Angiotensin-(1-7) (Ang 1-7) has been previously studied in combination with an antioxidant containing preparation as a cardioprotective reperfusion solution. In this study a stability improvement of aqueous Ang 1-7 solutions was observed. However, no data was provided on the responsibilities and causes of the noticed stability enhancement. Therefore, the influence of pH and pharmaceutical additives as well as the effect of the single specific agents present in the antioxidant preparation such as α-ketoglutaric acid (α-KG), 5-hydroxymethylfurfural (5-HMF), N-acetyl-seleno-l-methionine (NASeLM) and N-acetyl-l-methionine (NALM) on the stability was evaluated. Analyses were performed by an HPLC method with fluorescence detection. Crucial instability was found in a pH range of 5.0-7.5 without addition of the antioxidative mixture. Zetasizing confirmed the presence of microparticles and MS studies showed no degradation products within 25 days. 5-HMF was identified as main component for stability enhancement of Ang 1-7 solution. By adding this substance the stability of the cardioprotective peptide solution can be prolonged and appears as a promising approach for transplant purposes.

Angiotensin-(1-7) is an important active component in the renin-angiotensin-system. Due to its cardio protective effects it is now under investigation in combination with antioxidants as a reperfusion solution. The combination showed impressive effects on isolated hearts of male Wistar rats after induced ischemia. In this work a high performance liquid chromatography method with fluorescence detection was developed for the first time for in-process measurements as well as for stability tests of the peptide in the novel antioxidant-containing Karal® solution. For fluorescence detection of angiotensin-(1-7) fluorescamine as derivatization dye was applied. Under optimized conditions the method showed linearity over the range of 50 to 5000 ng/mL with R(2) of 0.9988 and an overall precision better than 5.0 %. LOD and LOQ were determined to be in the femtomol range on column. It was found that stability of angiotensin-(1-7) could be significantly improved in the antioxidant containing preparation compared to aqueous solutions.

Oxidative stress can lead to tissue damage in the body, resulting in conditions that may lead to heart failure. Carbonyl proteins and malondialdehyde are common markers for determining oxidative stress. In this study, a high-performance liquid chromatography with ultravioletvisible (HPLC-UV-Vis) method was developed for both carbonyl proteins and malondialdehyde and compared with common spectrophotometric methods. The HPLC-UV-Vis method for determining carbonyl proteins showed a linear correlation in a range of 0.05-1.6 mg. mL À1 (correlation coefficient: 0.9939). The limits of detection and quantitation were determined to be 1.7 and 5.1 pmol on-column, respectively. The HPLC-UV-Vis method for malondialdehyde exhibited a linear correlation in the range of 0.04-13.6 mg. mL À1 (correlation coefficient: 0.9982) with limits of detection and quantitation of 1.5 and 4.5 pmol on-column, respectively. As a proof of concept, the applicability of the methods for both marker substances was investigated in chicken heart tissue as a complex matrix sample. To simulate oxidative stress, the tissue was treated with hydrogen peroxide as the oxidizing agent. Samples prepared with hydrogen peroxide showed increased carbonyl protein and malondialdehyde levels when compared to nonoxidized samples and samples treated additionally with the antioxidant Trolox. Regarding oxidative stress, similar results were achieved when compared with common spectrophotometric methods. However, HPLC-UV-Vis presented higher reproducibility and accuracy. The developed methods for both markers enable rapid and simple investigation of possible antioxidant effects on tissue samples.

A multi-component solution, containing α-ketoglutaric acid (α-KG), 5-hydroxymethylfurfural (5-HMF), N-acetyl-seleno-l-methionine (NASeLM), and N-acetyl-l-methionine (NALM) as active ingredients, has been tested considering its supposed antioxidative effect with respect to heart transplantations. Oxidative stress was induced on isolated rat hearts through occlusion of a coronary artery and in chicken heart tissue through hydrogen peroxide. Both heart types were analyzed and the oxidative stress markers malondialdehyde (MDA) and carbonyl proteins (CPs) were determined via HPLC/UV–Vis. In both approaches, it was found that treatment with the multi-component solution led to a lower amount of MDA and CPs compared to a negative control treated with Krebs–Ringer solution (KRS). Further investigation on chicken heart tissue identified α-KG as antioxidative component in these experiments. However, numerous factors like arrhythmia, vessel dilatation, and minimization of oxidative stress effects play an important role for successful transplantation. Therefore, the investigated multi-component solution might be a novel approach against oxidative stress situations, for example at ischemia reperfusion injury during heart transplantations.

As progressive organ shortage in cardiac transplantation demands extension of donor criteria, effort is needed to optimize graft survival. Reactive oxygen and nitrogen species, generated during organ procurement, transplantation, and reperfusion, contribute to acute and late graft dysfunction. The combined application of diverse substances acting via different molecular pathways appears to be a reasonable approach to face the complex mechanism of ischemia reperfusion injury. Thus, an antioxidant solution containing α-ketoglutaric acid, 5-hydroxymethylfurfural, N-acetyl-L-methionine, and N-acetyl-selenium-L-methionine was combined with endogenous angiotensin-(1-7). Its capacity of myocardial protection was investigated in isolated Langendorff-perfused rat hearts subjected to warm and cold ischemia. The physiological cardiac parameters were assessed throughout the experiments. Effects were evaluated via determination of the oxidative stress parameters malondialdehyde and carbonyl proteins as well as immunohistochemical and ultrastructural tissue analyses. It was shown that a combination of 20% (v/v) antioxidant solution and 220 pM angiotensin-(1-7) led to the best results with a preservation of heart tissue against oxidative stress and morphological alteration. Additionally, immediate cardiac recovery (after warm ischemia) and normal physiological performance (after cold ischemia) were recorded. Overall, the results of this study indicate substantial cardioprotection of the novel combination with promising prospective for future clinical use.