Assays to predict atherosclerosis and dysfunctional high-density lipoprotein

An atherosclerosis and protein technology, which can be used in biological testing, material testing, etc., and can solve the problem of not knowing the pro-inflammatory HDL protein profile.

Inactive Publication Date: 2009-06-24
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although numerous proteins and enzyme activities have been associated with HDL, little is known about the specific protein profile associated with pro-inflammatory HDL

Method used

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  • Assays to predict atherosclerosis and dysfunctional high-density lipoprotein
  • Assays to predict atherosclerosis and dysfunctional high-density lipoprotein
  • Assays to predict atherosclerosis and dysfunctional high-density lipoprotein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

Detection of hemoglobin, haptoglobin, and hemopexin

[0146] ELISA methods for haptoglobin and hemopexin, and methods for preparation of HDL supernatants and apoA-I-binding immunosorbent proteins are shown below.

human hemoglobin ELISA

[0147] Table 1. Materials used for the hemoglobin ELISA

Note: Allow all reagents to come to room temperature before use.

HDL Separation Using Magnetic Bead Reagents

[0148] 1. Separate plasma or serum from the blood sample using a green top tube or a serum separation tube and centrifuge at 2300 rpm for 20 minutes at 5°C.

[0149] 2. Remove the supernatant (serum or plasma). Note: If the sample has been frozen, thaw the sample and centrifuge at 12,000 rpm in a centrifuge for 5 minutes at room temperature. This should settle any particles present in the serum or plasma that might affect the assay.

[0150] 3. Add supernatant (up to 250 μL / well) to a clear round bottom 96-well plate.

[0151] 4. Add 1 / 5 total volume of magneti...

Embodiment 2

[0338] We have reported that the inflammatory properties of HDL are a more sensitive indicator of atherosclerosis than HDL-cholesterol levels in both mice and humans. In this example, we describe the use of ProteinChip technology coupled to surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to identify HDL that distinguishes between normal mouse HDL and mouse HDL fed an atherogenic diet specific protein fingerprints. Feeding C57BL / 6J mice an atherogenic diet for 1 week resulted in decreased HDL-cholesterol levels, decreased paraoxonase activity, increased reactive oxygen species levels, and decreased ability of HDL to promote cholesterol efflux from macrophages. When the mice were switched back to normal chow for an additional 2 weeks, the pro-atherogenic features of HDL reverted to a normal phenotype. In total, we identified 88 SELDI peaks with p<0.05 that were differentially present in proinflammatory HDL of mice fed an atherogenic ...

Embodiment 3

in the serum of mice fed an atherosclerotic / hyperlipidemic diet HDL-bound hemoglobin

[0369] In Example 2, we identified 8 specific protein fingerprints using the strong anion-exchange SELDI ProteinChip technology that differentiates normal / anti-inflammatory HDL from pro-inflammatory HDL in a mouse model of atherosclerosis. Using microfluidics-tandem mass spectrometry, we identified the SELDI peaks representing m / z 14,900 and m / z 15,600 as mouse hemoglobin α chain (Hb-α, 14.9 kDa) and mouse hemoglobin β chain (Hb-β , 15.9 kDa). Western blot analysis confirmed differential binding of Hb to pro-inflammatory HDL when compared to normal HDL. Biochemical characterization of HDL-bound Hb further revealed that Hb bound to pro-inflammatory HDL has distinct physical and chemical properties, including reduced pI (pI4. High molecular weight complexes present in the HDL-containing fraction bind. The major form of hemoglobin found bound to HDL is oxyhemoglobin (oxyHb). Based on the...

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Abstract

This invention provides novel assays for the detection of dysfunctional HDL. The assays are good diagnostics and / or prognostics for atherosclerosis or other pathologies characterized by an inflammatory response. In certain embodiments the methods involve measurements of heme-related HDL-associated proteins (e.g., haptoglobin, hemopexin, etc.), and / or measurements of the relative distribution of HDL-associated proteins between HDL and the non-lipoprotein fractions of plasma / serum, and / or measurements of the ability of pro-inflammatory HDL to consume nitric oxide, and / or measurement of the ability of HDL to inhibit LDL aggregation.

Description

[0001] Cross References to Related Applications [0001] This application claims the benefit of and priority to USSN 60 / 843,213, filed September 7, 2006, and USSN 60 / 772,429, filed February 10, 2006, both of which are hereby incorporated by reference in their entirety in each case . Statement Regarding Inventor's Rights Under Federally Sponsored Research and Development [0002] This work was supported in part by grant numbers 1RO1HL7 1776 and HL30658 from the National Institutes of Health. The United States Government has certain rights in this invention. field of invention [0003] The present invention relates to the diagnosis of atherosclerosis. In particular, the present invention provides improved assays for detecting abnormally functioning HDL. Background of the invention [0004] Atherosclerosis is a chronic inflammatory disease of large and medium arteries and is a leading cause of morbidity and mortality in Western countries. The use of statins reduces mortal...

Claims

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Application Information

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IPC IPC(8): G01N33/53
Inventor A·M·富格尔曼S·T·雷迪
Owner RGT UNIV OF CALIFORNIA
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