with SAS Proc Mixed (version 9.2, mixed
linear models). The repeated-measures analyses were done separately for
the plasma and serum samples. A heterogeneous compound symmetry
variance-covariance form among the repeated measurements was assumed for
each outcome, and robust estimates of the standard errors of parameters
were used to do statistical tests and construct 95% confidence
intervals. T tests were used to compare the pairwise differences between
the model-based means (least squares means) at each protein level,
providing separates of the means by method (native or reinvestigated)
and protein level.
Native and re-investigated methods were used to evaluate the enzyme
activity of normal serum on the same 25 subjects. Each sample was
measured three times by each method. Similar studies were performed for
25 diabetic samples and osteoarthritis samples (data not shown).
Intra-sample coefficient of variation (CV) was used to assess the
repeatability of replications within each method. Inter-sample CV was
also calculated. Furthermore, the repeatability coefficient was
calculated to quantify the repeatability of each method from replicated
measurements obtained by the same method.
The repeatability coefficient (16) is defined as 1.96 [square root
of [2.sup.2]W] where [[sigma].sup.2.sub.w] is the within subject
variance for enzyme activity. Estimation of [[sigma].sup.2.sub.w] is
provided using the mean square error from an ANOVA model with 3
replicates from each sample (sample was the blocking factor for the
Additionally a plot of the mean difference (i.e., 1st minus 2nd
enzyme activity measurement) versus the mean of the two replicate enzyme
activity levels was included to graphically summarize the repeatability
of each method. The repeatability limits ([+ or -] 2.77 [S.sub.w]) were
added to the graph (as horizontal lines on the Y axis of Figure 2b). The
mean difference (i.e., mean bias) is not plotted on the graph since the
mean difference is statistically zero (as expected). Since the mean bias
for the other 2 plots (1st minus the 3rd measurement and 2nd minus the
3rd measurement) were very similar to the first plot these 2 plots were
not included in the manuscript.
RESULTS AND DISCUSSION
Figure 1a represents the estimation of standard NAG (Sigma-Aldrich,
St. Louis, USA) by native Reissig et al method (1) and re-investigated
Reissig et al method using 0.8 M and 2 M PTB reagent respectively. The
results show no difference in optical activity. The final colored
solution was clear with no signs of turbidity and no visible precipitate
was observed when centrifuged at 18000x g for 10 min in both the cases.
Thus, use of either 0.8 M or 2 M PTB reagent did not affect the
intensity of the chromogen generated.
[FIGURE 1a OMITTED]
Further, when standard NAG was estimated in presence of serum and
plasma samples of varied amounts of proteins (0 to 2000 [micro]g) using
native Reissig et al (1) method (0.8 M PTB), we observed the formation
of turbidity which required centrifugation prior to optical density
measurement. When comparing the reinvestigated and native methods using
serum protein, at protein 0 [micro]g, the estimated mean OD at 585 nm
using re-investigated method was 0.150 nm (95% CI: 0.147 to 0.153) and
at 2000 [micro]g protein this value was 0.138 nm (95% CI: 0.133 to
0.143). In contrast, the estimated mean OD at 585 nm using native method
at protein 0 [micro]g and 2000 [micro]g were 0.140 nm (95% CI: 0.137 to
0.144) and 0.043 nm (95% CI: 0.040 to 0.045) respectively. The mean OD
at 585 nm of serum of native method was significantly lower than that of
reinvestigated method (p<0.05) at all protein levels (Figure 1b).
Analyses of OD at 585 nm of plasma showed similar results (Figure 1c).
[FIGURE 1b OMITTED]
[FIGURE 1c OMITTED]
However, when similar estimation was done using 2 M PTB reagent
(re-investigated Reissig et al method); no visible turbidity formation
was seen. This modification was not only independent of centrifugation
but also did not reduce the color intensity (Fig. 1b, 1c). In addition
the estimation of NAG in the presence of total serum and plasma proteins
and in presence of other protein preparations such as bovine serum
albumin, casein and gelatin, the results obtained showed similar trend.
The protein content was determined by Biuret method (17) using bovine
serum albumin as standard.
In order to verify this finding, the normal serum HAase activity
was estimated. Figure 2a indicate the independent determination of HAase
activity in terms of the end product NAG estimation using 0.8 M (native)
and 2 M (re-investigated) PTB reagent. In presence of 0.8 M PTB, we
observed the formation of turbidity as well as reduction in the color
intensity. In addition, the color intensity was further reduced when we
attempted to obtain clearer supernatant by centrifugation. We observed a
drastic reduction in optical density. This poses a serious set back in
assessing the actual level of HAase activity in clinical samples. In
contrast, when we used 2 M PTB, the samples not only recorded an
increased optical density, the samples were clear with no traces of
turbidity and no prior centrifugation was required before measuring the
optical density. Thus, use of 2 M PTB instead of 0.8 M, ensures clearer
solutions even at higher protein levels of clinical samples with the
protein content as high as of about 2000 [micro]g, that is about 5 folds
more than that used (about 400 [micro]g) by the native Reissig et al
method. (1) However, further increase in the concentration of >2 M
PTB, did not have any influence on the turbidity nor the intensity of
the color formation (data not shown). All the serum samples used for the
study were procured by Government Ayurvedic Medical College, Mysore,
India with the prior consent from donors.
[FIGURE 2a OMITTED]
To access repeatability on the same sample, descriptive statistics
of intrasample CV (%) of normal serum was carried out. The mean
intrasample CVs for native and re-investigated methods were 0.9% and
0.5%, respectively. On the other hand, the intersample CV for the native
method was 1.7% while the number was 2.8% for the re-investigated
method. Repeatability coefficient of the native method was estimated as
1.96 [square root of 2 [S.sup.2]W] = 1.96 [square root of
2(0.000001139)] = 0.003 IU. In other words, 0.003 IU was the difference
that would be exceeded by only 5% of pairs of measurements made by the
native method on the same subject. Re-investigated method's
repeatability coefficient was estimated as 1.96 [square root of 2
[S.sup.2]W] = [square root of 2(0.000000627)] =0.002 IU. In other words,
0.002 IU was the difference that would be exceeded by only 5% of pairs
of measurements made by the native method on the same subject. Figure 2b
shows the repeatability limits of re-investigated ([+ or -] 0.003 IU)
and native methods ([+ or -] 0.002 IU) for normal serum. No 1st minus
2nd enzyme activity measurements exceeded the repeatability limits for
[FIGURE 2b OMITTED]
In conclusion, the estimation of HAase activity in clinical samples
appears to be tedious and results in anomaly. This is due to turbidity
interference when higher protein concentrations (>400 [micro]g) are
used. Also there is, the fact that underscores is the difficulty in
obtaining the measurable activity with lower protein concentrations in
samples. The re-investigated method we have tested and are proposing a
satisfactory and effective means of determining the HAase activity even
when high proteins are present in clinical samples. The reaction
mixtures were not turbid but clear, permitting a more accurate
estimation of the activity of the enzyme in clinical samples.
The authors thank Prof. Vasanth D Bhat, Regional Institute of
Education, Govt. of India, Mysore for his valuable suggestions during
the study. The authors are also thankful to Dr. Mangalgi and Dr. Aruna
Mangalgi, Department of Kayachikitsa, Govt. Ayurvedic Medical College,
Mysore for providing serum samples. Dr. S. Nagaraju thanks the Council
of Scientific and Industrial Research (CSIR), New Delhi, India for
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Shivaiah Nagaraju, MSc., PhD, Department of Medicine, Division of
Immunology and Rheumatology,
Stanford University, Stanford, CA, USA, Department of PG Studies
and Research in Biochemistry, Tumkur University, Tumkur
K Subbaiah Girish, MSc., PhD, Department of Biochemistry,
University of Mysore, Manasagangothri, Mysore, Karnataka State, India
Yi Pan, MS, PhD Departmentof Biostatistics and Bioinformatics,
Rollins School of Public Health, Emory University, Atlanta, GA
Kirk A Easely, MS, Department of Biostatistics and Bioinformatics,
Rollins School of Public Health, Emory University, Atlanta, GA
Kempaiah Kemparaju, MSc., PhD, Department of Biochemistry,
University of Mysore, Manasagangothri, Mysore, Karnataka State, India
Address for Correspondence: Dr. K. Kemparaju, Department of
Biochemistry, University of Mysore, Manasagangothri, Mysore-570 006,
India, +994599 6543, firstname.lastname@example.org