Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure Nω-(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation of Nε-(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I–IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen.

Although extracts of the roots and stems of Salacia chinensis have been used in folk medicines for chronic diseases such as rheumatism, irregular menstruation, asthma and diabetes mellitus, little is known about the mechanism by which Salacia chinensis extract (SCE) ameliorates these diseases. To clarify whether SCE ameliorates the progression of lifestyle-related diseases, the inhibitory effect of SCE on the formation of advanced glycation end products (AGEs) was analyzed in a rat model of streptozotocin-induced diabetes. Although the oral administration of SCE did not ameliorate the diabetes-induced decrease in body weight, it ameliorated the increase in glycoalbumin levels in diabetic rats. An analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) demonstrated that the levels of N(ε)-(carboxymethyl)lysine (CML) were highest in the femurs and that they increased by the induction of diabetes. The administration of SCE also ameliorated the decreased femur strength and the accumulation of CML. Furthermore, when all of the carbohydrates in the chow of diabetic rats were replaced with free glucose, the administration of SCE significantly ameliorated a diabetes-induced increase in glycoalbumin and decrease in serum creatinine level and body weight. This study provides evidence to support that SCE ameliorates diabetes-induced abnormalities by improving the uptake of glucose by various organs.

Trapa bispinosa Roxb. is an annual aquatic grass of the citrus family. Although its hot water extract displays antioxidative activity in vitro, little is known about its biological effectiveness. In the present study, we evaluated the extract's inhibitory effect on diabetic cataractogenesis and formation of advanced glycation end product. Lutein, which is beneficial for eye diseases, was administered concurrently. For short term administration, Trapa bispinosa Roxb. hot water extract and/or lutein were admin istered to type 1 diabetic rats. N ε (carboxymethyl)lysine and N ε (carboxyethyl)lysine were quantified in serum using mass spectrometry. The long term administration study was similar to the short term, except that the dosages were lower. In the short term study, co administration of the extract and lutein inhibited N ε (carboxymethyl)lysine and N ε (carboxyethyl)lysine in serum. However, in the long term study, only lutein inhibited N ε (carboxymethyl)lysine and N e (carboxyethyl)lysine in serum. These results suggest that lutein exerts its long term effect regardless of the concentration administered, while the extract exerts its effect when its concentration is increased. Relative to the consumption of the control diet, oral intake of the combination of the extract and lutein significantly inhibited the progression of cataracto genesis in the lens of diabetic rats, even at low doses, and the combination was more effective than individual treatments.

Two-photon absorption for diphenylacetylene derivatives with an electron-donating (ED) or electron-withdrawing (EW) group (DPA-Rs) was investigated by highsensitivity optical-probing photoacoustic spectroscopy. Twophoton absorption spectra and two-photon absorption cross sections σ (2) for DPA-Rs were successfully obtained. Two-photon absorption spectra of DPA-Rs with stronger ED or EW groups display more significant red-shifts and larger σ (2) values. Simulated two-photon absorption spectra, using time-dependent density functional theory within the Tamm−Dancoff approximation, compared well with the experimental spectra. Based on the three-state model, the substituent effect on the two-photon absorption for DPA-Rs was expected to manifest in the transition dipole moments and detuning energies. Information obtained from investigating the monosubstituent effect on two-photon absorption of DPA is critical for an improved understanding of two-photon absorption.

Summary Aphanothece sacrum (Sur.) Okada is a species of cyanobacteria found in Japan. Although it has been used in local cuisine in Kyushu, Japan, for 250 y, little is known about its beneficial effect as food. The daily intake of health beneficial phytochemicals is believed to be useful for preventing lifestyle-related diseases, such as diabetic cataracts. In this study, the inhibitory effect of freeze-dried A. sacrum (Asa) on the formation of diabetic cataracts (DCs) was evaluated. Type 1 diabetes was induced in mice using streptozotocin (STZ). The mice were divided into two groups: one was fed a normal diet (DM-control group) and the other was fed a diet containing 1% Asa (DM-Asa group). During the study, changes in blood glucose levels and the amount of food and water consumed were measured. After 3 mo, the amount of N ε -(carboxymethyl)lysine (CML), an oxidative stress marker, in the lens was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the blood glucose levels (pϭ0.91) and food consumption did not significantly change in any group, the oral administration of Asa tended to suppress CML accumulation (pϭ0.15) and significantly inhibited the progression of cataractogenesis in the diabetic lens compared with that reported for the normal diet (pϭ0.009). These results suggested that the daily intake of A. sacrum prevents the pathogenesis of cataracts, and indicated that may reduce the number of DC patients.