original article identification of advanced glycation ...5)92100.pdf92 introduction glycation is a...

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Introduction

Glycation is a non-enzymatic reaction between a protein and a reducing sugar, such as glucose 1,2). This reaction generates advanced glycation end products (AGEs) via a chain of intermediate reactions that produce Schiff bases and Amadori products. AGEs are implicated in the pathology of several age-related diseases, and many AGEs and their intermediate products, such as 3-deoxyglucosone (3DG), are thought to have strong relationship with aging and lifestyle diseases 1,3). Several AGEs exhibit a characteristic fluorescence at 370 nm/440 nm 4-6), and f luorescence intensity can be measured in vitro to examine the formation of AGEs under different treatments 7,8).

Fluorescence is also a potential method of measuring cumulative AGEs deposition in human skin non-invasively, in clinical studies 9-11). Previous studies have measured average fluorescence intensity between 420-600 nm, although Koetsier measured f luorescence at ex. 350-420 nm em. 420-600 nm

Original Article

Identification of Advanced Glycation Endproducts derived fluorescence spectrum in vitro and human skin

Keitaro Nomoto, Masayuki Yagi, Umenoi Hamada, Junko Naito, Yoshikazu Yonei

Anti-Aging Medical Research Center and Glycation Stress Research Center, Graduate School of Life and Medical Sciences, Doshisha University

AbstractObjective: Here, we compared the fluorescence spectra from in vitro models containing advanced glycation endproducts (AGEs) with human skin. Methods: The in vitro samples were prepared from glycated human serum albumin (HSA-Glc (+), (–)), pentosidine (a typical fluorescent AGE), and bovine skins. Fluorescence data from the skin of 11 healthy people were collected. We used a commercially available fluorometer to measure fluorescence at 370 nm excitation. The correlation of fluorescence intensity, age, and skin auto fluorescence was assessed using Pearson’s correlation coefficient.Results::The peak fluorescence of HSA- Glc (+) and pentosidine were close to 440 nm, and a peak fluorescence at 451 nm was observed in the spectra of human and bovine skin. The f luorescence intensity of human skin at 440 nm might be positively correlated with age (r = 0.582, p = 0.060) and skin AF (r = 0.832, p = 0.001).Conclusion: The in vitro results demonstrate that fluorescence intensity at 370/440 nm indicates fluorescent AGEs products. Since the fluorescence spectrum of glycated HSA and human skin are similar, it may be feasible to measure the degree of glycation in living tissue by measuring AGEs fluorescence.

Key words: advanced glycation endproducts (AGEs), pentosidine, fluorescence spectra, human serum albumin, skin

Received: Jul 16, 2013 Accepted: Aug. 25, 2013Published online: Oct. 30, 2013

Anti-Aging Medicine 10 (5) : 92-100, 2013(c) Japanese Society of Anti-Aging Medicine

Prof. Yoshikazu Yonei, M.D., Ph.D.Anti-Aging Medical Research Center and Glycation Stress Research Center,

Graduate School of Life and Medical Sciences, Doshisha University1-3, Tataramiyakodani, Kyotanabe-shi, Kyoto, 610-0321, Japan

Tel&Fax: +81-774-65-6394 / E-mail: [email protected]

on the skin of diabetes mellitus patients but did not find any specific fluorescence peak 12). Except for pentosidine (λmax: ex. 335 nm, em. 385 nm), no fluorescent AGEs have been detected on human skin.

The identification of specific fluorescence spectra derived from AGEs and emitted from skin is difficult because other fluorescent material, such as NADH, may also be present 13). A direct comparison of the fluorescence spectra from in vitro samples and in vivo human skin may help distinguish AGEs derived f luorescence. Here we compared the f luorescence spectra of specific proteins, skin glycation models, and human skin.

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Identification of Advanced Glycation Endproducts derived fluorescence spectrum

Subjects and Methods

Preparation and measurement of in vitro models AGEs were produced by incubating protein and bovine

skin samples in glucose reaction solutions for up to six days. Human Serum Albumin (HSA) (Sigma Chemical Co. Ltd, MO, USA) was incubated with and without glucose at 60 °C 7). The glucose (+) (G (+)) reaction solution contained 0.1 M phosphate buffer (PBS) (pH 7.4), 40mg/ml HSA, 2.0 M glucose solution, and distilled water at a 5:2:1:2 volume ratio. Earlier research has demonstrated this solution produces the pentosidine. The glucose (–) reaction (G (–)) solution contained 0.1 M PBS, 40 mg/mL HSA, and distilled water at a 5:2:3 volume ratio. Reaction solutions were incubated at 60 °C for 0, 3, 6 days.

Quinine sulfate solution were used as a positive controls for fluorescent material at 0, 5, 50, 500 µg/mL. The concentration of pentosidine produced by the HSA-Glc (+) model was measured by ELISA using the commercial kit ‘FSK Pentosidine’ (Fushimi Medicine Manufacture Place, Marugame, Kagawa, Japan) 8).

AGE positive or negative bovine skin, which contains collagen, were produced by incubating skin samples in a glucose positive (G (+)) solution (2 mol/L glucose and 0.1 M PBS) or a glucose negative (G (–)) solution, at 60 °C for 0, 2, 4 days 14).

Samples of bovine skin with known f luorescence were produced by adding 100 µL quinine sulfate to 50 mm2 samples of half-dried, untreated bovine skins, and the f luorescence spectrum measured after 30 minutes.

Fluorescence intensity of the all samples was measured at different wavelengths with an LS50 fluorometer (Perkin-Elmer Inc., USA). The samples were decanted into 1-cm cells and fluorescence measured at 10 nm slit-width and 200 nm/min.

Clinical studyThe skin fluorescence of 11 healthy men and women (five

men and six women mean age: 30.1 ± 11.6 years old) was measured with an AGE ReaderTM (DiagnOptics, Groningen, Netherlands) 15-17). Excitation light (wavelength 300-420 nm) was projected onto 1 cm2 of skin inside the upper arm, approximately 10 cm above the elbow, and the intensity of emitted light (420-600 nm, which is auto fluorescence (AF)) measured with a fluorometer. The skin AF (arbitrary units [AUs] × 100) was calculated from the mean value of the emitted light intensity divided by the excitation light intensity.

The f luorometer (model C10988MA, Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan) excitation light (370-380 nm) was produced by light-emitting diodes (LED) (Nitrode Semiconductors. Co., Ltd. Naruto, Tokushima, Japan) 18). Measurements are made by shining light at an angle of 45 degrees through a hole (6 mm diameter) for set periods, and measuring AF intensity in the dark.

The LED light source was fitted with visible light cut-off filters (IUV-365, Isuzu Glass Co., Osaka, Japan) to separate stray from excitation light. The f luorescence intensity of bovine skin samples was measured after 3,000 msec excitation. The difference between treated and untreated samples was calculated [check–?] (∆ Intensity: G (+) - G (–)). The intensity of the f luorescent-added skin samples was measured after 1,000 msec excitation, and calculated the difference between f luorescent-added and original samples was calculated ∆ Intensity).

Statistical analysisThe correlation of fluorescence intensity, age, and skin AF

was assessed using Pearson’s correlation coefficient (r), and r plotted against fluorescence wavelength.

Ethical approvalThe study followed the guidelines (‘The Ethical Principles

Concerning Epidemiologic Study’) published by the Japanese Minist ry of Health, Labour and Welfare; the Doshisha University Ethical Committee for Clinical Studies approved the study protocol (approval number #0832); all participants provided informed, written consent; data were not linked to subjects’ personal information.

Results

Fluorescence spectrum of solution samplesAt an excitation frequency of 370 nm, quinine sulfate

exhibited a peak fluorescence of 440 nm Fig. 1(a). The HSA-Glc (+) model exhibited a peak intensity at 430 nm, and the HSA-Glc (-) model exhibited low f luorescence intensity at 430 nm (Fig. 1(b)). At an excitation frequency of 370 nm, pentosidine exhibited a peak 444 nm (Fig. 2). When a sample of diluted pentosidine was measured to test the detection sensitivity of pentosidine, the detection sensitivity at 370/440 nm was about 1/42.6 times more than at 335/385 nm. The concentration of pentosidine in the HSA-Glc (+) reaction liquid was 0.161 µg/mL (measured by ELISA).

Exploratory experiment of fluorometer measurement condition

We did not observe characteristic spectrum of skin fluorescence between 400-600 nm after an excitation irradiation time 25 msec (Fig. 3(a)). But when the irradiation time was increased to 5,000 msec, the detection limit of the spectroscope was exceeded, and the spectra exhibited a characteristic peak between 451-485 nm. However, this spectrum may be affected by excitation light in the fluorescence region. When the visible light cut-off filter was added, the excitation light and fluorescence spectra were separated, and the peak of intensity exhibited peaks at 451 nm and 485 nm (Fig. 3(b)).

Fluorescence spectrum of bovine skinAfter two or four days, samples of bovine skin incubated

with glucose t u r ned brown (Fig. 4(a)) and exhibited fluorescence at 451 nm; the fluorescence intensity increased with incubation period (Fig. 4(b)).

In the fluorescent-added (quinine sulfate solution, HSA-Glc (+), pentosidine) bovine skin samples, the fluorescence intensity of between 420-600 nm increased with the concentration of fluorescence agent and incubation period (Fig. 5).

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Fig. 1. Fluorescence spectrum of quinine sulfate and HSA-GlcExcitation wavelength was at 370 nm.(a) Quinine sulfate ( positive control for fluorescence).(b) Human serum albumin incubated with glucose (G (+)) and no glucose (G (-)).

Fig. 2. Fluorescence spectrum of pentosidineThe maximum wavelength of 335/385 nm was observed the peak in the fluorescence wavelength of 444 nm at an excitation wavelength of 370 nm.

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Fig. 3. Exploratory experiment of preproduction of fluorometerSubject was 28 year-old Japanese man(a) Human skin fluorescence spectrum after irradiation by a normal LED for 25 msec and 5,000 msec(b) Human skin fluorescence spectrum after irradiation for 5,000 msec by an LED fitted with a visible light cut-off filter.

Measurement of CML content in skin stratum corneum

After two or four days, samples of bovine skin incubated with glucose turned brown (Fig. 4(a)) and exhibited fluorescence at 451 nm; the fluorescence intensity increased with incubation period (Fig. 4(b)).

In the fluorescent-added (quinine sulfate solution, HSA-Glc (+), pentosidine) bovine skin samples, the fluorescence intensity of between 420-600 nm increased with the concentration of fluorescence agent and incubation period (Fig. 5).

Clinical results for skin fluorescenceThe f luorescence exhibited by skin, after excitation

irradiation 5,000 msec, and salient difference of fluorescence intensity among 11 subjects were exhibited at 451 nm and 485 nm (Fig. 6(a)). The correlation coefficient of age and skin AF was positive (p < 0.05) (age (500-700 nm) and skin AF (420-650 nm) (Fig. 6(b)). In addition, the fluorescence intensity at 440 nm might be positively correlated with age (r = 0.582, p = 0.060) and with skin AF (r = 0.832, p = 0.001).

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Fig. 4. Relation between fluorescence intensity and incubation period with glucose (N = 3)(a) Appearance of bovine skin samples incubated with glucose in each time(b) Fluorescence spectrum of bovine skin samples after irradiation for 3000msec. Δ Intensity means the difference between sample incubated with glucose (G (+)) and no glucose (G (-)).

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Fig. 5. Results of fluorescent sample added tests (N = 3)(a) Quinine sulfate, (b) HSA-Glc (+), (c) pentosidine were added to bovine skin samples and incubated in glucose free solution. Δ Intensity means the difference in intensity between fluorescent-added and negative samples, irradiated at 1,000msec.

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Fig. 6. Fluorescence spectra of human skins and correlation coefficient of the intensity (N = 11)(a) Subjects were healthy men and women.(b) Pearson’s correlation coefficient (r) were plotted each wavelength on the graph.

p

DiscussionFluorescence Spectrum of solution samples

It has been previously repor ted that several AGEs physicochemical properties cause characteristic fluorescence at 370/440 nm 4). Here we found peak fluorescence at 440 nm (430 nm) in glycated HSA sample (Fig. 1(b)). HSA-Glc (–) exhibited less fluorescence at 430 nm, which may have been generated by the original HSA reagent. The detection sensitivity at 370/440 nm was 1/42.6 times compared to 335/385 nm pentosidine (Fig. 2). No previous research has detected pentosidine at 370/440 nm.

Paul reported that relative f luorescence intensity at 370/440 nm was positively correlated with pentosidine in human skins taken by skin biopsy 19). Our results suggest direct measurement of pentosidine at 370/440 nm may be possible. Although pentosidine is less common in plasma, the concentration of it increases with aging 2,20). Since pentosidine binds with collagens in the dermis, a non-invasive fluorescent measurement method may provide information on the relationship between aging and disease. These results indicate an excitation wavelength fluorescence at 370/440 nm is suitable to measure AGEs derived fluorescence intensity.

Hori reported that the protein derived from various living tissues, such as collagen, elastin, and keratin, exhibits a similar increase in fluorescence intensity to glucose incubated albumin, that depends on incubation period 7). The fluorescence at 370/440 nm may help measure glycation and the degree of aging in various living human tissues. We suggest the observed intensity at 370/440 nm represents ‘AGEs fluorescence’.

Fluorescence spectrum of bovine skin sampleData from the bovine skin samples showed the fluorescence

intensity at 451 nm was related to degree of skin glycation (Fig. 4(b)). Bachmann reported that the fluorescent substances detected from a living tissues at 360 nm of excitation light is

only the f luorescence originating in the collagen detected at 440-460 nm at present 21). The observed fluorescence intensity at 451 nm in the bovine skin sample is likely to be produced by reaction products from glycated collagen.

In the f luorescent-added samples, pentosidine derived fluorescence detected near 444 nm was also detected at 451 nm in the bovine skin sample (Fig. 5(c)). The result is significant because pentosidine is the only f luorescence AGEs reported from skin and is typical of AGEs linked with aging and some disease. Moreover, fluorometer used is unlikely to detect other lights, and can therefore be used to monitor AGEs fluorescence. In addition, the f luorescence spectrum of HSA-Glc (+) peak on the short wavelength side (near 430 nm) compared with the other fluorescence-added samples, implying fluorescence intensity at the short wavelength side had become a high value of intensity comparatively (Fig. 5(b)).

Fluorescence spectrum of human skinIn healthy people, differences in fluorescence are difficult

to detect at this detection sensitivity, although an AGE Reader can distinguish the f luorescence spectrum of healthy people

23), diabetes mellitus patients, and renal disease patients. Since the precise spectrum of fluorescent AGEs is not detectable in the human skins, a target cannot be extracted by the methods, so the mean intensity of the broad fluorescence region (420-600 nm) has been used to evaluate the degree of glycation in the body. This hinders the development of this field. Koetsier reported that a specific peak of intensity wasn't discovered for diabetes mellitus patients in ex. 350-420 em. 420-600 nm 12). Although the light intensity of a xenon lamp light source is stable and does not change with time, in a living body excitation light is easily dispersed and sensitivity falls. If a narrow-band of excitation light is used to avoid the dispersion, then the amount of excitation light will decline. The technical limit of the above measuring instruments is also mentioned as a subject.

The preproduction f luorometer was equipped with an

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4) Cerami A, Vlassara H, Brownlee M. Glucose and aging. Sci Am 256; 90-96: 1987

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7) Hori M, Yagi M, Nomoto K, et al: Experimental models for advanced glycation end product formation using albumin, collagen, elastin, keratin and proteoglycan. Anti-Aging Medicine 9(6); 125-134: 2012

8) Yagi M, Nomoto K, Hori M, et al: The effect of edible purple chrysanthemum extract on advanced glycation end products generation in skin: a randomized controlled clinical trial and in vitro study. Anti-Aging Medicine 9(2); 61-74: 2012

9) Momma H, Niu K, Kobayashi Y, et al: Skin advanced glycation end product accumulation and muscle strength among adult men. Eur J Appl Physiol 117(7); 1545-1552: 2011

10) Yue X, Hu H, Koetsier M, et al: Reference values for the Chinese population of skin autof luorescence as a marker of advanced glycation end products accumulated in tissue. Diabet Med 28(7); 818-823: 2011

11) Mulder DJ, Water TV, 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. 8(5); 523-535: 2006

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LED light source, and the half bandwidth of the emission spectrum is narrow because, and the f luorescence intensity higher than that provided by an AGE Reader. Moreover, the excitation light LED can be controlled by equipping with a visible light cut-off filter so that a pure fluorescence spectrum can be obtained. As a result, the spectra become detectable at peaks of 451 and 485 nm, which AGE Reader cannot detect in human skins (Fig. 3(b)). Thus, the preproduction fluorometer enabled a comparison between fluorescence AGEs produced in models and living tissue. However, one new problem is that the intensity of excitation light may exceed the gain value of a spectroscope used to measure f luorescence, and should be tested in future. Also, the intensity of excitation wavelength may need to be adjusted to adjust for differences in skin color

24,25).Among healthy people, we observed two peaks at 451

nm and 485 nm in human skins were found (Fig. 6(a)). Since correlation with skin AF is so strong and the f luorescence region is similar to spectrum obtained from HSA-Glc (+), we suggest the system is detecting fluorescence from skin AGEs. In future, most effective wavelength to monitor a degree of glycation in the body may be found by considering the relation between fluorescence spectrum and biological information.

ConclusionThe in vitro experimental result demonstrated that

fluorescence intensity detected at 370/440 nm was derived from AGEs. The fluorescence spectra of glycated HSA and human skin are similar, and thus measurement of AGEs fluorescence has potential to monitor the degree of glycation in the living tissue.

Conflicts of InterestThe authors have no conflicts of interest in this study.

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20) Sell DR, Nagaraj RH, Grandhee SK, et al: Pentosidine: a molecular marker for the cumulative damage to proteins in diabetes, aging, and uremia. Diabetes Metab Rev 7(4); 239-251: 1991 December

21) Bachmann L, Zezell DM, Ribeiro AC, et al: Fluorescence spect roscopy of biological t issue – A review. Applied Spectroscopy Reviews 41(6); 575-590: 2006

22) Dyer DG, Dunn JA, Thorpe SR, et al: Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 91(6); 2463-2469: 1993

23) Meerwaldt R, Graaff R, Oomen PHN, et al: Simple non-invasive assessment of advanced glycation endproduct accumulation. Diabetologia 47(7); 1324-1330: 2004

24) Draaijers IJ, Tempelman FR, Botman YA, et al: Colour evaluation in scars: tri-stimulus colorimeter, narrow-band simple reflectance meter or subjective evaluation? Burns 30(2); 103-107: 2004

25) Anderson RR, Parrish JA. The optics of human skin. J Invest Dermatol 77(1); 13-19: 1981

original article identification of advanced glycation ...5)92100.pdf92 introduction glycation is a...

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