Non-invasive measurement of skin autofluorescence as a beneficial surrogate marker for atherosclerosis in patients with type 2 diabetes. 

Sample Size: 68 patients with Type II Diabetes. 

Important methods: AGE accumulation was determined via a skin autofluorescence (AF) reader. Carotid intima-media thickness (max-IMT) was also collected using B-mode ultrasonography. Other measurements include: HbA1c, GA, pentosidine, age, and duration of diabetes. 

Statistical Analysis: Mann-Whitney U, Chi-squared, Spearman’s analysis, stepwise regression. For all, p<0.05. 

Study Type: Cross-sectional 

Results: Max-IMT was significantly correlated to age and AF. AF was significantly correlated to age and duration of diabetes diagnosis as well. Neither IMT nor AF were correlated to HbA1c, GA, or pentosidine. The stepwise regression showed that AF was also an independent determinant of Max-IMT. 

Conclusion: Skin autofluorescence maybe a used as a marker to non-invasively assess carotid atherosclerosis in Type II diabetics. 

Temma J, Matsuhisa M, Horie T, Kuroda A, Mori H, Tamaki M, Endo I, Aihara K, Abe M, Matsumoto T. Non-invasive Measurement of Skin Autofluorescence as a Beneficial Surrogate Marker for Atherosclerosis in Patients with Type 2 Diabetes. J Med Invest. 2015;62(3-4):126-9. doi: 10.2152/jmi.62.126. PMID: 26399335.

Autoimmune response to advanced glycosylation end-products of human LDL. 

Impact factor: 4.505 

Level of evidence: IV

Sample Size: 13 patients with Type I DM sera samples with purified AGE-LDL and lipoprotein antigen-antibody complexes were studied. 

Protocol: Blood was collected after a 12 hour fast and LDL was isolated and analyzed for content of (carboxymethyl)lysine (CML) and (carboxyethyl)lysine (CEL). AGE-LDL antibodies were isolated from the blood samples and loaded onto a Sepharose that was coupled to AGE-LDL. AGE-LDL antibody activity was measured by EIA. The immunoglobulin isotypes were determined by measuring IgG, IgA, and IgM by radial immunodiffusion (RID). The differences with unabsorbed and absorbed aliquots by EIA were considered indicative of the reactivity of the antibodies with the different LDL preparations. AGE-Antibody content was measured.

Statistical Analysis: Unpaired student T-test with Welch correction to compare specificity of AGE-LDL antibodies. Mann-Whitney test to compare CML-LDL and AGE-LDL reactivity. 

Study Type: In vitro. 

Results: The differences in the reactivity after absorption of the antibodies with AGE-LDL were significantly different from those observed after absorption with oxLDL (p=0.009) and native LDL (0.006). The differences in reactivity after absorbance of antibodies with CML-LDL and AGE-LDL were not significantly different.

Conclusion: Diabetics are at high risk of forming immune-complexes involving modified lipoproteins because chronic hyperglycemia leads to protein glycosylation. AGE-modified proteins have proven to be immunogenic and autoantibodies to these AGE-modified proteins has been found when studying AGE-Albumin, and it is thought that this could also happen to LDL that has been modified. IgG1 and IgG3 were the predominant isotype found in the plasma. AGE-LDL antibodies have a higher avidity for LDL than oxLDL antibodies, and this shows that may form stable immunocomplexes that can activate inflammatory cells.

Virella G, Thorpe SR, Alderson NL, Stephan EM, Atchley D, Wagner F, Lopes-Virella MF; DCCT/EDIC Research Group. Autoimmune response to advanced glycosylation end-products of human LDL. J Lipid Res. 2003 Mar;44(3):487-93. doi: 10.1194/jlr.M200370-JLR200. Epub 2002 Dec 1. PMID: 12562876.

Vascular effects of advanced glycation end-products: content of immunohistochemically detected AGEs in radial artery samples as a predictor for arterial calcification and cardiovascular risk in asymptomatic patients with chronic kidney disease.

Impact Factor: 2.137

Level of Evidence: III

Sample Size: 54 CKD patients (33 hemodialyzed, 21 predialyzed). 

Protocol: Examined parameters included BMI, incidence of diabetes, plasma fasting glucose, AGEs, soluble receptor for AGEs and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, serum C-reactive protein (hsCRP), plasminogen activator inhibitor-1 (PAI-1), and fetuin-A

Statistical Analysis: Shapiro-Wilk test for normality. 

Study Type: Experiment-based article. 

Results: Vascular content of AGEs was positively correlated with BMI, hsCRP, fetuin-A, PAI-1, and DPPH. Only fetuin-A was an independent predictor in multiple regression. There was a significant positive trend in the intensity of AGEs immunostaining among patients with grades 1, 2, and 3 calcifications, which predicted 3-year cardiovascular mortality irrespective of patient's age.

Discussion/Conclusion: The results of the study show a correlation between AGEs in the development of medial arterial calcification which can, in turn, impact the arterial AGE deposition on cardiovascular mortality in CKD patients. The intensity of AGE deposition in arterial media significantly predicted not only the advancement of medial arterial calcification but also cardiovascular mortality in CKD patients. 

Skin autofluorescence: a tool to identify type 2 diabetic patients at risk for developing microvascular complications

Impact factor: 16.019 

Evidence level: IV 

Type of study and any information related to it: Cohort  

Sample size: 881 

Important methods: An arm scanning autofluorescence reader was used to measure AGE levels in the blood. 

Statistical tests ran: One-way ANOVA with Bonferroni correction. p<0.05 was considered significant.  

Results: Kkin autofluorescence was higher in the patient groups who developed neuropathy or (micro)albuminuria compared with that in patients without these complications. At follow-up, newly developed neuropathy was diagnosed in 7.5% and newly developed (micro)albuminuria in 10.1% of patients; 12.5% of the population developed at least one microvascular complication. Skin autofluorescence at baseline was also significantly higher in the patient groups that developed any microvascular complication or who already had a microvascular complication at baseline compared with patients who did not develop any microvascular disease. Skin autofluorescence was significantly associated with the development of retinopathy (1.42 [1.01–1.99], P = 0.042), neuropathy (1.59 [1.15–2.19], P = 0.005), and (micro)albuminuria (1.73 [1.28–2.34], P < 0.001).  

Discussion/Conclusion: This study provides evidence that measurements of AGE levels using skin autofluorescence is a useful predictor of type 2 diabetes-related complications, including neuropathy, microvascular complications, and microalbuminuria.  Some of the benefits of this tool include ease of use, cost-effectiveness, and time-saving characteristics. Limitations to the study include a predominantly (95%) Caucasian cohort—advances in the AGE arm scanner technology aim to provide more accurate measurements in individuals with darker skin. Secondly, non-fluorescing AGE compounds will not be measured by the arm scanner, and other compounds that fluoresce similarly to AGE compounds will be measured by the arm scanner potentially leading to confounders. 

Gerrits EG, Lutgers HL, Kleefstra N, et al. Skin autofluorescence: a tool to identify type 2 diabetic patients at risk for developing microvascular complications. Diabetes Care. 2008;31(3):517-521. doi:10.2337/dc07-1755

Long-term physical activity leads to a significant increase in serum sRAGE levels: a sign of decreased AGE-mediated inflammation due to physical activity?

Impact factor: 1.618 

Level of Evidence: Level IV 

Study type: Prospective 

Study Sample size: 109 recruited and 98 completed the study 

Intro: Low levels of soluble receptor advanced glycation product (sRAGE) could be a predictor marker for cardiovascular disease (CVD). The goal of the study is to investigate the influence of 8 months of increased physical activity on serum sRAGE levels.   

Methods: Blood samples for the determination of sRAGE were taken at baseline, after 2, 6, and 8 months and analyzed by ELISA (enzyme-linked immunosorbent assay)   

Statistical Analysis: Spearman’s rho analysis, Backwards, multiple linear regression analysis for co-variables, Friedman test, all tests were performed in accordance with two-sided testing and p values ≤ 0.05 were considered significant.   

Results: The study shows that sRAGE serum level is inversely related with lipoprotein levels and BMI. Long-term physical activity leads to significant increase serum sRAGE levels. sRAGE increase was observed to be most significant in participants with initially low level of physical activity who have undergone increased physical activity throughout the experiment.   

Conclusion: The sports-mediated increase of sRAGE might be a sign of decreased AGE-mediated inflammation and highlight the protective effect of sports on CVD and other diseases which are at least partly mediated by an increased inflammation status. 

Sponder, M., Campean, I. A., Emich, M., Fritzer-Szekeres, M., Litschauer, B., Graf, S., Dalos, D., & Strametz-Juranek, J. (2018). Long-term physical activity leads to a significant increase in serum sRAGE levels: a sign of decreased AGE-mediated inflammation due to physical activity?. Heart and vessels, 33(8), 893–900. https://doi.org/10.1007/s00380-018-1125-5  

The association between skin auto-fluorescence of palmoplantar sites and microvascular complications in Asian patients with type 2 diabetes mellitus

Impact factor: 3.998 

Level of evidence: IV 

Type of study: in vivo, cross sectional 

Sample size: 166 Korean patients with type 2 diabetes 

Methods: Reflected skin auto-fluorescence (SAF) on the volar side of the forearms and transmitted SAF in the 1st dorsal interosseous muscle of the hand were measured with a currently developed multi-geometry optical system. The SAF emitted by the excitation beam and transmitted reference optical signals were collected by a multimode optical fiber on the opposite side of the muscle.   

Statistical analysis:  Among all T2DM subjects, a chi-square test was used for sex difference and a Mann-Whitney test was used for difference in DM duration between positive and negative subjects. All other statistical comparisons were made with an unpaired student’s t-test. One-way analysis of the variance test was used to compare SAF values varying with the number of associated complications. Pearson’s and Spearman’s correlation coefficients were calculated to assess correlation between quantitative and qualitative variables. Significance was defined as P < 0.05. 

Results: Complication group had significantly higher SAF values of palmoplantar sites and 1st dorsal interossei muscles of the hand than did the non-complication group. No differences were observed between the 2 groups in reflected SAF of non-palmoplantar sites. The transmitted SAF values of palmoplantar sites were increased in subjects with multiple complications and were tightly correlated with the duration of microvascular complications. 

Conclusion: SAF measurement in the palmoplantar sites with a non-invasive transmission-geometry optical system provided better microvascular complication screening performance compared to the SAF measurement of non-palmoplantar sites, particularly in Asian T2DM subjects. Palmoplantar SAF levels measured with a transmission-geometry optical system were tightly associated with duration and severity of microvascular complications in Korean patients with T2DM. Potentially, this new SAF detection method could be used as a monitoring method for microvascular complications, even in patients with darker skin tones. 

Kim JJ, Jeong B, Cho Y, Kwon MH, Lee YH, Kang U, Kang ES. The association between skin auto-fluorescence of palmoplantar sites and microvascular complications in Asian patients with type 2 diabetes mellitus. Sci Rep. 2018 Apr 20;8(1):6309. doi: 10.1038/s41598-018-24707-2. PMID: 29679014; PMCID: PMC5910431.

Advanced glycation end products (AGEs) in relation to atherosclerotic lipid profiles in middle-aged and elderly diabetic patients 

Impact factor: 2.922 

Level of evidence: IV 

Type of study: in vivo, cross sectional 

Sample size: 207 T2DM patients and 174 diabetic patients with proteinuria; all patients were ethnically Han Chinese 

Methods: Atherosclerotic profiles (total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)) were measured using standard methods. The following ratios were also calculated in order to determine significance of atherosclerosis: TC/HDL-C, LDL-C/HDL-C and TG/HDL-C. Calculating the total atherosclerotic index (AI) of each patient was on a point basis (TC ≤ 200 = 1 point, 200 < TC ≤ 240 = 2 points, TC > 240 = 3 points; TG ≤ 150 = 1 point, 150 < TG ≤ 200 = 2 points, TG > 200 = 3 points; HDL-C ≥ 50 = 1 point, 50 > HDL-C ≥ 40 = 2 points, HDL-C < 40 = 3 points; LDL-C ≤ 130 = 1 point, 130 < LDL-C ≤ 160 = 2 points, LDL-C > 160 = 3 points). AGEs were measured via enzyme-linked immunoassay method. 

Statistical analysis: t-test was used to assess the differences between the 2 groups. Spearman correlation coefficients were calculated for fasting glucose (GLU), glycated hemoglobin A1c (A1c), and AGEs in relation to atherosclerotic parameters. Multi-variable regression analyses were performed to examine the association between AGEs and atherosclerotic profiles after adjusting for potential confounders (age and BMI) and adjusting for GLU and A1c. Significance was defined as p < 0.05. 

Results: Females had higher AGEs, A1C, TC, HDL-C and LDL-C levels than males. Males had higher TC/HDL-C, LDL-C/HDL-C, and TG/HDL-C ratios compared to females. In both groups, AGEs levels were more positively correlated with TG and TC/LDL-C, LDL-C/HDL-C and TG/HDL-C ratios and negatively correlated with HDL-C levels. After adjusting for age, BMI, GLU and A1c levels, AGEs were positively associated with TC, TG, TC/HDL-C, LDL-C/HDL-C, TG/HDL-C and AI, and negatively associated with HDL-C levels in both sexes. 

Conclusion: AGEs were positively correlated with lipid profiles and atherosclerotic characteristics in both sexes, even after adjusting for age, BMI, GLU and/or A1c and other confounders. The patients with higher AGEs levels had worse lipid profiles, more adverse atherosclerotic characteristics, and higher AI. There was a stronger association between AGEs and lipid profiles and AI in the diabetic nephropathy group compared to the diabetic group. This could possibly be attributed to the fact that patients with diabetic nephropathies may have more serious illness and higher AGEs levels, making them more prone to atherosclerosis. AGEs could be a strong biomarker for evaluating the association between diabetes and atherosclerotic disorders in diabetic patients. 

Chang, JB., Chu, NF., Syu, JT. et al. Advanced glycation end products (AGEs) in relation to atherosclerotic lipid profiles in middle-aged and elderly diabetic patients. Lipids Health Dis 10, 228 (2011). https://doi.org/10.1186/1476-511X-10-228

Skin Autofluorescence is associated with Early-stage Atherosclerosis in Patients with Type 1 Diabetes

Impact factor: 3.876 

Level of evidence: IV 

Summary: The risk of atherosclerosis in Type One Diabetics and its tie to advanced end glycation products was determined by the skin autofluorescence. The study was done in Japan on 105 patients; 34 males and 71 women. They determined the amount of atherosclerosis by ultrasonic carotid intima-media thickness. The statistical analysis was done by a Pearson’s test. The results of the study showed that Type One Diabetic patients who had significantly higher skin autofluorescence ( p value of 0.001) had a much higher risk of atherosclerosis.  

Osawa S, Katakami N, Kuroda A, Takahara M, Sakamoto F, Kawamori D, Matsuoka T, Matsuhisa M, Shimomura I. Skin Autofluorescence is Associated with Early-stage Atherosclerosis in Patients with Type 1 Diabetes. J Atheroscler Thromb. 2017 Mar 1;24(3):312-326. doi: 10.5551/jat.35592. Epub 2016 Sep 2. PMID: 27592627; PMCID: PMC5383547.

Association between Fluorescent Advanced Glycation End-Products and Vascular Complications in Type 2 Diabetic Patients 

Impact factor: 2.276 

Level: I 

Summary: A clinical trial was done on diabetic patients to determine the correlation of oxidative plasma markers (including advanced end glycation products) and increased association of vascular complications. The study had 75 patients with poorly controlled type 2 diabetes (HbA1C over 7.5%) and 31 non-diabetic patients with no evidence of vascular disease. They used the level of fluorescence to determine the level of AGE’s. The results of this study showed that glycation products were significantly increased in the diabetic patients when compared to non diabetic patients (P value of 0.001, box and whisker). This provides evidence to monitor type 2 diabetic patients for the level of AGE’s to predict vascular events.  

Guerin-Dubourg A, Cournot M, Planesse C, Debussche X, Meilhac O, Rondeau P, Bourdon E. Association between Fluorescent Advanced Glycation End-Products and Vascular Complications in Type 2 Diabetic Patients. Biomed Res Int. 2017;2017:7989180. doi: 10.1155/2017/7989180. Epub 2017 Dec 6. PMID: 29362717; PMCID: PMC5736945.

Advanced Glycation End Products, Oxidation Products, and the Extent of Atherosclerosis During the VA Diabetes Trial and Follow-up Study

Impact Factor: 16.019 

Level: IV 

Summary: A study was done on 411 type 2 diabetic patients to determine the role of advanced end glycation products in atherosclerosis. Each of the patients was followed for ten years and scanned by CT of the coronary artery to determine the calcification levels. The study found that AGEs did affect the atherosclerosis of the arteries (p value of 0.02; box and whisker). With this information they were able to conclude that the AGEs play an important role in the development of sclerosed arteries. 

Saremi A, Howell S, Schwenke DC, Bahn G, Beisswenger PJ, Reaven PD; VADT Investigators. Advanced Glycation End Products, Oxidation Products, and the Extent of Atherosclerosis During the VA Diabetes Trial and Follow-up Study. Diabetes Care. 2017 Apr;40(4):591-598. doi: 10.2337/dc16-1875. Epub 2017 Feb 1. PMID: 28148544; PMCID: PMC5360279.

Role of advanced glycation end products with oxidative stress in resistance artery dysfunction in type 2 diabetic mice

Impact Factor: 6.604 

Level: IV 

Summary: A study was done on type two diabetic mice and nondiabetic mice over the course of three months to determine the role of advanced end glycation products in arterial resistance. They measured arterial resistance by arteriography and looked specifically at the mesenteric arteries. They found that the pressure induced myogenic tone was significantly increased in diabetic mice compared to nondiabetic mice ( p value 0.05). With this information the study was able to conclude that advanced end glycation products contributed to diabetic arterial complication and dysfunction.  

Su J, Lucchesi PA, Gonzalez-Villalobos RA, Palen DI, Rezk BM, Suzuki Y, Boulares HA, Matrougui K. Role of advanced glycation end products with oxidative stress in resistance artery dysfunction in type 2 diabetic mice. Arterioscler Thromb Vasc Biol. 2008 Aug;28(8):1432-8. doi: 10.1161/ATVBAHA.108.167205. Epub 2008 May 15. PMID: 18483403; PMCID: PMC2755261. 

Serum Levels of Advanced Glycation End Products Are Associated with In-Stent Restenosis in Diabetic Patients 

Impact Factor: 3.0 

Type of Study: Cross Sectional In Vivo Study  

Sample Size: 263   

Detailed Summary: In diabetic patients, the presence of AGEs within the body has been seen to cause inflammatory reactions. This study focused on the relationship of Advanced Age Glycation products and the formation of retinostensosis in patients who were diabetic. This study had taken blood samples from patients who had diabetes and who had the implantation of stents. The SAF fluorescence was used to measure the amount of AGEs that were in the patient's body. An angiogram was used to see the state of the stent that was placed in the body. Using multivariate analysis, the researchers were able to see that the amount of stent restenosis was much higher in patients who had a higher amount of serum AGEs in their blood. This may be significant when it comes to the pathology of diabetes and the other conditions that diabetes can manifest into. A clinician may be able to assess the risk of the patient developing an in-stent restenosis by measuring the serum AGE levels beforehand and use that information upon making decisions later in the treatment process. 

Rigo M, Lecocq M, Brouzeng C, Michelet M, Mohammedi K, Blanco L, Poupon P, Haissaguerre M, Monlun M, Foussard N, Larroumet A, Devouge AC, Ducos C, Bataglini Q, Liébart M, Rigalleau V. Skin autofluorescence, a marker of glucose memory in type 2 diabetes. Metabol Open. 2020 May 25;7:100038. doi: 10.1016/j.metop.2020.100038. PMID: 32812941; PMCID: PMC7424807.