Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study.

OBJECTIVES: Type 1 diabetes is a risk factor for coronary heart disease. The underlying mechanism behind the accelerated atherosclerosis formation is not fully understood but may be related to the formation of oxidation products and advanced glycation end-products (AGEs). We aimed to examine the associations between the collagen oxidation product methionine sulfoxide; the collagen AGEs methylglyoxal hydroimidazolone (MG-H1), glucosepane, pentosidine, glucuronidine/LW-1; and serum receptors for AGE (RAGE) with measures of coronary artery disease in patients with long-term type 1 diabetes.
METHODS: In this cross-sectional study, 99 participants with type 1 diabetes of ≥ 45-year duration and 63 controls without diabetes had either established coronary heart disease (CHD) or underwent Computed Tomography Coronary Angiography (CTCA) measuring total, calcified and soft/mixed plaque volume. Skin collagen methionine sulfoxide and AGEs were measured by liquid chromatography-mass spectrometry and serum sRAGE/esRAGE by ELISA.
RESULTS: In the diabetes group, low levels of methionine sulfoxide (adjusted for age, sex and mean HbA1c) were associated with normal coronary arteries, OR 0.48 (95% CI 0.27-0.88). Glucuronidine/LW-1 was associated with established CHD, OR 2.0 (1.16-3.49). MG-H1 and glucuronidine/LW-1 correlated with calcified plaque volume (r = 0.23-0.28, p<0.05), while pentosidine correlated with soft/mixed plaque volume (r = 0.29, p = 0.008), also in the adjusted analysis.
CONCLUSIONS: Low levels of collagen-bound methionine sulfoxide were associated with normal coronary arteries while glucuronidine/LW-1 was positively associated with established CHD in long-term type 1 diabetes, suggesting a role for metabolic and oxidative stress in the formation of atherosclerosis in diabetes.

1. Introduction

Patients with long-term type 1 diabetes have a high prevalence of coronary artery disease (CAD). The accelerated coronary atherosclerosis seen in diabetes is more severe and diffuse than in people without diabetes [1-3]. However, some people have normal coronary arteries after 50 years of type 1 diabetes as we have recently reported [4].

Why some people with long-term type 1 diabetes remain free of CAD is not known, but one theory by which hyperglycemia may lead to CAD is partly through the formation of oxidation products and advanced glycation end-products (AGEs) resulting in structural and functional alterations in the arterial wall [1]. High mean HbA1c level is a risk factor for future cardiovascular events [5, 6], and rodent studies suggest that reactive oxygen species (ROS) and carbonyl intermediates in AGEs formation play an essential role in linking hyperglycemia to cardiovascular disease [7, 8].

ROS are partially reduced unstable oxygen metabolites that easily react with other molecules in a cell. The oxidation product methionine sulfoxide (MetSO) can be formed through ROS-mediated protein methionine oxidation. This reaction may cause important structural and functional disruptions to the target protein, and it has been suggested that protein methionine oxidation may have a pathogenic role in vascular disease [9]. The collagen-bound MetSO has previously been linked to vascular complications in type 1 diabetes [10, 11].

AGEs are a heterogeneous group of compounds formed through several steps including the non-enzymatic reaction between a reducing sugar/reactive dicarbonyl and an amino group. Collagen-bound methylglyoxal hydroimidazolone 1 (MG-H1), pentosidine and glucuronidine/LW-1 are all examples of AGEs that have been linked with future subclinical macrovascular disease [12].

Previous studies in type 1 diabetes examining the associations between collagen oxidation products and AGEs with cardiovascular disease are based on either the presence of previous cardiovascular events or surrogate markers for CAD such as measurement of the carotid intima media thickness or coronary artery calcification score (CAC). Computer Tomography Coronary Angiography (CTCA) is a non-invasive technique that has the advantage of being able to differentiate between absence of CAD and gradually worsening degree of coronary atherosclerosis. The plaque morphology identified also provides valuable information [13]. The associations between MetSO and AGEs with CAD and plaque morphology as defined by CTCA in type 1 diabetes have not been reported previously.

We therefore aimed to explore associations between the collagen-bound oxidation product MetSO, four specific collagen-bound AGEs and serum receptors for AGE (RAGEs) with normal, plaque-free, coronary arteries on CTCA, and with measures of coronary artery disease including plaque morphology on CTCA and established coronary heart disease (CHD) in patients with long-term type 1 diabetes compared to controls.

2. Subjects, materials and methods

2.1 Study design and subjects

The Dialong study was a cross-sectional study of long-term survivors of type 1 diabetes conducted in 2015, including patients with type 1 diabetes for ≥ 45 years and an appropriate control group of similar age and sex distribution. The inclusion criteria have previously been set out in full [14]. Briefly, we invited all patients with type 1 diabetes diagnosed ≤ 1970 attending a state-funded specialized type 1 diabetes clinic in Oslo, Norway. Out of 136 eligible patients, 103 joined the CAD sub-study. The control group (n = 63) without diabetes consisted of spouses/friends of the patients with diabetes, and was included in the same time-period. First-degree relatives were excluded. The Norwegian Regional Committees for Medical and Health Research Ethics—South East (project no. 2014/851) approved the study, and all participants signed an informed consent.

2.2 Procedures

Background data were collected from patient charts at NDC, interviews and clinical examination [14]. All participants had a skin punch biopsy for measurements of collagen-linked AGEs and MetSO, fasting morning blood and urine samples, and retinal photos. The participants without established CHD (n = 88/103 in the diabetes group and n = 60/63 of controls) were referred to CTCA as previously described [4].

2.3 Outcomes

CTCA was performed on a Dual Source CT scanner (Somatom Definition Flash, Siemens, Erlangen, Germany), and the details for the investigation have been previously described [4].

Normal coronary arteries were defined as no plaque in any of the coronary arteries on CTCA. Non-obstructive CAD was defined as plaques resulting in a 1–50% diameter stenosis in any of the coronary arteries but none > 50%, and obstructive CAD as > 50% stenosis in any of the coronary arteries. Established CHD was defined as a previous episode of acute coronary syndrome or revascularization procedure, prior to enrolment in the study.

The volume (mm3) of all plaques in each patient was calculated and differentiated in categories based on plaque morphology; total plaque volume, calcified plaque volume and soft/mixed plaque volume. The plaque morphology was determined in each plaque by the amount of calcifications (i.e. density > 130 Hounsfield units) present. A plaque containing more than 90% calcifications was defined as a calcified plaque and 0–90% as a soft/mixed plaque [15].

CAC was calculated according to the Agatston method [15, 16].

Retinopathy was defined as either background or proliferative retinopathy changes based on retina photos (wide-angle camera based on SLO-technique (Optos Daytona)), which were all analyzed by one certified ophthalmologist [14].

2.4 Skin collagen AGEs and MetSO and skin autofluorescence

A 3 mm skin punch biopsy was obtained from each participant from the upper lateral part of the nates. The sample was immediately placed in a sterile container, flushed with nitrogen gas and transferred to a temperature of -80 degrees Celsius. Skin collagen bound MetSO and AGEs (glucosepane, pentosidine, glucuronidine/fluorophore LW-1, and MG-H1)were measured using liquid chromatography tandem mass spectrometry (LC/MS/MS) as previously described [14]. Briefly, samples of 50 ug insoluble delipidated skin collagen samples were exhaustively digested with proteolytic enzymes to release the free AGEs. The samples were spiked with isotopically labeled internal standards for each AGE and quantitated by LC/MS/MS. Each chromatography peak was individually analyzed in reference to the labeled peak. Collagen content was determined using the hydroxyproline assay as previously described [17]. Data are expressed in pmol AGE/mg collagen. The analyst was blinded regarding group affiliation. All participants also had skin intrinsic fluorescence measured non-invasively in the forearm by the autofluorescence reader (AGE reader) [18].

2.5 Serum sRAGE and esRAGE

Fasting blood samples without additives were separated within 1 hour by centrifugation at 2500xg for 10 min, and serum was transferred to a temperature of—80°C until analysis. Serum sRAGE and esRAGE were quantified using enzyme-linked immunosorbent assay (ELISA) kits (Quantikine ELISA, R&D Systems, Abingdon, Oxon, UK and B-Bridge International Inc., Cupertino, CA, USA respectively) as directed by the manufacturer’s instructions. Again, the analyst was blinded regarding group affiliation. The inter-assay coefficients of variation in our laboratory (at Oslo University Hospital, Ulleval) were 5.8% (sRAGE) and 5.7% (esRAGE).

2.6 Statistical analysis

The power analysis suggested we needed 77 participants with diabetes to detect a significant difference in glucosepane levels between patients with type 1 diabetes with obstructive CAD and patients with normal coronary arteries (power 90%, probability 0.05). This was based on a distribution in diabetic patients of 30%, 40% and 30% with absent CAD, non-obstructive CAD and obstructive CAD respectively and our own previous analysis showing mean glucosepane levels from the Oslo study to be 500 pmol/L higher in the group with obstructive CAD versus absent CAD [19, 20]. While our power analysis was based on only participants with obstructive disease and normal arteries, we decided to keep all the patients with non-obstructive CAD (60%) in our analyses to avoid loss of power. Cases with missing data were excluded, and we did a complete case analysis.

Clinical characteristics, CTCA findings and MetSO/AGEs/RAGEs levels were compared between the groups using two-tailed Student’s t-test or Mann Whitney U test for continuous data as appropriate and χ2 for categorical data. For bivariate outcomes, logistic regression analyses were performed to adjust for possible confounders (age, sex, and mean HbA1c). Statin use was not included in the analyses as all participants with established CHD were expected to be on statins. Standardized values for MetSO and the AGEs were calculated. We performed Spearman correlation analyses to assess for correlations between the markers and the plaque volume measures, and linear regression analyses to adjust for confounders. We assessed for collinearity between variables and only included variables with r < 0.7 in the same model. Any dependent variable that did not have normally distributed residuals, were natural log (ln)-transformed. A p-value ≤ 0.05 was considered significant. All analyses were performed using IBM SPSS Statistics version 25 (IBM SPSS Inc., Armonk, NY: IBM Corp.).

3. Results

3.1 Clinical characteristics

Table 1 shows the clinical characteristics, the CTCA findings and the AGEs, MetSO and RAGE levels in the diabetes and control groups. Four of the participants in the diabetes group did not have a punch biopsy performed (due to needle phobia and previous allergic reaction to local anesthesia) and were excluded from the analyses leaving a total of 99 participants with diabetes with complete data (4% missing data). Fifteen participants in the diabetes group had established CHD, and the remaining 84 completed the CTCA.

Table 1

Participant characteristics.

	Type 1 diabetes (n = 99)	Controls (n = 63)	P
Demographics/risk factors
Age	62.1 ± 7.1	62.7 ± 7.0	0.64
Sex, male	51 (51.5)	28 (44.4)	0.38
Daily smoker	4 (4)	6 (9.5)	0.16
LDL-cholesterol, mmol/L	2.7 (0.8)	3.8 (1.0)	< 0.001
Blood pressure systolic	146 ± 20	138 ± 20	0.012
diastolic	75 ± 8	81 ± 10	< 0.001
eGFRa	84 ± 19	82 ± 14	0.36
CRP, mg/L, median (IQR)	1.7 (0.8–3.5)	1.2 (0.6–2.6)	0.04
Diabetes related factors
EFD HbA1c %, mmol/mol	8.0 ± 0.8
	63.5 ± 8.6
Diabetes duration, years median (IQR)	49 (47–54)
Persistent albuminuria	17 (17.2)
Retinopathy None	5 (5.1)
Background	50 (50.5)
Proliferative	44 (44.4)
Neuropathy	63 (63.6)	11 (17.5)	< 0.001
Presence of CAD
Normal coronary arteries on CTCA	14 (14.1)	30 (47.6)	< 0.001
Non-obstructive CAD on CTCA	50 (50.5)	24 (38.1)
Obstructive CAD on CTCA	20 (20.2)	6 (9.5)
Established CHD	15 (15.2)	3 (4.8)	0.04
CTCA findings	n = 84	n = 60
Total plaque volume, mm3, median (IQR)	29.5 (3.90–95.8)	0.40 (0.0–7.4)	< 0.001
Calcified plaque volume, mm3, median (IQR)	20.8 (1.0–66.5)	0.15 (0.0–7.1)	< 0.001
Soft/mixed plaque volume, mm3, median (IQR)	0.0 (0.0–8.68)	0.0 (0.0–0.0)	0.001
CAC, Agatston units, median, (IQR)	124 (8–534)	1 (0–39)	< 0.001
MetSO, AGEs (pmol/mg) and RAGE
MetSO	61 ± 8	58 ± 9	0.020
Glucosepane	6480 ± 1254	3409 ± 607	< 0.001
Pentosidine	30 ± 10	18 ± 6	< 0.001
Glucuronidine/LW-1	1045 ± 645	461 ± 319	< 0.001
MG-H1	445 ± 193	299 ± 137	< 0.001
Skin autofluorescence arbitrary units	2.8 ± 0.53	2.2 ± 0.48	< 0.001
sRAGE pg/ml	1752 ± 973	1553 ± 541	0.14
esRAGE ng/ml	0.34 ± 0.29	0.28 ± 0.11	0.05

Data are mean ± SD or n (%) unless otherwise stated. There were 99 participants in the diabetes group and 63 participants in the control group, except for the CTCA findings as only the participants without established CHD were referred to CTCA. There were no missing data. CAD, coronary artery disease; IQR, inter-quartile range.

aestimated glomerular filtration rate calculated by MDRD formula

As previously described, the diabetes and control groups were of similar age and sex distribution, and the diabetes group had lower LDL-cholesterol levels, higher systolic blood pressure and lower diastolic blood pressure than the control group [4]. Five percent in the diabetes group had no retinopathy. The diabetes group had a significantly lower rate of normal coronary arteries than the control group (14.1% versus 47.6%) and a significantly higher rate of established CHD (15.2% versus 4.8%) (Table 1). CTCA also showed significantly higher rates of all plaque volume measures in the diabetes group compared to the control group (p≤ 0.001). All skin AGEs, MetSO, skin autofluorescence and the esRAGE were significantly higher in the diabetes group than in the control group (p ≤ 0.05 to < 0.0001) (Table 1 and S1 and S2 Figs).

3.2 Collagen MetSO, AGEs and serum RAGEs

In the diabetes group, all skin AGEs correlated with one another (r = 0.43–0.66, p < 0.001). MetSO correlated with MG-H1 (r = 0.42 p < 0.001) and glucosepane (r = 0.25, p = 0.02). Unlike MetSO and sRAGE/esRAGE, all four AGEs correlated with age (r = 0.25–0.48, p < 0.02) (Table 2). sRAGE and esRAGE correlated with eGFR (r = -.18, p = 0.02 and r = -0.26, p = 0.001 respectively). The other products did not correlate with eGFR, and there were no significant correlations with sex or disease duration. Among all markers, only skin glucosepane correlated significantly with mean HbA1c (r = 0.32, p = 0.003). In the control group, there was a significant correlation between glucosepane and pentosidine with age (r = 0.36 and 0.44, respectively, both p < 0.01), but not with HbA1c-levels.

Table 2

Coefficients of correlation (Spearman’s rho) in the diabetes group.

	M/SPV	CPV	CAC	age	Sex	Mean HbA1c	MetSO	MG-H1	glpane	pentos	LW-1	sRAGE	esRAGE
TPV	.342**	.893**	.901**	.368**	.326**	.172	.191	.243*	.185	.148	.268*	.035	.025
M/SPV	-	.002	.170	.106	.275*	.241*	.047	.126	.123	.291**	.214	-.067	-.110
CPV	-	-	.918**	.486**	.223*	.060	.169	.276*	.179	.131	.225*	.054	.056
CAC		-	-	.443**	.246*	.076	.233*	.307**	.159	.145	.256*	.154	.133
Age	-	-	-	-	-0.026	-.112	.182	.480**	.249*	.362**	.253*	.122	.196
Mean HbA1c	-	-	-	-	-	-	.144	.120	.318*	.055	.087	-.083	-.121
MetSO	-	-	-	-	-	-	-	.416**	.248*	.044	.023
MG-H1	-	-	-	-	-	-	-	-	.661**	.615**	.434**
Glpane	-	-	-	-	-	-	-	-	-	.648**	.580**
Pentos	-	-	-	-	-	-	-	-	-	-	.593**

* P < 0.05 (2-tailed).

**p < 0.01 (2-tailed).

TPV, Total plaque volume; M/SPV, Mixed/soft plaque volume; CPV, Calcified plaque volume; CAC, Coronary artery calcification score. MetSO, methionine sulfoxide; MG-H1, methylglyoxal hydroimidazolone; glpane, glucosepane; pentos, pentosidine; LW-1, glucuronidine/LW-1

3.3 Associations of collagen markers with CAD

In the diabetes group, the participants with normal coronary arteries had lower levels of MetSO compared to the participants with any degree of CAD (mean ± SD 55.7 ± 9.6 versus 62.4 ± 7.5, respectively, p = 0.003) (Figs 1 and 2). This association remained significant when adjusting for age, sex and mean HbA1c (OR per 1SD increase in MetSO (0.48 (95% CI 0.27–0.88), p = 0.02). None of the AGEs were significantly associated with having normal coronary arteries.

Fig 1

Association between levels of MetSO and having normal coronary arteries.

Scatter dot plot with line at median with interquartile range. MetSO, methionine sulfoxide. *p = 0.02 compared to “No”. Adjusted for age, sex and mean HbA1c.

Fig 2

Associations of MetSO, Glucuronidine /LW-1, MG-H1 and skin autofluorescence levels with categories of CAD in the diabetes group.

Concentrations with standard errors of the mean of: 2a, MetSO; 2b, Glucuronidine /LW-1; 2c, MG-H1; and 2d, skin autofluorescence with CAD categorized as normal, non-obstructive CAD, obstructive CAD and established CHD in the diabetes group. MetSO, methionine sulfoxide; MG-H1, methylglyoxal hydroimidazolone; CAD, coronary artery disease. CHD, coronary heart disease. * P < 0.02 versus the other three categories after adjusting for age, sex and mean HbA1c.

In the diabetes group, glucuronidine/LW-1 and skin autofluorescence were significantly higher in the group with established CHD compared to the participants without CHD entering the study. The mean ± SD glucuronidine/LW-1 levels in the patients with established CHD was 1495 pmol/mg ± 650 versus 965 pmol/mg ± 614, p = 0.003 in the participants without established CHD, and similarly for skin autofluorescence; 3.1 ± 0.66 versus 2.7 ± 0.48 (arbitrary units), p = 0.006. In the adjusted analyses (age, sex, mean HbA1c) only glucuronidine/LW-1 (OR per 1SD increase in glucuronidine/LW-1 2.0 (95% CI 1.2–3.5), p = 0.01) was significantly associated with established CHD (Fig 2b). We also found that MG-H1 (p = 0.03) and MetSO (p = 0.01) were associated with retinopathy (background or proliferative) in the diabetes group in univariate analyses. In the adjusted analysis, the ORadj for retinopathy per 1SD increase in MetSO was 3.2 (95% CI 1.1–9.0). The association with MG-H1 did not reach statistical significance in the adjusted analysis (p = 0.06).

In the diabetes group, MG-H1 and glucuronidine/LW-1 correlated with both total plaque volume and calcified plaque volume (all p < 0.05) (Table 2/Fig 3b and 3c), however when controlling for age and sex, none of the associations remained statistically significant. Only pentosidine correlated with soft/mixed plaque volume, and this association remained significant after controlling for age, sex and mean HbA1c (βln 0.06 (95% C.I. 0.02–0.09, p = 0.002). Glucosepane was not associated with any of the measures of CAD, and neither serum sRAGE nor esRAGE significantly correlated to any of the CAD measures (Table 2).

Fig 3

Associations of pentosidine, MG-H1 and Glucuronidine /LW-1 with plaque volume in the diabetes group.

Scatterplots with linear regression lines and 95% confidence bands showing correlations between a, pentosidine and soft/mixed plaque volume; b, MG-H1 and calcified plaque volume; and c, glucuronidine/LW-1 and total plaque volume in the diabetes group. All correlations had a 2-tailed significance level at p < 0.05. The association between pentosidine and soft/mixed plaque volume was the only significant association after adjusting for age, sex and HbA1c. MG-H1, methylglyoxal hydroimidazolone.

When performing the same analyses in the control group, we had the following significant findings: Pentosidine correlated with total plaque volume, also when adjusting for age and sex (βln 0.09 (0.03–0.15, p = 0.007). Pentosidine also correlated with calcified plaque volume in the adjusted analysis (βln 0.07 (0.01–0.13, p = 0.02). Skin autofluorescence correlated with total plaque volume only in the unadjusted analysis (r = 0.264, p = 0.04).

4. Discussion

In the present study cohort, CTCA allowed us to perform a detailed assessment of the coronary arteries, including plaque morphology. We demonstrated an association between low levels of the oxidation product MetSO and having normal coronary arteries in patients with long-term type 1 diabetes. We also showed an association between glucuronidine/LW1 with established CHD and with total plaque volume, in particular calcified plaques; however the correlation in the latter analysis was not significant when adjusting for age and sex. Pentosidine was associated with soft/mixed plaque volume, also in the adjusted analyses. In the control group, pentosidine was associated with total plaque volume, but there were no associations between MetSO and markers of CAD.

As expected, some patients with long-term type 1 diabetes remain free of CAD [4]. In the present study, we demonstrate that these patients have less accumulation of the collagen bound oxidation product MetSO in skin. This finding suggests that either oxidative collagen damage is associated with coronary atherosclerosis in type 1 diabetes or that MetSO is a marker of increased oxidative stress in the vascular bed. Protein methionine oxidation seems to have a role in vascular disease, and oxidation-sensitive methionine residues have been found in several proteins that are important in vascular biology, such as Apolipoprotein A-1, thrombomodulin and von Willebrand factor with resultant impaired or hyperactive protein function. [9] Interestingly, we found that low levels of collagen MetSO were not associated with having normal coronary arteries in the control group. However, we cannot exclude a type II error.

It is tempting to attribute the association between MetSO and CAD only in the participants with diabetes to chronic long-term hyperglycemia. Hyperglycemia increases the production of ROS in cultured bovine aortic endothelial cells, and the importance of oxidative stress in the formation of coronary atherosclerosis in diabetes has previously been identified in mechanistic studies in rodents [7, 21]. However, even though the half-life of skin collagen is about 15 years in individuals without diabetes [22], collagen bound MetSO did not correlate with mean 30-year HbA1c in our study. A reduced antioxidant defense in diabetes is supported by the significant association with pentosidine, the oxidation product of ascorbic acid. Alternatively, the impaired ability to respond to oxidative stress might explain why some patients with diabetes are more prone to developing coronary atherosclerosis [23].

No previous studies have reported on the association between collagen MetSO and detailed measures of coronary atherosclerosis assessed by CTCA. However, a study of 96 patients with type 1 diabetes showed an association between collagen MetSO and being prone to microvascular and macrovascular complications combined [10]. Also, the Oslo study demonstrated an association between collagen MetSO and arterial stiffness [11]. Conversely, a recent study showed a negative association between circulating MetSO levels and the incidence of cardiovascular events in type 2 diabetes [24]. Thus, there seemingly is paradoxical evidence in that circulating MetSO is negatively associated with cardiovascular disease while collagen accumulated MetSO is positively associated with cardiovascular disease. Data suggest that methionine-residues on the surface of proteins can act as endogenous antioxidants through red-ox reactions (methionine sulfoxide reductase), whereas methionine-residues that are buried in the proteins are less prone to oxidation and more prone to being inactivated following oxidation. As such, oxidation of collagen methionine may be more likely to result in functional alterations of the protein, and collagen methionine sulfoxide could thus be seen as a marker of cumulative oxidative tissue damage [25]. In that regard, the diabetes participants who were free of retinopathy also had lower collagen MetSO levels underlining the importance of this marker in long-term complications.

A number of studies examining the association between AGEs and vascular complications do not specify the different AGEs, but look upon them as a whole. Rather, AGEs are a heterogeneous group, and the individual products most likely exhibit somewhat different pathogenic effects. Also, several of the studies linking AGE products to cardiovascular disease are on circulating AGEs [26-28]. Regarding collagen AGEs, Monnier et al. identified collagen glucuronidine/LW-1, pentosidine and MG-H1 to be the most important AGE markers for future subclinical cardiovascular disease, measured as carotid intima media thickness [12]. Previous studies have demonstrated that AGEs increase with age and duration [29], and indeed, the levels of AGEs in our study were higher than in previous studies with younger participants, exemplified by glucosepane (the most abundant AGE) which had a mean level of 6480 pmol ± 1254 in the diabetes group in our study compared to up to 4000 pmol/mg in patients with diabetes at age 30–40 years [17]. In the present study, we found that pentosidine and mean HbA1c correlated with soft/mixed plaques while MG-H1 and glucuronidine/LW-1 correlated with calcified plaques (the latter two in unadjusted analyses). Calcifications are common in progressive atherosclerotic lesions and the coronary artery calcification burden is greater in type 1 diabetes than in healthy controls [3, 30]. Eighty-three percent of plaques measured in our population with diabetes were calcified [15]. Although not as prone to rupture as soft plaques, calcified plaques/CAC are clinically important due to an association with a higher risk of future coronary events [31].

Glucuronidine/LW-1 was associated with both established CHD and calcified plaques in our study signifying a role in advanced stages of CAD. Recently, partial structure elucidation of glucuronidine/LW-1 revealed that it contains a glucuronide, suggesting a glucuronidine/LW-1 precursor is glucuronidated in the liver or kidney to form a circulating reactive glucuronide precursor that glycates collagen [32]. While the structure of the precursor is yet unknown, our findings strengthen previous reports of research that glucuronidine/LW-1 is associated with microvascular and macrovascular complications in type 1 diabetes [32, 33]. However, more research is needed into the process of glucuronidation and how this may be linked to complications in diabetes. Conceivably, excess glucuronidation of vital neutriceuticals could weaken antioxidant defenses and account for faster vascular complication progression in selected individuals [32, 34].

Skin autofluorescence has previously been suggested to be associated with both subclinical and clinical atherosclerosis independent of diabetes [35]. While skin autofluorescence was associated with total plaque volume only in unadjusted analyses in the control group, it was associated with established CHD in the diabetes group, again signifying a role in advanced stages of the disease.

In the present study, pentosidine was associated with soft/mixed plaque content, but not with calcified plaques. As ascorbic acid is a precursor for pentosidine-formation, this could suggest that increased ascorbic acid oxidation correlates with higher volume of soft/mixed plaque [36, 37]. The soft (non-calcified) lipid-laden plaques are important as they are more vulnerable to rupture causing cardiac events and will also respond better to intensified medical treatment [30, 38]. Higher levels of circulating pentosidine have previously been associated with both incident fatal and non-fatal cardiovascular events and CAC in type 1 diabetes [27, 28]. The latter was not found in the present study. Our results add to the evidence that pentosidine is an important marker of CAD in type 1 diabetes.

Overall, the only similar findings in the diabetes and control groups were that pentosidine correlated with the total plaque volume in the control group and with mixed/soft plaque volume in the diabetes group. Measures of serum AGE levels, mainly reflecting N-(Carboxymethyl)lysine and to a smaller degree pentosidine, have previously been linked to severity of CAD on coronary angiography in participants without diabetes. However, the association between collagen pentosidine with measures of coronary atherosclerosis in persons without diabetes has to our knowledge, not been previously reported [39]. HbA1c did not correlate with pentosidine or with measures of coronary atherosclerosis in the control group. This indicates that the association between pentosidine and coronary plaques in the control group is at best indirectly related to hyperglycemia.

The present study was a cross-sectional study with its inherent limitations, and we can therefore not conclude that MetSO or the specific AGEs represent a cause or are a result of CAD. We did not find any significant associations between glucosepane and measures of CAD, on which the power analysis was based. Nevertheless, the power analysis was performed before data from the DCCT/EDIC study emerged, which identified other collagen AGEs (namely MG-H1, pentosidine and glucuronidine/LW-1) to be associated with measures of macrovascular disease [12]. Hence, while our findings must be interpreted as exploratory, they are in line with the data from the DCCT/EDIC study. The study is statistically relatively small and may be underpowered to detect weaker correlations. The control group were mostly spouses of the participants with diabetes, thereby having a similar lifestyle and diet, perhaps with similar intake of exogenous AGEs, which could possibly influence the total collagen AGE levels [40]. Still, all the AGEs were about twice the level in the diabetes group than the control group signifying the importance of hyperglycemia in endogenous AGE formation. Our biopsies were taken from skin collagen, and we do not know whether skin collagen is a good surrogate tissue for coronary arteries. However, based on previous observations that collagen glycation is more strongly correlated with long-term microvascular complications than hemoglobin glycation [12], accumulation of MetSO in long-lived skin collagen is expected to be a better reflection of arterial oxidative processes and damage than by similar processes in serum proteins. A further limitation is that we only have CTCA scans of the participants without established CHD who are selected from a survivor population. Hence, the correlations with plaque volume are therefore based on a population that probably has less CAD than the general type 1 diabetes population, possibly underestimating the effects of AGEs. Our participants with diabetes are from a single center, however as previously described, they had a similar HbA1c as the Norwegian population registered with the same duration [14]. Strengths of this study are a unique study population with very long-term type 1 diabetes with detailed measures of coronary atherosclerosis and chronic hyperglycaemia, a high inclusion rate of the eligible cohort and the presence of a control group.

5. Conclusions

In conclusion, we found an association between low levels of MetSO and normal coronary arteries on CTCA in long-term type 1 diabetes. There was also an association between high glucuronidine/LW-1 levels and advanced stages of CAD, i.e. established CHD and calcified plaques. Additionally, while MG-H1 and glucuronidine/LW-1 correlated with total and calcified plaque volume in univariate analyses, pentosidine correlated with soft/mixed plaques. Our results strengthen the knowledge of the importance of metabolic and oxidative stress on vascular cells in the formation of atherosclerosis in diabetes. Future research on the metabolic and oxidative damage on coronary arteries may reveal pharmaceutical targets that can decrease the risk of CAD in type 1 diabetes.

Scatter plots with linear regression lines showing the age-related accumulation of methionine sulfoxide (MetSO), glucuronidine/LW-1, methylglyoxal hydroimidazolone (MG-H1), pentosidine, glucosepane and skin autofluorescence in the type 1 diabetes group in red and controls in blue.

(PDF)

Click here for additional data file.

Scatter plots with linear regression lines showing the age-related accumulation of sRAGE og esRAGE in the type 1 diabetes group in red and controls in blue.

(PDF)

Click here for additional data file.

12 Feb 2020

PONE-D-20-00205

Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study.

PLOS ONE

Dear Dr. Holte,

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Ming-Chang Chiang

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Dr. Holte,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: In this cross-sectional study, the authors investigated the associations between the collagen oxidation products and advanced glycation end-products (AGEs) with measures of coronary artery diseases in individuals with type 1 diabetes with the duration of ≥ 45-years and controls. They found that Low levels of collagen-bound methionine sulfoxide were associated with normal coronary arteries while glucuronidine/LW-1 was positively associated with established CHD in long-term type 1 diabetes. The authors conclude that the dysregulation of metabolism and oxidative stress on vascular cells could be of importance in the formation of atherosclerosis in type 1 diabetes.

STRENGTHS:

(1) The unique study population with very long-term type 1 diabetes.

(2) Data on atherosclerosis in the long-standing T1D in populations are scarce.

COMMENTS AND QUESTIONS:

(1) Table 1. Have the authors measured other atherosclerosis risk factors, e.g. CRP? The subgroups of "Diabetes group" and "Controls without diabetes" may mislead the readers. I suggest the use of T1D instead of Diabetes. Are there eventual missing data? If yes, please specify it in the table note.

(2) Figure 2b. The axis title looks strange. I suggest inverting it.

Reviewer #2: The manuscript with a title of “Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery 2 disease in long-term survivors with type 1 diabetes. The Dialong study”. The authors have investigated the correlation between methionine sulfoxide and the collagen AGEs with coronary heart disease. There were more than 160 participants where HPLC and ELISA were used to measure the methionine sulfoxide and the collagen AGEs respectively. 99 participants of type 1 diabetes have coronary heart disease (CHD) or underwent Computed Tomography Coronary Angiography (CTCA). The novelty of this paper is mainly the correlation between MetSO and CAD and remaining correlations have been previously reported. L 427 says “Strengths of this study are a unique study population with very long-term type 1 diabetes with detailed measures of coronary atherosclerosis and chronic hyperglycaemia, a high inclusion rate of the eligible cohort and the presence of a control group”. Based on the that I believe this paper lacks novelty in the method and data section however the study population is unique although the small population used in this study! I recommend this work to be published in PLOSONE after addressing the below comments.

1. Add details for the MetSO, AGE’s and RAGE’s analysis method!

2. How the AGE’s in skin, RAGE’s in plasma are normalised?

3. L230: “all skin AGEs correlated with one another …” although this was found, only pentosidine correlation was found! Can author explain that!

4. L253: It seems that values of MetSO don’t match with table 1 - Can authors review explain?

5. L276: It seems that the glucuronidine/LW-1 and skin autofluorescence values don’t match with table 1 - Can authors review explain?

6. L349 “Thus, there seemingly is 349 paradoxical evidence in that circulating MetSO is negatively associated with cardiovascular 350 disease while collagen accumulated MetSO is positively associated with cardiovascular 351 disease” is it possible this applies for other AGE’s which will affect the correlation suggested by the authors?

7. L384 “In the present study, pentosidine was associated with soft/mixed plaque content, but 385 not with calcified plaques” the correlation between the plaque content with the pentosidine in skin are not direct because the circulating pentosidine was not measured? Can author elaborate in that?

8. Values in table 1, text and figures must be reviewed machining sure they all match.

9. L38 remove extra bracket.

**********

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Reviewer #1: No

Reviewer #2: No

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27 Mar 2020

We thank the academic editor and both the reviewers for thorough reviews of our manuscript and for the excellent comments. Please find the comments and our responses/action below.

Academic Editor’s comments and our responses:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We have reviewed PLOS ONE’s style requirements.

The name of the files have been changed/updated.

2. Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

We have changed the wording of our ethics statement on page 5: “The Norwegian Regional Committees for Medical and Health Research Ethics - South East (project no. 2014/851) approved the study, and all participants signed an informed consent. “

3. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

We believe there are ethical and legal restrictions on sharing the dataset as outlined in the cover letter.

An ethically compliant dataset may be provided by the authors who can be contacted through dr Elisabeth Qvigstad at eqwigsta@ous-hf.no / elisabeth.qvigstad@medisin.uio.no.

Reviewer #1: In this cross-sectional study, the authors investigated the associations between the collagen oxidation products and advanced glycation end-products (AGEs) with measures of coronary artery diseases in individuals with type 1 diabetes with the duration of ≥ 45-years and controls. They found that Low levels of collagen-bound methionine sulfoxide were associated with normal coronary arteries while glucuronidine/LW-1 was positively associated with established CHD in long-term type 1 diabetes. The authors conclude that the dysregulation of metabolism and oxidative stress on vascular cells could be of importance in the formation of atherosclerosis in type 1 diabetes.

STRENGTHS:
(1) The unique study population with very long-term type 1 diabetes.
(2) Data on atherosclerosis in the long-standing T1D in populations are scarce.

Reviewer #1 comments and our responses:

(1) Table 1.

-Have the authors measured other atherosclerosis risk factors, e.g. CRP?

We have measured CRP which is also an interesting factor. It was higher in the T1D group (median 1.7 vs 1.2), but it did not correlate with any of our outcomes in the diabetes group.

We have added the median CRP levels to table 1.

-The subgroups of "Diabetes group" and "Controls without diabetes" may mislead the readers. I suggest the use of T1D instead of Diabetes.

Thank you for pointing this out

We have changed the subgroups to “Type 1 diabetes” and “Controls”

-Are there eventual missing data? If yes, please specify it in the table note.

Thank you for this important point. All participants were interviewed, examined, had skin biopsies taken, blood tests drawn and had either CTCA or had known CHD. There were no missing data.

We have added to the table note: “There were no missing data”

(2) Figure 2b. The axis title looks strange. I suggest inverting it.

We agree that the title of figure 2b should be inverted.

The title now reads “Glucuronidine/LW-1”. We have also changed the label of the y-axis from pmol/mg to pmol/mg collagen” for Fig. 2a-2c.

Reviewer #2: The manuscript with a title of “Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery 2 disease in long-term survivors with type 1 diabetes. The Dialong study”. The authors have investigated the correlation between methionine sulfoxide and the collagen AGEs with coronary heart disease. There were more than 160 participants where HPLC and ELISA were used to measure the methionine sulfoxide and the collagen AGEs respectively. 99 participants of type 1 diabetes have coronary heart disease (CHD) or underwent Computed Tomography Coronary Angiography (CTCA). The novelty of this paper is mainly the correlation between MetSO and CAD and remaining correlations have been previously reported. L 427 says “Strengths of this study are a unique study population with very long-term type 1 diabetes with detailed measures of coronary atherosclerosis and chronic hyperglycaemia, a high inclusion rate of the eligible cohort and the presence of a control group”. Based on the that I believe this paper lacks novelty in the method and data section however the study population is unique although the small population used in this study! I recommend this work to be published in PLOSONE after addressing the below comments.

Reviewer #2 comments and our responses:

1. Add details for the MetSO, AGE’s and RAGE’s analysis method!

Thank you for pointing this out. We agree that we can provide more details on the analysis method.

We have changed/added the following details to page 7:

Paragraph 2.4: “Skin collagen bound MetSO and AGEs (glucosepane, pentosidine, glucuronidine/fluorophore LW-1, and MG-H1) were measured using liquid chromatography tandem mass spectrometry (LC/MS/MS) as previously described (14). Briefly samples of 50 ug insoluble delipidated skin collagen samples were exhaustively digested with proteolytic enzymes to release the free AGEs. The samples were spiked with isotopically labeled internal standards for each AGE and quantitated by LC/MS/MS. Each chromatography peak was individually analyzed in reference to the labeled peak. Collagen content was determined using the hydroxyproline assay as previously described (17). Data are expressed in pmol AGE/mg collagen.”

Paragraph 2.5: “Serum sRAGE and esRAGE were quantified using enzyme-linked immunosorbent assay (ELISA) kits (Quantikine ELISA, R&D Systems, Abingdon, Oxon, UK and B-Bridge International Inc., Cupertino, CA, USA respectively) as directed by the manufacturer’s instructions”.

2. How the AGE’s in skin, RAGE’s in plasma are normalised?

Raw data are displayed in each figure. No normalization was performed, but data were statistically adjusted for various variables as described in the legends to the Tables and Figures.

3. L230: “all skin AGEs correlated with one another …” although this was found, only pentosidine correlation was found! Can author explain that!

Thank you for this interesting point. To further elaborate on this, we have added the full Spearman correlation coefficients for AGE to AGE comparison in the bottom part of Table 2.

AGEs are all raised in patients with diabetes, but they are a heterogenous group of compounds. For example, previous data suggest that glucosepane and MG-H1 are more strongly associated with microvascular disease and MG-H1 and LW-1 are more strongly associated with macrovascular disease (Ref 12). In our paper, LW-1/glucuronidine was associated with established CHD. Also, MG-H1 and LW-1/glucuronidine were associated with more advanced (calcified) plaques in unadjusted analyses. On the other hand, pentosidine was associated with soft/mixed plaque volume, also in the adjusted analysis. As further detailed in the paper, LW-1/glucuronidine seems to be a glucuronidation product whereas collagen pentosidine could reflect increased ascorbic acid oxidation. Hence, these AGEs could reflect different pathological processes in the formation of coronary plaques.

See Table 2.

4. L253: It seems that values of MetSO don’t match with table 1 - Can authors review explain?

Table 1 compares the value of MetSO in subjects with and without type 1 diabetes while L253 describes the level of MetSo only in participants with diabetes where the levels of MetSO in those with normal coronary arteries are compared to the levels of MetSO in those with any degree of CAD.

5. L276: It seems that the glucuronidine/LW-1 and skin autofluorescence values don’t match with table 1 - Can authors review explain?

Similarly, table 1 compares the value of glucuronidine/LW-1 in subjects with and without type 1 diabetes while L276 describes the level of glucuronidine/LW-1 only in participants with diabetes with established CHD versus those without established CHD.

6. L349 “Thus, there seemingly is 349 paradoxical evidence in that circulating MetSO is negatively associated with cardiovascular 350 disease while collagen accumulated MetSO is positively associated with cardiovascular 351 disease” is it possible this applies for other AGE’s which will affect the correlation suggested by the authors?

Thank you for this interesting comment. Collagen-linked MetSO represents a cumulative measure of oxidative damage to tissue over several years, while, as we suggest in the paper, methionine-residues on the surface of proteins (circulating MetSO) can act as endogenous antioxidants through red-ox reactions (ref 24).

The relationship between collagen and circulating AGEs would be expected to be different. Further, the same inverse relationship has not been demonstrated with collagen and circulating AGEs. Indeed, while we found a positive correlation between collagen pentosidine and soft/mixed plaque volume, higher levels of circulating pentosidine have previously been associated with cardiovascular events (ref 26).

It would be interesting to study the association between collagen and circulating AGEs, but this was beyond the scope of our study.

7. L384 “In the present study, pentosidine was associated with soft/mixed plaque content, but 385 not with calcified plaques” the correlation between the plaque content with the pentosidine in skin are not direct because the circulating pentosidine was not measured? Can author elaborate in that?

Thank you for this interesting point. We have not measured serum pentosidine, but previous studies have found an association between circulating pentosidine and both incident CVD events and CAC in type 1 diabetes (ref 26 and 27).

It would have been interesting to have biopsies taken from the coronary arteries, and we do not know whether skin collagen AGE levels accurately represent collagen AGE levels in the vessel wall of the coronary arteries.

As skin collagen has a long half-life (ref 21), serum AGE levels and collagen AGE levels would be expected to differ and perhaps be different representations of pathological processes. Pentosidine is in all likelihood a marker of Vitamin C mishandling by the cell. Thus, elevated skin pentosidine may mean that chronic oxidative stress results in elevated levels of dehydroascorbate, i.e. the precursors of pentosidine. However, skin pentosidine can also form from Amadori product of glucose representing a cumulative form of glycemic insult. At this time, we do not have means to differentiate between ascorbic acid and glucose-derived pentosidine.

8. Values in table 1, text and figures must be reviewed machining sure they all match.

We have reviewed all the values in the tables, figures and texts to make sure they all match

In paragraph 3.3, we changed the p-value from 0.017 to 0.02 to match the number of decimals given in figure 1 (p=0.02).

9. L38 remove extra bracket.

Thank you

We have removed the extra bracket.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

3 Apr 2020

PONE-D-20-00205R1

Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study.

PLOS ONE

Dear Dr. Holte,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by May 18 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Ming-Chang Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

In general, findings presented here are novel, experiments seem to be well done, and paper describes an interesting topic. However, there are some major points that complicate acceptance of the paper in the present format.

The editor makes the following suggestions:

1. Provide fluorescence images of Fig 2.

2. Provide protein levels of RAGE.

3. The discussion should be improved correlating these results with the literature.

4. The manuscript is not linked to current conversations in the journal.

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

27 Apr 2020

We thank the editor for the thorough review of our manuscript and excellent comments. Please find our comments and changes to the manuscript below.

1. Provide fluorescence images of Fig 2.

Thank you for this suggestion. Figure 2 demonstrates the associations of a, MetSO, b, Glucuronidine/LW-1 c, MG-H1 and d, skin autofluorescence (measured using the AGE reader®) with categories of coronary artery disease in the diabetes group. MetSO, glucuronidine/LW-1 and MG-H1 were measured using LC-MS/MS. We agree that we could provide more raw data on the distribution of the levels of the variables, and we have now created scattergrams of the data versus age in both the diabetes and control groups as a supplement (Fig 1S).

2. Provide protein levels of RAGE.

Thank you for this suggestion. We measured sRAGE and esRAGE in serum using ELISA. They are expressed in pg/ml (Table 1). We are also providing the scattergram of these data in Fig 2S.

3. The discussion should be improved correlating these results with the literature.

We have added the following to the discussion: “Previous studies have demonstrated that AGEs increase with age and duration (29), and indeed, the levels of AGEs in our study were higher than in previous studies with younger participants, exemplified by glucosepane (the most abundant AGE) which had a mean level of 6480 pmol ± 1254 in the diabetes group in our study compared to up to 4000 pmol/mg in patients with diabetes at age 30–40 years (17).”

4. The manuscript is not linked to current conversations in the journal.

We have added the following to the discussion which refers to a study by den Dekker MA et al, PLoS One 2013: “Skin autofluorescence have previously been suggested to be associated with both subclinical and clinical atherosclerosis independent of diabetes (35). While skin autofluorescence was associated with total plaque volume only in unadjusted analyses in the control group, it was associated with established CHD in the diabetes group, again signifying a role in advanced stages of the disease.”

Submitted filename: Response to reviewers.docx

Click here for additional data file.

30 Apr 2020

Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study.

PONE-D-20-00205R2

Dear Dr. Holte,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Ming-Chang Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors complied with the experimental suggestions that now improve the experimental understanding and text. In the end I believe that the suggested modifications have been met making the work acceptable for publication.

Reviewers' comments:

4 May 2020

PONE-D-20-00205R2

Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study.

Dear Dr. Holte:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

With kind regards,

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on behalf of

Dr. Ming-Chang Chiang

Academic Editor

PLOS ONE