Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study
Andersson, Elin LU
; Tykesson, Emil
LU
; Lohmander, L Stefan
LU
; Karlsson, Niclas G
; Jin, Chunsheng
; Mirgorodskaya, Ekaterina
; Swärd, Per
LU
and Struglics, André
LU
(2023)
In Osteoarthritis and Cartilage Open
5(3).
p.1-11
- Abstract
- Objective
To validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples.
Design
Synovial fluid from osteoarthritis (OA, n = 25) and knee-injury (n = 13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry.
Results
Unsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in... (More) - Objective
To validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples.
Design
Synovial fluid from osteoarthritis (OA, n = 25) and knee-injury (n = 13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry.
Results
Unsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low.
Conclusions
The HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/09af97c0-eb06-426f-bdeb-898c4b8f7fa5
- author
- Andersson, Elin
LU
; Tykesson, Emil
LU
; Lohmander, L Stefan
LU
; Karlsson, Niclas G
; Jin, Chunsheng
; Mirgorodskaya, Ekaterina
; Swärd, Per
LU
and Struglics, André
LU
- organization
- publishing date
- 2023
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Osteoarthritis and Cartilage Open
- volume
- 5
- issue
- 3
- article number
- 100380
- pages
- 1 - 11
- publisher
- Elsevier
- external identifiers
-
- pmid:37426292
- scopus:85162537246
- ISSN
- 2665-9131
- DOI
- 10.1016/j.ocarto.2023.100380
- project
- The role of glycans in aggrecan degradation and as biomarkers in osteoarthritis
- language
- English
- LU publication?
- yes
- id
- 09af97c0-eb06-426f-bdeb-898c4b8f7fa5
- date added to LUP
- 2023-08-30 11:42:27
- date last changed
- 2023-08-31 04:00:38
@article{09af97c0-eb06-426f-bdeb-898c4b8f7fa5,
abstract = {{Objective<br/>To validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples.<br/><br/>Design<br/>Synovial fluid from osteoarthritis (OA, n = 25) and knee-injury (n = 13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry.<br/><br/>Results<br/>Unsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low.<br/><br/>Conclusions<br/>The HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.}},
author = {{Andersson, Elin and Tykesson, Emil and Lohmander, L Stefan and Karlsson, Niclas G and Jin, Chunsheng and Mirgorodskaya, Ekaterina and Swärd, Per and Struglics, André}},
issn = {{2665-9131}},
language = {{eng}},
number = {{3}},
pages = {{1--11}},
publisher = {{Elsevier}},
series = {{Osteoarthritis and Cartilage Open}},
title = {{Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study}},
url = {{http://dx.doi.org/10.1016/j.ocarto.2023.100380}},
doi = {{10.1016/j.ocarto.2023.100380}},
volume = {{5}},
year = {{2023}},
}