Name: cell-free biochemical fluorometric enzymatic assay for high-throughput measurement of lipid peroxidation in high density lipoprotein
Uploaded: 2017-10-12T13:00:00
Description: Watch this Scientific Journal Video about Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein at JoVE.com

The authors gratefully acknowledge the work of Dr Mohamad Navab, Alan Fogelman and Srinivasa Reddy for their key role in development of earlier iterations of this model. T.A.A. is supported by an RMIT University Vice-Chancellor&#39;s Postdoctoral Fellowship. AJ and AH are supported by NHMRC project grant 1108792. TK is supported by NIH grants NIH K08AI08272, NIH/NCATS Grant # &#181;L1TR000124.

All experiments using human biological samples were performed with ethics approval from the University of California Los Angeles, Los Angeles and the Alfred Hospital Human Ethics committee, Melbourne.
NOTE: There are many variations of the fluorochrome HDL function Assay (see discussion)32. Below we will describe the protocol that gives the most consistent and reproducible results. An overview of the assay is shown in Figure 2.
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The protocol described here offers a robust tool to answer important research questions regarding the role of HDL function in atherosclerosis and human disease. The assay quantifies the HDL lipid peroxide content per mg of HDL-C using enzymatic amplification (HRP). This approach avoids known limitations of prior HDL function assays (e.g. the cholesterol efflux assay) including significant heterogeneity with regards to types of cells used, type of readout reported, lack of standardization and confounding effects of trigly...

Atherosclerotic cardiovascular disease (CVD) is the leading cause of death worldwide1,2. Epidemiological studies have shown that low levels of high-density lipoprotein (HDL) cholesterol are generally inversely associated with the risk for the development of atherosclerosis1,2. Although several studies support an atheroprotective role for HDL1,2, the mechanism by which HDL attenuates the initiation and progression of atherosclerosis is complex 3,4. Thus, it has been suggested that the complex structure and function of HDL rather than absolute level may more accurately predict atherosclerosis 5,6,7,8. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. These i) reduce its cholesterol efflux potential 9, ii) decrease anti-inflammatory and increase HDL-associated pro-inflammatory proteins 6,7, iii) decrease antioxidant factor levels and activity and HDLs ability to inhibit oxidation of Low Density Lipoprotein (LDLox)10 and iv) increase lipid hydroperoxide content and redox activity (HDLox)9,11. Robust assays that evaluate the pleotropic functions of HDL (such as cholesterol efflux, antioxidant function) may complement determination of HDL-HDL-C in the clinic.
HDL function is usually assessed by cell-based methods such as the cholesterol efflux assay8,12,13,14. These methods have major limitations including significant heterogeneity with regards to types of cells used, type of readout reported, lack of standardization and confounding effects of triglycerides 7,15. These drawbacks pose difficulties for large clinical studies16. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. The cholesterol efflux is one of the most important functions of HDL but it can only be determined by cell-based assays. Other approaches to determine HDL function such as proteomics17,18,19,20,21,22,23,24 and cell-based monocyte chemotaxis assays of HDL function 17,22,25 have not been standardized and cannot be used in large scale human studies.
HDL has significant antioxidant atheroprotective effect5,6,7,8. The antioxidant function of HDL has been determined in the presence of LDL in previous cell free fluorometric assays 26. These biochemical fluorometric methods of HDL antioxidant function were originally developed by Mohamad Navab and Alan Fogelman and their colleagues26. Although many human studies have used these methods to determine HDL function 17,18,19,20,21,22,23,24, lipid (HDL)-lipid (LDL) and lipid-fluorochrome interactions may limit reproducibility of these cell free non-enzymatic biochemical assays of HDL function27,28.
Recent interest has focused on the functional consequences of HDL oxidation that is the result of oxidation of both lipids and proteins within HDL 27,29,30. Prior studies have shown that oxidation of HDL impairs HDL function 27,29,30. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Thus HDL lipid peroxide content can be used to determine HDL function 9,17,20,31 and given the known limitations of prior assays of HDL function7,15,27,32, we developed an alternative fluorometric method that quantifies HDL lipid peroxide content (HDLox) 32. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C 32. The biochemical principle of the assay is shown in Figure 1. We have shown that this fluorescence-based approach does not have the limitations of prior HDL function assays27,28. This assay has been further refined and standardized in our laboratory so that it can reliably be used in large scale human studies even with cryopreserved plasma 32,33,34,35,36,37,38,39,40,41,42. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction and associated cardiovascular and metabolic risk phenotypes32,33,34,35,36,37,38,39. Here, we describe this simple, yet robust method to measure HDL lipid peroxide content (HDLox). This assay can be used as a tool to answer important research questions regarding the role of HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis)32.

<table>
	<tbody>
		<tr>
			<td>Experimental Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDL PEG (Polyethylene Glycol) Precipitating Reagent</td>
			<td>Pointe Scientific</td>
			<td>H7511</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red reagent.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>DMSO.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>Horse Radish Peroxidase (HRP)</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>Cholesterol Esterase.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>&#160;Cholesterol Reference standard</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>&#160;Resorufin fluorescense Reference standard</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>5x Reaction Buffer.</td>
			<td>Life Technologies, Grand Island, NY</td>
			<td>A12216</td>
			<td>Amplex Red Cholesterol Assay Kit. 
			&#8226; &#8804;&#8211;20&#176;C &#8226; Desiccate 
			&#8226; Protect from light</td>
		</tr>
		<tr>
			<td>HDL Cholesterol Automated Reagent</td>
			<td>ThermoFisher Scientific Co., San Jose, CA, USA.</td>
			<td>TR39601</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasticware&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates (polypropylene, flat bottom, clear).</td>
			<td>Sigma Aldrich</td>
			<td>M0687</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates (polypropylene, flat bottom, black).</td>
			<td>Sigma Aldrich</td>
			<td>M9936</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>ClipTip 200, sterile</td>
			<td>ThermoFisher Scientific Co., San Jose, CA, USA.</td>
			<td>14-488-058</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Multichannel Pipettes, 8-channel, 125&#160;</td>
			<td>ThermoFisher Scientific Co., San Jose, CA, USA.</td>
			<td>&#160;14-387--955</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 2.01 software</td>
			<td>Biotek, Vermont, USA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 Plate reader</td>
			<td>Biotek, Vermont, USA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free biochemical fluorometric enzymatic assay for high-throughput measurement of lipid peroxidation in high density lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

50 &#181;L of each HDL sample are added into each well as in step 7.3. 50 &#181;L of HRP solution 5 U/mL (0.25 U) are then added into each well as in step 7.5. Samples are incubated for 30 min at 37 &#176;C as in step 7.6. 50 &#181;L of fluorochrome reagent are then added into each well as in step 7.7 (final concentration of 300 &#181;M). The fluorescent readout (in dark) is then assessed every minute over 120 minutes at 37 &#176;C with a fluorescent plate reader (530/590 nm filters). Rep...

Watch this Scientific Journal Video about Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein at JoVE.com

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

The overall goal of this experimental method is to determine the function of high density lipoprotein using a biochemical cell-free assay that measures the lipid peroxide content of a specific amount of HDL. This method can help answer key questions in several fields of medicine, such as the role of HDL function in cardiovascular disease metabolic abnormalities. The main advantage of this technique is that it is a cell-free biochemical method that can be used to reliably determine HDL function.Though this method can provide insight into the role of HDL function in cardiovascular disease and metabolic abnormalities, it can also be applied to other studies of disease, such as inflammatory diseases. To begin this procedure, prepare the working layout for the 96-well plate, including the appropriate controls, samples and replicates, as outlined in the text protocol. Calculate the volume of reagents needed based on the number of samples and replicates.Using a permanent marker, label tubes for each step in the assay. Next, combine 10 microliters from each sample to create a pooled control sample. Create a second pooled control sample for the clinical laboratory, in order to determine the HDL-C value.After preparing a large stock of HDL, cryo-preserve several aliquots to minimize freeze thaw cycles. Pool at least 10 different replicates to create a pooled quality control. Then, determine the average value of HDLox as outlined in the text protocol.Create a second pooled quality control for the clinical laboratory, in order to determine the HDL-C value. To begin, thaw a fresh plasma or serum sample. Mix together equal volumes of plasma and HDL cholesterol precipitating reagent.Pipette up and down several times to mix well. Centrifuge at a speed between 1000 and 2000 times G for 10 minutes. After this, aspirate the supernatant.Store the isolated HDL at four degrees Celsius until ready to perform the fluorochrome HDL function assay. If the clinical laboratory's HDL-C value is used to normalize HDLox, quantify the HDL-C from plasma as outlined in the text protocol. To begin, mix cholesterol and HRP, such that the prepared solutions contain five units per milliliter of HRP.Thaw a vial of fluorochrome reagent and a vial of DMSO to room temperature. After they are thawed, dissolve one milligram of fluorochrome reagent in 200 microliters of DMSO. Store this solution at a temperature below minus 20 degrees Celsius, while protected from light.Next, add 50 microliters of 1X reaction buffer to the sample plate as a negative control. Add 50 microliters of 20 millimolar hydrogen peroxide working solution as a positive control. After this, set out a fresh round bottom 96-well plate to be used as the preparation plate.Add 200 microliters of isolated HDL to each well of this preparation plate. Using a multichannel pipette, transfer 50 microliters of HDL from the preparation plate to the experimental plates, as outlined in the text protocol, with the number of experimental plates being equal to the number of replicates in the assay. After this, add 50 microliters of prepared HRP solution to each well.Incubate at 37 degrees Celsius for 30 minutes, then add 50 microliters of fluorochrome reagent to each well, for a total volume of 150 microliters per well. Mix well and protect from light. Using a fluorescent plate reader, assess the fluorescent readout at 37 degrees Celsius, in the dark, every minute for 120 minutes.Record the data using appropriate software, then calculate the mean value of fluorescence units as outlined in the text protocol. In this study, a cell-free biochemical fluorometric assay is developed to quantify the HDL lipid peroxide content per milligram of HDL-C. This is accomplished by enzymatic amplification using HRP.A fluorescent plate reader is then used to measure the fluorescence in the dark, every minute, for 120 minutes. Representative data for a patient known to have dysfunctional HDL is shown here, compared to the pooled control. The dysfunctional HDL is seen to have lipid peroxide levels two-fold higher than those seen in the pooled HDL from healthy participants.Next, the HDL is isolated and HDLox is determined in 50 healthy subjects and 100 HIV infected patients. The HIV infected patients are seen to have, on average, 60%higher HDL peroxide content per milligram of HDL-C than the healthy subjects. Once mastered, this technique can be done in under six hours if it is performed properly.While attempting this procedure, it's important to remember to prepare all reagents and controls upfront, be consistent, precise and always include the appropriate experimental control in each experimental plate. After watching this video, you should have a good understanding of how to isolate the HDL, determine the lipid peroxide content of a specific amount of HDL and to depredate our regarding HDL function. Don't forget that working with biospecimens can be extremely hazardous and precautions, such as gloves, should always be taken while performing this procedure.

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