Method for quantitation of collagen in tissue
View Patent ↗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.
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 by a method comprising:
(i) removing fat from the tissue sample;
(ii) dehydrating the tissue sample;
(iii) hydrolyzing the tissue sample to amino acids; and
(iv) derivatizating 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 the high-pressure liquid chromatography is performed on a C18 column.
10. The method of claim 9 , wherein the C18 column has a pH range of about 1 to about 12.
11. The method of claim 1 , wherein elution of the glycine, proline, and hydroxyproline during the high-pressure liquid chromatography is measured at 436 nm with a photodiode array detector.
12. The method of claim 1 , wherein the high-pressure liquid chromatography is reversed phase high-pressure liquid chromatography.
13. 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.
14. 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:
Amino
Acid
Conc
(
µg
/
mL
)
=
Analyte
Peak
Area
-
b
m
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:
Amino
Acid
Conc
(
µg
/
mg
)
=
(
Amino
Acid
Conc
(
µg
/
mL
)
dilution
factor
)
×
final
sample
vol
(
mL
)
Tissue
Wt
(
mg
)
.
15. 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:
Total
Collagen
=
(
∑
hydroxyproline
(
µg
/
mg
)
,
glycine
(
µg
/
mg
)
,
proline
(
µg
/
mg
)
in
tissue
)
C
wherein C is the sum of the percent composition of glycine, proline, and hydroxyproline in collagen divided by 100.
16. The method of claim 15 , wherein C equals 0.55.