CA2664005A1 - A method for quantitation of collagen in tissue - Google Patents

A method for quantitation of collagen in tissue Download PDF

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Publication number
CA2664005A1
CA2664005A1 CA002664005A CA2664005A CA2664005A1 CA 2664005 A1 CA2664005 A1 CA 2664005A1 CA 002664005 A CA002664005 A CA 002664005A CA 2664005 A CA2664005 A CA 2664005A CA 2664005 A1 CA2664005 A1 CA 2664005A1
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CA
Canada
Prior art keywords
tissue
proline
hydroxyproline
glycine
collagen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CA002664005A
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French (fr)
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CA2664005C (en
Inventor
Patricia A. Villanueva
Amy K. Mcnulty
Herbert D. Beniker
Kristine Kieswetter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KCI Licensing Inc
Original Assignee
Kci Licensing, Inc.
Patricia A. Villanueva
Amy K. Mcnulty
Herbert D. Beniker
Kristine Kieswetter
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Publication date
Application filed by Kci Licensing, Inc., Patricia A. Villanueva, Amy K. Mcnulty, Herbert D. Beniker, Kristine Kieswetter filed Critical Kci Licensing, Inc.
Publication of CA2664005A1 publication Critical patent/CA2664005A1/en
Application granted granted Critical
Publication of CA2664005C publication Critical patent/CA2664005C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/22—Injection in high pressure liquid systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Abstract

For successful wound healing to occur, the newly formed skin must generate tensile strength through collagen deposition. Measurement of collagen in the granulating wound bed may be predictive of successful wound healing. Existing methods for collagen measurement either require specialized equipment, or do not allow for discrimination of potential interfering molecules. Herein described is an accurate, specific and reliable method to extract and quantify collagen from tissue utilizing high pressure liquid chromatography techniques. The method is sensitive enough to measure the small amounts of collagen found in newly healing wounds.

Claims (17)

1. A method of assessing wound healing progress comprising:
(a) obtaining a tissue sample from a wound site of a subject;
(b) processing the tissue sample;
(c) separating glycine, proline, and hydroxyproline in the processed tissue sample by high-pressure liquid chromatography to obtain analyte peaks areas;
(d) determining concentrations of the glycine, proline, and hydroxyproline in the tissue;
(e) correlating the concentrations of the glycine, proline, and hydroxyproline in the tissue with an amount of total collagen in the tissue; and (f) comparing the amount of total collagen in the tissue at the wound site with the amount of collagen in non-wounded tissue to assess wound healing progress.
2. The method of claim 1, wherein the tissue is skin.
3. The method of claim 1, wherein the tissue is granulated.
4. The method of claim 1, wherein steps (a)-(f) are repeated at least once, and the tissue sample is obtained from the same wound site of the same subject for each repetition.
5. The method of claim 4, wherein the steps (a)-(f) are repeated at least once during a time period of between about 3 days and about 2 years.
6. The method of claim 4, further comprising comparing the amount of total collagen in the tissue at a first repetition with the amount of total collagen in the tissue at one or more subsequent repetitions.
7. The method of claim 1, wherein the subject is a mammal.
8. The method of claim 7, wherein the mammal is a human.
9. The method of claim 1 wherein processing includes the following steps:

(a) fat removal from the tissue sample;
(b) dehydration of the tissue sample;
(c) hydrolyzing the sample to amino acids; and (d) derivatization of the sample.
10. The method of claim 1, wherein the high-pressure liquid chromatography is performed on a C18 column.
11. The method of claim 10, wherein the C18 column has a pH range of about 1 to about 12.
12. The method of claim 1, wherein elution of the glycine, proline, and hydroxyproline during the high-pressure liquid chromatography is measured at nm with a photodiode array detector.
13. The method of claim 1, wherein the high-pressure liquid chromatography is reversed phase high-pressure liquid chromatography.
14. The method of claim 1, wherein the high-pressure liquid chromatography has a mobile phase of 70% 25 mM potassium phosphate, pH 11.0 buffer, and 30%
acetonitrile.
15. The method of claim 1, wherein determining the concentrations of the glycine, proline, and hydroxyproline in the tissue comprises:
(a) obtaining samples with known concentrations of glycine, proline, and hydroxyproline;
(b) separating the glycine, proline, and hydroxyproline in the samples by high-pressure liquid chromatography;
(c) plotting the known concentrations of glycine, proline, and hydroxyproline on a graph x-axis by analyte peak areas on the graph y-axis to devise a linear standard curve; and (d) calculating the concentrations of each of the glycine, proline, and hydroxyproline in the processed tissue sample using a formula:
wherein b is the y-intercept and m is the slope of the linear standard curve; and (e) calculating the concentrations of each of the glycine, proline, and hydroxyproline in the tissue using a formula:

16. The method of claim 1, wherein correlating the concentrations of the glycine, proline, and hydroxyproline in the tissue with the amount of total collagen in the tissue comprises using a formula:

wherein C is the sum of the percent composition of glycine, proline, and hydroxyproline in collagen divided by 100.
17. The method of claim 16, wherein C equals 0.55.
CA2664005A 2006-09-21 2007-09-21 A method for quantitation of collagen in tissue Expired - Fee Related CA2664005C (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US84625006P 2006-09-21 2006-09-21
US60/846,250 2006-09-21
US11/858,737 2007-09-20
US11/858,737 US7491541B2 (en) 2006-09-21 2007-09-20 Method for quantitation of collagen in tissue
PCT/US2007/079153 WO2008036891A2 (en) 2006-09-21 2007-09-21 A method for quantitation of collagen in tissue

Publications (2)

Publication Number Publication Date
CA2664005A1 true CA2664005A1 (en) 2008-03-27
CA2664005C CA2664005C (en) 2012-05-22

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Family Applications (1)

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CA2664005A Expired - Fee Related CA2664005C (en) 2006-09-21 2007-09-21 A method for quantitation of collagen in tissue

Country Status (12)

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US (2) US7491541B2 (en)
EP (1) EP2061898B1 (en)
JP (1) JP5065397B2 (en)
KR (1) KR20090068265A (en)
AU (1) AU2007299680B2 (en)
BR (1) BRPI0715287A2 (en)
CA (1) CA2664005C (en)
IL (1) IL197650A0 (en)
MX (1) MX2009003112A (en)
NO (1) NO20091433L (en)
RU (1) RU2452956C2 (en)
WO (1) WO2008036891A2 (en)

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