Hyperglycemia-related advanced glycation end-products is associated with the altered phosphatidylcholine metabolism in osteoarthritis patients with diabetes
Hyperglycemia-related advanced glycation end-products is associated with the altered phosphatidylcholine metabolism in osteoarthritis patients with diabetes
Weidong Zhang 0 1
Edward W. Randell 1
Guang Sun 1
Sergei Likhodii 1
Ming Liu 0 1
Andrew Furey 1
Guangju Zhai 0 1 2
0 Discipline of Genetics, Faculty of Medicine, Memorial University of Newfoundland , St John's, Newfoundland and Labrador , Canada , 2 School of Pharmaceutical Sciences, Jilin University , Changchun , P. R. China , 3 Department of Laboratory Medicine, Faculty of Medicine, Memorial University of Newfoundland , St John's, Newfoundland and Labrador , Canada , 4 Discipline of Medicine, Faculty of Medicine, Memorial University of Newfoundland , St John's, Newfoundland and Labrador , Canada , 5 Department of Surgery, Faculty of Medicine, Memorial University of Newfoundland , St John's, Newfoundland and Labrador , Canada
1 Editor: Lin Mei, Augusta University , UNITED STATES
2 Menzies Research Institute, University of Tasmania , Hobart, Tasmania , Australia
Data Availability Statement: The ethics approval
for the study reported in the manuscript did not
contain provisions for data sharing, therefore we
can't make the data available publicly. Health
Research Ethics Authority of Newfoundland and
Labrador, Canada is the authority that imposed the
restriction of data sharing on the study reported in
the manuscript. Interested, qualified researchers
may request these data by contacting Sandra
Veenstra, Ethics Director, Health Research Ethics
Authority, 95 Bonaventure Ave, Suite 200,
To test whether type 2 diabetic patients have an elevated level of advanced glycation
end-products (AGEs) and responsible for altered phosphatidylcholine metabolism, which
we recently found to be associated with osteoarthritis (OA) and diabetes mellitus (DM),
synovial fluid (SF) and plasma samples were collected from OA patients with and without
DM. Hyperglycemia-related AGEs including methylglyoxal (MG), free methylglyoxal-derived
hydroimidazolone (MG-H1), and protein bound N-(Carboxymethyl)lysine (CML) and
N-(Carboxyethyl)lysine (CEL) levels were measured in both SF and plasma samples using liquid
chromatography coupled tandem mass spectrometry methodology. The correlation
between these AGEs and phosphatidylcholine acyl-alkyl C34:3 (PC ae C34:3) and C36:3
(PC ae C36:3) were examined. Eighty four patients with knee OA, including 46 with DM and
38 without DM, were included in the study. There was no significant difference in plasma
levels of MG, MG-H1, CML, and CEL between OA patients with and without DM. However, the
levels of MG and MG-H1, but not CML and CEL in SF were significantly higher in OA
patients with DM than in those without (all p 0.04). This association strengthened after
adjustment for age, body mass index (BMI), sex and hexose level (p<0.02). Moreover,
the levels of MG-H1 in SF was negatively and significantly correlated with PC ae C34:3
(ρ = -0.34; p = 0.02) and PC ae C36:3 (ρ = -0.39; P = 0.03) after the adjustment of age, BMI,
sex and hexose level. Our data indicated that the production of non-protein bound AGEs
was increased within the OA-affected joint of DM patients. This is associated with changes
in phosphatidylcholine metabolism and might be responsible for the observed
epidemiological association between OA and DM.
Funding: This work was supported by the
Canadian Institutes of Health Research (CIHR);
Grant number:RNL-132178, GJZ (http://webapps.
cihr-irsc.gc.ca/cfdd/db_search); the Newfoundland
& Labrador Research and Development
Corporation and Memorial University of
Newfoundland (MUN). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Osteoarthritis (OA) is the most common joint disease worldwide, affecting 10% of men and
18% of women over 60 years of age [
]. Accumulating evidence suggests that OA is associated
with metabolic syndrome (MetS) related conditions, especially type 2 diabetes mellitus (DM)
[2±6]. While the potential mechanisms for the association between OA and DM remain
unclear, we [
] recently found that phosphatidylcholine acyl-alkyl C34:3 (PC ae C34:3) and
C36:3 (PC ae C36:3) were associated with the concurrence of OA and DM, suggesting that altered phosphatidylcholine metabolism might be responsible for the observed association between OA and DM.
There is substantial evidence to suggest that intracellular glucose toxicity in DM may be
mediated through increased production of highly reactive α-ketoaldehydes [8±10]. The
nonenzymatic reaction of α-ketoaldehydes with protein produces advanced glycation
end-products (AGEs). The accumulation of AGEs may play a causal role in the development of
complications of DM including OA. AGEs have also been implicated in OA [
AGEs can modify normal macromolecules functions directly or by generating reactive
oxygen species (ROS) either independently, or by activation of the receptor for AGEs (RAGE) [13±
15]. It has been reported that ROS can result in membrane lipid peroxidation [
peroxidation is the process in which free radicals "steal" electrons from the lipids in cell membranes,
resulting in cell damage. This process proceeds by a free radical chain reaction mechanism. It
often affects polyunsaturated fatty acids, because they contain multiple double bonds in between
which lie methylene bridges (-CH2-) that possess especially reactive hydrogen atoms. PC ae
C34:3 and PC ae C36:3 belong to a special but sub-group of unsaturated phosphatidylcholines
which comprise an ether linkage to one alkyl chain and one polyunsaturated fatty acid and have
unique bioactivity, including a role in generation of lipid second messengers, but also providing
a protective effect against oxidative stress [
]. The decreased concentrations of both
unsaturated PC metabolites in OA with DM may be related to an increased potential for lipid
peroxidation with AGE potentially playing a pathogenic role. We therefore undertook this study to
examine whether plasma and synovial levels of AGEs were increased in OA patients with DM,
and determined how this might be related to the levels of PC ae C34:3 and PC ae C36:3.
Patients and methods
The study was part of the ongoing Newfoundland Osteoarthritis Study (NFOAS) . Patients
with OA were recruited from those who underwent total knee replacement surgery due to
primary OA between November 2011 and December 2014 in St. Clare's Mercy Hospital and
Health Science Centre General Hospital in St. John's, the capital of Newfoundland and
Labrador, Canada. Demographic and medical information was collected by a self-administered
general questionnaire with the assistance of research staff if necessary. DM status of the patients
were determined by the self-reported general questionnaire and confirmed by their hospital
diagnosis records. The study was approved by the Health Research Ethics Authority of
Newfoundland and Labrador (reference number is 11.311) and written consent was obtained from
all the participants.
Measurement of PC ae C34:3 and PC ae C36:3 in plasma and synovial
Blood samples were collected after overnight fast of at least 8 h, and SF samples were collected during joint replacement surgeries. Plasma and SF samples were processed as previous
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]. Metabolic profiling in both plasma and SF were performed by the Waters
XEVO TQ MS system (Waters Ltd.) using the Biocrates AbsoluteIDQ p180 kit as part of a previous project . Data on PC ae C34:3 and PC ae C36:3 were retrieved from the metabolic profile data for the purposes of the current study.
Measurement of methylglyoxal (MG) and hydroimidazolone (MG-H1) in
plasma and SF
MG was measured by liquid chromatography coupled with tandem mass spectrometry
(LC-MS/MS) using methodology previously developed by our group [
]. Briefly, small
aliquots of samples (~100 μL; SF and plasma) was derivatized using 2, 3-diaminonaphthalene (2,
3-DAN) and extracted, and dried under nitrogen gas. The dried extracts were reconstituted
with acetonitrile for LC-MS/MS analysis. The 2, 3-DAN derivative of methylglyoxal was
separated in an isocratic solvent system consisting of 0.1% formic acid and acetonitrile (35/65, v/v)
using a C8 column (3.5 μm, 2.1×100 mm, Waters, Massachusetts, USA) at a flow rate of
0.30 mL/min at a temperature of 25ÊC. The derivative of methylglyoxal was determined by
selective reaction monitoring of the transition m/z 195>168 and using 5-methylquinoline
(m/z 145>91) as an internal standard.
MG-H1 was measured by LC-MS/MS using methodology previously developed in our labo
]. Briefly, samples (synovial and plasma) will be extracted by strong anion exchange
solid phase extraction columns (Oasis MCX 1cc 30 mg, Waters, Massachusetts, USA). The
dried extracts containing MG-H1 was reconstituted with methanol for LC-MS/MS by the
Water Alliance HT 2795-Micromass Quattro Ultima LC-MS/MS system. MG-H1 was sepa
rated in a gradient solvent system consisting of 10 mM formic acid in methanol, water, and
acetonitrile at a flow rate of 0.6 mL/min at ambient temperature on a Hilic Sillica column
(Atlantis1 Hilic Slica, 3 μm, 2.1×50 mm, Waters Corporation, Massachusetts, USA).
Measurement of N-(Carboxymethyl)lysine (CML) and N-(Carboxyethyl)
lysine (CEL) in plasma and SF
CML and CEL were measured by LC-MS/MS using methodology previously developed by
Teerlink et al.  but with minor modifications to the gradient system to optimize separation
using our system. Briefly, sample were analyzed at ambient temperature by reversed-phase
HPLC on a Symmetry C18 column (5μm, 3.9×150mm, Waters, Massachusetts, USA) and
using nonafluoropentanoic acid (NFPA) as the ion pairing agent in a two solvent gradient
separation involving acetonitrile as the organic phase. Analyses were performed in positive-ion
mode but on the Water Alliance HT 2795-Micromass Quattro Ultima LC-MS/MS system. For
both CML and CEL, the product ion at m/z 84.1 was used for quantification and the product
ion at m/z 130.1 for confirmation. In each series, five calibration samples spanning the
concentration ranges of 0±2.5 μmol/L for CML and 0±1.0 μmol/L for CEL were included.
The distribution of the concentrations of PC ae C34:3, PC ae C36:3, MG, MG-H1, CML, and
CEL in plasma and SF samples were examined by qq plot and transformed by log10
transformation where necessary. The missing values (no data) were replaced by half of the minimum
value found in the dataset [
]. The comparisons of the four AGEs between OA with and
without DM were tested by t- test, and visualized by boxplots. The correlation between each of the
four AGEs and PC ae C34:3 and PC ae C36:3 were examined by Spearman correlation
coefficient. Statistically significant results were identified by p 0.05. The potential confounders
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including age, sex, body mass index (BMI) and hexose level (>90% is glucose) were adjusted
by linear regression modelling. All the statistical analysis was carried out using PCIT package
and ggplot2 Graphics packages implemented in R (version 3.1.1 for Windows).
In total, 84 knee OA patients, including 46 with DM and 38 without DM, were included in
the study. Basic descriptive demographics of the study population are presented in Table 1.
The number of males and females in the DM and non-DM groups were similar, but OA
patients with DM were older and had a higher BMI than OA patients without DM (Table 1;
all p 0.01). We measured the total hexose levels in both plasma and SF samples, which is
mainly represented by glucose. The plasma and SF hexose levels in OA with DM were 4383.6
±3075.5 μM and 5034.5±5153.9 μM, respectively, 2815.2±2224.6 μM, and 1841.8±1082.7μM
for OA without DM. The difference in hexose levels between DM and non-DM patients were
significant regardless of the type of samples as expected (all P<0.04). The hexose level in the
SF of DM patients was higher than that in the plasma, but was not statistically significant.
The concentrations of MG, MG-H1, CML, CEL, PC ae C34:3 and PC ae C36:3 were mea
sured in both plasma and SF samples. Because all the measurements were not normally
distributed, data was log10 transformed for subsequent analyses.
None of the plasma concentrations of MG, MG-H1, CML and CEL was significantly
different between OA with and without DM. However, the SF concentration of MG-H1 in OA
patients with DM was 2.53±2.29 (log10 ng/ml), which was significantly higher than that in OA
without DM patients (2.29±2.06, log10 ng/ml) (P = 0.01) (Fig 1). Similarly, the SF
concentration of MG in OA patients with DM (2.07±1.49, log10 ng/ml) was also significantly higher than
that in OA patients without DM (2.01±1.41, log10 ng/ml; P = 0.03) (Fig 1). The statistical
significances of the differences were maintained after adjustment for age, BMI, sex, and hexose
level (P = 0.03 for MG-H1 and P = 0.02 for MG). However, the SF concentrations of CML and
CEL were not significantly different between OA with and without DM (Fig 1).
Similar to our previous findings , the concentrations of PC ae C34:3 and PC ae C36:3 in
SF and plasma were significant lower (all p<0.05) in OA patients with DM than that in OA
patients without DM (Fig 2). For SF analysis, that the concentration of PC ae C34:3 and PC ae
C36:3 in OA patients with DM were 0.015±0.18 (log10 ng/ml) and 0.003±0.21 (log10 ng/ml),
respectively, which were 23% and 19% significantly lower (p = 0.04) than that in OA patients
without DM whose concentrations were 0.018±0.15 (log10 ng/ml) and 0.010±0.15 (log10 ng/
ml), respectively (Fig 2). The overall plasma concentrations of both metabolites were higher
than that in SF (Fig 2), but similar to the SF results, OA patients with DM had a concentration
reduced by 34% and 27%, respectively for PC ae C34:3 and PC ae C36:3, compared to OA
without DM (all p<0.03; Fig 2). The differences remained statistically significant after
adjustment of sex, age and BMI (P<0.05).
OA+DM (n = 46)
OA (n = 38)
Fig 1. The concentrations of MG-H1, MG, CEL and CML in SF of OA and OA+DM patients. (DM: diabetes). P values were adjusted for sex, age and
Further, we found that MG-H1concontration in SF was negatively correlated with PC ae
C34:3 (ρ = -0.34, p = 0.02) and PC ae C36:3 (ρ = -0.39, p = 0.028), but none of the other three
AGEs under study showed significant correlation with the two PCs. The correlation remained significant after adjustment for age, sex, BMI, hexose level (p<0.05).
Fig 2. The concentrations of PC ae C34:3 and PC ae C36:3 in SF (A and B) and plasma (C and D) of OA and OA+DM patients. (DM: diabetes). P
values were adjusted for sex, age and BMI.
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To the best of our knowledge, this is the first study examining levels of AGE precursor MG,
and AGEs including MG-H1, CML, and CEL in both plasma and SF of OA patients with and
without DM. We demonstrated that increased glycation, evidenced by higher levels of MG and
MG-H1 in SF, was associated with altered phosphatidylcholine metabolism, specifically certain plasmalogens within the joint. These relationships may provide insights into the underlying pathogenic process mediating the association between OA and DM observed in epidemiological studies.
MG, a potent glycation agent, is a highly reactive α-ketoaldehyde formed by both enzymatic
and non-enzymatic processes using glycolytic intermediates or sugar [
]. MG-H1 and CEL
result from MG-mediated damage of protein arginine and lysine residues, respectively.
MGmodified proteins undergo cellular proteolysis and releasing free MG-H1 for excretion.
Therefore, MG-H1 found in body fluids most likely originates from cells releasing small molecule
waste into circulation for disposal. Based on this assumption, SF MG-H1 levels could reflect
more closely the in situ breakdown of glycated intracellular and extracellular proteins by cells
and tissues in immediate contact and surrounding the joint. MG-H1 is the major AGE in
proteins of tissues and body fluids [
]. It increases in DM and is associated with vascular
complications, renal failure, arthritis (OA and rheumatic arthritis) and ageing [25±28]. In the
current study, SF levels of both MG and MG-H1 were higher in OA with DM than that in OA
without DM, presenting evidence in support of a putative mechanism explaining the
relationship between OA and DM in epidemiological studies. That is, the accumulation of MG and
MG-H1 in the SF may be evidence of a role intracellular or extracellular protein glycation in
the joint area, which may be higher in DM, mediating down-stream tissue damage in the local
environment. Interestingly, plasma concentrations of MG and MG-H1 were not significantly
different between both groups. While, the reason for this is not known, we speculate that a
modifying effect of antidiabetic drugs on plasma glycemia, glycation of blood plasma proteins,
and major tissues giving rise to free AGEs, like MG-H1, that eventually appear in blood for
excretion may be at work. The most frequently used drugs for the treatment of DM include
suppressors of hepatic gluconeogenesis (metformin) [
], insulin-sensitizing PPAR agonists
], and insulin secretagogues (sulfonylureas) [
]. Many studies have reported
the inhibiting effect of these drugs on AGE formation [32±35]. In addition, in a study involved
22 patients with PCOS and 22 healthy women, Diamanti-Kandarakis et al. [
] also found that
plasma AGEs levels were reduced significantly after 6 months of metformin administration in
women with polycystic ovary syndrome. Thus, we speculate that potential antidiabetic drugs
may have immediate effects on reducing blood glucose level and inhibiting the production of
AGEs in the blood, but the effect on reducing tissue glucose levels and non-enzymatic
glycation reactions in the joint and SF may be lagging, leading to elevated production and
accumulation of AGEs, particularly MG and MG-H1. While we did not measure glucose levels in the
same samples used in the study, we measured hexose levels, which represent more than 90% of
glucose. Indeed, SF hexose levels in OA patients with DM were higher than that in plasma and
all OA with DM patients had higher hexose levels than those without in both SF and plasma,
supporting this hypothesis.
In cell and animal studies, AGEs have been implicated as key pathogenic factors in initia
tion and progression of OA and DM [
]. The main mechanisms involve altered function
of many intra- and extracellular proteins and generation of reactive oxygen species (ROS) by
the activation of the receptor for AGEs (RAGE) . Increased ROS leads to the induction of
lipid peroxidation. Lipid peroxidation is considered as the main molecular mechanisms
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involved in the oxidative damage to cell structures and in the toxic process that leads to cell
]. Acyl-alkyl-phosphatidylcholines has been shown to have antioxidant properties by
preventing plasma lipoprotein oxidation [
]. In the present study, MG-H1 negatively
correlates with the levels of PC ae C34:3 and PC ae C36:3, suggesting that decreased levels of these
two polyunsaturated lipids in OA and DM patients could be due to the over lipid oxidation
caused by AGEs, but also loss of an important antioxidant with potential to protect from free
radical damage. Unsaturated phosphatidylcholines are the main constituent phospholipids
that cover on the surface of the cartilage tissue and have an important role in lubrication ,
load-bearing function [
] and maintaining normal physiological functions of joint cartilage
]. Reduced levels of the antioxidant potential of the PC plasminogen would have deleterious
effects on initiation and progression of OA.
CML and CEL are also major protein bound AGEs found in tissue and blood and have
been established as ligands of RAGE [
]. We measured their plasma and SF levels in the
current study, but did not find a significant difference between the OA patients with and
without DM. This may be attributed to that CML and CEL in blood are mainly coming from
proteins with relatively short half-life and are reflecting relatively short-term changes in glycemia
and events occurring in circulation. Moreover, the SF proteome shares considerable similarity
with that of plasma, no doubt because most of these proteins originate from plasma . From
this perspective, CML and CEL levels should reflect changes in glycation event occurring in
There are some limitations in the study. Firstly, the sample size was relatively small, but a
post hoc power calculation suggested that we had 98.5% study power to detect the observed
difference with the given sample size. Secondly, we assessed only four major AGEs in the
current study, thus we may miss other AGEs that might also play a role in OA with DM.
However, AGEs form by non-enzymatic processes and the major AGEs formed were
covered in this study. Thirdly, patients were on various medications that have a potential
modifying effect on inflammatory processes and glycemic conditions that may profoundly
modify the AGE response. While these effects can be significant these treatments do not
normalize glycemia, but may require larger patient populations and greater powered studies
to reveal differences that may be present. It is worth noting that formation of AGE
precursors like MG and AGEs involve mechanisms more complex than just glycemia alone, and
the effects of drugs like metformin on AGE levels involve interactions that are independent
of direct effects of the drug on glycemia [
]. Fourthly, Diabetes could cause many
complications that may have indirect effects on phosphatidylcholine metabolism. However, very
few subjects had diabetic complications including cardiovascular diseases (CVD), renal
damage, and eye damage. Thus we did not have sufficient power to examine the
confounding effects of these diabetic complications on the observed association. Fifthly, the results
would be more conclusive if we had negative controls. However, obtaining SF samples from
healthy subjects are nearly impossible ethically. However, obtaining SF samples from
healthy subjects are nearly impossible ethically. And lastly, the study was cross-sectional; a
longitudinal study is required to establish a causal relationship for the observed association
in the current study.
In summary, we demonstrated that both MG-H1 and MG concentrations in SF were ele
vated in OA patients with DM and associated with the levels of PC ae C34:3 and PC ae C36:3.
These findings might assist to establish a relationship between AGE mediated tissue damage especially in the joint, and derangement in phosphotidylcholine metabolism particularly affecting PC plasmalogens. These relationships might help form the mechanistic relationship explaining the epidemiological association between OA and DM.
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We thank all the study participants who made this study possible and the staff from the operation room who helped us in collecting samples of the NFOAS.
Data curation: Weidong Zhang.
Formal analysis: Weidong Zhang, Guangju Zhai.
Funding acquisition: Guangju Zhai.
Investigation: Weidong Zhang, Guangju Zhai.
Resources: Guangju Zhai.
Supervision: Guangju Zhai.
Validation: Edward W. Randell, Guang Sun, Guangju Zhai.
Writing ± original draft: Weidong Zhang.
Writing ± review & editing: Edward W. Randell, Guang Sun, Sergei Likhodii, Ming Liu,
Andrew Furey, Guangju Zhai.
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