Advanced Glycation End Products Are Associated with Diabetes Status and Physical Functions in Patients with Cardiovascular Disease

Advanced glycated end products (AGEs) accumulate systemically and cause diabetes complications. However, whether noninvasive measurable AGEs are associated with diabetes status and physical functions remains unclear. One hundred and ten patients with cardiovascular disease (CVD) who underwent outpatient cardiac rehabilitation were included. AGEs scores, using AGEs sensors, were evaluated concomitantly with a physical evaluation, including testing the isometric knee extension strength (IKES) and 6 min walking distance (6MWD). Thirty-three (30%) patients had a history of diabetes mellitus (DM). The AGEs score was not different in the presence of DM history (0.52 ± 0.09 vs. 0.51 ± 0.09, p = 0.768) and was not correlated with blood glucose (r = 0.001, p = 0.995). The AGEs score was positively correlated with hemoglobin A1c (HbA1c, r = 0.288, p = 0.004) and negatively correlated with physical functions (IKES, r = −0.243, p = 0.011; 6MWD, r = −0.298, p = 0.002). The multivariate analysis demonstrated that 6MWD was independently associated with a high AGEs score (>0.52). The AGEs score was associated with HbA1c, IKES, and 6MWD in patients with CVD. The AGEs score might be a useful indicator for evaluating not only glycemic control but also physical functions.


Introduction
Diabetes mellitus (DM) affects all-cause mortality in patients with cardiovascular disease (CVD) [1]. DM is an important factor in the secondary prevention and management of CVD. When hyperglycemia continues, serum advanced glycation end-products (AGEs) are produced during glucose metabolism [2]. Serum AGEs induce oxidative stress, which causes nitric oxide inactivation, inflammatory responses, thrombus formation, and progression of arteriosclerosis [3,4]. The accumulation of AGEs leads to the development of CVD. AGEs were evaluated by using a noninvasive physical method that uses the forearm and as a blood sampling test [5]. Skin autofluorescence (sAF) measured using the forearm was strongly correlated with AGEs from skin biopsy [6], although sAF did not match either serum or urine AGEs [7]. This sAF was associated with the duration of DM [8][9][10] and hemoglobin A1c [6,8,11]. Moreover, sAF was associated not only with DM but also with physical functions, including muscle strength and exercise capacity [11]. Furthermore, skin AGEs in patients undergoing cardiac rehabilitation are predictive of all-cause mortality and hospitalization for heart failure [12]. A meta-analysis reported that sAF levels measured by the forearm might be useful in assessing mortality risk in patients with CVD [13]. The most important aspect of CVD management is the noninvasive assessment of AGEs. Yamanaka et al. developed an AGEs sensor that can easily and quickly evaluate sAF by using a fingertip [14]. Several studies that used this new device showed that the AGEs score measured by the AGEs sensor was associated with serum AGEs [14] levels and glycation stress [15]. However, the relationship between AGEs score and clinical characteristics, including DM, has not been completely evaluated. Furthermore, the relationship between AGEs score and physical functions remains unclear. Thus, the purpose of this study was to (1) investigate the relationship between AGEs score and DM, (2) evaluate the relationship between AGEs score and glycemic control, and (3) assess the relationship between AGEs score and physical functions in patients with CVD.

Study Population
We conducted a single-center retrospective observational study between August 2020 and November 2021 at the cardiac rehabilitation center of Kitasato University Kitasato Institute Hospital. We enrolled 149 patients with CVD who underwent cardiac rehabilitation. CVD diagnosis included ischemic heart disease (myocardial infarction, angina pectoris, and vasospastic angina), heart failure, valvular heart disease, and atrial fibrillation. After excluding patients aged < 65 years (n = 33) and those who had difficulty measuring physical functions due to a decline in cognitive function or orthopedic disease (n = 6), 110 patients were finally included in the study (Figure 1). The Kitasato Institute Hospital Research Ethics Committee approved the study protocol (clinical trial registration number 21028).
Nutrients 2022, 14, x FOR PEER REVIEW 2 of 12 uses the forearm and as a blood sampling test [5]. Skin autofluorescence (sAF) measured using the forearm was strongly correlated with AGEs from skin biopsy [6], although sAF did not match either serum or urine AGEs [7]. This sAF was associated with the duration of DM [8][9][10] and hemoglobin A1c [6,8,11]. Moreover, sAF was associated not only with DM but also with physical functions, including muscle strength and exercise capacity [11]. Furthermore, skin AGEs in patients undergoing cardiac rehabilitation are predictive of all-cause mortality and hospitalization for heart failure [12]. A meta-analysis reported that sAF levels measured by the forearm might be useful in assessing mortality risk in patients with CVD [13]. The most important aspect of CVD management is the noninvasive assessment of AGEs. Yamanaka et al. developed an AGEs sensor that can easily and quickly evaluate sAF by using a fingertip [14]. Several studies that used this new device showed that the AGEs score measured by the AGEs sensor was associated with serum AGEs [14] levels and glycation stress [15]. However, the relationship between AGEs score and clinical characteristics, including DM, has not been completely evaluated. Furthermore, the relationship between AGEs score and physical functions remains unclear. Thus, the purpose of this study was to (1) investigate the relationship between AGEs score and DM, (2) evaluate the relationship between AGEs score and glycemic control, and (3) assess the relationship between AGEs score and physical functions in patients with CVD.

Study Population
We conducted a single-center retrospective observational study between August 2020 and November 2021 at the cardiac rehabilitation center of Kitasato University Kitasato Institute Hospital. We enrolled 149 patients with CVD who underwent cardiac rehabilitation. CVD diagnosis included ischemic heart disease (myocardial infarction, angina pectoris, and vasospastic angina), heart failure, valvular heart disease, and atrial fibrillation. After excluding patients aged < 65 years (n = 33) and those who had difficulty measuring physical functions due to a decline in cognitive function or orthopedic disease (n = 6), 110 patients were finally included in the study (Figure 1). The Kitasato Institute Hospital Research Ethics Committee approved the study protocol (clinical trial registration number 21028).

Assessment of AGEs Score
To estimate the AGEs score, cardiac rehabilitation measurements of sAF levels were performed by using an AGEs sensor (SHARP, Kobe, Japan). AGEs have the property of emitting fluorescence when the specific excitation light irradiates them. The AGEs sensor irradiates the fingertips with excitation light, acquires percutaneous fluorescence of the fingertips, and measures skin autofluorescence [4]. The sAF levels were measured by using the middle finger of the left hand, in which the least amount of skin melanin was present [14]. We performed sAF measurements twice before cardiac rehabilitation and used the mean values for the analysis. The measured AGEs were expressed as the AGEs score in arbitrary units with an upper limit of 10.0 and a lower limit of 0.0. According to a recent manufacturer survey, 0.5 is an arbitrary unit that approximately corresponds to the average score of healthy Japanese patients aged 50 years. The AGEs sensor displayed a value when the coefficient of variation was less than 1%. A previous study has demonstrated that AGEs sensor is useful for the noninvasive assessment of glycation stress [15].

Assessment of Physical Functions
We evaluated handgrip strength (HGS), isometric knee extension strength (IKES), and 6 min walking distance (6MWD) as physical functions and used a dynamometer to measure HGS (TKK 5401; Takei, Tokyo, Japan). The patients performed two maximal isometric voluntary contractions of both hands for 3 s each while seated on a bench with the elbow flexed at 90 • . The width of the dynamometer handle was adjusted for each patient to match their hand size. The highest strength measurement (kg) was used for the analysis. IKES was measured by using a handheld dynamometer to determine leg strength (µ-Tas; ANIMA, Tokyo, Japan). Briefly, with the patient seated in a chair with a non-extensible strap connecting the ankle to a strain gauge, 5 s of maximal isometric voluntary contractions of the quadriceps was collected twice for both legs, with the hip joint at approximately 90 • flexion. Consecutive measurements were obtained for the right and left quadriceps muscles. The highest strength values on the right or left side were expressed as absolute values (kg) and relative to the body mass (%BM). The 6MWD was determined according to the guidelines of the American Thoracic Society, under the supervision of technicians. The patients were instructed to walk at their own pace along a straight, flat hallway from chair to chair, and the distance (in meters) was recorded after six minutes.

Definition
Hypertension was defined as an arterial blood pressure of ≥140/90 mmHg or the use of antihypertensive medication. Dyslipidemia (DL) was defined as low-density lipoprotein cholesterol ≥ 140 mg/dL, triglyceride ≥ 150 mg/dL, or the use of medication for DL. DM was defined as symptoms of diabetes plus random plasma glucose concentration ≥ 200 mg/dL, fasting plasma glucose concentration ≥ 126 mg/dL, or use of medication for DM. AGEs score, physical function, laboratory data, and clinical information were obtained within 2 weeks.

Statistical Analysis
Continuous variables with normal distribution were expressed as mean ± standard deviation (SD), whereas the median value with interquartile range was reported when the data were not normally distributed. The basic characteristics of patients with or without DM were compared. We analyzed the correlation between the AGEs score and patient characteristics. Continuous variables were analyzed by using a t-test. Categorical variables were reported as counts (%) and analyzed by using the chi-squared test. Multivariate regression analysis was performed to identify the factors associated with the presence of high AGEs score among variables with p < 0.050 in the univariate logistic regression analysis. Statistical significance was defined at p < 0.050. SPSS 27 version (IBM Corporation, Armonk, NY, USA) was used to perform all statistical analyses. Since related studies were limited, the cutoff value of the AGEs score is unknown. In this study, the AGEs score was normally distributed (Figure 2). We defined values above the median high as high AGEs score and below the median low as low AGEs score. Thus, the median AGEs score (0.52) was classified into two groups (high-and low-AGE patients) and subsequently analyzed. normally distributed ( Figure 2). We defined values above the median high as high AGEs score and below the median low as low AGEs score. Thus, the median AGEs score (0.52) was classified into two groups (high-and low-AGE patients) and subsequently analyzed.

Comparison of Clinical Characteristics between High and Low AGEs Score
The number of patients with a high and low AGEs score was fifty-three (48%) and fifty-seven (52%), respectively. Table 3 shows the comparison of clinical characteristics between the high AGE and low AGE score. All the clinical characteristics were similar between the two groups, except for HbA1c (6.4 ± 0.8 vs. 6.1 ± 0.7%, p = 0.044), HGS (21.1 ± 7.6 vs. 24.8 ± 8.8 kg, p = 0.023), IKES (36.5 ± 12.0 vs. 42.8 ± 13.5%BM, p = 0.013), and 6MWD

Physical Function and Presence of High AGEs Score
The univariate analysis showed that 6MWD (odds ratio (OR) 0.996; 95% confidence interval (CI) 0.993-0.999; p = 0.015) was significantly associated with the presence of a high AGEs score (>0.52) ( Table 4). The multivariate analysis demonstrated that the 6MWD was independently associated with a high AGEs score (>0.52) ( Table 5). Compared to the factors previously associated with AGEs [16][17][18], 6MWD was independently associated with a high AGEs score (Tables 6 and 7).

Discussion
The main findings of this study were as follows: (1) there was no significant difference in AGEs score of patients with or without DM; (2) the AGEs score was significantly correlated with HbA1c but not with blood glucose; and (3) the AGEs score was also significantly correlated with physical functions, including IKES and 6MWD. In particular, the 6MWD was independently associated with a high AGEs score (>0.52).

AGEs Score and DM
In this study, AGEs score was not associated with a history of DM. Our results corroborated the findings of a previous study, which reported that AGEs measured by the forearm were not associated with a history of DM [12]. AGEs are metabolites of blood glucose and may not directly reflect the history of DM. AGEs include a variety of substances, such as pentosidine, carboxymethyl lysine, and pyrraline, which are produced by metabolites of blood glucose [19]. AGEs measured in the forearm have been shown to correlate with pentosidine levels [8]. AGEs measured at the fingertip were correlated with methylglyoxal 5-hydro-5-methylimidazolones [15]. Both are metabolites of blood glucose and may reflect glucose metabolism, which is not directly related to the history of DM. Furthermore, AGEs are not necessarily related to blood glucose because they are produced not only from glucose but also from fructose and aldehydes [20,21]. In contrast, it has also been reported that patients with DM have higher serum AGEs than those without DM [22]. These differences might be caused by variations in glucose metabolism, DM treatment, and subsequent DM status. Thus, the relationship between the AGEs score and a history of DM remains controversial. This small number of studies indicates that further data accumulation is needed in the future.

AGEs Score and HbA1c
The AGEs score was significantly correlated with HbA1c and was not correlated with blood glucose levels in this study. Previously, the serum AGEs were also strongly associated with HbA1c [22] but were not associated with blood glucose levels [11]. This is because AGEs are metabolized relatively slowly over weeks to months [23], reflecting the status of glycemic control over the medium-to-long-term from the vein to the skin [24]. Furthermore, HbA1c is an Amadori rearrangement substance produced by the same process as that of AGEs [25]. Therefore, AGEs are thought to reflect mid-to-long-term glycemic control rather than current glycemic control. Our study showed that the AGEs score may be useful as an indicator of noninvasive glycemic control in patients with CVD.

AGEs Score and Physical Functions
In this study, the AGEs score was also significantly correlated with physical functions, including IKES and 6MWD. The 6MWD was independently associated with a high AGEs score (>0.52). Previous studies have shown that forearm AGEs are significantly associated with reduced exercise tolerance [11] and physical functions, such as handgrip strength [26] and walking speed [27]. Accumulated AGEs increase muscle stiffness, reduce the viscoelastic properties of muscles, and impair muscle function [28]. In endothelial cells, AGEs affect endothelial dysfunction and loss of muscle mass and strength [29]. Clinically, serum AGEs are associated with evaluated endothelial function by brachial flow-mediated vasodilation [30]. Similarly, AGEs decrease exercise tolerance in the myocardium by inducing myocardial stiffness and diastolic dysfunction [3,31]. Therefore, patients with high AGEs have weaker HGS, IKES, and 6MWD than those with lower AGEs score. Furthermore, high physical function reduces the accumulation of AGEs. Since muscles consume glucose, AGEs accumulate less because of high muscle strength and exercise tolerance [32]. Several studies have clarified the relationship between physical activity and AGEs [33,34]. It has been reported that patients with higher physical activity have lower forearm AGEs [33]. Furthermore, AGEs are influenced by lifestyle habits such as physical activity, sleeping time, and cognitive function [35]. Individuals with low physical activity have been reported to accumulate more AGEs than those with high physical activity [34]. As mentioned above, the accumulation of AGEs affects muscle strength and exercise tolerance, whereas decreased physical activity accelerates the accumulation of AGEs. However, the causal relationship between AGEs and physical function remains unclear. A recent study suggests that long-term exercise may be reduced AGEs [36]. Further longitudinal studies are required to elucidate the causal relationship between AGEs accumulation and physical functions.

Limitations
This study had several limitations. First, this was a retrospective and observational study conducted at a single center with a limited number of patients. Second, there was no control group without CVD, because we focused on patients with CVD. Therefore, the results should be interpreted cautiously. Third, there was no complete removal of potential confounding factors that might affect the AGEs score (e.g., temperature, time, and season). Fourth, the AGEs score index may vary among the CVD types. Fifth, the results might have changed if a different cutoff AGEs score was applied. Sixth, there were no data on the diagnosis of DM in these patients. Seventh, this study did not consider the effects of dietary guidance or lifestyle. Eighth, three were no data of blood, plasma, or serum AGEs concentration. Thus, the interpretation needs caution about the association between the AGEs score and the presence of DM history. Ninth, this study included limited patients with chronic heart failure (52%), ischemia heart disease who underwent percutaneous coronary intervention (28%), valvular disease (6%), and atrial fibrillation (30%) who were outpatients for cardiac rehabilitation. The other cardiovascular disease could not speculate the AGEs score by sAF. Lastly, the findings of this observational study did not clarify the causal relationship between AGEs score and DM.

Conclusions
The AGEs score was associated with HbA1c level and physical functions in patients with CVD. The AGEs score might be a useful indicator for evaluating not only glycemic control but also physical functions.