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 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).
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.
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.
1. Specimen Processing
2. Day 0-Preparation for the assay
3. Day 1-Preparation of controls
4. Day 1-Separation of HDL Cholesterol using HDL precipitation
NOTE: Use a commercially available standardized HDL Cholesterol precipitating reagent to isolate apoB depleted serum according to the manufacturer's instructions. These reagents are widely used in colorimetric assays to determine HDL cholesterol levels.
5. Day 1-Determination of HDL-C in isolated HDL
NOTE: This is optional if the value of HDL-C from the clinical laboratory is used to normalize HDLox by HDL-C amount.
6. Day 2-Preparation of reagents
7. Day 2-Fluorochrome Assay
8. Day 3-Data analysis
50 µL of each HDL sample are added into each well as in step 7.3. 50 µL 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 °C as in step 7.6. 50 µL of fluorochrome reagent are then added into each well as in step 7.7 (final concentration of 300 µM). The fluorescent readout (in dark) is then assessed every minute over 120 minutes at 37 °C with a fluorescent plate reader (530/590 nm filters). Representative fluorescence data for blank, pooled control, sample with known dysfunctional HDL and sample with normal HDL are shown in Figure 4. Raw representative data and step by step analysis of results using the equations described in section 8 of the protocol are shown in Table 1. In this example, fresh HDL samples were used and higher HDLox values are generally obtained with fresh HDL. For example, HDL known to be dysfunctional based on independent assays of HDL function and from HIV-infected person had approximately 2-fold relatively higher amount of lipid peroxide compared to pooled HDL from healthy participants. Figure 5 shows representative results from a study with subjects known to have impaired HDL function28,32. The HIV-infected persons had approximately 60% higher mean HDL-lipid peroxide content (per mg of HDL-C) compared to the uninfected persons. In our prior published studies with cryopreserved HDL, this method was able to demonstrate at least 10% relative differences in HDLox compared to HDL from control groups (without disease e.g. chronic HIV infection)36,37,38.
Figure 1: Determination of HDL lipid peroxide content per specific amount of HDL-C.
In states of systemic inflammation and oxidative stress HDL has increased lipid peroxides (LOOH) (HDLox) that are associated with impaired HDL function. HRP catalyzes the oxidation of non-fluorescent fluorochrome to fluorescent resorufin red. This oxidation can be driven by both endogenous peroxides present in the reaction (OH-) and HDL-lipid peroxides (LOOH-). Without cholesterol oxidase, resorufin (with HRP) can quantify the intrinsic HDL lipid peroxide content of a specific amount of HDL cholesterol. The background production of OH- as a result of air oxidation of the buffer is subtracted from the fluorescent readout of each well. Resorufin has minimal autofluorescence in most samples.This figure has been modified 32. Please click here to view a larger version of this figure.
Figure 2: Overview of the workflow for the fluorochromeassay of HDL lipid peroxidation.
Day 0: Preparation for the assay: A) design 96 well plate layouts, estimate volume of needed samples (plasma/serum) and reagents (HRP enzyme, fluorochrome Reagent, buffers, PEG reagent), label tubes
Day 1: Preparation for the assay and HDL isolation: A) preparation of controls (study specific-pooled control, quality controls, positive and negative controls) B) HDL precipitation using PEG reagent.
Day 2: Preparation for the assay (if not done in Day 1) and fluorochrome assay: A) preparation of controls ( study specific-pooled control, quality controls, positive and negative controls) A) addition of HDL samples: To minimize experimental variability and ensure that addition of reagents and samples will be done consistently and in a timely manner it is recommended that all additions of samples (e.g. 160 µL) are first done in a separate 96 well round-bottom, clear, polystyrene or polypropylene plate (Plate 1). Then a multichannel pipette can be used to transfer specific volumes (e.g. 50 µL) into 3 96-well plates (Plates 2-4: polypropylene, flat bottom, black plates) (that have identical layout). B) addition of HRP: Add 50 µL of 5 U/mL (0.25 U) to each well using a multichannel pipette C) Incubate at 37 °C for 30 min D) Add 50 µL of 300 µM /well of fluorochrome Reagent to each well using a multichannel pipette E) Assess the fluorescent readout (in dark) every minute over 120 minutes at 37 °C with a fluorescent plate reader (530/590 nm filters).
Day 3: Data analysis. Please click here to view a larger version of this figure.
Figure 3: Representative layout of 96 well plates that are typically used in the fluorochrome HDL function Assay.
Differences in timing of addition of HDL samples into wells of a plate can lead to differences in spontaneous oxidation between different wells. To minimize experimental variability, avoid variable spontaneous oxidation between different wells and ensure that addition of reagents and samples will be done consistently and in a timely manner, it is recommended that all additions of samples (e.g. 160 µL) are first done in a separate 96 well round-bottom, clear, polystyrene or polypropylene plate (Plate 1). Then a multichannel pipette can be used to transfer specific volumes (e.g. 50 µL) into 3 96-well plates (Plates 2-4: polypropylene, flat bottom, black plates) (that have identical layout). Please click here to view a larger version of this figure.
Figure 4: Representative data of HDL lipid peroxidation assay.
The fluorescent readout (in the dark) is then assessed every minute over 120 minutes at 37 °C with a fluorescent plate reader (530/590 nm filters). Representative data (arbitrary units) are shown for blank (no HDL; negative controls), positive control (H2O2), pooled HDL control and HDL from patient known to have dysfunctional HDL (based on two independent HDL function assays-cholesterol efflux and monocyte chemotaxis assay). Please click here to view a larger version of this figure.
Figure 5: The lipid peroxide assay of HDL function can detect high lipid peroxide content per specific amount of HDL-C in vivo.
HDL was isolated and HDLox was determined as described in the Protocol in 50 healthy subjects and 100 patients with HIV infection. The HIV-infected persons had higher HDLox (1.59±0.53) compared to the controls (1.01±0.31) (p<0.001). This figure has been modified 32. Please click here to view a larger version of this figure.
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 triglycerides7,15,32. Determination of biochemical rather than biological properties (e.g. cholesterol efflux) of HDL may be more reproducible. The inter-assay experimental variability of <10% compares favorably with the cell-based assays of HDL function, where interassay experimental variability is often >15% (or not reported). In addition, this approach may limit biochemical interactions between lipids and fluorescent probes32. Determination of HDL oxidation is relevant in the context of cardiovascular diseases given the key role of oxidative stress within the arterial wall in pathogenesis of atherogenesis 46. We have used the fluorochrome HDL function assay to detect impaired HDL function in states of chronic oxidative stress such as atherosclerosis and Human Immunodeficiency Virus infection (HIV)32. Using biochemical assay of HDL function, several studies have confirmed the association of HDLox with obesity and cardiovascular disease 19,21,27,34,35,36,47. Significantly, in human pilot studies we demonstrated associations of HDLox with validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction and associated cardiovascular and metabolic risk phenotypes32,36. Although this assay has been used in several studies to address the role of HDL function in different diseases such as chronic HIV infection32,38, atherosclerosis 32 and obesity36, it remains to be validated in large scale studies with available clinical endpoints of CVD (e.g. heart attacks).
The reaction of the fluorochrome with peroxides in the presence of HRP to produce highly fluorescent resorufin is well established 48,49. HRP catalyzes the oxidation of non-fluorescent fluorochrome to fluorescent resorufin red50,51. This oxidation can be driven by both endogenous peroxides present in the reaction (OH-) and HDL-lipid peroxides (LOOH-). Without cholesterol oxidase, resorufin (with HRP) can quantify the intrinsic HDL lipid peroxide content of a specific amount of HDL cholesterol. The background production of OH- as a result of air oxidation of the buffer is subtracted from the fluorescent readout of each well. Resorufin has minimal autofluorescence in most samples. Addition of catalase (1-4 U/mL) in the reaction buffer can quickly attenuate endogenous peroxides so that the increase in the fluorescent readout over time is driven by HDL lipid peroxides. The assay is versatile and can assess lipid peroxide content in HDL cholesteryl esters versus free HDL cholesterol32,48,49 .
There are many variations of the fluorochrome HDL function Assay32. Briefly there are at least 3 major approaches: a) add a specific volume of HDL (e.g. 50 µL) per well and later normalize the HDL lipid peroxidation value by the level of HDL-C as determined in the clinical laboratory; b) determine the concentration of HDL-C in each sample based on standard colorimetric cholesterol assays and then add a specific amount of HDL-C (e.g. 1 µg) per well; and c) normalize the HDL function readout by HDL or apoA-I protein content32. There are also at least 3 major approaches on how to isolate HDL for HDL lipid peroxidation assays: a) HDL Cholesterol Precipitation e.g. with polyethylene glycol; b) Immunoaffinity capture; and c) other standard not high-throughput methods for HDL isolation such as µLtracentrifugation32. In addition, the assay is versatile and can assess lipid peroxide content in HDL cholesteryl esters versus free HDL cholesterol 32. There are several ways to report the results e.g. arbitrary fluorescence units, standardized resorufin fluorescence units, normalized ratio to a pooled control32. Herein we present the most reproducible approach.
If plasma is used it is important to prepare plasma in tubes that have sodium citrate as the anticoagulant 27,28. EDTA40 and heparin sulfate can interfere with oxidation reactions 41,42. Heparin sulfate can interfere with biochemical assays of HDL function 27,28. Although cryopreserved serum and plasma are suboptimal for determination of lipids, HDL function and HDL lipid peroxidation, cryopreserved samples can quantify relative differences in HDL lipid peroxidation per mg of HDL. It is important to process all samples within a specific study in the exact same way (e.g. same freeze-thaw cycles) and include a pooled control in each plate to account for potential confounders related to differences in sample processing among different studies. Long-term cryopreservation can compromise the results of HDL function assays43 but relative differences in HDL lipid peroxidation among samples within one study may still be assessed if an appropriate pooled control is used 27,28.
Importantly, prior HDL function assays are not standardized. Here we describe an approach where use of appropriate controls ensures standardization of the readout. More specifically there are several known parameters that may affect the readout of biochemical assays of HDL function such as matrix effects (serum vs method of plasma preparation, presence of albumin and other proteins), cryopreservation, freeze-thawing32. The HDL function fluorochrome assay can be standardized with the use of commercially available standards and experimental reagents32. However, there are also several unknown (or poorly characterized) confounders in each study and among different persons that may affect determination of HDL function results. Thus, in each plate we use a pooled HDL control prepared from the specific specimens of interest within a given study (that have been processed identically) that minimizes artifacts and confounders32. Use of plasma blood bank samples and HDL-C values from the clinical laboratory can further standardize the assay32.
We have previously shown that different methods of HDL isolation can significantly affect the readout of biochemical assays of HDL function 27,28,32 but the fluorochrome can reliably measure HDLox irrespective of the HDL isolation 32. HDL isolation methods are a major limiting factor for high-throughput studies of HDL function. Ultracentrifugation is considered the reference method today, but remains a time consuming method52. Electrophoresis is imprecise and not standardized 53. HDL precipitation methods involve the use of polyanions such as polyethylene glycol (PEG) to precipitate low density lipoproteins leaving the HDL in the supernatant 54. Although these methods displayed certain drawbacks such as variable results with lipemic serums and interferences with enzymatic cholesterol procedures 55 prior modifications on the original procedure using standardized commercially available reagents yield a simple, reliable and accurate procedure56. Although PEG precipitation is commonly used method to isolate HDL21,57 from patient serum, a major issue is the presence of non-HDL proteins such as albumin58. Immunoaffinity isolation can be used to minimize the effect of albumin on HDL function 32. Although relative differences in HDLox for a specific capture method can be assessed, specific antibodies against HDL may not fully capture complex HDL structures. Using appropriate controls as described in this protocol, relative differences in HDLox among HDL specimens (isolated by PEG precipitation) can be reliably determined.
This assay, like all other assays of HDL function, has key limitations. In vivo oxidation of HDL in the arterial wall is quite complex and heterogeneous and lipid peroxide content may only partially reflect HDLox46. HDL structure and function continuously changes in vivo and measurement of HDL function at one timepoint may not reflect the impact of HDL dysfunction in end organ disease over time59. It is not known whether the HDLox readout should be normalized by HDL-C, or HDL protein 22. Future clinical studies should validate the importance of the readout of the assay (HDL lipid peroxide content per mg of HDL-C).
In conclusion, the fluorochrome HDL function assay is a reliable method for determination of the HDL lipid peroxide content per specific amount of HDL (HDLox). Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction and associated cardiovascular and metabolic risk phenotypes 19,21,27,32,34,35,36,37,38,39,47. This method offers a convenient, yet robust tool for examining the role of HDL function in human disease.
The authors have nothing to disclose.
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's Postdoctoral Fellowship. AJ and AH are supported by NHMRC project grant 1108792. TK is supported by NIH grants NIH K08AI08272, NIH/NCATS Grant # µL1TR000124.
Experimental Reagents | |||
HDL PEG (Polyethylene Glycol) Precipitating Reagent | Pointe Scientific | H7511 | |
Amplex Red reagent. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
DMSO. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
Horse Radish Peroxidase (HRP) | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
Cholesterol Esterase. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
Cholesterol Reference standard | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
Resorufin fluorescense Reference standard | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
5x Reaction Buffer. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
HDL Cholesterol Automated Reagent | ThermoFisher Scientific Co., San Jose, CA, USA. | TR39601 | |
Name | Company | Catalog Number | コメント |
Plasticware | |||
96-well plates (polypropylene, flat bottom, clear). | Sigma Aldrich | M0687 | |
96-well plates (polypropylene, flat bottom, black). | Sigma Aldrich | M9936 | |
1.5 mL Eppendorf tubes | Eppendorf | 0030 125.150 | |
ClipTip 200, sterile | ThermoFisher Scientific Co., San Jose, CA, USA. | 14-488-058 | |
Thermo Scientific Multichannel Pipettes, 8-channel, 125 | ThermoFisher Scientific Co., San Jose, CA, USA. | 14-387–955 | |
Name | Company | Catalog Number | コメント |
Software | |||
Gen5 2.01 software | Biotek, Vermont, USA | NA | |
Name | Company | Catalog Number | コメント |
Equipment | |||
Gen5 Plate reader | Biotek, Vermont, USA | NA |