Novel approach over template molecule elimination in molecularly imprinted polymers by using heat activated persulfate.

This study proposes a new alternative for template removal from molecularly imprinted polymers by heat activated persulfate. It is known that trace amounts of template molecule remains in the polymer network after extraction by current methodologies leading to bleeding and incomplete removal of template which could compromise final determination of target analytes especially in trace analysis. A previously developed molecularly imprinted polymer specially designed for Coenzyme Q10 (CoQ10) extraction was employed as a model to test this template elimination approach. This polymer is based on methacrylic acid and ethylene glycol dimethylacrylate as monomers and Coenzyme Q0 as template. This coenzyme has the same quinone group as the CoQ10. Selectivity was analyzed comparing the recovery of CoQ10 and ubichromenol, a CoQ10 related substance. Chemical degradation using heat-activated persulfate allows the elimination of the template molecule with a high level of efficiency, being a simple and ecological methodology, yielding a polymer that exhibits comparable selectivity and imprinting effect with respect to traditional extraction methods.

Diffusion Coefficients of Variable-Size Amphiphilic Additives in a Glass-Forming Polyethylene Matrix.

Diffusion of additives in polymers is an important issue in the plastics industry since migratory-type molecules are widely used to tune the properties of polymeric composites. Predicting the diffusional behavior of new additives can minimize the need for repetitive experiments. This work presents molecular dynamics simulations at the microsecond time scale and uses the MARTINI force field to estimate self-diffusion coefficients, D, of six monounsaturated amides and their analogs carboxylic acids in polyethylene matrices (PE, MW = 5600 Da). The results are strongly influenced by the glass-forming properties of the PE matrix, which we characterize by three distinct temperatures. The metastability region (T < 325 K), the glass transition temperature (Tg = 256-260 K), and the end of the transition (T ≅ 200 K). Self-diffusion mechanisms are inferred from the results of the dependence of D on the molecular mass of the additive, observing a Rouse-like behavior at high temperatures and deviations from it within the metastability region of the matrix. Interestingly, D values are nonsensitive to the nature of the considered polar head for additives of similar size. The temperature-dependent behavior of D follows, at fixed additive size, a linear Arrhenius pattern at high temperatures and a super Arrhenius trend at lower temperatures, which is well represented with a power law equation as predicted by the Mode Coupling Theory (MCT). We offer a conceptual explanation for the observed super-Arrhenius behavior. This explanation draws on Truhlar and Kohen's interpretation of the available energies at both the initial and the transition states along the diffusion pathway. The matrix's mobility significantly affects solute self-diffusion, yielding equal activation enthalpies for the Arrhenius region or the same power law parameters for the super-Arrhenius regime. Finally, we establish a one-to-one time-equivalence of the self-diffusion processes between CG and all-atom systems for the largest additives and the PE matrix in the high-temperature regime.

Experimental glaucoma triggers a pro-oxidative and pro-inflammatory state in the rat cornea.

BACKGROUND: Increasing evidence suggests that glaucoma affects the ocular surface. We aimed to investigate the cellular mechanisms underlying the glaucoma-associated corneal alterations in an animal model. METHODS: Wistar rats underwent the cauterization of two episcleral veins of the left eye to elevate the intraocular pressure (ipsilateral, G-IL). Control animals received a sham procedure (C-IL). Contralateral eyes did not receive any procedure (G-CL or C-CL). Enzymes related to the redox status, oxidative damage to macromolecules, and inflammatory markers were assessed in corneal lysates. RESULTS: Compared to C-IL, NOX4, NOX2, and iNOS expression was increased in G-IL (68%, p < 0.01; 247%, p < 0.01; and 200%, p < 0.001, respectively). We found an increase in SOD activity in G-IL (60%, p < 0.05). The GSH/GSSG ratio decreased in G-IL (80%, p < 0.05), with a decrease in GR activity (40%, p < 0.05). G-IL displayed oxidative (90%, p < 0.01) and nitrosative (40%, p < 0.05) protein damage, and enhanced lipid peroxidation (100%, p < 0.01). G-IL group showed an increased in CD45, CD68 and F4/80 expression (50%, p < 0.05; 190%, p < 0.001 and 110%, p < 0.05, respectively). G-CL displayed a higher expression of Nrf2 (60%, p < 0.001) and increased activity of SOD, CAT, and GPx (60%, p < 0.05; 90%, p < 0.01; and 50%, p < 0.05, respectively). CONCLUSIONS: Glaucoma induces a redox imbalance in the ipsilateral cornea with an adaptive response of the contralateral one. GENERAL SIGNIFICANCE: Our study provides a possible mechanism involving oxidative stress and inflammation that explains the corneal alterations observed in glaucoma. We demonstrate that these changes extend not only to the ipsilateral but also to the contralateral cornea.

Bioavailability of coenzyme Q10 loaded in an oleogel formulation for oral therapy: Comparison with a commercial-grade solid formulation.

Coenzyme Q10 (CoQ10) is essential in mitochondrial bioenergetics and is a potent endogenous antioxidant. Low CoQ10 levels are associated with neurodegenerative, metabolic, muscular and cardiovascular disorders. Early treatment with high doses (5-50 mg/kg/day) demonstrated to limit the onset and progression of neuropathology. Recently, we developed an oleogel matrix able to support a high dose of oil-dissolved CoQ10, easy to swallow by CoQ10-deficient patients who suffer from secondary dysphagia. In the present study, we evaluated the bioavailability of oleogel-dissolved CoQ10 and plasma antioxidant status in healthy adults in single-dose and repeated-dose studies. The single-dose study demonstrated that, in terms of CoQ10 bioavailability, 1 g CoQ10/5g oleogel-disk was equivalent to the solid form (1 g CoQ10/three 00-size-capsules), whereas the repeated-dose study (14-days-administration) demonstrated a significantly higher increase in plasma CoQ10 when administered through the oleogel, which could be compatible with the levels necessary to achieve an adequate therapeutic response. Also, a trend to a higher plasma apparent half-life (greater than24 h) was observed for the oleogel-loaded-CoQ10. In conclusion, the oleogel matrix does not compromise the oil-dissolved CoQ10 bioavailability and can prevent the non-adherence to this vital supplementation in patients with high CoQ10 requirements. No significant variation in the plasma antioxidant status (vitamins A, E and C, glutathione and TBARs) was observed.

Thioredoxin-1 is required for the cardioprotecive effect of sildenafil against ischaemia/reperfusion injury and mitochondrial dysfunction in mice.

Sildenafil is a phosphodiesterase type 5 inhibitor which confers cardioprotection against myocardial ischaemia/reperfusion (I/R) injury. The aim of this study was to determine if Trx1 participates in cardioprotection exerted by sildenafil in an acute model of I/R, and to evaluate mitochondrial bioenergetics and cellular redox status. Langendorff-perfused hearts from wild type (WT) mice and a dominant negative (DN-Trx1) mutant of Trx1 were assigned to placebo or sildenafil (0.7 mg/kg i.p.) and subjected to 30 min of ischaemia followed by 120 min of reperfusion. WT + S showed a significant reduction of infarct size (51.2 ± 3.0% vs. 30 ± 3.0%, p < .001), an effect not observed in DN-Trx. After I/R, sildenafil preserved state 3 oxygen consumption from WT, but had a milder effect in DN-Trx1 only partially protecting state 3 values. Treatment restored respiratory control (RC) after I/R, which resulted 8% (WT) and 24% (DN-Trx1) lower than in basal conditions. After I/R, a significant increase in H2O2 production was observed both for WT and DN-Trx (WT: 1.17 ± 0.13 nmol/mg protein and DN-Trx: 1.38 ± 0.12 nmol/min mg protein). With sildenafil, values were 21% lower only in WT I/R. Treatment decreased GSSG levels both in WT and DN-Trx1. In addition, GSSG/GSH2 ratio was partially restored by sildenafil. Also, an increase in p-eNOS/eNOS even before the myocardial ischaemia was observed with sildenafil, both in WT (14%, p > .05) and in DN-Trx (35%, p < .05). Active Trx1 is required for the onset of the cardioprotective effects of sildenafil on I/R injury, together with the preservation of cellular redox balance and mitochondrial function.

Advances in drug delivery, gene delivery and therapeutic agents based on dendritic materials.

Dendrimers are synthetic polymers that grow in three dimensions into well-defined structures. Their morphological appearance resembles a number of trees connected by a common point. Dendritic nanoparticles have been studied for a large number of pharmaceutical and biomedical applications including gene and drug delivery, clinical diagnosis and MRI. Despite the application of dendrimers, research is still in its childhood in comparison with liposomes and other nanomaterials. They are now playing a key role in several therapeutic strategies, with dendrimer-based products in clinical trials. The aim of this review is to describe the state-of-the-art of biomedical applications of dendrimers - and dendrimer conjugates - such as drug and gene delivery and antiviral activity.

Modified ribavirin analogues as antiviral agents against Junín virus.

In this work, several ribavirin analogues were synthesized and incorporated into a multivalent arrangement. Both were subsequently modified by the addition of polyhydroxylated residues. Their antiviral activity was tested against Junín virus, etiological agent responsible of Argentine hemorrhagic fever. Some compounds inhibited Junín virus in the range of 13.2-389.1 µM. Two modified ribavirin analogues presented an effective concentration comparable to ribavirin but with a higher selectivity index.

Game-changing restraint of Ros-damaged phenylalanine, upon tumor metastasis.

An abrupt increase in metastatic growth as a consequence of the removal of primary tumors suggests that the concomitant resistance (CR) phenomenon might occur in human cancer. CR occurs in murine tumors and ROS-damaged phenylalanine, meta-tyrosine (m-Tyr), was proposed as the serum anti-tumor factor primarily responsible for CR. Herein, we demonstrate for the first time that CR happens in different experimental human solid tumors (prostate, lung anaplastic, and nasopharyngeal carcinoma). Moreover, m-Tyr was detected in the serum of mice bearing prostate cancer (PCa) xenografts. Primary tumor growth was inhibited in animals injected with m-Tyr. Further, the CR phenomenon was reversed when secondary implants were injected into mice with phenylalanine (Phe), a protective amino acid highly present in primary tumors. PCa cells exposed to m-Tyr in vitro showed reduced cell viability, downregulated NFκB/STAT3/Notch axis, and induced autophagy; effects reversed by Phe. Strikingly, m-Tyr administration also impaired both, spontaneous metastasis derived from murine mammary carcinomas (4T1, C7HI, and LMM3) and PCa experimental metastases. Altogether, our findings propose m-Tyr delivery as a novel approach to boost the therapeutic efficacy of the current treatment for metastasis preventing the escape from tumor dormancy.

Synthesis and characterization of molecularly imprinted polymer nanoparticles for coenzyme Q10 dispersive micro solid phase extraction.

Molecularly imprinted polymer nanoparticles (MIPNPs) with the ability to recognize coenzyme Q10 (CoQ10) were synthesised in order to be employed as sorbent in a dispersive micro-solid phase extraction (DMSPE) for the determination of CoQ10 in a liver extract. CoQ10 is a redox-active, lipophilic substance integrated in the mitochondrial respiratory chain which acts as an electron carrier, shuttling electrons from complex I (NADH-ubiquinone oxidoreductase) and II (succinate-ubiquinone oxidoreductase) to complex III (ubiquinol-cytochrome c reductase), for the production of cellular energy. The MIPNPs were synthesised by precipitation polymerization using coenzyme Q0 as the dummy template, methacrylic acid as the functional monomer, an acetonitrile: water mixture as the porogen, ethylene glycol dimethacrylate as the crosslinker and potassium persulfate as initiator. The nanoparticles were characterized by microscopy, capillary electrophoresis, dynamic light scattering, N2 adsorption-desorption isotherms, and infrared spectroscopy. The MIPNPs demonstrated the presence of selective cavities complementary to the quinone nucleus of CoQ10, leading to a specific recognition of CoQ10 compared with related compounds. In the liver extract the relative CoQ10 peak area (CoQ10 area/total peak area) increased from 4.6% to 25.4% after the DMSPE procedure. The recovery percentage of CoQ10 from the liver matrix was between 70.5% and 83.7% quantified against CoQ10 standard processed under the same conditions. The DMSPE procedure allows the elution of almost all the CoQ10 retained (99.4%) in a small volume (200µL), allowing the sample to be concentrated 2.5 times (LOD: 1.1µgg(-1) and LOQ: 3.7µgg(-1) of tissue). The resulted clean up of the sample, the improvement in peak shape and baseline and the reduction of interferences, evidence that the MIPNPs could potentially be applied as sorbent in a DMSPE with satisfactory results and with a minimum amount of sorbent (1mg).

New analytical strategies applied to the determination of Coenzyme Q10 in biological matrix.

In the last few years the importance of Coenzyme Q10 (CoQ10) determination has gained clinical relevance. CoQ10 is a redox-active, lipophilic substance integrated in the mitochondrial respiratory chain which acts as an electron carrier for the production of cellular energy. In addition, it is recognized as a primary regenerating antioxidant playing an intrinsic role against oxidative damage. There are some reports of low CoQ10 levels in a number of disorders, such as cancer, muscular, neurodegenerative, cardiological, and reproductive diseases. Therefore, it is a priority to develop analytical methodologies for evaluating CoQ10 in matrices of greater importance for the correct diagnosis of diseases, simple enough to be used in routine clinical laboratories. In this chapter two recently developed techniques, capillary electrophoresis and microHPLC, for the analysis of CoQ10 in biological matrices, are studied.

The use of coenzyme Q0 as a template in the development of a molecularly imprinted polymer for the selective recognition of coenzyme Q10.

In this work, a novel molecularly imprinted polymer (MIP) for use as a solid phase extraction sorbent was developed for the determination of coenzyme Q10 (CoQ10) in liver extract. CoQ10 is an essential cofactor in mitochondrial oxidative phosphorylation and a powerful antioxidant agent found in low concentrations in biological samples. This fact and its high hydrophobicity make the analysis of CoQ10 technically challenging. Accordingly, a MIP was synthesised using coenzyme Q0 as the template, methacrylic acid as the functional monomer, acetonitrile as the porogen, ethylene glycol dimethacrylate as the crosslinker and benzoyl peroxide as the initiator. Various parameters affecting the polymer preparation and extraction efficiency were evaluated. Morphological characterisation of the MIP and its proper comparison with C18 as a sorbent in solid phase extraction were performed. The optimal conditions for the molecularly imprinted solid phase extraction (MISPE) consisted of 400 µL of sample mixed with 30 mg of MIP and 600 µL of water to reach the optimum solution loading. The loading was followed by a washing step consisting of 1 mL of a 1-propanol solution (1-propanol:water, 30:70,v/v) and elution with 1 mL of 1-propanol. After clean-up, the CoQ10 in the samples was analysed by high performance liquid chromatography. The extraction recoveries were higher than 73.7% with good precision (3.6-8.3%). The limits of detection and quantification were 2.4 and 7.5 µg g(-1), respectively, and a linear range between 7.5 and 150 µg g(-1) of tissue was achieved. The new MISPE procedure provided a successful clean-up for the determination of CoQ10 in a complex matrix.

Coenzyme Q in pregnant women and rats with intrahepatic cholestasis.

BACKGROUND & AIMS: Intrahepatic cholestasis of pregnancy is a high-risk liver disease given the eventual deleterious consequences that may occur in the foetus. It is accepted that the abnormal accumulation of hydrophobic bile acids in maternal serum are responsible for the disease development. Hydrophobic bile acids induce oxidative stress and apoptosis leading to the damage of the hepatic parenchyma and eventually extrahepatic tissues. As coenzyme Q (CoQ) is considered an early marker of oxidative stress in this study, we sought to assess CoQ levels, bile acid profile and oxidative stress status in intrahepatic cholestasis. METHODS: CoQ, vitamin E and malondialdehyde were measured in plasma and/or tissues by HPLC-UV method whereas serum bile acids by capillary electrophoresis in rats with ethinyl estradiol-induced cholestasis and women with pregnancy cholestasis. RESULTS: CoQ and vitamin E plasma levels were diminished in both rats and women with intrahepatic cholestasis. Furthermore, reduced CoQ was also found in muscle and brain of cholestatic rats but no changes were observed in heart or liver. In addition, a positive correlation between CoQ and ursodeoxycholic/lithocholic acid ratio was found in intrahepatic cholestasis suggesting that increased plasma lithocholic acid may be intimately related to CoQ depletion in blood and tissues. CONCLUSION: Significant CoQ and vitamin E depletion occur in both animals and humans with intrahepatic cholestasis likely as the result of increased hydrophobic bile acids known to produce significant oxidative stress. Present findings further suggest that antioxidant supplementation complementary to traditional treatment may improve cholestasis outcome.

Bile acids content in brain of common duct ligated rats.

INTRODUCTION: Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Bile duct ligated rats constitute an interesting model to study the mechanism of cholestasis, and its action on several organs and tissues, including the brain. AIM: To analyze brain bile acids individually in ligated rats to evaluate if its profile is altered towards a more toxic condition in cholestasis. MATERIAL AND METHODS: Male Wistar rats were used and separated in two groups: bile duct ligated rats and sham operated rats (n = 5 in each group). Bile acid profile was assessed in brain homogenates. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase determinations, bilirubin and ammonia plasma concentration were also measured in both groups. RESULTS: Although the total amount of bile acids in control animal brains showed a higher concentration than in bile duct ligated rats, the bile acid profile in this group was found more toxic composition than in controls. Lithocholic acid was present in brain in higher concentration (87.4 % of total brain bile acids) in ligated rats and absent in controls. Alkaline phosphatase, bilirubin and ammonia were significantly higher in bile duct ligated rats than in control group. CONCLUSION: It was found a toxic brain bile acid profile during hepatic cholestasis which could be one of the explanations of hepatic encephalopathy observed in cholestatic diseases.

In search of an accurate evaluation of intrahepatic cholestasis of pregnancy.

Until now, biochemical parameter for diagnosis of intrahepatic cholestasis of pregnancy (ICP) mostly used is the rise of total serum bile acids (TSBA) above the upper normal limit of 11 µM. However, differential diagnosis is very difficult since overlapped values calculated on bile acids determinations, are observed in different conditions of pregnancy including the benign condition of pruritus gravidarum. The aim of this work was to determine the better markers in ICP for a precise diagnosis together with parameters associated with severity of symptoms and treatment evaluation. Serum bile acid profiles were evaluated using capillary electrophoresis in 38 healthy pregnant women and 32 ICP patients and it was calculated the sensitivity, specificity, accuracy, predictive values and the relationships of certain individual bile acids in pregnant women in order to replace TSBA determinations. The evaluation of the results shows that LCA and UDCA/LCA ratio provided information for a more complete and accurate diagnosis and evaluation of ICP than calculation of solely TSBA levels in pregnant women.

Novel and highly sensitive mixed-polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples.

A mixed-polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric ß-CD, 0.4% w/v tetronic(®) 1107 and 400 mM tris-phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 50 cm of total length and 75 µm id, an applied voltage of -7 kV, a temperature of 30°C and 200 nm UV-detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 µg/mL) (OSCS) and 0.1% w/w (0.1 µg/mL) (Der), and values of LOQ 0.2% w/w (0.2 µg/mL) (OSCS) and 0.3% w/w (0.3 µg/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.

Simultaneous determination of nine endogenous steroids in human urine by polymeric-mixed micelle capillary electrophoresis.

A new CE system based on the use of polymeric-mixed micelles (cholic acid, SDS and the poloxamine Tetronic(®) 1107) was developed for the simultaneous determination of nine steroids in human urine. This method allows the baseline separation and quantitation of cortisol, androstenedione, estriol, dehydroepiandrosterone sulfate, testosterone, dehydroepiandrosterone, estrone, progesterone and estradiol in less than 25 min showing to be sensitive enough to detect low concentrations of these steroids in urine samples (5-45 ng/mL). The optimized electrophoretic conditions were performed using a 50 cm × 75 µm capillary, 18 kV, 25°C, with 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic(®) 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran in 5 mM borate - 5 mM phosphate buffer (pH=8.0) as a background electrolyte and a dual 210/254 UV-detection. The method can simultaneously determine 0.1-120 µg/mL, which corresponds to 5-6000 ng/mL of steroids in 2 mL urine. The recoveries ranged between 82.4 and 101.5%. Due to its simplicity, speed, accuracy and reliability, the proposed method could be a potential alternative to the traditional methodologies used with clinical purposes.

A capillary electrophoretic system based on a novel microemulsion for the analysis of coenzyme Q10 in human plasma by electrokinetic chromatography.

A new analytical method for determination of coenzyme Q10 (2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone, CoQ10) in human plasma was developed based on CE using a double tensioactive microemulsion. CoQ10 was quantitatively extracted into 1-propanol/hexane and quantified by MEEKC. The microemulsion was prepared by mixing 1.4% w/w sodium bis(2-ethylhexyl) sulfosuccinate, 4% w/w cholic acid, 1% w/w octane, 8.5% w/w butanol, 0.1% w/w PVA and 85% w/w 10 mM Tris buffer at pH 9.0. The optimized electrophoretic conditions included the use of an uncoated silica capillary of 60 cm total length and 75 mum id, an applied voltage of 20 kV, room temperature and 214 nm ultraviolet detection. Selectivity, linearity, LOD, LOQ, precision and accuracy were evaluated as the parameters of validation. Owing to its simplicity and reliability, the proposed method can be an advantageous alternative to the traditional methodology for the quantitation of CoQ10 in human plasma with good accuracy and precision.

Lithocholic acid as a biomarker of intrahepatic cholestasis of pregnancy during ursodeoxycholic acid treatment.

BACKGROUND: The diagnosis and treatment of intrahepatic cholestasis of pregnancy (ICP) has important implications on fetal health. The biochemical parameter commonly used in the diagnosis of ICP is the determination of the concentration of total serum bile acids (TSBA). However, bile acid profile, especially lithocholic acid (LCA) analysis is a more sensitive and specific biomarker for differential diagnosis of this pathology and also could be an alternative to evaluate the efficiency of ursodeoxycholic acid (UDCA) for ICP treatment. METHODS: Serum bile acid (SBA) profiles including LCA determination, were studied in 28 ICP patients using a capillary electrophoresis method. The effects of UDCA treatment on bile acid profile, were analysed in 23 out of 28 ICP patients and the two samples obtained before and 15 days after treatment were compared. Two samples taken as controls were also obtained from each of five patients without therapy. RESULTS: A dramatic decrease in LCA concentrations and maintenance of TSBA concentrations were found in all patients after UDCA therapy, whereas SBA profiles together with LCA values did not change in patients without therapy. CONCLUSION: We propose LCA as an alternative biomarker and a more sensitive parameter than TSBA to evaluate the effectiveness of UDCA treatment, at least in ICP patients from Argentina.