Background:
Cumulative evidence indicates that plasma homocysteine (Hcy) level is positively correlated with cardiovascular mortality in Type 2 diabetic patients.
Aims:
To explore the effects and mechanisms of elevated plasma Hcy level[[Unable to Display Character: ―]] hyperhomocysteinemia (HHcy) on endothelial function in db/db mice.
Methods and Results:
HHcy was induced in diabetic db/db and non-diabetic db/+ mice fed with a high methionine diet (HM, 2% methionine) for 8 weeks (plasma tHcy=54.31±5.4 and 34.21±4.15 μM). Endothelial function was examined in small mesenteric arteries (SMA) using myographs. In non-diabetic mice, HHcy did not change vascular relaxation to acetylcholine (Ach); whereas, nitric oxide (NO)- and prostacyclin (PGI2)-mediated relaxation to Ach were impaired. Interestingly, endothelium-derived hyperpolarization factor (EDHF)-mediated relaxation to Ach was improved. In diabetic mice, HHcy potentiated the impairment of NO-, PGI2- and EDHF-mediated relaxation to Ach. Moreover, sodium hydrogen sulfide (NaHS), a donor of hydrogen sulfide (H2S), induced EDHF-mediated relaxation which was impaired in diabetic mice and potentiated by HHcy. NS309, a non-specific calcium-activated potassium channel (Kca) activator, significantly improved H2S- and Ach-induced EDHF-mediated relaxation in diabetic mice with HHcy. Tram-34, an intermediate conductance Kca (IK) blocker, but not small conductance Kca blocker apamin, diminished HHcy-induced impairment of EDHF-mediated relaxation in diabetic mice, suggesting that IK inactivation plays a major role. Free sulfide was decreased in plasma and SMA of diabetic mice which was potentiated with HHcy. Superoxide generation was increased and potentiated by HHcy in lung ECs from diabetic mice. Moreover, PEG-SOD improved vascular relaxation in diabetic mice with HHcy. Finally, tyrosine nitration of IK was increased in human cardiac microvascular endothelial cells (ECs) treated with either D-glucose (25 mM) or DL-Hcy (500 μM) for 48h, which was potentiated by a combination of D-glucose and DL-Hcy.
Conclusions:
H2S is a major EDHF in resistant arteries in mouse. H2S-contributed EDHF-mediated vascular relaxation was impaired in diabetes and was potentiated by HHcy via oxidative stress and IK inactivation.
The measurement of hydrogen sulphide (H2S) has held the attention of the analytical community due to its unique physiological and pathophysiological roles in biological systems. This work reports fabrication and characterization of screen‐printed electrode (SPE) integrated lab‐on‐a‐chip (LOAC) for H2S detection. The device has been designed to detect all forms of H2S present in the plasma; however, current work is a proof of concept of device operation for free sulphide. The device consists of three distinct layers for H2S separation and SPE to detect H2S. It operates with pH‐dependent liberation and trapping from samples introduced. The first layer, the releasing layer, consists of a releasing chamber for the liberation of H2S. The second layer, a silicone membrane, is where gas diffuses from the sample layer to the third layer. The third layer, the trapping layer, is integrated with SPE to determine the concentration of H2S. The device uses an electrochemical technique. First, electrode performances are tested on the cell vial to establish suitability for subsequent LOAC incorporation. Common metal electrodes are compared with boron‐doped ultra‐nano‐crystalline diamond (BDUNCD) electrode and SPE. The range of detection, detection limit, and sensitivity of the electrode are characterized. In conclusion, a proof of concept of an electrochemical sensing of H2S in a LOAC is reported. The device is portable, robust and can easily be fabricated. Transfer data indicated that 10% sulphide detected into the trapping chamber in a reproducible manner at 20 min. The SPE integrated LOAC shows a huge advantage over conventional techniques.
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