ICURT PROCEEDINGS
25-Hydroxyvitamin D and Advanced Glycation Endproducts in Healthy and Hypertensive Subjects: Are There Interactions? ebekova, DSc, MD,† Gholamreza Fazeli, PhD,* Michael St€urmer, MD, PhD,* Katarına S Udo Bahner, PhD, MD,‡ Franz St€ab, Dr.rer.nat.,§ and August Heidland, PhD, MD* Advanced glycation endproducts (AGEs) accumulate during aging. Skin is the single organ of vitamin D synthesis, induced by ultraviolet B light. Accumulation of AGEs in the skin could interfere with synthesis of the vitamin, whereas the microinflammation and oxidative stress (associated with hypovitaminosis D) could amplify both the toxic effects of AGEs and their production. Clinical data on potential interactions between vitamin D3 deficiency and AGE accumulation are sparse. Here we investigated potential associations between levels of circulating vitamin D3 and those of AGEs in blood and skin with regard to markers of inflammation and oxidative stress in nondiabetic subjects. In a cross-sectional study, 146 subjects (119 healthy persons and 27 hypertensive patients; 73 male and 73 female; mean age, 57.0 6 15.5 years) were included. Skin autofluorescence (SAF) and plasma levels of vitamin D3, AGE-associated fluorescence, high-sensitivity C-reactive protein level, and advanced oxidation protein products as well as renal function (estimated glomerular filtration rate) were determined. In a subgroup of 61 patients, Nε-carboxymethyllysine, soluble receptor of AGEs, and soluble vascular adhesion protein-1 were additionally analyzed. Vitamin D3 level averaged 22.5 6 8.9 ng/mL. Prevalence of vitamin D insufficiency (2029 ng/mL) was 43%, and that of deficiency (,20 ng/mL) 37%. The age-dependent rise in SAF was steeper in smokers and in subjects presenting arterial hypertension. No association between SAF and hypovitaminosis D was revealed. Among smokers, an inverse relationship manifested between vitamin D3 and plasma AGE-associated fluorescence as well as soluble vascular adhesion protein-1. Our data suggest that in nondiabetic adults, hypovitaminosis D does not enhance toxicity and accumulation of AGEs. Only in smokers interactions are conceivable. Ó 2015 by the National Kidney Foundation, Inc. All rights reserved.
A
Introduction
DVANCED GLYCATION ENDPRODUCTS (AGEs) are heterogeneous compounds, formed via the Maillard reaction (nonenzymatic reaction of a sugar ketone or aldehyde group with a free amino group of proteins, lipids, and nucleic acids) or alternatively under conditions of oxidative and carbonyl stress.1,2 Moreover, accumulation of AGEs is increased in chronic kidney disease (CKD), in part because of an impaired renal *
University of W€urzburg, W€urzburg, Germany. Comenius University, Medical Faculty, Bratislava, Slovakia. ‡ KfH Nierenzentrum W€urzburg, W€urzburg, Germany. § Beiersdorf AG, Hamburg, Germany. Financial Disclosure: A.H. received honoraria for lectures from gbo Medizintechnik AG and U.B. from Fresenius Medical Care. F.S. is an employee of Beiersdorf AG, Hamburg, Germany. The other authors declare that they have no relevant financial interests. Support: This study was supported by a grant from Beiersdorf AG, Hamburg, Germany to the Verein zur Bek€ampfung der Hochdruck-und Nierenkrankheiten W€urzburg e.V. Address correspondence to August Heidland, PhD, MD, Department of Internal Medicine, University of W€urzburg and KfH Kidney Center, HansBrandmann-Weg-1, 97080, W€urzburg, Germany. E-mail: August. †
[email protected] Ó
2015 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2014.10.027
Journal of Renal Nutrition, Vol -, No - (-), 2015: pp 1-8
removal,3 consumption of thermally processed food rich in glycotoxins,4,5 and tobacco smoke.6 Adverse effects of AGEs result from modifications of proteins and lipids leading to structural and functional alterations of plasma and extracellular or intracellular proteins. In particular, crosslinking of collagen, thickening of basement membranes, and trapping of proteins (low-density lipoprotein) induce vascular stiffness and an enhanced vascular permeability. AGE modifications of plasma or tissue proteins are associated with impaired protein degradation.7 Interaction of AGEs with their receptors (particularly RAGE) induces cell activation via nuclear transcription factors, for example, NF-kB, with reduced nitric oxide formation, enhanced generation of oxygen radicals, synthesis of proinflammatory cytokines or chemokines, fibrogenic growth factors (transforming growth factor beta 1), vascular adhesion molecules, and cell proliferation.8 RAGE are located on the surface of multiple cells including endothelial, smooth muscle, mesangial and tubule cells, monocytes, macrophages, and neurons.9 AGEs disturb endothelial functions in particular by lowering nitric oxide levels, which promotes the adherence of leukocytes. In this way, AGEs are implicated in the aging process, in particular of atherosclerosis, in the manifestation and progression of renal disease and in diabetic patients in the macroangiopathy and microangiopathy (nephropathy, retinopathy, and 1
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neuropathy).10 AGEs also induce genomic damage11 and may be associated with an enhanced cancer incidence.12 In the risk assessment of AGE accumulation, determination of their circulating blood components such as pentosidine, N3-carboxymethyllysine (CML), and AGEassociated fluorescence (AGE-Fl) are used. They are related to severity and prognosis of chronic heart failure13 and to coronary and peripheral artery disease in diabetic patients.14 Enhanced plasma AGE levels are independent predictors of total, cardiovascular, and coronary mortality in women with type 2 diabetes15 and of mortality in hemodialysis patients.16 Soluble RAGE (sRAGE) represents the truncated form of the receptor, which may neutralize the toxic effect of AGEs.17 A relatively novel marker of AGE accumulation is the noninvasive determination of skin autofluorescence (SAF). Particularly, skin collagen is a target for AGE modification.18 SAF reflects the accumulation of AGEs in the tissues and hence the ‘‘long-term cumulative metabolic and oxidative stress.’’ Because of the substantially longer halflife of collagen in comparison with plasma proteins, SAF provides information on the long-term cumulative burden, whereas plasma AGEs reflect more that of the recent past. SAF is an independent predictor of cardiovascular complications, morbidity, and mortality.19-21 In moderate renal failure, SAF was inversely correlated to circulating endothelial progenitor cells.22 The skin is the single organ producing the steroid hormone vitamin D with pleiotropic actions on most tissues and cells in the body.23 Low serum concentrations of 25OH vitamin D (an estimate of vitamin D3 stores) represent a worldwide problem.24 It may cause rickets, osteoporosis, osteomalacia, and fractures, as well as muscle weakness,23 in part as a result of secondary hyperparathyroidism.25 Numerous prospective studies revealed associations of low vitamin D3 levels with hypertension (via stimulation of renin-angiotensin-aldosterone system),26 cardiovascular complications,27 infectious diseases (lowered cathelicidin levels), insulin resistance and type 2 diabetes,28 progression of CKD, and autoimmune diseases (type 1 diabetes, multiple sclerosis, and rheumatoid arthritis).23 Up till now, there are no data about potential relationships between vitamin D3 and AGEs in clinical medicine. In vitro studies suggested that the deleterious impact of AGE-modified human serum albumin on endothelial cells could be blunted by addition of calcitriol [1,25(OH)2D3].29 In diabetic rats, treatment with vitamin D (cholecalciferol) reduced the deposition of AGEs (CML) in the aortic wall and the systemic oxidative stress.30 In the present article, we raised the following questions: (1) Is an enhanced accumulation of AGEs in the skin associated with an impaired synthesis of vitamin D? (2) Does hypovitaminosis D contribute to an amplification of the AGEinduced microinflammation and oxidative stress? (3) Does hypovitaminosis D accelerate the formation of AGEs?
Subjects and Methods This single-center observational study was performed in a practice of Internal Medicine (Dr Werner St€ urmer) in W€ urzburg and conducted in accordance to the principles of the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of the Medical Faculty of the University of W€ urzburg. All subjects signed an informed consent to participate. In the cross-sectional study we included a total of 146 nondiabetic subjects (aged, 14-96 years) on occasion of their routine medical examination. Of them, 73% were apparently healthy, whereas 27% showed arterial hypertension. Fifteen percent of the participants were current smokers. All investigated subjects were without dermatosis, scars, and pigment disorders. Exclusion criteria comprised any acute illness, diabetes mellitus and HbA1c levels .6%, autoimmune diseases, malignancies, moderate-to-severe CKD (estimated glomerular filtration rate [eGFR] ,60 mL/min/1.73 m2), infections, use of glucocorticoids, vitamin D supplements, regular visits of a solarium, and use of tanning cream (during the last 14 days). Anthropometric measurements to calculate body mass index were performed. SAF was measured on the volar side of the forearm using the AGE Reader (Diagnoptics BV, Groningen, the Netherlands).19 eGFR was calculated using the abbreviated Modification of Diet in Renal Disease formula. Handgrip muscle strength was measured using the Baseline hydraulic hand dynamometer (Fabrication Enterprises Inc., White Plains, New York). Blood was collected in the morning hours after overnight fasting from antecubital vein. Routine blood chemistry was determined with an automatic analyzer. HbA1c was analyzed using high-pressure liquid chromatography (ADAM A1cHA-8180 FAST; Axonlab, Baden, Switzerland). Aliquots of plasma were stored at 280 C until special analyses. High-sensitivity C-reactive protein (hs-CRP) levels were measured nephelometrically using Siemens reagent (Labor Limbach, Heidelberg, Germany) and vitamin D3 by an electrochemiluminescence immunoassay from Roche (Labor Limbach, Heidelberg, Germany. Analysis of AGE-Fl in plasma was performed according to M€ unch et al.31 and of advanced oxidation protein products (AOPPs) according to Witko-Sarsat et al.32 In a subgroup of randomly selected 61 patients, CML (Microcoat, Bernried, Germany), sRAGE (R&D Systems, Minneapolis, Minnesota), and soluble vascular receptor adhesion protein-1 (sVAP-1; Bender Med System Inc., Vienna, Austria) were determined using commercial enzyme-linked immunosorbent assay kits according to manufacturer’s instructions.
Statistical Analysis Skewed data were log transformed for statistical analyses, but for simplicity, means and standard deviations are given.
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VITAMIN D3 AND SKIN AUTOFLUORESCENCE
The Student t test (2-sided) was used to compare 2 groups. Comparisons among 3 groups were performed using analysis of variance with Scheffe post hoc test, when necessary. Categorical data were evaluated using chi-square test. Pearson correlation coefficients were calculated. Multivariate analysis was performed using the general linear model. P ,.05 was considered significant. Statistical program SPSS, version 16 (SPSS Inc., Chicago, IL) was used for statistical analyses.
Results Cohort characteristics are given in Table 1. The subgroup in which additional markers were also determined did not differ significantly from the rest of the cohort except for higher plasma AGE-Fl’s (P 5 .002) and AOPPs (P 5.036; Table 1).
Skin Autofluorescence In the whole group, a significant linear correlation was revealed only between SAF and age (r 5 0.464; P , .001) or HbA1c (r 5 0.223; P 5.037). The association between SAF and eGFR did not reach significance (r 5 20.151; P 5 .16). Multivariate analysis with age, body mass index, eGFR, hs-CRP, AGE-Fl, AOPPs, smoking status, and presence of hypertension entered as independent variables indicated age and current smoking as independent variables affecting SAF (corrected model: P , .001; age: P , .001; smoking: P 5 .011; R2: 27%). Comparison between groups did not reveal a significant difference in SAF between smokers and nonsmokers (2.4 6 0.7 vs. 2.3 6 0.5 arbitrary units [AU]; P 5 .57),
stemming probably from the fact that the smokers were significantly younger (45.8 6 12.1 vs. 58.9 6 115.1 years; P ,.001) and presented higher eGFR (94 6 16 vs. 83 6 16 mL/min/ 1.73 m2; P ,.02) and lower HbA1c compared with the nonsmokers (5.3 6 0.3 vs. 5.5 6 0.3, p , .012). No significant association between SAF and other variables except for age (Fig. 1) was revealed if smokers and nonsmokers were evaluated separately. There was also a significant relationship between plasma AGE-associated fluorescence and age (Fig. 2). Subjects with hypertension presented higher SAF and plasma AGE-Fl in comparison to normotensive subjects (2.5 6 0.5 vs. 2.3 6 0.5; P 5 .018 and 315 6 86 vs. 273 6 60 AU; P 5.007, respectively), but they were also significantly older (64.8 6 12.2 AU vs. 54.0 6 15.5 years; P , .001; Table 2). Other variables did not differ significantly between normotensive and hypertensive subjects. Vitamin D3 level averaged in the cohort to 22.5 6 8.9 ng/mL (Table 1). Males and females did not differ significantly (22.6 6 9.1 ng/mL vs. 22.3 6 8.8 ng/mL; P 5 .84). 43% of all participants presented a vitamin D3 deficiency (concentration ,20 ng/ mL), 37% a vitamin D3 insufficiency (concentration 2029 ng/mL), whereas sufficient vitamin D3 levels (concentration . 30 ng/mL) were revealed in 20% of the subjects. Between-group comparisons did not show any other significance (Table 3). In the whole cohort, no significant relationship between vitamin D3 and SAF (Fig. 3), or any other variable, was revealed. Multivariate analysis neither showed any
Table 1. Demographic and Laboratory Characteristics of the Subjects Characteristic n M/F, n (%) Age (y), mean 6 SD BMI (kg/m2), mean 6 SD eGFR (mL/min/1.73 m2), mean 6 SD HbA1c (%), mean 6 SD Vitamin D3 (ng/mL), mean 6 SD SAF (AU), mean 6 SD Fl-AGEs (AU), mean 6 SD CML (ng/mL), mean 6 SD sRAGE (pg/mL), mean 6 SD hs-CRP (mg/L), mean 6 SD sVAP-1 (ng/mL), mean 6 SD AOPPs (mmol/L), mean 6 SD Hypertension, N/Y (%), mean 6 SD Current smokers, N/Y (%) Grip strength (pounds), mean 6 SD
All
Special Variables Subgroup
Rest From All
P
146 73/73 (50/50) 57.0 6 15.5 27.0 6 3.6 85 6 16 5.4 6 0.3 22.5 6 8.9 2.3 6 0.5 285 6 70 NA NA 2.0 6 2.2 NA 175 6 118 106/40 (73/27) 124/22 (85/15) 84 6 30
61 30/31 (49/51) 57.9 6 15.0 26.7 6 3.9 85 6 17 5.5 6 0.4 22.1 6 8.0 2.4 6 0.6 307 6 81 1,066 6 374 999 6 352 2.0 6 2.3 399 6 162 203 6 170 42/19 (69/31) 51/10 (84/16) 83 6 28
85 43/42 (51/49) 56.3 6 15.7 27.2 6 3.3 85 6 15 5.4 6 0.3 22.7 6 9.6 2.3 6 0.5 268 6 57 NA NA 1.9 6 2.1 NA 155 6 47 64/21 (75/25) 73/12 (86/14) 85 6 15
.87* .53 .49 .96 .28 .71 .10 .002 NA NA .81 NA .036 .65* .70* .91
AF, autofluorescence; AOPPs, advanced oxidation protein products; AU, arbitrary units; BMI, body mass index; CML, Nε-carboxymethyllysine; eGFR, estimated glomerular filtration rate; F, females; Fl-AGEs, advanced glycation endproducts associated fluorescence of plasma; hs-CRP, high-sensitivity C-reactive protein; HbA1c, glycated hemoglobin A1c; M, males; N, not; NA, not applicable; SAF, skin autofluorescence; SD, standard deviation; sRAGE, soluble receptor for advanced glycation endproducts; sVAP-1, soluble vascular adhesion protein-1; Y, yes. Italics represent statistics calculated on log transformed data. Bold represent statistical significance. *Chi-square.
€ STURMER ET AL
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Figure 1. Relationship between skin autofluorescence (SAF) and age (years). AU, arbitrary units; NS-HT, hypertensive nonsmokers; NS-NT, normotensive nonsmokers; S-HT, hypertensive smokers; S-NT, normotensive smokers. Full line represents the regression line in the nonsmokers cohort. The dotted line represents the regression line in the smokers cohort.
independent significant contributor. Mean vitamin D3 levels did not differ between smokers and nonsmokers (23.2 6 9.4 vs. 22.3 6 8.9 ng/mL; P 5.66). Also between subjects with and without hypertension, no difference was observed (22.7 6 8.4 vs. 21.8 6 10.3 ng/mL; P 5 .59). Among smokers, an inverse relationship between vitamin D3 concentrations and plasma AGE-Fl (r 5 20.551; P 5 .008) was observed (Fig. 4). There was also an inverse relationship between sVAP-1 and plasma AGE-Fl (r 5 0.633; P 5.049; Fig. 5A) and a direct relationship between sVAP-1 and vitamin D3 (r 5 20.696; P 5.026; Fig. 5B).
Discussion Our findings reveal, for the first time, that in apparently healthy subjects, vitamin D deficiency may not contribute to the formation of AGEs. Moreover, accumulation of
AGEs in the skin may not impair the cutaneous photosynthesis of vitamin D. In line with earlier investigations, we found an agedependent rise of SAF and plasma AGE-Fl.19,21,33,34 The increase was more pronounced in subjects with hypertension. Moreover, active smokers showed higher SAF and plasma AGE-Fl levels, which is in agreements with earlier observations.33-35 In smokers we also revealed a direct relationship between plasma AGE-Fl and sVAP-1. Endothelial sVAP-1 is an adhesion molecule possessing the enzyme activity of semicarbazide-sensitive aminooxidase, which degrades primary amines into corresponding aldehydes, producing hydrogen peroxide and ammonia.36 Reactive aldehydes are potent glycating agents, whereas hydrogen peroxide contributes to oxidative stress and thereby accelerates the formation of AGEs. In humans, hyperglycemia-
Figure 2. Relationship between age and plasma advanced glycation endproducts–associated fluorescence (AGE-Fl). AU, arbitrary units; NS, nonsmokers; S, smokers.
5
VITAMIN D3 AND SKIN AUTOFLUORESCENCE Table 2. Characteristics of Subjects According to Absence or Presence of Hypertension Characteristic n* M/F, n (%) Age (y), mean 6 SD Vitamin D3 (ng/mL), mean 6 SD Skin AF (AU), mean 6 SD AGE-Fl (AU), mean 6 SD CML (ng/mL), mean 6 SD sRAGE (pg/mL), mean 6 SD hs-CRP (mg/L), mean 6 SD sVAP-1 (ng/mL), mean 6 SD AOPPs (mmol/L), mean 6 SD Smokers, N/Y (%) Grip strength (pounds), mean 6 SD
Normotensive Subjects
Hypertensive Subjects
P
107 50/57 (47/53) 54.0 6 15.5 22.1 6 8.4 2.3 6 0.5 273 6 60 1,128 6 403 1,008 6 341 1.8 6 2.0 397 6 184 175 6 110 90/17 (84/16) 84 6 31
39 23/16 (59/41) 64.8 6 12.2 21.8 6 10.3 2.5 6 0.5 315 6 86 929 6 256 980 6 384 2.4 6 2.7 402 6 104 176 6 140 34/5 (87/13) 82 6 25
.19† ,.001 .59 .018 .007 .054 .78 .15 .92 .51 .65† .73
AF, autofluorescence; AOPPs, advanced oxidation protein products; AU, arbitrary units; CML, Nε-carboxymethyllysine; F, females; Fl-AGEs, advanced glycation endproducts–associated fluorescence of plasma; hs-CRP, high-sensitivity C-reactive protein; M, males; N, not; P, significance (Student t test); SD, standard deviation; sRAGE, soluble receptor for advanced glycation endproducts; sVAP-1, soluble vascular adhesion protein-1; Y, yes. Italics represent statistics calculated on log transformed data. Bold represent statistical significance. *CML, sVAP-1, and sRAGE levels given for 42 normotensive and 19 hypertensive subjects. †Chi-square.
induced rise in circulation sVAP-1 level directly correlates with plasma AGE levels,37 and sVAP-1 is associated with subclinical atherosclerotic manifestations and the increased risk of cardiovascular events and mortality.38 Moreover, tobacco smoke has been shown to contain reactive glycation products which can react with proteins to form AGEs.6 It is also conceivable that chronic passive smoking could contribute to an enhanced AGE formation. Unfortunately, this important aspect has not been studied as yet.
A positive relationship between SAF and HbA1c suggests that even in nondiabetic subjects long-term control of glycemia affects tissue accumulation of AGEs. The lacking relationship between SAF and eGFR might be explained by the fact that only subjects with normal or slightly reduced renal function were included in our study. Moderate-tosevere CKD was exclusion criterion. Markers of inflammation and oxidative stress (hs-CRP and AOPPs, which are formed mainly during oxidative burst of phagocytes via
Table 3. Demographic and Laboratory Data According to Vitamin D3 Status Characteristic n M/F (n) Age (y), mean 6 SD Vitamin D3 (ng/mL), mean 6 SD Skin AF (AU), mean 6 SD AGE-Fl (AU), mean 6 SD CML (ng/L), mean 6 SD sRAGE (pg/mL), mean 6 SD hs-CRP (mg/L), mean 6 SD sVAP-1 (ng/mL), mean 6 SD AOPPs (mmol/L), mean 6 SD Hypertension (N/Y) Current smokers (N/Y) Grip strength (pounds), mean 6 SD
Vitamin D3 ,20 ng/mL
Vitamin D3 20-29 ng/mL
Vitamin D3 .30 ng/mL
P (ANOVA)
63 32/31 57.1 6 16.5 14.6 6 4.0 2.4 6 0.6 286 6 87 1,046 6 369 1,023 6 379 2.2 6 2.5 417 6 138 189 6 132 44/19 53/10 82 6 30
54 26/28 54.2 6 13.9 24.5 6 2.6* 2.3 6 0.5 281 6 59 1,083 6 315 942 6 329 2.0 6 2.2 386 6 169 146 6 76 42/12 46/8 87 6 31
29 15/14 59.9 6 15.1 35.9 6 4.9*,† 2.3 6 0.5 283 6 49 976 6 384 1,022 6 387 1.6 6 1.3 410 6 201 200 6 145 21/8 25/4 80 6 25
— .94‡ .44 ,.001 .55 .86 .58 .99 .92 .91 .68 .62‡ .97‡ .75
AF, autofluorescence; AOPPs, advanced oxidation protein products; AU, arbitrary units; CML, Nε-carboxymethyllysine; F, females; Fl-AGEs, advanced glycation endproducts associated fluorescence of plasma; hs-CRP, high-sensitivity C-reactive protein; M, males; N, not; SD, standard deviation; sRAGE, soluble receptor for advanced glycation endproducts; sVAP-1, soluble vascular adhesion protein-1; Y, yes. Italics represent statistics calculated on log transformed data. Bold represent statistical significance. *P . .001 vs. vitamin D3–deficient subjects (i.e., plasma concentration of vitamin D3 ,20 ng/mL). †P . .001 vs. subjects with insufficient vitamin D3 levels (i.e., plasma concentrations between 20 and 29 ng/mL). ‡Chi-square.
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Figure 3. Relationship between skin autofluorescence (SAF) and plasma vitamin D3 concentrations in the whole cohort. AU, arbitrary units; NS-NT, normotensive nonsmokers; S-HT, hypertensive smokers; S-NT, normotensive smokers.
the myeloperoxidase reaction) did not show a correlation with SAF levels. This suggests that the accumulation of AGEs was particularly induced by the age of the patients and less by inflammation and oxidative stress. Potential confounding factors of the biochemical consequences of AGE accumulation in the hypertensive patients are the treatment with various blood pressure–lowering drugs such as angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers. According to in vitro and in vivo studies, they exert anti-inflammatory, antioxidative, and anti-AGE forming effects.11,39 In addition, calcium receptor antagonist and angiotensin II receptor blockers decrease the level of AGEs.40,41 Furthermore, various statins were shown to modulate both the microinflammation and the formation of AGEs,42 in part by increasing sRAGE levels.43 However,
the number of included hypertensive subjects was too few, not allowing for valid testing of drug interactions. Of our subjects, 80% presented vitamin D insufficiency or deficiency. This high prevalence is in agreement with numerous other studies.23,24 Vitamin D3 concentration showed no relationship to SAF. Even in subjects with the highest SAF concentrations, the vitamin D3 levels were not more reduced. This observation suggests that high SAF does not hinder the formation of vitamin D3. In line with this assumption are investigations in hemodialysis patients after repeated exposures to ultraviolet B radiation. Despite the well-known high levels of skin AGEs in these patients, the increase of vitamin D3 concentration was even higher (42%) than in healthy control subjects (14%).44 On the other hand, the enhanced vitamin D3 formation after ultraviolet B radiation was relatively short
Figure 4. Relationship between plasma advanced glycation endproducts–associated fluorescence (AGE-Fl) and vitamin D3 concentrations. AU, arbitrary units; NS, nonsmokers; S, smokers.
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VITAMIN D3 AND SKIN AUTOFLUORESCENCE
Figure 5. (A) Correlation between plasma advanced glycation endproducts–associated fluorescence (AGE-Fl) and concentration of soluble vascular adhesion molecule 1 (sVAP-1). (B) Inverse correlation between vitamin D3 and sVAP-1. AU, arbitrary units.
lasting, suggesting that in hemodialysis patients, the half-life of vitamin D3 is shorter than in healthy subjects. Surprisingly, the low vitamin D3 levels in our cohort were not associated with markers of inflammation or oxidative stress. We only observed a trend to higher hs-CRP levels in subjects with vitamin D3 deficiency. However, in the small subgroup of smokers, a different response was observed. There was an inverse relationship between plasma AGE-Fl and vitamin D3. Moreover, the concentration of sVAP-1 increased significantly in subjects with vitamin D3 deficiency. These findings suggest that in smokers, low levels of vitamin D3 could aggravate toxicity of smoking as indicated by the rise of sVAP-1 and plasma AGE fluorescence. Because hypovitaminosis D is frequently associated with muscle wasting (sarcopenia) and weakness,23 we also measured muscle strength with the handgrip test. In line with the unchanged markers of inflammation, there was no difference between subjects with low and normal vitamin D3 levels. The fact that in our cohort the markers of oxidative stress and inflammation were in the normal range despite vitamin D3 deficiency suggests that the frequently observed activation of these markers in various disease states is not induced directly by vitamin D3 deficiency. Correspondingly in various controlled studies, the application of vitamin D did not improve the microinflammation and oxidative stress.45 Our negative findings are in line with a recent statement (2014) of the US Preventive Services Task Force, which concluded that routine vitamin D screening for healthy adults cannot be recommended because of insufficient evidence for a pathologic role in healthy subject. However, hypovitaminosis D may be associated with a secondary hyperparathyroidism.46 Parathyroid hormone (PTH) excess induces not only harmful effects on the skeleton but also numerous extraskeletal alterations.47 Recently, it has been shown that in patients with vitamin D3 deficiency and concomitant high PTH levels, the glucose homeostasis was markedly disturbed as indicated by a poor beta cell function and decreased insulin sensitivity.48 Therefore, PTH is of interest in patients with vitamin D3 deficiency.
In summary, our cross-sectional study shows that in relatively healthy subjects with vitamin D3 insufficiency or deficiency there is no modulation of the elevated AGE levels in skin and plasma. Hypovitaminosis D was also not associated with a rise in the levels of markers of microinflammation and oxidative stress. Only in the subgroup of active smokers, an inverse relationship between vitamin D3 deficiency and plasma AGE fluorescence was observed. Smokers also demonstrated enhanced levels of circulating sVAP-1. With regard to the negative results of our crosssectional study, there is a need for controlled longitudinal studies focusing on the effects of vitamin D supplementation on skin and plasma AGEs as well as markers of microinflammation and oxidative stress to exclude a potential relationship between vitamin D status and AGEs accumulation, and their interaction in potentiating of toxic effects.
Practical Application In nondiabetic, apparently healthy adults, age-dependent rise in SAF—representing long-term burden of cumulative metabolic and oxidative stress—does not interfere with vitamin D production in the skin. Vitamin D deficiency does not seem to enhance formation of AGEs via induction of oxidative stress or microinflammation. However, the previously mentioned interactions might not be unequivocally excluded in the smokers. In this regard, smokers and subjects exposed to passive smoking definitely require further attention.
References 1. Brownlee M. Lilly Lecture 1993. Glycation and diabetic complications. Diabetes. 1994;43:836-841. 2. Miyata T, Wada Y, Cai Z, et al. Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int. 1997;51:1170-1181. 3. Gugliucci A, Bendayan M. Renal fate of circulating advanced glycated end products (AGE): evidence for reabsorption and catabolism of AGEpeptides by renal proximal tubular cells. Diabetologia. 1996;39:149-160. 4. Uribarri J, Woodruff S, Goodman S, et al. Advanced glycation end products in foods and a practical guide to their reduction in the diet. J Am Diet Assoc. 2010;110:911-916 e912. 5. Sebekova K, Klenovics KS, Boor P, et al. Behaviour and hormonal status in healthy rats on a diet rich in Maillard reaction products with or without solvent extractable aroma compounds. Physiol Behav. 2012;105:693-701.
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6. Cerami C, Founds H, Nicholl I, et al. Tobacco smoke is a source of toxic reactive glycation products. Proc Natl Acad Sci USA. 1997;94:13915-13920. 7. Sebekova K, Schinzel R, Ling H, et al. Advanced glycated albumin impairs protein degradation in the kidney proximal tubules cell line LLC-PK1. Cell Mol Biol (noisy-le-grand). 1998;44:1051-1060. 8. Thornalley PJ. Cell activation by glycated proteins. AGE receptors, receptor recognition factors and functional classification of AGEs. Cell Mol Biol (noisy-legrand). 1998;44:1013-1023. 9. Schmidt AM, Stern DM. RAGE: a new target for the prevention and treatment of the vascular and inflammatory complications of diabetes. Trends Endocrinol Metab. 2000;11:368-375. 10. Makita Z, Radoff S, Rayfield EJ, et al. Advanced glycosylation endproducts in patients with diabetic nephropathy. N Engl J Med. 1991;325:836-842. 11. Stopper H, Schinzel R, Sebekova K, Heidland A. Genotoxicity of advanced glycation end products in mammalian cells. Cancer Lett. 2003;190:151-156. 12. Sebekova K, Wagner Z, Schupp N, Boor P. Genomic damage and malignancy in end-stage renal failure: do advanced glycation end products contribute? Kidney Blood Press Res. 2007;30:56-66. 13. Hartog JW, Voors AA, Bakker SJ, Smit AJ, van Veldhuisen DJ. Advanced glycation end-products (AGEs) and heart failure: pathophysiology and clinical implications. Eur J Heart Fail. 2004;9:1146-1155. 14. Lapolla A, Piarulli F, Sartore G, et al. Advanced glycation end products and antioxidant status in type 2 diabetic patients with and without peripheral artery disease. Diabetes Care. 2007;30:670-676. 15. Kilhovd BK, Juutilainen A, Lehto S, et al. Increased serum levels of advanced glycation endproducts predict total, cardiovascular and coronary mortality in women with type 2 diabetes: a population-based 18 year follow-up study. Diabetologia. 2007;50:1409-1417. 16. Wagner Z, Molnar M, Molnar GA, et al. Serum carboxymethyllysine predicts mortality in hemodialysis patients. Am J Kidney Dis. 2006;47:294-300. 17. Raucci A, Cugusi S, Antonelli A, et al. A soluble form of the receptor for advanced glycation endproducts (RAGE) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10). FASEB J. 2008;22:3716-3727. 18. Monnier VM, Bautista O, Kenny D, et al. Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional therapy of type 1 diabetes—relevance of glycated collagen products versus HbA(1c) as markers of diabetic complications. Diabetes. 1999;48:870-880. 19. Mulder DJ, Van de Water T, Lutgers HL, et al. Skin autofluorescence, a novel marker for glycemic and oxidative stress-derived advanced glycation endproducts: an overview of current clinical studies, evidence, and limitations. Diabetes Technol Ther. 2006;8:523-535. 20. Meerwaldt R, Zeebregts CJ, Navis G, Hillebrands JL, Lefrandt JD, Smit AJ. Accumulation of advanced glycation end products and chronic complications in ESRD treated by dialysis. Am J Kidney Dis. 2009;53:138-150. 21. Meerwaldt R, Graaff R, Oomen PHN, et al. Simple non-invasive assessment of advanced glycation endproduct accumulation. Diabetologia. 2004;47:1324-1330. 22. McIntyre NJ, Fluck RJ, McIntyre CW, Taal MW. Skin autofluorescence and the association with renal and cardiovascular risk factors in chronic kidney disease stage 3. Clin J Am Soc Nephrol. 2011;6:2356-2363. 23. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-281. 24. Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am J Clin Nutr. 2008;87:1080S-1086S. 25. Visser M, Deeg DJH, Lips P. Low vitamin D and high parathyroid hormone levels as determinants of loss of muscle strength and muscle mass (sarcopenia): the Longitudinal Aging Study Amsterdam. J Clin Endocrinol Metab. 2003;88:5766-5772. 26. Pilz S, Tomaschitz A, Ritz E, Pieber TR. Vitamin D status and arterial hypertension: a systematic review. Nat Rev Cardiol. 2009;6:621-630. 27. Schleithoff SS, Zittermann A, Tenderich G, Berthold HK, Stehle P, Koerfer R. Vitamin D supplementation improves cytokine profiles in patients with congestive heart failure: a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr. 2006;83:754-759.
28. Mitri J, Muraru MD, Pittas AG. Vitamin D and type 2 diabetes: a systematic review. Eur J Clin Nutr. 2011;65:1005-1015. 29. Talmor Y, Golan E, Benchetrit S, et al. Calcitriol blunts the deleterious impact of advanced glycation end products on endothelial cells. Am J Physiol Ren Physiol. 2008;294:F1059-F1064. 30. Salum E, Kals J, Kampus P, et al. Vitamin D reduces deposition of advanced glycation end-products in the aortic wall and systemic oxidative stress in diabetic rats. Diabetes Res Clin Pract. 2013;100:243-249. 31. Munch G, Keis R, Wessels A, et al. Determination of advanced glycation end products in serum by fluorescence spectroscopy and competitive ELISA. Eur J Clin Chem Clin Biochem. 1997;35:669-677. 32. Witko-Sarsat V, Friedlander M, Nguyen Khoa T, et al. Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure. J Immunol. 1998;161:2524-2532. 33. Koetsier M, Lutgers HL, de Jonge C, Links TP, Smit AJ, Graaff R. Reference values of skin autofluorescence. Diabetes Technol Ther. 2010;12:399-403. 34. Klenovics KS, Kollarova R, Hodosy J, Celec P, Sebekova K. Reference values of skin autofluorescence as an estimation of tissue accumulation of advanced glycation end products in a general Slovak population. Diabet Med. 2014;31:581-585. 35. Yue X, Hu H, Koetsier M, Graaff R, Han C. Reference values for the Chinese population of skin autofluorescence as a marker of advanced glycation end products accumulated in tissue. Diabet Med. 2011;28:818-823. 36. Lyles GA, Chalmers J. The metabolism of aminoacetone to methylglyoxal by semicarbazide-sensitive amine oxidase in human umbilical artery. Biochem Pharmacol. 1992;43:1409-1414. 37. Li HY, Lin MS, Wei JN, et al. Change of serum vascular adhesion protein-1 after glucose loading correlates to carotid intima-medial thickness in non-diabetic subjects. Clin Chim Acta. 2009;403:97-101. 38. Aalto K, Havulinna AS, Jalkanen S, Salomaa V, Salmi M. Soluble vascular adhesion protein-1 predicts incident major adverse cardiovascular events and improves reclassification in a Finnish prospective cohort study. Circ Cardiovasc Genet. 2014;7:529-535. 39. Forbes JM, Cooper ME, Thallas V, et al. Reduction of the accumulation of advanced glycation end products by ACE inhibition in experimental diabetic nephropathy. Diabetes. 2002;51:3274-3282. 40. Nangaku M, Miyata T, Sada T, et al. Anti-hypertensive agents inhibit in vivo the formation of advanced glycation end products and improve renal damage in a type 2 diabetic nephropathy rat model. J Am Soc Nephrol. 2003;14:1212-1222. 41. Sebekova K, Gazdikova K, Syrova D, et al. Effects of ramipril in nondiabetic nephropathy: improved parameters of oxidatives stress and potential modulation of advanced glycation end products. J Hum Hypertens. 2003;17:265-270. 42. Jinnouchi Y, Yamagishi S, Takeuchi M, Ishida S, Jinnouchi J, Imaizumi T. Atorvastatin decreases serum levels of advanced glycation end products (AGEs) in patients with type 2 diabetes. Clin Exp Med. 2006;6:191-193. 43. Quade-Lyssy P, Kanarek AM, Baiersdorfer M, Postina R, Kojro E. Statins stimulate the production of a soluble form of the receptor for advanced glycation end products. J Lipid Res. 2013;54:3052-3061. 44. Ala-Houhala MJ, Vahavihu K, Hasan T, et al. Narrow-band ultraviolet B exposure increases serum vitamin D levels in haemodialysis patients. Nephrol Dial Transplant. 2012;27:2435-2440. 45. Autier P, Boniol M, Pizot C, Mullie P. Vitamin D status and ill health: a systematic review. Lancet Diabetes Endocrinol. 2014;2:76-89. 46. Pepe J, Romagnoli E, Nofroni I, et al. Vitamin D status as the major factor determining the circulating levels of parathyroid hormone: a study in normal subjects. Osteoporos Int. 2005;16:805-812. 47. Massry S. Parathyroid hormone as uremic toxin. In: Massry and Glassock’s Textbook of Nephrology. 4th ed. Philadelphia, PA: Lippincott Williams and Wilkins; 2001:1221-1244. 48. Kramer CK, Swaminathan B, Hanley AJ, et al. Prospective associations of Vitamin D status with beta-cell function, insulin sensitivity and glycemia: the impact of parathyroid hormone status. Diabetes. 2014. in press.
25-Hydroxyvitamin D and Advanced Glycation Endproducts in Healthy and Hypertensive Subjects: Are There Interactions? ebekova, DSc, MD,† Gholamreza Fazeli, PhD,* Michael St€urmer, MD, PhD,* Katarına S Udo Bahner, PhD, MD,‡ Franz St€ab, Dr.rer.nat.,§ and August Heidland, PhD, MD* Advanced glycation endproducts (AGEs) accumulate during aging. Skin is the single organ of vitamin D synthesis, induced by ultraviolet B light. Accumulation of AGEs in the skin could interfere with synthesis of the vitamin, whereas the microinflammation and oxidative stress (associated with hypovitaminosis D) could amplify both the toxic effects of AGEs and their production. Clinical data on potential interactions between vitamin D3 deficiency and AGE accumulation are sparse. Here we investigated potential associations between levels of circulating vitamin D3 and those of AGEs in blood and skin with regard to markers of inflammation and oxidative stress in nondiabetic subjects. In a cross-sectional study, 146 subjects (119 healthy persons and 27 hypertensive patients; 73 male and 73 female; mean age, 57.0 6 15.5 years) were included. Skin autofluorescence (SAF) and plasma levels of vitamin D3, AGE-associated fluorescence, high-sensitivity C-reactive protein level, and advanced oxidation protein products as well as renal function (estimated glomerular filtration rate) were determined. In a subgroup of 61 patients, Nε-carboxymethyllysine, soluble receptor of AGEs, and soluble vascular adhesion protein-1 were additionally analyzed. Vitamin D3 level averaged 22.5 6 8.9 ng/mL. Prevalence of vitamin D insufficiency (2029 ng/mL) was 43%, and that of deficiency (,20 ng/mL) 37%. The age-dependent rise in SAF was steeper in smokers and in subjects presenting arterial hypertension. No association between SAF and hypovitaminosis D was revealed. Among smokers, an inverse relationship manifested between vitamin D3 and plasma AGE-associated fluorescence as well as soluble vascular adhesion protein-1. Our data suggest that in nondiabetic adults, hypovitaminosis D does not enhance toxicity and accumulation of AGEs. Only in smokers interactions are conceivable. Ó 2015 by the National Kidney Foundation, Inc. All rights reserved.
A
Introduction
DVANCED GLYCATION ENDPRODUCTS (AGEs) are heterogeneous compounds, formed via the Maillard reaction (nonenzymatic reaction of a sugar ketone or aldehyde group with a free amino group of proteins, lipids, and nucleic acids) or alternatively under conditions of oxidative and carbonyl stress.1,2 Moreover, accumulation of AGEs is increased in chronic kidney disease (CKD), in part because of an impaired renal *
University of W€urzburg, W€urzburg, Germany. Comenius University, Medical Faculty, Bratislava, Slovakia. ‡ KfH Nierenzentrum W€urzburg, W€urzburg, Germany. § Beiersdorf AG, Hamburg, Germany. Financial Disclosure: A.H. received honoraria for lectures from gbo Medizintechnik AG and U.B. from Fresenius Medical Care. F.S. is an employee of Beiersdorf AG, Hamburg, Germany. The other authors declare that they have no relevant financial interests. Support: This study was supported by a grant from Beiersdorf AG, Hamburg, Germany to the Verein zur Bek€ampfung der Hochdruck-und Nierenkrankheiten W€urzburg e.V. Address correspondence to August Heidland, PhD, MD, Department of Internal Medicine, University of W€urzburg and KfH Kidney Center, HansBrandmann-Weg-1, 97080, W€urzburg, Germany. E-mail: August. †
[email protected] Ó
2015 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2014.10.027
Journal of Renal Nutrition, Vol -, No - (-), 2015: pp 1-8
removal,3 consumption of thermally processed food rich in glycotoxins,4,5 and tobacco smoke.6 Adverse effects of AGEs result from modifications of proteins and lipids leading to structural and functional alterations of plasma and extracellular or intracellular proteins. In particular, crosslinking of collagen, thickening of basement membranes, and trapping of proteins (low-density lipoprotein) induce vascular stiffness and an enhanced vascular permeability. AGE modifications of plasma or tissue proteins are associated with impaired protein degradation.7 Interaction of AGEs with their receptors (particularly RAGE) induces cell activation via nuclear transcription factors, for example, NF-kB, with reduced nitric oxide formation, enhanced generation of oxygen radicals, synthesis of proinflammatory cytokines or chemokines, fibrogenic growth factors (transforming growth factor beta 1), vascular adhesion molecules, and cell proliferation.8 RAGE are located on the surface of multiple cells including endothelial, smooth muscle, mesangial and tubule cells, monocytes, macrophages, and neurons.9 AGEs disturb endothelial functions in particular by lowering nitric oxide levels, which promotes the adherence of leukocytes. In this way, AGEs are implicated in the aging process, in particular of atherosclerosis, in the manifestation and progression of renal disease and in diabetic patients in the macroangiopathy and microangiopathy (nephropathy, retinopathy, and 1
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neuropathy).10 AGEs also induce genomic damage11 and may be associated with an enhanced cancer incidence.12 In the risk assessment of AGE accumulation, determination of their circulating blood components such as pentosidine, N3-carboxymethyllysine (CML), and AGEassociated fluorescence (AGE-Fl) are used. They are related to severity and prognosis of chronic heart failure13 and to coronary and peripheral artery disease in diabetic patients.14 Enhanced plasma AGE levels are independent predictors of total, cardiovascular, and coronary mortality in women with type 2 diabetes15 and of mortality in hemodialysis patients.16 Soluble RAGE (sRAGE) represents the truncated form of the receptor, which may neutralize the toxic effect of AGEs.17 A relatively novel marker of AGE accumulation is the noninvasive determination of skin autofluorescence (SAF). Particularly, skin collagen is a target for AGE modification.18 SAF reflects the accumulation of AGEs in the tissues and hence the ‘‘long-term cumulative metabolic and oxidative stress.’’ Because of the substantially longer halflife of collagen in comparison with plasma proteins, SAF provides information on the long-term cumulative burden, whereas plasma AGEs reflect more that of the recent past. SAF is an independent predictor of cardiovascular complications, morbidity, and mortality.19-21 In moderate renal failure, SAF was inversely correlated to circulating endothelial progenitor cells.22 The skin is the single organ producing the steroid hormone vitamin D with pleiotropic actions on most tissues and cells in the body.23 Low serum concentrations of 25OH vitamin D (an estimate of vitamin D3 stores) represent a worldwide problem.24 It may cause rickets, osteoporosis, osteomalacia, and fractures, as well as muscle weakness,23 in part as a result of secondary hyperparathyroidism.25 Numerous prospective studies revealed associations of low vitamin D3 levels with hypertension (via stimulation of renin-angiotensin-aldosterone system),26 cardiovascular complications,27 infectious diseases (lowered cathelicidin levels), insulin resistance and type 2 diabetes,28 progression of CKD, and autoimmune diseases (type 1 diabetes, multiple sclerosis, and rheumatoid arthritis).23 Up till now, there are no data about potential relationships between vitamin D3 and AGEs in clinical medicine. In vitro studies suggested that the deleterious impact of AGE-modified human serum albumin on endothelial cells could be blunted by addition of calcitriol [1,25(OH)2D3].29 In diabetic rats, treatment with vitamin D (cholecalciferol) reduced the deposition of AGEs (CML) in the aortic wall and the systemic oxidative stress.30 In the present article, we raised the following questions: (1) Is an enhanced accumulation of AGEs in the skin associated with an impaired synthesis of vitamin D? (2) Does hypovitaminosis D contribute to an amplification of the AGEinduced microinflammation and oxidative stress? (3) Does hypovitaminosis D accelerate the formation of AGEs?
Subjects and Methods This single-center observational study was performed in a practice of Internal Medicine (Dr Werner St€ urmer) in W€ urzburg and conducted in accordance to the principles of the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of the Medical Faculty of the University of W€ urzburg. All subjects signed an informed consent to participate. In the cross-sectional study we included a total of 146 nondiabetic subjects (aged, 14-96 years) on occasion of their routine medical examination. Of them, 73% were apparently healthy, whereas 27% showed arterial hypertension. Fifteen percent of the participants were current smokers. All investigated subjects were without dermatosis, scars, and pigment disorders. Exclusion criteria comprised any acute illness, diabetes mellitus and HbA1c levels .6%, autoimmune diseases, malignancies, moderate-to-severe CKD (estimated glomerular filtration rate [eGFR] ,60 mL/min/1.73 m2), infections, use of glucocorticoids, vitamin D supplements, regular visits of a solarium, and use of tanning cream (during the last 14 days). Anthropometric measurements to calculate body mass index were performed. SAF was measured on the volar side of the forearm using the AGE Reader (Diagnoptics BV, Groningen, the Netherlands).19 eGFR was calculated using the abbreviated Modification of Diet in Renal Disease formula. Handgrip muscle strength was measured using the Baseline hydraulic hand dynamometer (Fabrication Enterprises Inc., White Plains, New York). Blood was collected in the morning hours after overnight fasting from antecubital vein. Routine blood chemistry was determined with an automatic analyzer. HbA1c was analyzed using high-pressure liquid chromatography (ADAM A1cHA-8180 FAST; Axonlab, Baden, Switzerland). Aliquots of plasma were stored at 280 C until special analyses. High-sensitivity C-reactive protein (hs-CRP) levels were measured nephelometrically using Siemens reagent (Labor Limbach, Heidelberg, Germany) and vitamin D3 by an electrochemiluminescence immunoassay from Roche (Labor Limbach, Heidelberg, Germany. Analysis of AGE-Fl in plasma was performed according to M€ unch et al.31 and of advanced oxidation protein products (AOPPs) according to Witko-Sarsat et al.32 In a subgroup of randomly selected 61 patients, CML (Microcoat, Bernried, Germany), sRAGE (R&D Systems, Minneapolis, Minnesota), and soluble vascular receptor adhesion protein-1 (sVAP-1; Bender Med System Inc., Vienna, Austria) were determined using commercial enzyme-linked immunosorbent assay kits according to manufacturer’s instructions.
Statistical Analysis Skewed data were log transformed for statistical analyses, but for simplicity, means and standard deviations are given.
3
VITAMIN D3 AND SKIN AUTOFLUORESCENCE
The Student t test (2-sided) was used to compare 2 groups. Comparisons among 3 groups were performed using analysis of variance with Scheffe post hoc test, when necessary. Categorical data were evaluated using chi-square test. Pearson correlation coefficients were calculated. Multivariate analysis was performed using the general linear model. P ,.05 was considered significant. Statistical program SPSS, version 16 (SPSS Inc., Chicago, IL) was used for statistical analyses.
Results Cohort characteristics are given in Table 1. The subgroup in which additional markers were also determined did not differ significantly from the rest of the cohort except for higher plasma AGE-Fl’s (P 5 .002) and AOPPs (P 5.036; Table 1).
Skin Autofluorescence In the whole group, a significant linear correlation was revealed only between SAF and age (r 5 0.464; P , .001) or HbA1c (r 5 0.223; P 5.037). The association between SAF and eGFR did not reach significance (r 5 20.151; P 5 .16). Multivariate analysis with age, body mass index, eGFR, hs-CRP, AGE-Fl, AOPPs, smoking status, and presence of hypertension entered as independent variables indicated age and current smoking as independent variables affecting SAF (corrected model: P , .001; age: P , .001; smoking: P 5 .011; R2: 27%). Comparison between groups did not reveal a significant difference in SAF between smokers and nonsmokers (2.4 6 0.7 vs. 2.3 6 0.5 arbitrary units [AU]; P 5 .57),
stemming probably from the fact that the smokers were significantly younger (45.8 6 12.1 vs. 58.9 6 115.1 years; P ,.001) and presented higher eGFR (94 6 16 vs. 83 6 16 mL/min/ 1.73 m2; P ,.02) and lower HbA1c compared with the nonsmokers (5.3 6 0.3 vs. 5.5 6 0.3, p , .012). No significant association between SAF and other variables except for age (Fig. 1) was revealed if smokers and nonsmokers were evaluated separately. There was also a significant relationship between plasma AGE-associated fluorescence and age (Fig. 2). Subjects with hypertension presented higher SAF and plasma AGE-Fl in comparison to normotensive subjects (2.5 6 0.5 vs. 2.3 6 0.5; P 5 .018 and 315 6 86 vs. 273 6 60 AU; P 5.007, respectively), but they were also significantly older (64.8 6 12.2 AU vs. 54.0 6 15.5 years; P , .001; Table 2). Other variables did not differ significantly between normotensive and hypertensive subjects. Vitamin D3 level averaged in the cohort to 22.5 6 8.9 ng/mL (Table 1). Males and females did not differ significantly (22.6 6 9.1 ng/mL vs. 22.3 6 8.8 ng/mL; P 5 .84). 43% of all participants presented a vitamin D3 deficiency (concentration ,20 ng/ mL), 37% a vitamin D3 insufficiency (concentration 2029 ng/mL), whereas sufficient vitamin D3 levels (concentration . 30 ng/mL) were revealed in 20% of the subjects. Between-group comparisons did not show any other significance (Table 3). In the whole cohort, no significant relationship between vitamin D3 and SAF (Fig. 3), or any other variable, was revealed. Multivariate analysis neither showed any
Table 1. Demographic and Laboratory Characteristics of the Subjects Characteristic n M/F, n (%) Age (y), mean 6 SD BMI (kg/m2), mean 6 SD eGFR (mL/min/1.73 m2), mean 6 SD HbA1c (%), mean 6 SD Vitamin D3 (ng/mL), mean 6 SD SAF (AU), mean 6 SD Fl-AGEs (AU), mean 6 SD CML (ng/mL), mean 6 SD sRAGE (pg/mL), mean 6 SD hs-CRP (mg/L), mean 6 SD sVAP-1 (ng/mL), mean 6 SD AOPPs (mmol/L), mean 6 SD Hypertension, N/Y (%), mean 6 SD Current smokers, N/Y (%) Grip strength (pounds), mean 6 SD
All
Special Variables Subgroup
Rest From All
P
146 73/73 (50/50) 57.0 6 15.5 27.0 6 3.6 85 6 16 5.4 6 0.3 22.5 6 8.9 2.3 6 0.5 285 6 70 NA NA 2.0 6 2.2 NA 175 6 118 106/40 (73/27) 124/22 (85/15) 84 6 30
61 30/31 (49/51) 57.9 6 15.0 26.7 6 3.9 85 6 17 5.5 6 0.4 22.1 6 8.0 2.4 6 0.6 307 6 81 1,066 6 374 999 6 352 2.0 6 2.3 399 6 162 203 6 170 42/19 (69/31) 51/10 (84/16) 83 6 28
85 43/42 (51/49) 56.3 6 15.7 27.2 6 3.3 85 6 15 5.4 6 0.3 22.7 6 9.6 2.3 6 0.5 268 6 57 NA NA 1.9 6 2.1 NA 155 6 47 64/21 (75/25) 73/12 (86/14) 85 6 15
.87* .53 .49 .96 .28 .71 .10 .002 NA NA .81 NA .036 .65* .70* .91
AF, autofluorescence; AOPPs, advanced oxidation protein products; AU, arbitrary units; BMI, body mass index; CML, Nε-carboxymethyllysine; eGFR, estimated glomerular filtration rate; F, females; Fl-AGEs, advanced glycation endproducts associated fluorescence of plasma; hs-CRP, high-sensitivity C-reactive protein; HbA1c, glycated hemoglobin A1c; M, males; N, not; NA, not applicable; SAF, skin autofluorescence; SD, standard deviation; sRAGE, soluble receptor for advanced glycation endproducts; sVAP-1, soluble vascular adhesion protein-1; Y, yes. Italics represent statistics calculated on log transformed data. Bold represent statistical significance. *Chi-square.
€ STURMER ET AL
4
Figure 1. Relationship between skin autofluorescence (SAF) and age (years). AU, arbitrary units; NS-HT, hypertensive nonsmokers; NS-NT, normotensive nonsmokers; S-HT, hypertensive smokers; S-NT, normotensive smokers. Full line represents the regression line in the nonsmokers cohort. The dotted line represents the regression line in the smokers cohort.
independent significant contributor. Mean vitamin D3 levels did not differ between smokers and nonsmokers (23.2 6 9.4 vs. 22.3 6 8.9 ng/mL; P 5.66). Also between subjects with and without hypertension, no difference was observed (22.7 6 8.4 vs. 21.8 6 10.3 ng/mL; P 5 .59). Among smokers, an inverse relationship between vitamin D3 concentrations and plasma AGE-Fl (r 5 20.551; P 5 .008) was observed (Fig. 4). There was also an inverse relationship between sVAP-1 and plasma AGE-Fl (r 5 0.633; P 5.049; Fig. 5A) and a direct relationship between sVAP-1 and vitamin D3 (r 5 20.696; P 5.026; Fig. 5B).
Discussion Our findings reveal, for the first time, that in apparently healthy subjects, vitamin D deficiency may not contribute to the formation of AGEs. Moreover, accumulation of
AGEs in the skin may not impair the cutaneous photosynthesis of vitamin D. In line with earlier investigations, we found an agedependent rise of SAF and plasma AGE-Fl.19,21,33,34 The increase was more pronounced in subjects with hypertension. Moreover, active smokers showed higher SAF and plasma AGE-Fl levels, which is in agreements with earlier observations.33-35 In smokers we also revealed a direct relationship between plasma AGE-Fl and sVAP-1. Endothelial sVAP-1 is an adhesion molecule possessing the enzyme activity of semicarbazide-sensitive aminooxidase, which degrades primary amines into corresponding aldehydes, producing hydrogen peroxide and ammonia.36 Reactive aldehydes are potent glycating agents, whereas hydrogen peroxide contributes to oxidative stress and thereby accelerates the formation of AGEs. In humans, hyperglycemia-
Figure 2. Relationship between age and plasma advanced glycation endproducts–associated fluorescence (AGE-Fl). AU, arbitrary units; NS, nonsmokers; S, smokers.
5
VITAMIN D3 AND SKIN AUTOFLUORESCENCE Table 2. Characteristics of Subjects According to Absence or Presence of Hypertension Characteristic n* M/F, n (%) Age (y), mean 6 SD Vitamin D3 (ng/mL), mean 6 SD Skin AF (AU), mean 6 SD AGE-Fl (AU), mean 6 SD CML (ng/mL), mean 6 SD sRAGE (pg/mL), mean 6 SD hs-CRP (mg/L), mean 6 SD sVAP-1 (ng/mL), mean 6 SD AOPPs (mmol/L), mean 6 SD Smokers, N/Y (%) Grip strength (pounds), mean 6 SD
Normotensive Subjects
Hypertensive Subjects
P
107 50/57 (47/53) 54.0 6 15.5 22.1 6 8.4 2.3 6 0.5 273 6 60 1,128 6 403 1,008 6 341 1.8 6 2.0 397 6 184 175 6 110 90/17 (84/16) 84 6 31
39 23/16 (59/41) 64.8 6 12.2 21.8 6 10.3 2.5 6 0.5 315 6 86 929 6 256 980 6 384 2.4 6 2.7 402 6 104 176 6 140 34/5 (87/13) 82 6 25
.19† ,.001 .59 .018 .007 .054 .78 .15 .92 .51 .65† .73
AF, autofluorescence; AOPPs, advanced oxidation protein products; AU, arbitrary units; CML, Nε-carboxymethyllysine; F, females; Fl-AGEs, advanced glycation endproducts–associated fluorescence of plasma; hs-CRP, high-sensitivity C-reactive protein; M, males; N, not; P, significance (Student t test); SD, standard deviation; sRAGE, soluble receptor for advanced glycation endproducts; sVAP-1, soluble vascular adhesion protein-1; Y, yes. Italics represent statistics calculated on log transformed data. Bold represent statistical significance. *CML, sVAP-1, and sRAGE levels given for 42 normotensive and 19 hypertensive subjects. †Chi-square.
induced rise in circulation sVAP-1 level directly correlates with plasma AGE levels,37 and sVAP-1 is associated with subclinical atherosclerotic manifestations and the increased risk of cardiovascular events and mortality.38 Moreover, tobacco smoke has been shown to contain reactive glycation products which can react with proteins to form AGEs.6 It is also conceivable that chronic passive smoking could contribute to an enhanced AGE formation. Unfortunately, this important aspect has not been studied as yet.
A positive relationship between SAF and HbA1c suggests that even in nondiabetic subjects long-term control of glycemia affects tissue accumulation of AGEs. The lacking relationship between SAF and eGFR might be explained by the fact that only subjects with normal or slightly reduced renal function were included in our study. Moderate-tosevere CKD was exclusion criterion. Markers of inflammation and oxidative stress (hs-CRP and AOPPs, which are formed mainly during oxidative burst of phagocytes via
Table 3. Demographic and Laboratory Data According to Vitamin D3 Status Characteristic n M/F (n) Age (y), mean 6 SD Vitamin D3 (ng/mL), mean 6 SD Skin AF (AU), mean 6 SD AGE-Fl (AU), mean 6 SD CML (ng/L), mean 6 SD sRAGE (pg/mL), mean 6 SD hs-CRP (mg/L), mean 6 SD sVAP-1 (ng/mL), mean 6 SD AOPPs (mmol/L), mean 6 SD Hypertension (N/Y) Current smokers (N/Y) Grip strength (pounds), mean 6 SD
Vitamin D3 ,20 ng/mL
Vitamin D3 20-29 ng/mL
Vitamin D3 .30 ng/mL
P (ANOVA)
63 32/31 57.1 6 16.5 14.6 6 4.0 2.4 6 0.6 286 6 87 1,046 6 369 1,023 6 379 2.2 6 2.5 417 6 138 189 6 132 44/19 53/10 82 6 30
54 26/28 54.2 6 13.9 24.5 6 2.6* 2.3 6 0.5 281 6 59 1,083 6 315 942 6 329 2.0 6 2.2 386 6 169 146 6 76 42/12 46/8 87 6 31
29 15/14 59.9 6 15.1 35.9 6 4.9*,† 2.3 6 0.5 283 6 49 976 6 384 1,022 6 387 1.6 6 1.3 410 6 201 200 6 145 21/8 25/4 80 6 25
— .94‡ .44 ,.001 .55 .86 .58 .99 .92 .91 .68 .62‡ .97‡ .75
AF, autofluorescence; AOPPs, advanced oxidation protein products; AU, arbitrary units; CML, Nε-carboxymethyllysine; F, females; Fl-AGEs, advanced glycation endproducts associated fluorescence of plasma; hs-CRP, high-sensitivity C-reactive protein; M, males; N, not; SD, standard deviation; sRAGE, soluble receptor for advanced glycation endproducts; sVAP-1, soluble vascular adhesion protein-1; Y, yes. Italics represent statistics calculated on log transformed data. Bold represent statistical significance. *P . .001 vs. vitamin D3–deficient subjects (i.e., plasma concentration of vitamin D3 ,20 ng/mL). †P . .001 vs. subjects with insufficient vitamin D3 levels (i.e., plasma concentrations between 20 and 29 ng/mL). ‡Chi-square.
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Figure 3. Relationship between skin autofluorescence (SAF) and plasma vitamin D3 concentrations in the whole cohort. AU, arbitrary units; NS-NT, normotensive nonsmokers; S-HT, hypertensive smokers; S-NT, normotensive smokers.
the myeloperoxidase reaction) did not show a correlation with SAF levels. This suggests that the accumulation of AGEs was particularly induced by the age of the patients and less by inflammation and oxidative stress. Potential confounding factors of the biochemical consequences of AGE accumulation in the hypertensive patients are the treatment with various blood pressure–lowering drugs such as angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers. According to in vitro and in vivo studies, they exert anti-inflammatory, antioxidative, and anti-AGE forming effects.11,39 In addition, calcium receptor antagonist and angiotensin II receptor blockers decrease the level of AGEs.40,41 Furthermore, various statins were shown to modulate both the microinflammation and the formation of AGEs,42 in part by increasing sRAGE levels.43 However,
the number of included hypertensive subjects was too few, not allowing for valid testing of drug interactions. Of our subjects, 80% presented vitamin D insufficiency or deficiency. This high prevalence is in agreement with numerous other studies.23,24 Vitamin D3 concentration showed no relationship to SAF. Even in subjects with the highest SAF concentrations, the vitamin D3 levels were not more reduced. This observation suggests that high SAF does not hinder the formation of vitamin D3. In line with this assumption are investigations in hemodialysis patients after repeated exposures to ultraviolet B radiation. Despite the well-known high levels of skin AGEs in these patients, the increase of vitamin D3 concentration was even higher (42%) than in healthy control subjects (14%).44 On the other hand, the enhanced vitamin D3 formation after ultraviolet B radiation was relatively short
Figure 4. Relationship between plasma advanced glycation endproducts–associated fluorescence (AGE-Fl) and vitamin D3 concentrations. AU, arbitrary units; NS, nonsmokers; S, smokers.
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VITAMIN D3 AND SKIN AUTOFLUORESCENCE
Figure 5. (A) Correlation between plasma advanced glycation endproducts–associated fluorescence (AGE-Fl) and concentration of soluble vascular adhesion molecule 1 (sVAP-1). (B) Inverse correlation between vitamin D3 and sVAP-1. AU, arbitrary units.
lasting, suggesting that in hemodialysis patients, the half-life of vitamin D3 is shorter than in healthy subjects. Surprisingly, the low vitamin D3 levels in our cohort were not associated with markers of inflammation or oxidative stress. We only observed a trend to higher hs-CRP levels in subjects with vitamin D3 deficiency. However, in the small subgroup of smokers, a different response was observed. There was an inverse relationship between plasma AGE-Fl and vitamin D3. Moreover, the concentration of sVAP-1 increased significantly in subjects with vitamin D3 deficiency. These findings suggest that in smokers, low levels of vitamin D3 could aggravate toxicity of smoking as indicated by the rise of sVAP-1 and plasma AGE fluorescence. Because hypovitaminosis D is frequently associated with muscle wasting (sarcopenia) and weakness,23 we also measured muscle strength with the handgrip test. In line with the unchanged markers of inflammation, there was no difference between subjects with low and normal vitamin D3 levels. The fact that in our cohort the markers of oxidative stress and inflammation were in the normal range despite vitamin D3 deficiency suggests that the frequently observed activation of these markers in various disease states is not induced directly by vitamin D3 deficiency. Correspondingly in various controlled studies, the application of vitamin D did not improve the microinflammation and oxidative stress.45 Our negative findings are in line with a recent statement (2014) of the US Preventive Services Task Force, which concluded that routine vitamin D screening for healthy adults cannot be recommended because of insufficient evidence for a pathologic role in healthy subject. However, hypovitaminosis D may be associated with a secondary hyperparathyroidism.46 Parathyroid hormone (PTH) excess induces not only harmful effects on the skeleton but also numerous extraskeletal alterations.47 Recently, it has been shown that in patients with vitamin D3 deficiency and concomitant high PTH levels, the glucose homeostasis was markedly disturbed as indicated by a poor beta cell function and decreased insulin sensitivity.48 Therefore, PTH is of interest in patients with vitamin D3 deficiency.
In summary, our cross-sectional study shows that in relatively healthy subjects with vitamin D3 insufficiency or deficiency there is no modulation of the elevated AGE levels in skin and plasma. Hypovitaminosis D was also not associated with a rise in the levels of markers of microinflammation and oxidative stress. Only in the subgroup of active smokers, an inverse relationship between vitamin D3 deficiency and plasma AGE fluorescence was observed. Smokers also demonstrated enhanced levels of circulating sVAP-1. With regard to the negative results of our crosssectional study, there is a need for controlled longitudinal studies focusing on the effects of vitamin D supplementation on skin and plasma AGEs as well as markers of microinflammation and oxidative stress to exclude a potential relationship between vitamin D status and AGEs accumulation, and their interaction in potentiating of toxic effects.
Practical Application In nondiabetic, apparently healthy adults, age-dependent rise in SAF—representing long-term burden of cumulative metabolic and oxidative stress—does not interfere with vitamin D production in the skin. Vitamin D deficiency does not seem to enhance formation of AGEs via induction of oxidative stress or microinflammation. However, the previously mentioned interactions might not be unequivocally excluded in the smokers. In this regard, smokers and subjects exposed to passive smoking definitely require further attention.
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