Milan Nikolic

Lipid status, anti-oxidant enzyme defence and haemoglobin content in the blood of long-term clozapine-treated schizophrenic patients.

OBJECTIVE Despite clozapines unique effectiveness in patients with schizophrenia, a number of adverse effects have been recognised including abnormalities in lipid and glucose metabolisms. A high clozapine level in red blood cells (RBCs) and disturbed anti-oxidant enzyme activities in blood from schizophrenic patients prompted us to investigate lipid status and anti-oxidant enzyme defence in the blood of chronic schizophrenic patients on long-term clozapine therapy. METHODS Plasma lipids, RBC anti-oxidant enzyme activities and haemoglobin (Hb) content were measured using established procedures in a group of eighteen chronically-medicated (average 630 days of therapy) schizophrenic patients receiving clozapine (average dose of 295 mg/day) and data were compared with those from a group of eighteen well-matched normal controls. RESULTS Significantly higher levels of plasma triglycerides (by 47%, p<0.01) and total cholesterol and phospholipids (by 8% and 11%, respectively p<0.05) in patients were found. CuZn-superoxide dismutase (SOD1) activity was markedly higher (by 35%, p<0.001) while selenium-dependent glutathione peroxidase (GSH-Px1) activity was markedly lower (by 41%, p<0.001) in patients. In addition, metHb and HbA1c levels in patients were significantly higher (by 58% and 25%, respectively p<0.001). SOD1 activity was negatively correlated (p<0.001) to GSH-Px1 activity in patients. CONCLUSIONS The findings support the view that ongoing oxidative stress may be a mechanism by which clozapine induces some adverse effects that increase the risk of diabetes and metabolic syndrome. If valid, this would indicate that in parallel with long-term clozapine treatment, schizophrenic patients could be encouraged to make some lifestyle changes to limit the detrimental effects of the medication.

Structure and antioxidant activity of β-lactoglobulin-glycoconjugates obtained by high-intensity-ultrasound-induced Maillard reaction in aqueous model systems under neutral conditions.

Sonication is a new processing technology in the dairy industry. The aim of this study was to test glycation of β-lactoglobulin (BLG) in Maillard reaction (MR) induced by high-intensity ultrasound in aqueous solution under neutral conditions at 10-15 °C, which is not favourable for the MR. BLG was sonicated in the presence of glucose, galactose, lactose, fructose, ribose and arabinose. Formation of Maillard reaction products (MRPs) was monitored by mass spectrometry, spectrophotometry and fluorimetry. Ultrasound treatment resulted in formation of MRPs with all tested carbohydrates. Ribose induced the highest degree of modification resulting in 76% of BLG modified and an average of three anhydroribose units attached. Circular dichroism spectra analyses indicated only minor alterations in secondary and tertiary structures. MRP obtained by ultrasound exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and possessed increased iron-chelating activity and reducing power. High-intensity ultrasound efficiently promotes BLG-glycoconjugates formation by MR in aqueous solutions under non-denaturing conditions.

Effect of atypical antipsychotics on antioxidant enzyme activities in human erythrocytes (in vitro study)

This study was set out to examine the impact of atypical antipsychotic drugs: aripiprazole, clozapine, ziprasidone, olanzapine, quetiapine, sertindole and amisulpride on the activity of antioxidant defence enzymes in human erythrocytes in vitro.

Characterization and effects of binding of food-derived bioactive phycocyanobilin to bovine serum albumin

Phycocyanobilin (PCB) is a blue tetrapyrrole chromophore of C-phycocyanin, the main protein of the microalga Spirulina, with numerous proven health-related benefits. We examined binding of PCB to bovine serum albumin (BSA) and how it affects protein and ligand stability. Protein fluorescence quenching and microscale thermophoresis demonstrated high-affinity binding (Ka=2×106M-1). Spectroscopic titration with molecular docking analysis revealed two binding sites on BSA, at the inter-domain cleft and at subdomain IB, while CD spectroscopy indicated stereo-selective binding of the P conformer of the pigment to the protein. The PCB protein complex showed increased thermal stability. Although complex formation partly masked the antioxidant properties of PCB and BSA, a mutually protective effect against free radical-induced oxidation was found. BSA could be suitable for delivery of PCB as a food colorant or bioactive component. Our results also highlight subtle differences between PCB binding to bovine vs. human serum albumin.

Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin, a blue-colored biliprotein of microalga Spirulina.

C-phycocyanin, the major protein of cyanobacteria Spirulina, possesses significant antioxidant, anti-cancer, anti-inflammatory and immunomodulatory effects, ascribed to covalently attached linear tetrapyrrole chromophore phycocyanobilin. There are no literature data about structure and biological activities of released peptides with bound chromophore in C-phycocyanin digest. This study aims to identify chromopeptides obtained after pepsin digestion of C-phycocyanin and to examine their bioactivities. C-phycocyanin is rapidly digested by pepsin in simulated gastric fluid. The structure of released chromopeptides was analyzed by high resolution tandem mass spectrometry and peptides varying in size from 2 to 13 amino acid residues were identified in both subunits of C-phycocyanin. Following separation by HPLC, chromopeptides were analyzed for potential bioactivities. It was shown that all five chromopeptide fractions have significant antioxidant and metal-chelating activities and show cytotoxic effect on human cervical adenocarcinoma and epithelial colonic cancer cell lines. In addition, chromopeptides protect human erythrocytes from free radical-induced hemolysis in antioxidative capacity-dependant manner. There was a positive correlation between antioxidative potency and other biological activities of chromopeptides. Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore.

Anion–π interactions in protein–porphyrin complexes

In this work, we have analyzed the influence of anion–π interactions on the stability of high resolution protein–porphyrin complex crystal structures. The anion–π interactions are distance and orientation dependent. Results of ab initio calculations of stabilization energies showed that they lie mostly in the range from −2 to −4 kcal mol−1 with some of the anion–π interactions having stabilization energies of up to −16 kcal mol−1. In the anionic group, the numbers of anion–π interactions involving Asp and Glu are similar, while His is more often involved in these interactions than other aromatic residues. Furthermore, our study showed that in the dataset used about 70% of the investigated anion–π interactions are in fact multiple anion–π interactions. Our results suggest that interacting residues are predominantly located in buried and partially buried regions. The secondary structure of the anion–π interaction involving residues shows that most of the interacting residues preferred to be in helix conformations. Significant numbers of aromatic residues involved in anion–π interactions have one or more stabilization centers, providing additional stability to the protein–porphyrin complexes. The conservation patterns indicate that more than half of the residues involved in these interactions are evolutionarily conserved, indicating that the contribution of the anion–π interaction is an important factor for the structural stability of the investigated protein–porphyrin complexes.

Phycocyanobilin, a bioactive tetrapyrrolic compound of blue-green alga Spirulina, binds with high affinity and competes with bilirubin for binding on human serum albumin

Human serum albumin (HSA) is an important regulator of the pharmacokinetic properties of bioactive compounds. Phycocyanobilin is a blue tetrapyrrole chromophore of C-phycocyanin with proven health-promoting activities. Despite its structural similarity to bilirubin, the conformation it adopts in aqueous solution is different and the pigment is more soluble than bilirubin. The aim of our study was to examine binding of phycocyanobilin for HSA and to investigate its competition with bilirubin. Based on a computational approach, we demonstrated two putative high-affinity binding pockets on HSA of virtually identical energies for the neutral and anion forms of bilirubin, but with slightly favorable predictions for anion forms of phycocyanobilin. Computational prediction of phycocyanobilin pKa values suggested a monoanion form to be the most stable form at physiological conditions. The computationally predicted binding sites for phycocyanobilin were identical to the two previously identified binding sites for bilirubin (subdomains IB and IIA). Results obtained by protein and pigment fluorescence measurements, circular dichroism, and competition experiments confirmed high affinity (binding constant of 2.2 × 106 M−1 at 25 °C), stereo-selective binding of phycocyanobilin M-conformer to HSA and its competition with bilirubin, warfarin and hemin. Our experimental data confirm that phycocyanobilin binds to IB and IIA binding site of HSA with an affinity similar to bilirubin. In conditions characterized by an increased bilirubin plasma concentration, or intake of drugs binding to IB or IIA binding site, pharmacokinetics of phycocyanobilin may also be changed.

Stabilization of Human Serum Albumin by the Binding of Phycocyanobilin, a Bioactive Chromophore of Blue-Green Alga Spirulina: Molecular Dynamics and Experimental Study

Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma.

Dietary lipid intake influences the level of cholesterol bound to haemoglobin in human erythrocytes

BackgroundBlood cholesterol levels are affected by diet and in particular by the type of fat intake. We originally showed that a significant but variable amount of cholesterol is firmly bound to haemoglobin (Hb) yielding the Hb-lipid adduct (Hb-Ch) in erythrocytes isolated from normo-lipidemic males.Aim of the studyTo establish whether dietary lipids affect the level of Hb-Ch in human erythrocytes.MethodsSeventy-four healthy free-living adults were separated according to their serum cholesterol levels into two groups: normo-cholesterolemic (LDL cholesterol <3.4 mmol/l and total cholesterol <5.2 mmol/l) (NC) and hyper-cholesterolemic (LDL cholesterol ≥3.4 mmol/l) (HC). Habitual dietary information was used to classify subjects in both study groups into sub-groups of low-fat (≤30% total energy as fat) and high-fat consumers (>30% total energy as fat). The NC low-fat consumers were placed on a high-lipid (high-fat and high-cholesterol) diet whereas the HC subjects with high-fat intake were assigned to a low-lipid (low-fat and low-cholesterol) diet. Both types of dietary intervention were allowed to continue for 6 weeks. The main variable under scrutiny was the Hb-Ch concentration.ResultsIn both study groups low-fat intake subjects had low levels of Hb-Ch (approx. 0.35 mmol/l RBC) compared with high-fat intake subjects (approx. 0.60 mmol/l RBC), and serum cholesterol was not correlated with Hb-Ch. The two dietary interventions produced substantial changes in the Hb-Ch level that paralleled variation in the serum cholesterol concentration. A high-lipid diet (35% fat, 15% saturated; 580 mg cholesterol) increased Hb-Ch (by approximately 47%, P < 0.001) in subjects with low Hb-Ch at onset, whereas a low-lipid diet (28% fat, 9% saturated; 280 mg cholesterol) decreased Hb-Ch (by approximately 40%, P < 0.001) in subjects with high Hb-Ch at onset.ConclusionHigh consumption of dietary lipids, including saturated fat and cholesterol, has an important influence on the level of Hb-Ch in human erythrocytes.

Effects of antipsychotic drug administration on antioxidative defense enzymes in male rat kidney

ABSTRACT The use of atypical antipsychotic drugs (APD) was reported to be associated with adverse effects on the kidneys. Thus, the aim of this study was to examine whether APD exerted their adverse effects by interfering with the renal antioxidant defense system. Male 3-mo-old Wistar rats were treated for 28 d with ziprasidone (ZIP), clozapine (CLO), or sertindole (SER) using a daily dose recommended for antipsychotic drug therapy. The expression and activities of antioxidant enzymes superoxide dismutase (SOD) type 1 and type 2, catalase (CAT), glutathione reductase (GR), and glutathione S-transferases (GSTs) activity were measured in the kidneys. Changes in the kidneys were also evaluated histologically. Ziprasidone, CLO, and SER reduced renal SOD type 1 and type 2 activities. Decreased CAT activity was observed only in SER-treated rats. An inhibition in GR activity and increased activity of GST was found only after treatment with CLO. Histological analysis showed dilatation of proximal tubules in kidneys with all three drugs. In conclusion, data indicate that redox disturbances may contribute to renal morphologic alterations in proximal tubules in rats treated with all APD.