Skin autofluorescence in the general population: associations and prediction
van Waateringe, Robert Paul
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associations and prediction
Robert P. van Waateringe
Robert P. van Waateringe
Thesis, University of Groningen, The Netherlands ISBN: 978-94-6375-308-1
Cover design: Stijn Eikenaar, persoonlijkproefschrift.nl Lay-out: Stijn Eikenaar, persoonlijkproefschrift.nl Printing: Ridderprint BV | www.ridderprint.nl Copyright © 2019 Robert P. van Waateringe
All rights reserved. No part of this thesis may be reproduced, stored or transmitted in any way or by any means without the prior permission of the author, or when applicable, of the publishers of the scientific papers.
This work was supported by the BioSHaRE-EU project (Biobank Standardisation and Harmonisation for Research Excellence in the European Union) under grant agreement n 261433, receiving funds from the National Consortium for Healthy Ageing, and from the European Union’s Seventh Framework Program (FP7/2007-2013).
The LifeLines Cohort Study is supported by the Netherlands organization of Scientific Research NWO (grant 175.010.2007.006, the Ministry of Economic Affairs, the Ministry of Education, Culture and Science, the Ministry of Health, Welfare and Sports, the Northern Netherlands Collaboration of Provinces (SNN), the Province of Groningen, University Medical Center Groningen, University of Groningen.
Financial support for printing of this thesis was kindly provided by The Endocrinology Fund (as part of the Ubbo Emmius Fund, Graduate School of Medical Sciences, University Medical Center Groningen, University of Groningen.
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op maandag 13 mei 2019 om 12.45 uur
door
Robert Paul van Waateringe geboren op 9 juli 1987
Copromotor Dr. H.L. Lutgers
Beoordelingscommissie Prof. dr. J.A.M. Zeebregts Prof. dr. H.P. Hammes Prof. dr. C. Schalkwijk
Skin autofluorescence in the general population: associations and prediction 1. Hoewel bekend is dat het drinken van koffie de kans op het ontstaan van type 2
diabetes en hart- en vaatziekten vermindert, is hogere koffie consumptie wel geassocieerd met een hogere huid autofluorescentie waarde. (dit proefschrift) 2. Het duurt meer dan 10 jaar voordat de ongunstige effecten van roken op accumulatie
van AGEs zijn verdwenen. (dit proefschrift)
3. De huid autofluorescentie waarde is verhoogd in mensen met het metabool syndroom en neemt toe met het aantal individuele componenten. (dit proefschrift)
4. Bij mensen met het metabool syndroom geeft een grotere middelomtrek een hogere huid autofluorescentie waarde ten opzichte van mensen met een normale middelomtrek implicerend dat de hoeveelheid visceraal vet belangrijker bijdraagt aan de huid autofluorescentie waarde en cardiovasculair risico dan het metabool syndroom op zich. (dit proefschrift)
5. De huid autofluorescentie waarde is een sterke en onafhankelijke voorspeller voor het 4-jaars risico op het ontstaan van type 2 diabetes, hart- en vaatziekten en overlijden in de algemene bevolking. (dit proefschrift)
6. Een lage vrij-T4 waarde is onafhankelijk geassocieerd met nieuw vastgestelde type 2 diabetes. (dit proefschrift)
7. Vermindering van de hoeveelheid AGEs in de voeding vermindert het ontstaan van type 2 diabetes en hart- en vaatziekten (Vlassara & Uribarri, Curr Diab Rep 2014) maar ook de kwaliteit van leven.
8. Toevoeging van de huid autofluorescentie bepaling aan de UKPDS risico score verbetert de risico-inschatting op cardiovasculaire morbiditeit en mortaliteit in mensen met type 2 diabetes (Lutgers et al. Diabetologia, 2009)
9. Als gevolg van de nieuwe wetgeving omtrent het verplicht vermelden van het BIG nummer zal de voorkant van het proefschrift belangrijke veranderingen ondergaan. 10. Bij de diagnostiek naar een feochromocytoom dient eerst de biochemische diagnose
rond te zijn, alvorens afbeeldend onderzoek naar de bijnieren aan te vragen. 11. Rookvrije zones rondom ziekenhuizen en andere openbare ruimtes moeten zoveel
mogelijk worden uitgebreid.
12. Voetballen is heel simpel, maar het moeilijkste wat er is, is simpel voetballen. (Johan
Chapter 1 Introduction and aim of the thesis 11
Chapter 2 Lifestyle and clinical determinants of skin autofluorescence in a
population-based cohort study
Eur J Clin Invest. 2016 May;46(5)
21
Chapter 3 The association between various smoking behaviors, cotinine biomarkers
and skin autofluorescence, a marker for advanced glycation end product accumulation
PLoS One. 2017 Jun 20;12(6)
47
Chapter 4 Skin autofluorescence, a non-invasive biomarker for advanced glycation
end products, is associated with the metabolic syndrome and its individual components
Diabetol Metab Syndr. 2017 May; 30;9:42
73
Chapter 5 Skin autofluorescence predicts incident type 2 diabetes, cardiovascular
disease and mortality in the general population
Diabetologia. 2018 Nov;
101
Chapter 6 Serum free thyroxine has opposite effects on incident type 2 diabetes and
cardiovascular disease in the general population
Submitted
133
Chapter 7 Skin autofluorescence, a non-invasive biomarker for advanced glycation
end products, is not related to the number of pregnancies
Journal of Diabetes. 2018 Nov;10 (11)
169
Chapter 8 Influence of Storage and Inter- and Intra-Assay Variability on the
Measurement of Inflammatory Biomarkers in Population-Based Biobanking
Biopreserv Biobank. 2017 Dec;15(6)
181
Introduction and aim of the thesis
Introduction
Advanced glycation end products
Advanced glycation end products (AGEs) are a group of very heterogeneous compounds formed by non-enzymatic glycation of proteins, lipids and nucleic acids under conditions of glycaemic, oxidative or carbonyl stress (1). This mechanism of AGE formation is also called the Maillard or browning reaction (2). In short, the interaction between the carbonyl groups of reducing sugars and free amino groups of proteins leads to the formation of a Schiff base. Rearrangement of the Schiff base results in Amadori products. A well-known example of an Amadadori product is glycated haemoglobin which is commonly used in clinical practice to asses long-term diabetes control. The slow process of oxidation of the Amadori products leads to reactive carbonyl compounds and subsequently to the formation of AGEs (3, 4). Reactive oxygen species (ROS) are also involved in the lipid peroxidation pathway, altering lipids in reactive carbonyl compounds. This reaction results in the formation of advanced lipoxidation end products (ALEs) (5, 6).
Next to endogenous formation of AGEs, there are also exogenous sources of AGEs such as tobacco smoke and certain foods. Tobacco smoke itself contains high levels reactive glycation products (7). In addition, tobacco smoke causes oxidative stress, which in turn can enhance the formation of AGEs. High levels of AGEs were observed in lenses and blood vessels of tobacco smokers (8). A second exogenous source of AGEs is the modern diet. Intake of food containing high levels of AGEs may lead to an increase of AGEs. Uncooked, animal derived food, rich of protein and fad, generally contain high levels of AGEs (9). However, more important is the way how food is processed. Especially the preparation and prolonged heating of food plays an key role in the generation of AGEs, including grilling, roasting and boiling (9, 10). It has been estimated that approximately 10% of AGEs from food are absorbed from the gastro-intestinal tract into the circulation (11).
AGEs are known to accumulate in the body during ageing (12). The lifetime of AGEs depends on the specific protein on which the AGEs are cross-linked. It has been reported that AGEs reflect long-term (~ 15 years) accumulation of glycaemic and oxidative stress, and might therefore be considered as a marker of ‘metabolic memory’ (13). In people with diabetes, both the formation and accumulation of AGEs is increased as a result of chronic hyperglycaemia (14, 15). In individuals with impaired renal function, the formation of AGEs may be a result of oxidative or carbonyl stress (16, 17), while decreased renal clearance of serum AGEs also contributes to increased accumulation of AGEs (16, 18).
In diabetes, accumulation of AGEs is associated with both micro- and macrovascular complications (19). Accumulation of AGEs in the kidney may cause thickening of the capillary basement membrane and sclerosis of the glomeruli (20). Increased accumulation of AGEs in the axoplasm of myelinated and unmyelinated neurons and Schwann cells supports its involvement in the development and progression of diabetic neuropathy (21). Accumulation of AGEs induce the production of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and transforming growth factor-b (TGF-b), which are key cytokines related to the development of diabetic retinopathy (22, 23). Cross-linking of proteins within the vascular wall will reduce vascular elasticity and increase arterial stiffness (24, 25). In contrast, treatment with AGEs breakers for 1-3 weeks has been shown to effectively reverse arterial stiffness in rats with streptozotocin-induced diabetes (26). AGEs accumulation have also been found in carotid atherosclerotic plaques which underlines that accumulation of AGEs is involved in the development of atherosclerosis (27).
Furthermore, binding of AGEs to its receptor (RAGE) might play a role in the development of diabetes cardiovascular complications. Activation of RAGE causes intracellular signaling which leads to a cascade of pro-inflammatory cytokines including nuclear factor-kappa B, interleukin 6 and tumor necrosis factor alpha (28, 29). Furthermore, it stimulates smooth muscle cell proliferation, increases oxidative stress and causes endothelial dysfunction (28, 30).
Non-invasive measurement of skin AGEs
In the past, several investigators have tried to assess AGE levels in the blood and in tissues obtained by (skin) biopsies. However, this method is invasive, time-consuming and associated with high costs. Measurement of AGEs is nowadays performed using a device that is called the AGE Reader (Diagnoptics). This device is developed in Groningen, The Netherlands. It estimated skin AGE content non-invasively based on its fluorescent characteristics of many AGE compounds (31). Therefore, measuring skin AGEs using its fluorescent capacities is called skin autofluorescence (SAF). The measurement of SAF is easily performed in the office on the volar side of the forearm, 10cm below the elbow. The AGE Reader illuminates a skin surface of approximately 4 cm², guarded against surrounding light, with an excitation light source with a wavelength between 300 and 420nm (peak intensity at ~ 370nm). Emission light and reflected excitation light from the skin are measured with an internal spectrometer in the range 300 to 600nm. SAF is calculated by dividing the average emitted light intensity per nanometer in the range of 420-600 nm by the average excitated light intensity per nanometer in the range
420 nm and multiplied by 100. SAF levels are expressed in arbitrary units and increase or decrease per arbitrary unit (AU). It has been shown that SAF levels correlate well with tissue AGEs measured in skin biopsies from subjects with and without diabetes (31, 32). Intra-individual variance among diabetic and control subjects was estimated at around 5% when repeated SAF measurement were taken over a single day (31). SAF levels positively correlate with ageing and are higher among subjects with diabetes and impaired renal function (33).
Skin autofluorescence and previous studies
Over the past 10 years, many studies have been performed using SAF as a biomarker for several clinical conditions, including type 2 diabetes, renal failure and several cardiovascular diseases. Among subjects with type 2 diabetes, higher SAF levels were associated with both micro-and macrovascular complications, and with the severity of diabetes-related complications (34, 35). Furthermore, SAF have been demonstrated to be a strong predictor for cardiac mortality among subjects with type 2 diabetes (36). Addition of SAF measurement to the United Kingdom Prospective Diabetes Study (UKPDS) risk score resulted in re-classification of around 25% of the study population from the low-risk to the high-low-risk group (37). The clinical value of SAF has also been studied in subjects with renal failure. A Japanese study reported that high SAF levels are associated with the progression of chronic kidney disease (38). Other studies have shown that SAF predicts both overall and cardiovascular mortality among (pre)hemodialysis patients (39-41). Furthermore, SAF independently predicts chronic graft dysfunction in renal transplant recipients (42). Only a small number of studies have investigated SAF in subjects without diabetes or impaired renal function. It has been shown that SAF levels are elevated in subjects with (sub-clinical) atherosclerosis and acute ST elevated myocardial infarction, whereas a SAF above the median was of predictive value for future cardiovascular events (43, 44). SAF levels are elevated in subjects with stable coronary artery heart disease and associated with carotid artery intima media thickness (45, 46). Furthermore, SAF levels are higher in individuals with carotid artery stenosis and peripheral artery disease and associated with 5-years risk of mortality and cardiovascular events (47, 48).
Outline of thesis
Most previous research on SAF has focused on populations with either type 2 diabetes, peripheral artery disease, or (end-stage) renal failure. In this thesis, we aim to describe the association between skin autofluorescence and cardiometabolic risk factors in the general population. For these studies, we use data collected in people participating in
the Dutch LifeLines Cohort Study. In addition, we evaluate whether skin autofluorescence is able to predict incident type 2 diabetes, cardiovascular disease and overall mortality within the general population.
The formation and accumulation of AGEs is a complex pathway including either endogenous as well as exogenous pathways. Moreover, when measuring skin autofluorescence, many factors have to be taken into account. Therefore, we describe both clinical and lifestyle determinants of skin autofluorescence among subjects with and without type 2 diabetes in chapter 2. Tobacco smoking is an exogenous source of AGEs accumulation and higher SAF levels are shown in individuals who smoke. However, the effect of secondhand smoking and smoking cessation on AGEs accumulation needs to be determined. The results of this study are presented in chapter 3. The metabolic syndrome is a combination of several cardiometabolic abnormalities associated with a higher risk of both cardiovascular disease and type 2 diabetes. Explorative associations between skin autofluorescence and the individual metabolic syndrome components are shown in chapter 4. In chapter 5 we evaluate the predictive value of skin autofluorescence as a biomarker for incident type 2 diabetes, cardiovascular disease and overall mortality. Next, we assess the association between thyroid hormone levels and incident type 2 diabetes, cardiovascular disease and overall mortality in the general population and whether the association is independent of SAF. The results are shown in chapter 6. Inflammation plays an essential role in the development of insulin resistance, type 2 diabetes and atherosclerosis. Preliminary data from our group showed that SAF levels increase during pregnancy, and do not return to levels found before pregnancy (Groen et al. unpublished data). This may be caused by increased oxidative and glycemic stress during pregnancy. In chapter 7 we examined the relationship between SAF and the number of pregnancies. In chapter 8 we assessed the reproducibility and standardization of several inflammatory biomarkers, including hsCRP, hsIL6 and TNFα, taking into account the influence of assay variability, the reproducibility of a specific measurement between laboratories as well as the influence of sample storage and storage time. In chapter 9 we summarize and discuss the separate chapters and reflect on possible future research on SAF in the general population, and its current application in screening for incident type 2 diabetes, cardiovascular disease and mortality.
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45. Mulder DJ, van Haelst PL, Gross S, de Leeuw K, Bijzet J, Graaff R, et al. Skin autofluorescence is elevated in patients with stable coronary artery disease and is associated with serum levels of neopterin and the soluble receptor for advanced glycation end products. Atherosclerosis. 2008 Mar;197(1):217-23.
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Lifestyle and clinical determinants of skin autofluorescence
in a population-based cohort study
R.P. van Waateringe, S.N. Slagter, M.M. van der Klauw, J.V. van Vliet-Ostaptchouk. R. Graaff, A.D. Paterson, H.L. Lutgers, B.H.R. Wolffenbuttel
European Journal of Clinical Investigation. 2016 May; 46(5)
Abstract
Background Skin autofluorescence (SAF) is a non-invasive marker of advanced glycation end products (AGEs). In diabetes, higher SAF levels has been positively associated with long-term complications, cardiovascular morbidity and mortality. Because little is knoCliwn about the factors which influence SAF in non-diabetic individuals, we assessed the association of clinical and lifestyle parameters with SAF as well as their interactions in a large-scale, non-diabetic population and performed the same analyses in a type 2 diabetic subgroup.
Methods In a cross-sectional study in participants from the LifeLines Cohort Study, extensive clinical and biochemical phenotyping, including SAF measurement, was assessed in 9009 subjects of whom 314 (3.5%) subjects with type 2 diabetes.
Results Mean SAF was 2.04 ± 0.44 arbitrary units (AU) in non-diabetic individuals and 2.44 ± 0.55 AU in type 2 diabetic subjects (p<0.0001). Multivariate backward regression analysis showed that in the non-diabetic population, SAF was significantly and independently associated with age, BMI, HbA1c, creatinine clearance, genetic polymorphism in NAT2 (rs4921914), current smoking, pack-years of smoking and coffee consumption. In the type 2 diabetic group, a similar set of factors was associated with SAF, except for coffee consumption.
Conclusions In addition to the established literature on type 2 diabetes, we have demonstrated that SAF levels are associated with several clinical and lifestyle factors in the non-diabetic population. These parameters should be taken into consideration when using SAF as a screening or prediction tool for populations at risk for cardiovascular disease and diabetes.
Introduction
Accumulation of advanced glycation end products (AGEs) is one of the pathophysiological mechanisms associated with aging (1). The formation and accumulation of AGEs is increased in age-related diseases such as diabetes (2), renal insufficiency (3) and dementia (4). AGEs are formed when proteins are chemically modified by reducing sugars (5) or by reactive carbonyl compounds (6) and represent cumulative exposure to metabolic and oxidative stress (7). Skin autofluorescence (SAF) is a marker for AGE accumulation in the body and can be assessed non-invasively with a device known as the AGE Reader.
It has been demonstrated that SAF predicts cardiovascular morbidity and mortality in diabetes and end-stage renal failure (8-10). Furthermore, higher SAF levels have been reported to be associated with carotid artery intima media thickness (11) and peripheral artery disease (12, 13), independent of diabetes and renal failure.
SAF may be influenced by both clinical and lifestyle factors. Previous studies have shown that smokers have higher SAF levels compared to non-smokers (9, 14) since tobacco smoke causes oxidative stress and is an exogenous source of reactive glycation products (15). Recently, caffeine consumption was found to be associated with higher skin intrinsic fluorescence (SIF) levels in type 1 diabetes (16). However, more research is needed to determine whether this association also exists in non-diabetic and type 2 diabetic subjects. From a genetic perspective, we have shown a strong association between N-acetyltransferase 2 (NAT2) acetylator polymorphism and SAF both in subjects with type 1 and 2 diabetes as well as in those without diabetes (17). These findings demonstrate that genetic variation is an important modulator of SAF.
It is important to determine if SAF is a predictor of cardiovascular morbidity in an aging population not affected by diabetes or renal disease. However, data about factors that influence SAF in non-diabetic individuals are scarce. Therefore, we assessed the association between several clinical and lifestyle factors and SAF, along with their interactions in a large-scale, non-diabetic population and performed the same analyses in a subpopulation of individuals with type 2 diabetes.
Materials and methods
Participants
Subjects included were participants from the LifeLines Cohort Study (18), a large prospective population-based cohort study examining the interaction between genetic and environmental factors in the development of chronic diseases and healthy ageing. Between 2006 and 2013, individuals from the northern region of the Netherlands were invited to participate in the study through their general practitioner. Baseline data including physical examination and extensive questionnaires have been collected from more than 167,000 participants. Follow up visits are scheduled every five years in order to collect information on biochemical measures, lifestyle behavior and psychological factors contributing to health and disease (19). All participants provided written informed consent before participating in the study. The study has been approved by the Medical Ethical review Committee of the University Medical Center Groningen.
For the current analyses, we evaluated participants 18-80 years of age, from whom SAF measurements and genetic data were available. This is the same LifeLines cohort sample as in our previous study on the NAT2 polymorphism (17). We have excluded subjects with type 1 diabetes (n=12) and with severely impaired renal function, defined as serum creatinine >140 µmol/L (n= 29). This resulted in 9009 subjects for analyses, of whom 314 (3.5%) had type 2 diabetes. Of the latter subjects, 212 were already known to have diabetes and another 102 were newly diagnosed by a single fasting blood plasma glucose level (≥7.0 mmol/L) at their baseline visit at the LifeLines research site.
Skin autofluorescence
Skin autofluorescence (SAF) was assessed using the AGE Reader (DiagnOptics Technologies BV, Groningen, the Netherlands). The method has been described in detail previously (14, 20). In short, the AGE Reader illuminates a skin surface of approximately 4 cm², guarded against surrounding light, with an excitation light source whose wavelength is between 300 and 420nm (peak intensity at ~ 370nm). Emission light and reflected excitation light from the skin are measured with an internal spectrometer in the range 300 to 600nm. Measurements were performed on the volar side of the forearm, 10cm below the elbow, at room temperature. SAF was calculated by dividing the average emitted light intensity per nanometer in the range of 420-600 nm by the average excitated light intensity per nanometer in the range 300-420 nm and multiplied by 100. SAF levels are expressed in arbitrary units and increase or decrease per arbitrary unit (AU).
Clinical and lifestyle data
The following clinical data were collected: age, gender, body mass index (BMI), systolic and diastolic blood pressure, serum lipids, HbA1c, diabetes duration, creatinine clearance and use of medication. Participants were asked to complete an extensive questionnaire which included structured questions about smoking behavior and coffee consumption. Subjects were classified according to smoking status at baseline as never smoker, ex-smoker or current ex-smoker. Coffee consumption was recorded as the number of cups of coffee per day. We were not able to distinguish between caffeinated and decaffeinated coffee consumption.
Anthropometry
Weight was measured to the nearest 0.1 kg and height to the nearest 0.5 cm by trained technicians using calibrated measuring equipment, with participants wearing light clothing and no shoes. Body mass index (BMI) was calculated as weight divided by height squared (kg/m²). Systolic and diastolic blood pressure were measured every minute for 10 minutes using an automated Dinamap Monitor (GE Healthcare, Freiburg, Germany). The average of the last three readings was recorded for each blood pressure parameter.
Biochemical measures and genotyping
Blood was collected in the fasting state between 8.00 and 10.00 a.m. and transported to the LifeLines laboratory facility at room temperature or at 4°C, depending on the sample requirements. On the day of collection, HbA1c (EDTA-anticoagulated) was analyzed using a NGSP-certified turbidimetric inhibition immunoassay on a Cobas Integra 800 CTS analyzer (Roche Diagnostics Nederland BV, Almere, the Netherlands). Serum creatinine was measured on a Roche Modular P chemistry analyzer (Roche, Basel Switzerland), and creatinine clearance was calculated with the Cockcroft-Gault formula (21). Total and high density lipoprotein (HDL) cholesterol were measured using an enzymatic colorimetric method, triglycerides using a colorimetric UV method, and low density lipoprotein (LDL) cholesterol using an enzymatic method, also on a Roche Modular P chemistry analyzer (Roche, Basel, Switzerland). Fasting blood glucose was measured using a hexokinase method.
In the analysis, we included the single nucleotide polymorphism (SNP) at the
NAT2 locus (rs4921914) which was previously reported to be associated with SAF (17).
The rs4921914 genotypes were imputed (imputation score 0.89) after exclusion of low-quality samples and SNPs before imputation of the data derived from Illumina CytoSNP
12v2 assay (Illumina, San Diego, CA, USA). Full details on genotyping platform, quality control, other filters applied to SNPs, and the imputation are described elsewhere (17).
Statistical analyses
SPSS (version 22, IBM, Armonk, NY, USA) was used for statistical analyses. Data are shown as mean ± standard deviation (SD) or median and interquartile range (IQR) in case of non-normally distributed data. Student´s t test or Mann-Whitney U test was performed to compare groups. SAF Z-scores were calculated based on the total population in order to correct for age differences. Linear regression analyses were performed to determine the association between clinical and lifestyle determinants and SAF. First a baseline model with only age was assessed. Next, the other determinants were added separately to that model in order to assess their individual contributions. A backward-stepwise method was used including all clinical and lifestyle determinants to derive a final prediction model for SAF, including only determinants that remained significant. We assessed possible effect modification between clinical and lifestyle factors in their effect on SAF which is shown in a final interaction model including only significant determinants. To determine whether associations for SAF differed between subjects with and without diabetes, we additionally tested for the interaction between diabetes and clinical and lifestyle factors. A final prediction model for the total population was assessed including significant determinants only. P< 0.05 (two-tailed) was considered statistically significant.
Results
Table 1 provides the clinical characteristics of the study population. Mean age of the non-diabetic population was 49 years, 10 years younger than the type 2 non-diabetic subgroup (p<0.0001). SAF levels were significantly higher in the type 2 diabetic population (2.44 ± 0.55 AU) than in the non-diabetic subgroup (2.04 ± 0.44 AU) (p<0.0001).
Table 1. Clinical characteristics of the non-diabetic population and type 2 diabetic group
Parameters Non-diabetes Type 2 diabetes
N 8695 314
Age (years) 49 ± 11 59 ± 11 **
Gender (male/female) n (%) 3570 (41) / 5125 (59) 168 (53) / 150 (47)
Body mass index (kg/m²) 26.4 ± 4.2 30.5 ± 5.4 **
Systolic blood pressure (mmHg) 129 ± 16 137 ± 17 *
Diastolic blood pressure (mmHg) 75 ± 9 77 ± 9
Total cholesterol (mmol/L) 5.1 ± 0.9 4.7 ± 1.2
HDL cholesterol (mmol/L) 1.4 ± 0.4 1.2 ± 0.3
LDL cholesterol (mmol/L) 3.3 ± 0.9 2.9 ± 1.0
Triglycerides (mmol/L) 1.05 (0.8-1.5) 1.41 (1.0-1.4)
Creatinine clearance (ml/min) 113 ± 31 119 ± 45 *
HbA1c (%)
HbA1c (mmol/mol) 5.5 ± 0.337 ± 3.3 6.8 ± 1.2 **51 ± 13
Estimated diabetes duration (years) n.a. 6.4 (3.2-11.0)
Oral agents/insulin, % n.a. 47/15 †
Statins, % 6.2 46.2 ** NAT2 polymorphism, n (%) TT CT CC 5685 (65) 2706 (31) 304 (4) 209 (67) 96 (30) 9 (3) Smoking status, n (%) ‡ Never smokers Ex-smokers Current smokers 3508 (41) 3174 (37) 1914 (22) 106 (34) 147 (48) 56 (18) Pack-years in ex- and current smokers 11 (4.6-19.0) 18 (8.5-29.4) * Coffee consumption (cups per day) 3.8 (2.3-5.2) 3.8 (1.9-5.5)
SAF (AU) 2.04 ± 0.44 2.44 ± 0.55 **
Data are presented as means ± standard deviation, or median (interquartile range) and number (%). Creatinine clearance (Cockcroft-Gault formula); SAF, skin autofluorescence; AU, arbitrary units; † 29 subjects used oral agents + insulin, ‡ Missing values for smoking status (n=104) * p <0.001 ** p <0.0001
Univariate associations with SAF
The univariate associations between clinical and lifestyle determinants and SAF showed that in the non-diabetic population, age, male gender, BMI, HbA1c, total cholesterol, LDL cholesterol, triglycerides, current smoking, ex-smoking, pack-years of smoking and coffee consumption were positively associated with SAF (online Supplemental Table S1). Negative associations were found for creatinine clearance and the fast acetylator allele of
NAT2. In the type 2 diabetic group, age, HbA1c, current smoking, pack-years of smoking
and coffee consumption were positively associated with SAF. Creatinine clearance, total cholesterol, LDL cholesterol and the fast acetylator allele of NAT2 were negatively associated with SAF (online Supplemental Table S1).
Multivariate associations with SAF
A baseline model including age explained 28.5% of the variance in SAF in the non-diabetic population (Table 2). Pack-years of smoking (4.0%), current smoking (3.7%), coffee consumption (3.6 %), and NAT2 polymorphism (2.1%) had the highest additional contribution. In the type 2 diabetic population, 23.8% of the variance in SAF could be explained by age. Current smoking had the highest additional contribution (8.9%) to the baseline model, followed by pack-years of smoking (4.4%) and NAT2 polymorphism (2.7%).
Table 2. Multivariate linear regression model for skin autofluorescence (SAF) in the non-diabetic
population and type 2 diabetic group
Determinants Coefficient β SE P Value R² (%)
Non-diabetes (N=8695)
Baseline model
Age 0.021 3.6 x 10 -4 1.0 x 10 -200 28.5
Clinical and lifestyle parameters ΔR² (%)
Male gender 0.053 0.008 4.1 x 10 -11 0.4
Body mass index 0.004 0.001 1.5 x 10 -4 0.1
HbA1c 0.082 0.014 3.2 x 10 -9 0.3
Creatinine clearance (ml/min) 2.0 x 10 -4 3.1 x 10 -4 3.2 x 10 -8 0.3
Total cholesterol 0.001 0.004 0.893 0.0
LDL cholesterol 0.004 0.005 0.396 0.0
HDL cholesterol -0.084 0.010 6.7 x 10 -16 0.5
Triglycerides 0.030 0.005 2.5 x 10 -9 0.3
NAT2 polymorphism (CC vs CT and TT) -0.114 0.007 1.2 x 10 -57 2.1 Current smoking vs never smoking 0.205 0.009 5.4 x 10 -101 3.7
Ex-smoking vs never smoking -0.029 0.009 0.001 0.1
Pack-years 0.007 4.6 x 10 -4 4.2 x 10 -56 4.0
Coffee consumption (cups per day) 0.076 0.002 3.6 x 10 -108 3.6
Type 2 diabetes (N=314)
Baseline model
Age 0.025 0.003 3.8 x 10 -20 23.8
Clinical and lifestyle parameters ΔR² (%)
Male gender 0.151 0.054 0.005 1.9
Body mass index 0.003 0.005 0.589 0.1
HbA1c 0.064 0.023 0.006 1.8
Creatinine clearance (ml/min) 0.005 0.002 0.011 1.6
Total cholesterol -0.063 0.023 0.007 1.8
LDL cholesterol -0.072 0.026 0.006 1.6
HDL cholesterol -0.250 0.086 0.004 1.8
Triglycerides 0.036 0.021 0.087 0.5
Estimated diabetes duration 0.005 0.004 0.246 0.6
NAT2 polymorphism (CC vs CT and TT) -0.167 0.050 0.001 2.7 Current smoking vs never smoking 0.436 0.069 7.0 x 10 -10 8.9
Ex-smoking vs never smoking -0.032 0.056 0.570 0.1
Pack-years 0.006 0.002 0.001 4.4
Coffee consumption (cups per day) 0.029 0.012 0.018 1.6
NAT2, N-acetyltransferase 2; SE, standard error; R²: explained variance in SAF (%); ΔR²:
additional explained variance of clinical and lifestyle parameters on top of baseline model
In the non-diabetic population, the multivariate regression model showed that age, BMI, HbA1c, creatinine clearance, NAT2 polymorphism, current smoking, pack-years of smoking and coffee consumption were independent predictors of SAF explaining 33.8% of the variance in SAF (Table 3). In the type 2 diabetic population, a similar set of predictors excluding coffee consumption explained 46.8% of the variance in SAF.
Table 3. Prediction model for skin autofluorescence (SAF) in the non-diabetic population and the
type 2 diabetic group
Predictors Coefficient β SE P Value R² (%)
Non-diabetes (n=8695) 33.8
Age 0.018 0.001 1.3 x 10 -116
Body mass index 0.006 0.002 0.001
HbA1c 0.061 0.019 0.001
Creatinine clearance (ml/min) -0.001 2.8 x 10 -4 7.9 x 10 -7 NAT2 polymorphism (CC vs CT and TT) -0.119 0.009 1.0 x 10 -35 Current smoking vs never smoking 0.115 0.013 9.6 x 10 -20
Pack-years 0.004 4.9 x 10 -4 7.0 x 10 -20
Coffee consumption (cups per day) 0.032 0.002 3.0 x 10 -40
Type 2 diabetes (N=314) 46.8
Age 0.017 0.006 0.004
Body mass index 0.020 0.010 0.039
HbA1c 0.112 0.035 0.002
Creatinine clearance (ml/min) -0.005 0.002 0.001
NAT2 polymorphism (CC vs CT and TT) -0.210 0.069 0.003
Current smoking vs never smoking 0.327 0.101 0.002
Pack-years 0.005 0.002 0.018
NAT2, N-acetyltransferase 2; SE, standard error; R²: explained variance in SAF (%)
The effect of coffee consumption on SAF
In the non-diabetic population, coffee consumption was significantly and dose-dependently associated with higher SAF Z-scores (p<0.001) whereas for the type 2 diabetic population a non-significant trend was found (p=0.104) (Figure 1). The association between coffee consumption and SAF was modified by NAT2 polymorphism (p=0.001) (online Supplemental Table S2). Among subjects having a TT genotype, a mean intake of one cup of coffee per day was associated with 0.052 AU increase in SAF (0.032 AU and 0.029 AU for CT respectively CC genotype) (online Supplemental Table S3). In the type 2 diabetic population, a mean intake of one cup of coffee per day was associated with 0.049 AU increase among subjects with a TT genotype. The associations for CT (0.016 AU) and CC genotype (0.005 AU) were not significant (p=0.380 respectively p=0.948)
Figure 1. The effect of coffee consumption on SAF in the non-diabetic population and type 2 diabetic group
Dots (type 2 diabetes) and squares (non-diabetes) show mean SAF Z-scores ± SEM, r2 correlation
coefficient. Sample size per category: non-diabetes, 0 cups of coffee per day (n=563), type 2 di-abetes, 0 cups of coffee per day (n=11); non-didi-abetes, 1-2 cups of coffee per day (n=1491), type 2 diabetes, 1-2 cups of coffee per day (n=43); non-diabetes, 3-4 cups of coffee per day (n=3044), type 2 diabetes, 3-4 cups of coffee per day (n=79); non-diabetes, 5-6 cups of coffee per day (n=2263), type 2 diabetes, 5-6 cups of coffee per day (n=63); non-diabetes, >6 cups of coffee per day (n=1061), type 2 diabetes, >6 cups of coffee per day (n=35). SAF, skin autofluorescence
Smoking and SAF
Figure 2 shows the age-adjusted SAF Z-scores for different smoking groups. Within each smoking group, subjects from the non-diabetic population had significantly lower SAF Z-scores compared to subjects with type 2 diabetes (never smokers, p<0.05; ex-smokers, p<0.0001; current smokers, p<0.0001). In both groups, current and ex-smokers had higher SAF Z-scores compared to never smokers (p<0.05 - p<0.0001 ). Furthermore, SAF levels increased with the number of pack-years smoked, indicating a dose-dependent effect (online Supplemental Figure S1).
Figure 2. Age-adjusted SAF Z-scores stratified for smoking status in the non-diabetic population and type 2 diabetic group
Boxes show mean, minimum and maximum SAF Z-scores, whiskers represent the 5th and 95th
per-centile. Sample size per category: non-diabetes, never smoker (n=3516), type 2 diabetes, never smoker (n=106); non-diabetes, ex-smoker (n=3186), type 2 diabetes, ex-smoker (n=150); non-di-abetes, current smoker (n=1919), type 2 dinon-di-abetes, current smoker (n=57). SAF, skin autofluores-cence. * p <0.05, ** p <0.0001
Clinical - lifestyle interactions
Next, we evaluated effect modification for clinical and lifestyle determinants in their effect on SAF. A significant interaction between current smoking and age (p<0.0001) was observed showing that one year increase in age was associated with an additional 0.002 AU increase in SAF, which implies a 12% increase in age-dependency (data not shown). The final interaction model for the non-diabetic population, including significant determinants only, is presented in online Supplemental Table S2.
Finally, we assessed whether associations with SAF differed between subjects with and without diabetes. Effect modification was observed for type 2 diabetes and HbA1c (p<0.0001) (data not shown) with HbA1c having a larger effect on SAF in subjects with type 2 diabetes compared to individuals without diabetes. Also, a significant interaction between current smoking and type 2 diabetes (p=0.04) in their association with SAF was found (data not shown). Current smoking had a larger effect on SAF in subjects with type 2 diabetes compared to non-diabetic individuals which was already demonstrated in Table 2 and Figure 2. The final prediction model for SAF for the total population is given in online Supplemental Table S4.
Discussion
This is the first study to report an integrated analysis of both lifestyle and clinical factors that influence SAF in a large-scale non-diabetic population, as well as in a subpopulation with type 2 diabetes. We have shown that SAF is significantly and independently associated with age, BMI, HbA1c, creatinine clearance, NAT2 polymorphism, current smoking, pack-years of smoking and coffee consumption explaining 33.8% of the variance in SAF. In the type 2 diabetic population, SAF was associated with the same factors with the exception of coffee consumption and explained 46.8% of the variance in SAF.
Coffee consumption was dose-dependently associated with higher SAF levels both in the non-diabetic population as well as in subgroup of type 2 diabetic individuals. Recently, Eny et. al. reported a positive association between caffeine consumption and SIF in type 1 diabetes (16). However, a previous Dutch study examining the association between dietary habits and SAF found no association between coffee consumption and SAF among 147 elderly subjects (22). The average amount of coffee consumed daily was comparable to our study (mean 3.4 cups compared to a median of 3.8 cups) however, the subjects in our study were on average 10 years younger. Another -more likely- explanation for the different findings might be that their study was underpowered due to the small sample size. Factors that could explain elevated SAF levels in coffee consumers may be fluorescent substances in coffee (fluorophores) or indirectly as a consequence of the roasting process of coffee beans, which can be considered as a Maillard reaction, leading to the formation of browning products such as melanoidins (23). Also, it might be that coffee consumers are more likely to smoke which would result in a positive association between coffee consumption and SAF. Nevertheless, we found that both determinants were independently associated with SAF in the multivariate model whereas no effect modification was observed.
Coffee is one of the most consumed beverages around the world and many studies have examined its association with health and disease. Moderate amounts of coffee consumption have been reported to be protective against cardiovascular disease (24) and type 2 diabetes (25, 26). In addition, a recent study has demonstrated that coffee consumption was associated with a lower risk of overall mortality (27). Coffee is a major source of the phenolic antioxidant chlorogenic acid and its daily intake from coffee consumption is estimated to be 0.5 - 1 gram (28). Part of the beneficial effects of coffee consumption might be attributed to chlorogenic acid (No Reference Selected) which reduces oxidative stress and inhibits hydrolysis of glucose-6-phosphatase, leading
to lower plasma glucose concentrations [29]. Interestingly, chlorogenic acid has been reported to inhibit AGE formation in vitro [30,31]. During the roasting process of coffee beans however, a significant amount of chlorogenic acid is lost [32]. Hereby, the inhibitory effect of chlorogenic acid on AGE formation might be attenuated. The roasting process of coffee beans further leads to profound changes in the chemical composition of coffee brew, including the formation of melanoidins as the end products of the Maillard reaction [23]. This may explain how coffee consumption, as an exogenous source of AGE, can contribute to increased SAF levels. Overall, the present study shows that coffee consumption is associated with higher SAF levels. When using SAF to predict cardiovascular events, this may lead to overestimation of true risk in those with high coffee consumption.
Previously, we have shown that N-acetyltransferase 2 (NAT2) acetylator polymorphism was significantly associated with SAF [17]. NAT2 is a drug-metabolizing enzyme for which certain gene polymorphisms have been associated with increased risk of several cancers [33]. Interestingly, in the present study effect modification was observed for NAT2 polymorphism and coffee consumption in their effect on SAF. The effect of coffee consumption on SAF was strongest for individuals with the slow acetylator genotype and weakest for individuals with the fast acetylator genotype. Previous studies have shown that NAT2 is involved in the metabolic pathway of caffeine [34,35]. Since caffeine has fluorescent properties[36], it may be that the association between NAT2 and SAF was influenced by fluorescence of caffeine present in the skin. However, both
NAT2 polymorphism and coffee consumption were also independently associated with
SAF when analyzed together.
Our results showing significantly higher SAF levels in current smokers compared to never smokers are in agreement with earlier studies performed in type 2 diabetes [9,14]. In addition, a higher number of pack-years was associated with higher SAF levels, which was also found in a study among patients with chronic obstructive pulmonary disease [37]. Theoretically, many years of smoking – and thus exposure to long-term oxidative stress – may contribute either directly or indirectly to increased accumulation of AGEs throughout life. Since smoking enhances the risk for diabetes-related cardiovascular complications [38], it might be that the larger effect of smoking on SAF in type 2 diabetic subjects translates into higher cardiovascular risk of smoking in diabetes. Future follow-up studies are needed to confirm this hypothesis.
As expected, age was significantly and independently associated with SAF in both the non-diabetic population and type 2 diabetic subgroup. In general, mean SAF was significantly higher in the type 2 diabetic subgroup compared to the non-diabetic
population but it should be emphasized that the former group was on average 10 years older. Ageing has been thought to be a key factor in non-enzymatic glycation of proteins [1,5] which has been confirmed in several studies using the AGE Reader, all showing a linear relationship between increasing age and higher SAF [14,20].
The pathway of endogenous AGE formation might be different between subjects with and without diabetes. In diabetes, AGE formation is based on a combination of chronic hyperglycaemia and oxidative stress [6,7] as well as through lipid-derived intermediates, resulting in advanced lipoxidation end products (ALEs) [39]. In addition, since serum AGEs are cleared by the kidney [3], changes in renal function may also influence AGE accumulation [40]. In subjects without diabetes however, ageing is thought to be the most important factor contributing to endogenous AGE formation [1,5], as can be deduced from our results.
HbA1c, a measure of semi-recent glycaemic status, was significantly associated with higher SAF levels, with a larger effect among type 2 diabetic individuals. A study among type 2 diabetic individuals concluded that SAF was poorly predicted by HbA1c level [42]. In type 1 diabetes, SAF was associated with long-term mean HbA1c, but not with most recent HbA1c [43]. Another study among Japanese type 1 diabetes patients showed that SAF significantly correlated with HbA1c over the past 15 years which probably is a better measure of long-term glycaemic load [44]. An earlier study showed that Hb-AGE was a better indicator for long-term blood glucose control compared to HbA1c [45]. In clinical practice, HbA1c represents the average glycaemic control over the last 5 to 6 weeks [45]. HbA1c levels in diabetes can fluctuate over short-time periods [46], which makes it plausible that the association with SAF, reflecting a much longer-term metabolic memory (~ 15 years) [47], is inconsistent. In non-diabetic subjects, HbA1c levels are expected to show less fluctuations which could explain a more consistent relationship with SAF in the population without diabetes.
Our study has some strengths and limitations. First, the large majority of our study population were individuals without diabetes making this SAF study unique. Moreover, due to the large number of participants, we were able to perform analyses for different smoking statuses and the variety in the amount of coffee consumption typically found in the general population. Because clinical data was obtained at the same time of the SAF measurement, the associations found are highly reliable. A limitation of our study is a potential misclassification of some individuals with regard to their smoking status as we cannot rule out misreporting of smoking habits or history. Secondly, diagnoses of type 2 diabetes was made by a single fasting plasma glucose only. Unfortunately, we
were not able to include in our analyses the use of other caffeine-rich beverages, such as tea or soft drinks, or caffeine from foods.
In addition to the established literature in type 2 diabetes, we have demonstrated that SAF is influenced by clinical and lifestyle factors, including smoking and coffee consumption in a large-scale non-diabetic population. These parameters need to be taken into consideration when using SAF as a screening or prediction tool for populations at risk for cardiovascular disease and diabetes.
Author´s contributions
Conceived and designed the study: RPW MMvdK BHRW. Performed the statistical analyses and analyzed the data: RPW BHRW. Data interpretation: RPW BHRW MMvdK HLL JVO RG SNS ADP. Drafting the manuscript: RPW. Intellectual contributions to the manuscript, helped drafting the manuscript and have read and approved the final version: all authors.
Acknowledgments
This work was supported by Netherlands Consortium for Healthy Ageing (NCHA), Bio-SHaRE-EU, Biobank Standardisation and Harmonization for research excellence in the European Union. Bioresource research impact factor BRIF4568. The manuscript is based on data from the LifeLines cohort study. LifeLines adheres to standards for open data availability.
The data catalogue of LifeLines is publicly accessible on www.lifelines.net.
All international researchers can apply for data at the LifeLines research office (LLscience@ umcg.nl). The LifeLines system allows access for reproducibility of the study results. We thank Sally Hill for providing scientific medical writing services.
Conflict of interests
RG is founder and shareholder of DiagnOptics BV, Groningen, the Netherlands, manufacturing autofluorescence readers (http://www.diagnoptics.com/) which has been used in the present study. All other authors declare that they have no competing interests.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Prior presentations
Parts of this study were presented in abstract form at the American Diabetes Association (ADA), June 2014, San Francisco (USA) and at the European Association for the Study of Diabetes (EASD), September 2014, Vienna (Austria).
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SUPPLEMENTAL DATA
Additional Table S1. Univariate linear regression model for skin autofluorescence (SAF) in the
non-diabetic population and type 2 diabetic group
Determinants Coefficient β SE P Value
Non-diabetes (N=8695)
Age 0.021 3.6 x 10 -4 1.0 x 10 -200
Male gender 0.059 0.010 8.5 x 10 -10
Body mass index 0.009 0.001 2.0 x 10 -14
HbA1c 0.359 0.015 2.7 x 10 -127
Creatinine clearance (ml/min) -0.004 1.5 x 10 -4 6.3 x 10 -159
Total cholesterol 0.076 0.005 5.0 x 10 -58
LDL cholesterol 0.071 0.005 6.7 x 10 -42
HDL cholesterol -0.012 0.012 0.333
Triglycerides 0.046 0.006 9.4 x 10 -15
NAT2 polymorphism (CC vs CT and TT) -0.115 0.008 4.0 x 10 -42
Current smoking vs never smoking 0.124 0.011 8.3 x 10 -28
Ex-smoking vs never smoking 0.076 0.010 1.2 x 10 -14
Pack-years 0.011 0.001 8.8 x 10 -93
Coffee consumption (cups per day) 0.044 0.002 1.2 x 10 -103
Type 2 diabetes (N=314)
Age 0.025 0.003 3.8 x 10 -20
Male gender 0.119 0.062 0.055
Body mass index -0.004 0.006 0.436
HbA1c 0.063 0.027 0.019
Estimated diabetes duration 0.009 0.005 0.058
Creatinine clearance (ml/min) -0.004 0.001 4.9 x 10 -9
Total cholesterol -0.066 0.026 0.013
LDL cholesterol -0.086 0.030 0.004
HDL cholesterol -0.028 0.097 0.774
Triglycerides 3.9 x 10 -4 0.024 0.987
NAT2 polymorphism (CC vs CT and TT) -0.180 0.057 0.002
Current smoking vs never smoking 0.262 0.080 0.001
Ex-smoking vs never smoking 0.073 0.063 0.243
Pack-years 0.009 0.002 1.2 x 10 -5
Coffee consumption (cups per day) 0.030 0.013 0.025
