Biomarkers related with a supra-nutritional metabolic state of selenium and diagnosis method in which said biomarkers are identified

Patent metadata

Field	Value
Publication number	US-9764319-B2
Application number	US-201214123451-A
Country	US
Kind code	B2
Filing date	Aug 1, 2012
Priority date	Jun 2, 2011
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

The disclosure provides methods and kits for diagnosing the nutritional state of selenium, using six proteins as biomarkers for which the expression increases when the metabolic state is supra-nutritional.

First claim

What is claimed is:

1. A process for the preparation of an electrophoretic pattern of a reference protein associated with a supranutritional metabolic state of the trace element selenium, the process comprising:
I) fractionating plasma proteins by cation exchange to retain proteins complement factor H, plasminogen isoform CRA-f, LOC 366747 protein, complement component 4a, albumin, Da1-24, Ig Gamma-C region 2C chain, H4 heavy chain inter alfa inhibitor, Ig kappa precursor and apolipoprotein H;
II) electrophoretically separating the retained plasma proteins into bands; and
III) determining the identity and relative abundance of at least a band of complement factor H, thereby providing the electrophoretic pattern of the reference protein associated with a supranutritional metabolic state of the trace element selenium.

2. The process of claim 1 , wherein fractionating plasma proteins comprises:
a) adding a cation exchange resin to a plasma sample then resuspending the cationic exchange resin until complete homogenization is attained;
b) taking a volume of the suspension;
c) mixing the volume of the suspension with a 1:9 proportional amount of buffer pH 7.5;
d) washing the resin with pH 7.5 buffer plus 0.1 M NaCl;
e) elute proteins adsorbed onto the resin with a buffer 1 M NaCl pH 7.5, then removing the resin from the supernatant, and recovering the supernatant.

3. The process of claim 2 , wherein electrophoretically separating the retained plasma proteins and determining the identity and relative abundance comprises:
a) preparing a 12% polyacrylamide gel;
b) mixing a portion of the supernatant obtained at the point e) in step I) with of a loading buffer;
c) denaturing proteins in the mixture of supernatant and loading buffer by heat;
d) electrophoresing the denatured mixture in a lane of the gel;
e) staining the gel with Coomassie blue dye;
g) acquiring an image of the stained gel digitally, and
h) quantifying densitometrically at least the band of complement factor H, as it is defined by the position of the band in the gel relative to other defined bands in the gel.

4. The process of claim 1 , wherein determining the identity of the band comprises:
a) cutting the band from the gel;
b) digesting the band with trypsin for 12 hours;
c) identification of peptide masses by Matrix-Assisted-Laser Desorption-Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS); and
d) determining internal sequences of the ions obtained.

5. The process of claim 1 , wherein the bands comprise Bands R-2 to R-15 as follows:

Band
Protein

R-2
complement factor H

R-3
complement factor H

R-4
plasminogen isoform CRA-f

R-5
LOC 366747 Protein

R-6
complement component 4a

R-7
Albumin

R-8
Da1-24

R-9
Ig Gamma-C region 2C chain

R-10
Beta 2 glycoprotein 1 precursor

R-11
Ig gamma-2A region C chain

R-12
complement component 4a

R-13
H4 heavy chain Inter alfa inhibitor

R-14
Ig kappa precursor

R-15
Apolipoprotein H,

wherein the relative abundance of at least bands R-3 and at least one of R-10, R-11, R-12, R-13 and R-15 is determined.

6. The process of claim 5 , wherein the relative abundance of at least bands R-3, R-10, R-11, R-12, R-13 and R-15 is determined.

7. A method of determining the supranutritional metabolic state of the trace element selenium in a mammalian subject, comprising:
acquiring a blood plasma sample from the subject;
fractionating blood plasma proteins in the blood plasma sample by cation exchange to retain proteins complement factor H, plasminogen isoform CRA-f, LOC 366747 protein, complement component 4a, albumin, Da1-24, Ig Gamma-C region 2C chain, H4 heavy chain inter alfa inhibitor, Ig kappa precursor and apolipoprotein H;
analyzing the fractions by denaturing electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE) to determine the relative abundance of at least complement factor H represented by band R-3, and optionally one or all peptides represented by the bands R-2 and R-4 to R-15:

Band
Protein

R-2
complement factor H

R-3
complement factor H

R-4
plasminogen iso form CRA-f

R-5
LOC 366747 Protein

R-6
complement component 4a

R-7
Albumin

R-8
Da1-24

R-9
Ig Gamma-C region 2C chain

R-10
Beta 2 glycoprotein 1 precursor

R-11
Ig gamma-2A region C chain

R-12
complement component 4a

R-13
H4 heavy chain Inter alfa inhibitor

R-14
Ig kappa precursor

R-15
Apolipoprotein H

Assignees

• Mahn Andrea
• Univ Santiago Chile

Inventors

• Mahn Andrea

Classifications

• G01N2030/8831

  involving peptides or proteins · CPC title

• C07K1/26

  Electrophoresis · CPC title

• G01N27/44756

  Apparatus specially adapted therefor · CPC title

• B01J39/26 Primary

  Cation exchangers for chromatographic processes · CPC title