Name: measurement of chitinase activity in biological samples
Uploaded: 2019-08-22T15:00:00
Description: Watch this Scientific Journal Video about Measurement of Chitinase Activity in Biological Samples at JoVE.com

This research was supported by American Lung Association and American Thoracic Society awards to LS. Authors sincerely want to thank Dr. Jack Elias and Dr. Chun Geun Lee for providing transgenic mouse strains.

All animal procedures were performed under an IACUC-approved protocol at Yale University School of Medicine.
1. Mouse Sample Collection
<ol>
	<li>Anesthetization
	<ol>
		<li>Anesthetize mice using ketamine and xylazine (100 mg/kg of ketamine and 10 mg/kg of xylazine).</li>
	</ol>
	</li>
	<li>Blood sample collection
	<ol>
		<li>Confirm the depth of anesthesia by non-responsiveness to a toe pinch.</li>
		<li>Surgically open the chest cavity to expose the heart...

<ol>
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A., Ahmad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitinases:+An+update.">Chitinases: An update.</a> Journal of Pharmacy and Bioallied Sciences. 5 (1), 21-29 (2013).</li><li>Sharma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+Role+of+Chitotriosidase+during+Lung+Infection+with+Klebsiella+pneumoniae.">Regulation and Role of Chitotriosidase during Lung Infection with Klebsiella pneumoniae.</a> Journal of Immunology. 201 (2), 615-626 (2018).</li><li>Kanneganti, M., Kamba, A., Mizoguchi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+chitotriosidase+(chitinase+1)+under+normal+and+disease+conditions.">Role of chitotriosidase (chitinase 1) under normal and disease conditions.</a> Journal of Epithelial Biology &#38; Pharmacology. 5, 1-9 (2012).</li><li>Lee, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Chitin+and+Chitinase/Chitinase-Like+Proteins+in+Inflammation,+Tissue+Remodeling,+and+Injury.">Role of Chitin and Chitinase/Chitinase-Like Proteins in Inflammation, Tissue Remodeling, and Injury.</a> Annual Review of Physiology. (73), 479-501 (2011).</li><li>Jeuniaux, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitinase:+An+Addition+to+the+List+of+Hydrolases+in+the+Digestive+Tract+of+Vertebrates.">Chitinase: An Addition to the List of Hydrolases in the Digestive Tract of Vertebrates.</a> Nature. 192 (4798), 135-136 (1961).</li><li>Boot, R. 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J., Morroll, S., Tighe, P., G&#246;tz, F., Falcone, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chitotriosidase+is+expressed+in+the+eye+and+lacrimal+gland+and+has+an+antimicrobial+spectrum+different+from+lysozyme.">Human chitotriosidase is expressed in the eye and lacrimal gland and has an antimicrobial spectrum different from lysozyme.</a> Microbes and Infection. 10 (1), 69-78 (2008).</li><li>Kim, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMCase+is+a+crucial+regulator+of+type+2+immune+responses+to+inhaled+house+dust+mites.">AMCase is a crucial regulator of type 2 immune responses to inhaled house dust mites.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (22), e2891-e2899 (2015).</li><li>Olkhovych, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitotriosidase+activity+as+additional+biomarker+in+the+diagnosis+of+lysosomal+storage+diseases.">Chitotriosidase activity as additional biomarker in the diagnosis of lysosomal storage diseases.</a> Ukrainian Biochemical Journal. 88 (1), 69-78 (2016).</li><li>Bouayadi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease:+An+Underdiagnosed+Pathology+in+the+Eastern+Moroccan+Population.">Disease: An Underdiagnosed Pathology in the Eastern Moroccan Population.</a> eJIFCC. 30 (1), 82-87 (2019).</li><li>Tasci, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+serum+chitotriosidase+activity+in+early+treatment+of+patients+with+active+tuberculosis+and+a+negative+sputum+smear.">Efficacy of serum chitotriosidase activity in early treatment of patients with active tuberculosis and a negative sputum smear.</a> Therapeutics and Clinical Risk Management. 8, 369-372 (2012).</li><li>Barone, R., Simpor&#233;, J., Malaguarnera, L., Pignatelli, S., Musumeci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+chitotriosidase+activity+in+acute+Plasmodium+falciparum+malaria.">Plasma chitotriosidase activity in acute Plasmodium falciparum malaria.</a> Clinica Chimica Acta. 331 (1-2), 79-85 (2003).</li><li>Kitamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitinase+inhibition+promotes+atherosclerosis+in+hyperlipidemic+mice.">Chitinase inhibition promotes atherosclerosis in hyperlipidemic mice.</a> American Journal of Pathology. 183 (1), 313-325 (2013).</li><li>Kzhyshkowska, J., Gratchev, A., Goerdt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Chitinases+and+Chitinase-Like+Proteins+as+Indicators+for+Infl+Ammation+and+Cancer.">Human Chitinases and Chitinase-Like Proteins as Indicators for Infl Ammation and Cancer.</a> Biomark Insights. 2, 128-146 (2007).</li></ol>

Chitinase activity has emerged as an important biomarker for predicting disease severity, disease progression, therapeutic effectiveness and the presence of specific pathogens18. Although many of the long-postulated theories about the role of chitinases have not been experimentally proven19, new studies have provided important insights into the role of chitinases and chitinase like proteins in various diseases2,9<su...

Chitin is the second most abundant polysaccharide on the earth after cellulose, serving as a major structural component of a variety of organisms including the exoskeleton of insects, fungi, yeast, and algae; some vertebrates also have chitin1. Chitinases are a family of enzymes that are capable of breaking down chitin and are highly conserved throughout evolution in species ranging from bacteria to mammals2,3. In addition to chitinases, mammals also have chitinase-like proteins which are similar to chitinases in their ability to bind chitin but differ in that they lack enzymatic ability to cleave the chitin4.
Although chitin and chitinases have long been studied&#8212;with investigations dating back to the early 1900&#8217;s&#8212;the main focus has been on their role in insects and other invertebrates. In fact, it was not until the 1960s when vertebrates were found to have chitinases at all. Using a chitobiase-based assay, chitinases were shown to be found in the digestive tract of a number of vertebrates including lizards and blackbirds&#8212;an observation hypothesized to be due to the consumption of chitin-containing organisms such as insects5.
Mammals have two enzymatically active forms of chitinase: acidic mammalian chitinase (AMCase; also known as CHIT2 and CHIA) and chitotriosidase (CHIT1)4. Both of these proteins are capable of hydrolyzing chitin. However, CHIT1 is more enzymatically active in humans, where almost all of chitinase activity is derived from CHIT1.4 In mice, both Chit1 and AMCase almost equally contribute to the overall chitinase activity6. On the other hand, chitinase-like proteins lack chitinase activity.
Chit1 is mainly secreted by macrophages and is often considered to be an immune response to chitin-containing pathogens7. The enzyme has also been shown to be involved in the maturation of monocytes into both M1 and M2 macrophage subtypes, even without the presence of the substrate chitin8. Furthermore, it may also be involved in the maturation of other immune cells including T helper type 2 (Th2) cells and eosinophils&#8212;as was shown to be the case in cryptococcal lung infection9. These studies point to a complex role of chitinases in the immune system.
In recent years, Chit1 levels have been found to serve as an important biomarker of progression for over 40 different human diseases including lysosomal storage diseases, infectious diseases, respiratory diseases, endocrinological diseases, cardiovascular diseases, neurological diseases, and others (reviewed10). In many of these diseases, the levels of Chit1 are a strong predictor of disease severity and therapeutic effectiveness10.
As chitinases have gained a reputation as a biomarker for numerous medical conditions including Gaucher Disease, tools and assays have been developed to facilitate testing for chitinase presence. Older methods include Schales&#8217; procedure, a protocol adapted from a blood glucose test, and the 3,5 dinitrosalicylic acid (DNS) method. However, these methods are often time sensitive and technically difficult11. The procedure for both of these tests requires the reduction of inorganic oxidants, ferricyanide in the case of the Schales&#8217; procedure for example, producing a color change that can only be measured spectrophotometrically. Additionally, both tests involve a heating or boiling step that is both time consuming and necessary for the color to develop12,13.
Described here is a quick and simple fluorimetric assay to determine chitinase levels in mammalian samples14,15. Two of the samples used here include serum and bronchoalveolar lavage fluids (BAL); chitinase activity has also been measured in breast milk and urine samples, and the technique can be performed in those type of samples as well as in any other biological fluid16,17.

<table>
	<tbody>
		<tr>
			<td>4-methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td>Standard: commonly used in flourimetric assays for determination of enzyme activity</td>
		</tr>
		<tr>
			<td>4MU-GlcNAc2</td>
			<td>Sigma</td>
			<td>M9763</td>
			<td>fluorescent chitinase substrate for use in mouse samples</td>
		</tr>
		<tr>
			<td>4MU-GlcNAc3</td>
			<td>Sigma</td>
			<td>M5639</td>
			<td>fluorescent chitinase substrate for use in human samples</td>
		</tr>
		<tr>
			<td>Citric Acid-monohydrate</td>
			<td></td>
			<td></td>
			<td>for use in McIlvain Buffer</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td></td>
			<td></td>
			<td>for use in Stop Buffer at a concentration of 0.3 M</td>
		</tr>
		<tr>
			<td>Na2HPO4xH2O</td>
			<td></td>
			<td></td>
			<td>for use in McIlvain Buffer</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td></td>
			<td></td>
			<td>for use in Stop Buffer at a concentration of 12 g/L</td>
		</tr>
		<tr>
			<td>Vision Plate: Non-sterile, untreated black 96 well plate</td>
			<td>4titude</td>
			<td>4ti-0224</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of chitinase activity in biological samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

The results shown here are from a study measuring the chitinase activity in serum and BAL samples of wild type (C57BL/6) and Chit1 transgenic mice (on C57BL/6 background) that overexpress the Chit1 gene on a doxycycline promoter. Our data show that both serum and BAL samples have detectable chitinase activity at baseline 698.2 &#177; 189.9 nmol/mL&#183;h and 485.7 &#177; 114 nmol/ml&#183;h, respectively. Overexpression of the Chit1 gene using doxycycline in drinking water for 4 weeks resulted in the elevation of chitinas...

Watch this Scientific Journal Video about Measurement of Chitinase Activity in Biological Samples at JoVE.com

Measurement of Chitinase Activity in Biological Samples

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

This method allows a more effective way to quantify chitinase activity in biological samples. Chitinase activity has been shown to serve as a good marker for diagnosis and therapeutic efficacy in numerous conditions and diseases. Earlier methods for the measurement of chitinase activity were often time consuming and difficult to perform, both problems that have been solved by the assay shown here.This technique will help fulfill the potential chitinases have as a diagnostic and therapeutic marker by making it easier to measure chitinase activity;for example, it can be used to monitor the treatment of Gaucher disease because chitinase activity has been shown to decrease during effective treatment. Begin by preparing substrates, standards, and solutions for the assay. Prepare 1x McIlvain buffer according to the manuscript directions and use it to dilute chitin substrates to 500 micromolar each.Dissolve 4-methylumbelliferone, the standard, to a concentration of 0.1 millimolar in stop buffer to create a standard curve stock solution. For every 10 samples, mix 2.17 milliliters of 1x McIlvain buffer with 100 microliters of the chitin substrate to create a working solution, then pipette 95 microliters of the working solution into each well of a 96-well plate. Add five microliters of the test sample to each well and mix it into the working solution.Cover the plate and shake it for five seconds, then incubate the plate at 37 degrees Celsius for 15 minutes to allow the enzymatic reaction to take place. Meanwhile, dilute the stock solution of 4-methylumbelliferone to make a five-micromolar working standard solution and create a standard curve by preparing a series of dilutions according to the manuscript. Start with the five-micromolar concentration and mix each dilution well.After the incubation, add 200 microliters of stop buffer to each well to stop the reaction. Add 300-microliter duplicates of each standard to the same plate, then read the plate at an excitation of 360 nanometers and an emission of 455 nanometers. This protocol was successfully used to measure chitinase activity in bronchoalveolar lavage and serum fluid of wild type and chitotriosidase over-expressing transgenic mice.The assay confirms that overexpression of the CHIT-1 gene in mice results in increased chitinase activity. A standard curve was used to determine chitinase based on fluorescence of the samples. The most important thing to remember when first attempting the protocol is to add the samples as quickly and efficiently as possible while still mixing thoroughly.When you first start out, it may be beneficial to do fewer samples per run to ensure accuracy in measurements, due to a shorter incubation period.

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