Estimation of Serum Hyaluronidase Activity Overcoming the Turbidity Interference

Shivaiah Nagaraju; K Subbaiah Girish; Yi Pan; Kirk A Easely; Kempaiah Kemparaju

doi:10.29074/ascls.24.3.172

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Shivaiah Nagaraju, MSc., PhD
1. 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
1. Department of Biochemistry, University of Mysore, Manasagangothri, Mysore, Karnataka State, India
Yi Pan, MS, PhD
1. Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, GA
Kirk A Easely, MS
1. Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, GA
Kempaiah Kemparaju, MSc., PhD⇑
1. 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, nagarajubiochem{at}gmail.com

Abstract

The assay of mammalian hyaluronidases (HAases) is important in understanding the role of the hyaluronan-hyaluronidase (HA-HAase) system in various pathophysiological processes. Despite several quantitative assay method options, the Morgan-Elson colorimetric method modified by Reissig et al [1] is considered the best for determining the activity in clinical samples. However, the sensitivity of the method was greatly limited by presence of protein above 400 μg due to turbidity interference that led to chromogen quenching. Therefore, an effort has been made to reinvestigate the Reissig et al method. In the re-investigated method, a standardized optimal 0.32 M potassium tetraborate (PTB) was used against 0.13 M (native) to overcome the turbidity interference. The estimated mean OD at 585 nm of serum for native method was 0.043 (95% CI: 0.040 to 0.045), while that for the re-investigated method was 0.138 (95% CI: 0.133 to 0.143, p<0.0001). The mean OD at 585 nm of serum of native method was significantly lower than that of re-investigated method (p<0.05) at all protein levels. This was also true for estimated mean OD at 585 nm of plasma. The mean intrasample CVs for native and re-investigated methods were 0.9% and 0.5%, respectively, for normal serum. Furthermore, the repeatability coefficient of normal serum for native was 0.003 IU, while re-investigated method experienced that of 0.002 IU.