Oceanview program

Excitation-emission spectra of the urine samples were recorded using a FLAME-S-VIS-NIR-ES spectrometer (Ocean Optics, Largo, FL, USA) with a light source LLS-365 and an INSTMA-20 slit (slit width: 200 µm). One mL urine was transferred into a quartz cuvette with a 1 cm path length (Carl Zeiss, Oberkochen, Germany). The reflection probe (QR400-7-VIS-BX, premium 400 µm, VIS/NIR) (Ocean Optics, Largo, FL, USA) was positioned against the cuvette and a black background was used. The OceanView program (Ocean Optics, Largo, FL, USA) was set with an integration time of 10 ms and an average of 128 scans. Using an excitation wavelength of 365 nm, the fluorescence spectra of urinary AGEs were recorded at a 400–620 nm emission range. After background correction, the fluorescence signal of each urine sample was measured. Normalized fluorescence spectra were prepared by dividing the relative fluorescence intensity at each wavelength by the (maximum) relative fluorescence intensity at the (corresponding) peak wavelength. As the urinary concentration of AGEs depends on the urine volume, the relative fluorescence intensity (expressed in arbitrary units) was adjusted for the urinary creatinine concentration.

For the detection and measurement of AGE-related autofluorescence (excitation 370 nm, emission 390–700 nm) in the human skin samples, a Flame miniature spectrometer (FLAME-S-VIS-NIR-ES, 350–1000 nm, Ocean Optics, Dunedin, FL, USA) coupled to an LSS-LED light source (365 nm, Ocean Optics) and a reflection probe (QR400-7-IS-BX, Ocean Optics) was used. For the AF measurements, first, the background signal was corrected, after which the skin samples were transferred to a black 96-well plate (SPL Lifesciences, Pochon, Republic of Korea), and fluorescence measurements were taken at a 90° angle. Each measurement was repeated three to five times. The intensity of the light source was manually set to an excitation peak with an intensity of approximately 60,000. The AF measurements were processed using the OceanView program (Ocean Optics, Largo, FL, USA), which was set to an average of 128 scans and an integration time of 10 ms. The AF values were calculated by dividing the mean light intensity emitted per nm for a range of 407–677 nm by the mean light intensity per nm for a range of 342–407 nm [35 (link),48 (link)].