Different RONS Generation in MTC-SK and NSCL Cells Lead to Varying Antitumoral Effects of Alpha-Ketoglutarate + 5-HMF.

BACKGROUND: Carbonylated proteins (CPs) serve as specific indicators of increased reactive oxygen and nitrogen species (RONS) production in cancer cells, attributed to the dysregulated mitochondrial energy metabolism known as the Warburg effect. The aim of this study was to investigate the potential of alpha-ketoglutarate (aKG), 5-hydroxymethylfurfural (5-HMF), and their combination as mitochondrial-targeting antioxidants in MTC-SK or NCI-H23 cancer cells. METHODS: MTC-SK and NCI-H23 cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of aKG, 5-HMF, and the combined aKG + 5-HMF solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of MTC-SK and NCI-H23 cells. RESULTS: The mitochondrial activity of MTC-SK cells exhibited a concentration- and time-dependent reduction upon treatment with aKG, 5-HMF, or the combined aKG + 5-HMF. The half-maximal inhibitory concentration (IC50%) for mitochondrial activity was achieved at 500 µg/mL aKG, 200 µg/mL 5-HMF, and 200 µg/mL aKG + 66.7 µg/mL 5-HMF after 72 h. In contrast, NCI-H23 cells showed a minimal reduction (10%) in mitochondrial activity even at the highest combined concentration of aKG + 5-HMF. The CP levels in MTC-SK cells were measured at 8.7 nmol/mg protein, while NCI-H23 cells exhibited CP levels of 1.4 nmol/mg protein. The combination of aKG + 5-HMF led to a decrease in CP levels specifically in MTC-SK cells. The correlation between mitochondrial activity and CP levels in the presence of different concentrations of combined aKG + 5-HMF in MTC-SK cells demonstrated a linear and concentration-dependent decline in CP levels and mitochondrial activity. Conversely, the effect was less pronounced in NCI-H23 cells. Cell growth of MTC-CK cells was reduced to 60% after 48 h and maintained at 50% after 72 h incubation when treated with 500 µg/mL aKG (IC50%). Addition of 500 µg/mL 5-HMF inhibited cell growth completely regardless of the incubation time. The IC50% for 5-HMF on MTC-CK cell growth was calculated at 375 µg/mL after 24 h incubation and 200 µg/mL 5-HMF after 72 h. MTC-SK cells treated with 500 µg/mL aKG + 167 µg/mL 5-HMF showed no cell growth. The calculated IC50% for the combined substances was 250 µg/mL aKG + 83.3 µg/mL 5-HMF (48 h incubation) and 200 µg/mL aKG + 66.7 µg/mL 5-HMF (72 h incubation). None of the tested concentrations of aKG, 5-HMF, or the combined solution had any effect on NCI-H23 cell growth at any incubation time. Caspase-3 activity increased to 21% in MTC-CK cells in the presence of 500 µg/mL aKG, while an increase to 59.6% was observed using 500 µg/mL 5-HMF. The combination of 500 µg/mL aKG + 167.7 µg/mL 5-HMF resulted in a caspase-3 activity of 55.2%. No caspase-3 activation was observed in NCI-H23 cells when treated with aKG, 5-HMF, or the combined solutions. CONCLUSION: CPs may serve as potential markers for distinguishing between cancer cells regulated by RONS. The combination of aKG + 5-HMF showed induced cell death in high-RONS-generating cancer cells compared to low-RONS-generating cancer cells.

Alpha-Ketoglutarate or 5-HMF: Single Compounds Effectively Eliminate Leukemia Cells via Caspase-3 Apoptosis and Antioxidative Pathways.

BACKGROUND: We recently showed that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) has a solid antitumoral effect on the Jurkat cell line due to the fact of its antioxidative, caspase-3 and apoptosis activities, but no negative effect on human fibroblasts was obtained. The question arises how the single compounds, aKG and 5-HMF, affect peroxynitrite (ONOO-) and nitration of tyrosine residues, Jurkat cell proliferation and caspase-activated apoptosis. METHODS: The ONOO- luminol-induced chemiluminescence reaction was used to measure the ONOO- scavenging function of aKG or 5-HMF, and their protection against nitration of tyrosine residues on bovine serum albumin was estimated with the ELISA technique. The Jurkat cell line was cultivated in the absence or presence of aKG or 5-HMF solutions between 0 and 3.5 µM aKG or 0 and 4 µM 5-HMF. Jurkat cells were tested for cell proliferation, mitochondrial activity and caspase-activated apoptosis. RESULTS: aKG showed a concentration-dependent reduction in ONOO-, resulting in a 90% elimination of ONOO- using 200 mM aKG. In addition, 20 and 200 mM 5-HMF were able to reduce ONOO- only by 20%, while lower concentrations of 5-HMF remained stable in the presence of ONOO-. Nitration of tyrosine residues was inhibited 4 fold more effectively with 5-HMF compared to aKG measuring the IC50%. Both substances, aKG and 5-HMF, were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time. The aKG effectively reduced Jurkat cell growth down to 50% after 48 and 72 h of incubation using the highest concentration of 3.5 µM, and 1, 1.6, 2, 3 and 4 µM 5-HMF inhibited any cell growth within (i) 24 h; 1.6, 2, 3 and 4 µM 5-HMF within 48 h (ii); 2, 3 and 4 µM 5-HMF within 72 h (iii). Furthermore, 4 µM was able to eliminate the starting cell number of 20,000 cells after 48 and 72 h down to 11,233 cells. The mitochondrial activity measurements supported the data on aKG or 5-HMF regarding cell growth in Jurkat cells, in both a dose- and incubation-time-dependent manner: the highest concentration of 3.5 µM aKG reduced the mitochondrial activity over 24 h (67.7%), 48 h (57.9%) and 72 h (46.8%) of incubation with Jurkat cells compared to the control incubation without aKG (100%). 5-HMF was more effective compared to aKG; the mitochondrial activity in the presence of 4 µM 5-HMF decreased after 24 h down to 68.4%, after 48 h to 42.9% and after 72 h to 32.0%. Moreover, 1.7 and 3.4 µM aKG had no effect on caspase-3-activated apoptosis (0.58% and 0.56%) in the Jurkat cell line. However, 2 and 4 µM 5-HMF increased the caspase-3-activated apoptosis up to 22.1% and 42.5% compared to the control (2.9%). A combined solution of 1.7 µM aKG + 0.7 µM 5-HMF showed a higher caspase-3-activated apoptosis (15.7%) compared to 1.7 µM aKG or 2 µM 5-HMF alone. In addition, 3.5 µM µg/mL aKG + 1.7 µM 5-HMF induced caspase-activated apoptosis up to 55.6% compared to 4.5% or 35.6% caspase-3 activity using 3.5 µM aKG or 4 µM 5-HMF. CONCLUSION: Both substances showed high antioxidative potential in eliminating either peroxynitrite or nitration of tyrosine residues, which results in a better inhibition of cell growth and mitochondrial activity of 5-HMF compared to aKG. However, caspase-3-activated apoptosis measurements revealed that the combination of both substances synergistically is the most effective compared to single compounds.

Investigation of antioxidative effects of a cardioprotective solution in heart tissue.

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.

Determination of Angiotensin-(1-7) with HPLC/Fluorescence-Detection.

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.

The Influence of N-Acetyl-selenomethionine on Two RONS-Generating Cancer Cell Lines Compared to N-Acetyl-methionine.

N-acetyl-selenomethionine (NASeLM), a representative of the selenium compounds, failed to convince in clinical studies and cell cultures that it neither inhibits cancer growth nor has a chemoprotective effect. This study aims to find out whether NASeLM shows a growth-inhibiting property compared to the carrier substance N-Acetyl-L-methionine (NALM) on two different cancer cells, namely Jurkat cells and MTC-SK cells. METHODS: Jurkat and MTC-SK cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of NASeLM and NALM solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of Jurkat and MTC-SK cells. RESULTS: Both substances, NASeLM and NALM, were similarly able to inhibit cell growth and mitochondrial activity of Jurkat cells in a concentration-dependent and time-dependent manner up to 70%. Only the determination of caspase activity showed that only NASeLM was able to increase this to almost 40% compared to the control as well as the same lack of NALM. However, the experiments on MTC-SK cells showed a clear difference in favor of NASeLM compared to NALM. While NASeLM was able to reduce cell growth to up to 55%, the same amount of NALM was only at around 15%, which turned out to be highly significant (p < 0.001). The same could also be measured for the reduction in MTC-SK mitochondrial activity. Time dependence could also be recognized: the longer both substances, NASeLM and NALM, were incubated, the higher the effect on cell growth and mitochondrial activity, in favour of NASeLM. Only NASeLM was able to increase caspase-3 activity in MTC-SK cells: at 250 µg/mL NASeLM, caspase-3 activity increased significantly to 28% after 24 and 48 h compared to the control (14%) or the same NALM concentration (14%). After 72 h, this could still increase to 37%. A further increase in the NASeLM concentration did not result in higher caspase-3 activity. CONCLUSION: NASeLM could clearly increase caspase-3 activity in both cell types, Jurkat or MTC-SK cells, and thus induce cell death. NALM and NASeLM showed a reduction in cell growth and mitochondrial activity in both cell lines: While NALM and NASeLM showed almost identical measurements on Jurkat cells, NASeLM was much more effective on MTC-SK than the non-selenium-containing carrier, indicating that it has additional anti-chemoprotective effects.

Determination of metabolites of 5-hydroxymethylfurfural in human urine after oral application.

5-Hydroxymethylfurfural (5-HMF) is a natural occurring substance taken up by everyday food. In former studies it was shown that 5-HMF is completely decomposed in the body after oral or intravenous application resulting in three main metabolites named 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine, and possibly a forth metabolic substance, termed 5-sulphoxymethylfurfural, is formed. Determination is possible via HPLC using a hydrophilic interaction chromatography (HILIC) column with an appropriate gradient system (ACN/ammonium formate 100 mM, pH 2.35). Urine samples were purified by use of an SPE method beforehand working with ScreenA cartridges. This cleaning procedure was validated based on ICH guidelines in terms of linearity, quantification, and detection limit, as well as precision, repeatability, and accuracy. Analysis of real-life samples coming from two healthy probands and one cancer patient, who all received 240 mg 5-hydroxymethylfurfural orally once a day, showed dicarboxylic acid and the glycine conjugate in their urine samples. Recovery of the initial compound in form of transformed metabolites was up to 90% within 48 h. Potentially toxic 5-sulphoxymethylfurfural could not be found.

Development and validation of an HPLC method to determine metabolites of 5-hydroxymethylfurfural (5-HMF).

The food component 5-hydroxymethylfurfural is supposed to have antioxidative properties and is therefore used as an acting agent in a novel anticancer infusion solution, named Karal®, and an oral supplementation. Previous studies showed that after oral and intravenous application, the substance is completely decomposed to its metabolites: 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine. The formation of a fourth metabolite, namely 5-sulphoxymethylfurfural, is still not clarified according to literature. Due to commercial unavailability, synthesis of 5-sulphoxymethylfurfural was conducted and a synthesis procedure for N-(hydroxymethyl)furoyl glycine had to be developed. Identification of the synthesised compounds was proven by LC-MS and NMR. An appropriate HPLC method was established to obtain good separation of the four possible metabolic substances and 5-hydroxymethylfurfural within 12 min via a HILIC column (150 × 4.6 mm, 5 µm) using a gradient grade system switching from mobile phase A (ACN/ammonium formate 100 mM, pH 2.35, 95:5, v/v) to mobile phase B (ACN/ammonium formate 100 mM, pH 2.35, 85:15, v/v). The procedure was afterward validated following ICH guidelines in terms of selectivity, linearity, precision, LOD, and LOQ.

Method development and validation for the analysis of a new anti-cancer infusion solution via HPLC.

A fast and simple HPLC method has been developed and validated for the quantification of a completely new anti-cancer drug during the manufacturing process. The combination of four compounds including α-ketoglutaric acid, hydroxymethylfurfural, N-acetyl-L-methionine and N-acetyl-L-selenomethionine, administered intravenously, is still in test phase but has already shown promising results in cancer therapy. HPLC separation was achieved on an RP-18 column with a gradient system. However, the highly different concentrations of the compounds required a variation in the detection wavelength within one run. In order to produce a chromatogram where peaks were comparable on a similar range scale, detection at absorption maxima for the two most concentrated components was avoided. After optimization of the gradient program it was possible to detect all four substances within 14 min in spite of their strongly different chemical structure. The method developed was validated for accuracy, repeatability, reproducibility and robustness in relation to temperature and pH of buffer. Linearity as well as the limit of detection and quantification were determined. This HPLC method was found to be precise, accurate and reproducible and can be easily used for in-line process control during the manufacture of the anti-tumour infusion solution.

Alpha-Ketoglutarate: A Potential Inner Mitochondrial and Cytosolic Protector against Peroxynitrite and Peroxynitrite-Induced Nitration?

The generation of peroxynitrite (ONOO-) is associated with several diseases, including atherosclerosis, hypertension, neurodegeneration, cancer, inflammation, and sepsis. Alpha-ketoglutarate (αKG) is a known potential highly antioxidative agent for radical oxidative species such as peroxides. The question arises as to whether αKG is also a potential scavenger of ONOO- and a potential protector against ONOO--mediated nitration of proteins. NMR studies of 1 mM αKG in 100 mM phosphate-buffered saline at pH 7.4 and pH 6.0 were carried out in the presence or absence of a final concentration of 2 mM ONOO-. An ONOO--luminol-induced chemiluminescence reaction was used to measure the scavenging function of several concentrations of αKG; quantification of αKG was performed via spectrophotometric enzymatic assay of αKG in the absence or presence of 0, 1, or 2 mM ONOO-. The nitration of tyrosine residues on proteins was measured on ONOO--treated bovine serum albumin (BSA) in the presence or absence of 0-24 mM αKG by an ELISA technique using a specific anti-IgG against nitro-tyrosine. The addition of ONOO- to αKG led to the formation of succinic acid and nitrite at pH 7.0, but not at pH 6.0, as αKG was stable against ONOO-. The absorbance of enzymatically estimated αKG at the time point of 30 min was significantly lower in favour of ONOO- (1 mM: 0.21 ± 0.03, 2 mM: 0.12 ± 0.05 vs. 0 mM: 0.32 ± 0.02; p < 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO- and αKG concentrations (y = -2 × 105 ln(x) + 1 × 106; r2 = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, p < 0.001) compared to the control (474,401 ± 18,259); for 20 and 200 mM αKG, there were further reductions to 163,546 ± 26,196 cps (p < 0.001) and 12,658 ± 1928 cps (p < 0.001). Nitrated tyrosine residues were estimated using the ELISA technique. A negative linear correlation was obtained by estimating nitrated tyrosine residues in the presence of αKG (r2 = 0.94): a reduction by half of nitrated tyrosine was estimated using 12 mM αKG compared to the control (326.1 ± 39.6 nmol vs. 844.5 ± 128.4 nmol; p < 0.001).

Alpha-Ketoglutarate and 5-HMF: A Potential Anti-Tumoral Combination against Leukemia Cells.

We have recently shown that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) might have anti-tumoral potential due to its antioxidative activities. The question arises if these substances have caspase-3- and apoptosis-activating effects on the cell proliferation in Jurkat and HF-SAR cells. Antioxidative capacity of several combined aKG + 5-HMF solution was estimated by cigarette smoke radical oxidized proteins of fetal calf serum (FCS) using the estimation of carbonylated proteins. The usage of 500 µg/mL aKG + 166.7 µg/mL 5-HMF showed the best antioxidative capacity to inhibit protein modification of more than 50% compared to control measurement. A Jurkat cell line and human fibroblasts (HF-SAR) were cultivated in the absence or presence of combined AKG + 5-HMF solutions between 0 µg/mL aKG + 0 µg/mL 5-HMF and different concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Aliquots of Jurkat cells were tested for cell proliferation, mitochondrial activity, caspase activity, apoptotic cells and of the carbonylated protein content as marker of oxidized proteins in cell lysates after 24, 48, and 72 h of incubation. The combined solutions of aKG + 5-HMF were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time, with the greatest reductions using 500 µg/mL aKG + 166.7 µg/mL 5-HMF after 24 h of incubation compared to 24 h with the control (22,832 cells vs. 32,537 cells), as well as after 48 h (21,243 vs. 52,123 cells) and after 72 h (23,224 cells). Cell growth was totally inhibited by the 500 µg/mL AKG + 166.7 µg/mL solution between 0 and 72 h of incubation compared to 0 h of incubation for the control. The mitochondrial activity measurements supported the data on cell growth in Jurkat cells: The highest concentration of 500 µg/mL aKG + 166.7 µg/mL 5-HMF was able to reduce the mitochondrial activity over 24 h (58.9%), 48 h (28.7%), and 72 h (9.9%) of incubation with Jurkat cells compared not only to the control incubation, but also to the concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 375 µg/mL aKG 125 µg/mL 5-HMF, which were able to significantly reduce the mitochondrial activity after 48 h (28.7% or 35.1%) and 72 h (9.9% or 18.2%) compared to 24 h with the control (100%). A slight increase in cell proliferation was found in HF-SAR using the highest concentration (500 µg/mL aKG + 166.7 µg/mL 5-HMF) between 0 h and 72 h incubation of 140%, while no significant differences were found in the mitochondrial activity of HF-SAR in the absence or presence of several combined aKG + 5-HMF solutions. The solutions with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF showed a significantly higher caspase activity (51.6% or 13.5%) compared to the control (2.9%) in addition to a higher apoptosis rate (63.2% or 31.4% vs. control: 14.9%). Cell lysate carbonylated proteins were significantly higher in Jurkat cells compared to HF-SAR cells (11.10 vs. 2.2 nmol/mg). About 72 h incubation of Jurkat cells with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF reduced significantly the carbonylated protein content down to 5.55 or 7.44 nmol/mg whereas only the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution showed a significant reduction of carbonylated proteins of HF-SAR (1.73 nmol/mg).

Dose- and Sex-Dependent Changes in Hemoglobin Oxygen Affinity by the Micronutrient 5-Hydroxymethylfurfural and α-Ketoglutaric Acid.

5-Hydroxymethylfurfural (5-HMF) is known to increase hemoglobin oxygen affinity (Hb-O2 affinity) and to induce a left shift of the oxygen dissociation curve (ODC). It is under investigation as a therapeutic agent in sickle cell anemia and in conditions where pulmonary oxygen uptake is deteriorated or limited (e.g., various clinical conditions or altitude exposure). The combination of 5-HMF and α-ketoglutaric acid (αKG) is commercially available as a nutritional supplement. To further elucidate dose effects, ODCs were measured in vitro in venous whole blood samples of 20 healthy volunteers (10 female and 10 male) after the addition of three different doses of 5-HMF, αKG and the combination of both. Linear regression analysis revealed a strong dose-dependent increase in Hb-O2 affinity for 5-HMF (R2 = 0.887; p < 0.001) and the commercially available combination with αKG (R2 = 0.882; p < 0.001). αKG alone increased Hb-O2 affinity as well but to a lower extent. Both the combination (5-HMF + αKG) and 5-HMF alone exerted different P50 and Hill coefficient responses overall and between sexes, with more pronounced effects in females. With increasing Hb-O2 affinity, the sigmoidal shape of the ODC was better preserved by the combination of 5-HMF and αKG than by 5-HMF alone. Concerning the therapeutic effects of 5-HMF, this study emphasizes the importance of adequate dosing in various physiological and clinical conditions, where a left-shifted ODC might be beneficial. By preserving the sigmoidal shape of the ODC, the combination of 5-HMF and αKG at low (both sexes) and medium (males only) doses might be able to better maintain efficient oxygen transport, particularly by mitigating potentially deteriorated oxygen unloading in the tissue. However, expanding knowledge on the interaction between 5-HMF and Hb-O2 affinity in vitro necessitates further investigations in vivo to additionally assess pharmacokinetic mechanisms.

Simultaneous quantitative determination of alpha-ketoglutaric acid and 5-hydroxymethylfurfural in human plasma by gas chromatography-mass spectrometry.

Alpha-ketoglutaric acid (alpha-KG) and 5-hydroxymethylfurfural (5-HMF) are currently under investigation as promising cancer cell damaging agents. A method for the simultaneous quantitative determination of alpha-KG and 5-HMF in human plasma was established for screening these compounds in human plasma. Plasma samples were directly treated with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride to form the corresponding oximes, thus facilitating subsequent liquid-liquid extraction. After formation of the trimethylsilyl ethers, samples were analyzed by gas chromatography with electron ionization mass spectrometry. Stable isotope labeled standards were used, the preparation of (13)C(6)-5-HMF is described. Limits of quantitation were set to 0.938 microg/mL for alpha-KG and 0.156 microg/mL for 5-HMF. Inter-day accuracy was < or = 93.7% (alpha-KG) and < or = 92.8% (5-HMF). Inter-day precision was < or = 6.0% (alpha-KG) and < or = 4.6% (5-HMF). The method has been successfully applied to pharmacokinetic profiling of the compounds after intravenous application.

Carbonyl proteins as a clinical marker in Alzheimer's disease and its relation to tryptophan degradation and immune activation.

BACKGROUND: The question arises whether oxidative stress is connected with systemic immune activation in Alzheimer's disease (AD) and mild cognitive impairment (MCI). During immune response interferon-gamma stimulates the kynurenine (Kyn) pathway, a major route of L-tryptophan (Trp) degradation. METHODS: Plasma Kyn, Trp and the Kyn to Trp ratio (Kyn/Trp), carbonyl proteins (CP) as oxidative stress parameter and homocysteine, neopterin, folate and vitamin B12 were measured from patients with AD and MCI (n = 16: 6 females and 4 males with AD, 3 females and 3 males with MCI; 63.3 +/- 13.7 years), and an age matched healthy control group (n = 15: 11 females and 4 males; 62.8 +/- 3.6 years). We correlated the oxidative stress parameter CP with the degradation of Trp creating a new quotient CP/Trp and calculated the sensitivity, specificity, and cut-off values for CP, Trp, CP/Trp, and Kyn/Trp using discriminate analysis. RESULTS: CP was significantly higher in AD/MCI (930 +/- 265 pmol/mg; p < 0.001) compared to controls (300 +/- 120 pmol/mg), Trp was significantly lower in AD/MCI (48.9 +/- 9.0 micromol/L; p < 0.001) than controls (65.2 +/- 10.7 micromol/L). While Kyn showed no significant difference between AD/MCI (1.72 +/- 0.56 micromol/L) and controls (1.53 +/- 0.29 micromol/L), Kyn/Trp was significantly higher in AD/MCI (35.2 +/- 8.8 micromol/mmol; p < 0.001) than in controls (23.7 +/- 4.2 micromol/mmol). CP/Trp ratio was more than 4 fold higher in the AD/MCI group (19.8 +/- 7.76 [(pmol/mg)/(micromol/L)]; p < 0.001) compared to controls (4.79 +/- 2.26 [(pmol/mg)/(micromol/L)]). Homocysteine, folate, vitamin B12, and neopterin showed no significant difference. Discriminant analysis provided CP alone as the best clinical marker with highest sensitivity and highest specificity for AD/MCI followed by the ratio of CP/Trp. ROC curve analysis provided the best result for CP/Trp. CONCLUSIONS: These preliminary results support the hypothesis that oxidative damage to proteins is directly connected with Trp degradation and Kyn pathway in the systemic immune activation.

Antioxidant Solution in Combination with Angiotensin-(1-7) Provides Myocardial Protection in Langendorff-Perfused Rat Hearts.

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.

The role of nanoparticle size in hemocompatibility.

It is expected that nanoparticular matters will be increasingly used for industrial and medical applications. Since it is known that nanoparticles exhibit unique and potential hazardous properties due to their small size, toxicity studies, risk assessment and risk management are of great interest. We focussed on adverse effects on human blood. Processes which warrant special attention are clotting, reactions triggering inflammatory and immune responses and hemolysis. Starting with the determination of size and surface charge in different media we assessed the effect of size and surface charge on induction of coagulation, thrombocyte activation, complement activation, granulocyte activation and hemolysis. We used polystyrene particles as model because they are available in different sizes but constant surface charges. The presence of salts and of protein in the dispersion solution increased particle size and neutralized surface charge. Positively charged particles formed aggregates in buffered solution. Interference of the particles with assays based on fluorescence associated cell sorting was identified. Positive surface charge induced activation of complement. Small size caused thrombocyte and granulocyte activation, and hemolysis. A characterization of particle size and surface charge in the solutions used for the experiments appears important for interpretation of the results. The size dependency of adverse effects in human blood is not linear; negatively charged particles larger than 60 nm hydrodynamic diameter appear to be considerably less hematotoxic than smaller ones.

Using tolerance intervals in pre-study validation of analytical methods to predict in-study results. The fit-for-future-purpose concept.

It is recognized that the purpose of validation of analytical methods is to demonstrate that the method is suited for its intended purpose. Validation is not only required by regulatory authorities, but is also a decisive phase before the routine use of the method. For a quantitative analytical method the objective is to quantify the target analytes with a known and suitable accuracy. For that purpose, first, a decision about the validity of the method based on prediction is proposed: a method is declared proper for routine application if it is considered that most of the future results generated will be accurate enough. This can be achieved by using the "beta-expectation tolerance interval" (accuracy profile) as the decision tool to assess the validity of the analytical method. Moreover, the concept of "fit-for-purpose" is also proposed here to select the most relevant response function as calibration curve, i.e. choosing a response function based solely on the predicted results this model will allow to obtain. This paper reports four case studies where the results obtained with quality control samples in routine were compared to predictions made in the validation phase. Predictions made using the "beta-expectation tolerance interval" are shown to be accurate and trustful for decision making. It is therefore suggested that an adequate way to conciliate both the objectives of the analytical method in routine analysis and those of the validation step consists in taking the decision about the validity of the analytical method based on prediction of the future results using the most appropriate response function curve, i.e. the fit-for-future-purpose concept.

Interdependency of the oxidizability of lipoproteins and peroxidase activity with base excess, HCO3, pH and magnesium in human venous and capillary blood.

As continuous production of free radicals and reactive oxygen species is a normal metabolic process, increased metabolism during exercise/workload should increase free radical generation and oxidative stress. Oxidative stress intensity should then depend on the intensity of metabolic stress effects. Intensity of stress is usually reflected in norepinephrine (NE) levels, which correlate linearly and significantly with changes in blood gases, blood buffer systems, blood electrolytes, blood glucose and lactate [Porta, S., Leitner, G., Heidinger, D., Lang, T., Weiss, U., Smolle, K.H., Hasiba, K., 1997. Magnesium während der Alpinausbildung bringt um 30% bessere Energieverwertung. Magnesium-Bulletin 19(2), 59-61]. Those parameters were used in an open study design to screen 64 subjects for metabolic stress effects along with their antioxidative capacity using both venous and capillary blood. To compare venous and capillary blood, we took venous blood samples from 12 healthy volunteers and capillary blood from 52 other healthy subjects. To show whether free radical changes indeed go along with metabolic stress effects, we tried to quantify relations between metabolic stress effects and oxidative stress by linear correlations. In conclusion, both venous and capillary blood are suitable for determining at least those parameters of the oxidative state that we used. All significant correlations of peroxidase activity and oxidation lag time (OLT) with pH, bicarbonate (HCO3), base excess (BE) and magnesium (Mg) indicate that free radical production increases with metabolism. Those relationships could help to evaluate the oxidative state more precisely.

Development of a high-performance liquid chromatographic method with electrochemical detection for the determination of hyperforin.

An HPLC method with electrochemical detection for the determination of hyperforin extracts without using additional sample precleaning has been developed and validated. The hyperforin solutions were separated isocratically using a mobile phase consisting of 10% ammonium acetate buffer (0.5 M, pH 3.7)-MeOH-acetonitrile (10:40:50, v/v) at a flow rate of 0.8 mL/min. Hyperforin was detected amperometrically with a glassy carbon electrode at a potential of +1.1 V versus Ag/AgCl/3 M potassium chloride reference electrode. Under these conditions, a plot of integrated peak area versus concentration of hyperforin was found to be linear over the range of 0.054-5.4 microg/mL, with a relative standard deviation of 2.2-8.6%. The limit of detection was 0.050 ng on column. The determination of the hyperforin content in a commercially available St. John's Wort preparation exhibited a mean content of 1.56 mg. Recovery experiments led to a mean recovery rate of 97 +/- 5.8%. The proposed method is not time-consuming, sensitive and reproducible and is therefore suitable for routine analysis of hyperforin in herbal medicinal products.

Rapid screening of triazines and quantitative determination in drinking water.

A sensitive, rapid and inexpensive analysis method has been developed for the triazines most frequently used in Palestine; the method includes fluorodensitometric screening and densitometric determination of the individual substances. Terbutryn as a model substance was derivatized with dansyl chloride in sodium hydrogen-carbonate or phosphate buffer solution to yield a green-blue fluorescent compound. Derivatization occurred at 120 degrees C within maximum of 10-min reaction time. The fluorescent compound formed was separated from excess reagent and other by-products on silica gel TLC plates and was then determined fluorodensitometrically. A linearity range between 20 and 1200 pg/spot was achieved. The method was also applied to other triazine herbicides such as ametryn, atrazine, propazine, terbuthylazine and simazine. Drinking water samples spiked with triazines were extracted using RP-C18 polar plus cartridges, and the extract could be then dansylated as a total. Recoveries were between 88% and 95%; the detection limit was 10 pg/spot and could be further improved to 2 pg/spot by a dipping solution. For quantification, each of the six triazines can be separated on one of three different stationary phases after solid phase extraction and measured densitometrically. The LOD for each individual triazine was 100 ng/l.

Influence of several Styrian wines on bovine coronary artery contractions induced by endogenous agents.

In the current study, seven Styrian white wine varieties, as well as their corresponding grape skin extracts (GSE), were tested for possible antagonistic effects on several endogenous vasoconstrictors known to be involved in the pathogenesis of cardiovascular diseases via tissue contraction experiments. Results were compared to those of Zweigelt, the most cultivated Styrian red wine. Bovine coronary artery strips were attached to a force transducer connected to a bridge amplifier, and isometric force was recorded on a multipen recorder. Preincubation of the vessels with the dealcoholized wines (DAW; 330 microl/ml) inhibited the constrictions to histamine and serotonin (5-HT) NO-dependently in the range of 5-80% and 30-90%, respectively (Zweigelt 47% and 90%, respectively). The corresponding GSE (20 mg/ml) inhibited the coronary constrictions to histamine in the range of 40% to 80% (Zweigelt 80%). Additionally, all DAW antagonized the contractile responses to the thromboxane-mimetic U46619 and the isoprostane 8-iso-PGF2alpha, indicating a nonspecific inhibition. To characterize the vasoactive component(s), the Traminer GSE was separated representatively using ultrafiltration, solid phase extraction (SPE) on Isolute C18(EC), and Isolute SCX-2, as well as column chromatography (CC) on polyamide. The resulting fractions were subsequently bioassayed for vasorelaxing activity. Preliminary results refer to a very hydrophilic, nonpolyphenolic basic compound with a MW<1000 Da. Our findings demonstrate that certain Styrian wines have remarkable antagonistic effects on several endogenous agonists in vitro, and that also nonpolyphenolic components in grapes may contribute to cardiovascular protection. Further separation steps as well as phytochemical and pharmacological investigations are in progress.