3,4-Dihydroxy-l-phenylalanine as a biomarker of oxidative damage in proteins: improved detection using cloud-point extraction and HPLC.

Oxidized protein adducts are formed under conditions of oxidative stress and may represent a valuable biomarker for a variety of diseases which share this common aetiology. A suitable candidate biomarker for oxidized proteins is protein-bound 3,4-dihydroxyl-l-phenylalanine (l-DOPA), which is formed on 3'-hydroxylation of tyrosine residues by hydroxyl radicals. Existing methodologies to measure protein-bound l-DOPA employ lengthy acid hydrolysis steps (ca. 16h) which may cause artifactual protein oxidation, followed by HPLC with detection based on the intrinsic fluorescence of l-DOPA. We report a novel method for the measurement of protein-bound l-DOPA which involves rapid hydrolysis followed by pre-column concentration of 6-aminoquinolyl-derivatives using cloud-point extraction. The derivatized material is resolved by reversed-phase HPLC in less than 30min and has derivatization chemistry compatible with both UV and fluorescent detection, providing detection down to the femtomole level. The method provides identical results to those found with highly specific ELISA-based techniques and requires only basic instrumentation. The stability of the 6-aminoquinolyl-derivatives together with the fast and sensitive nature of the assay will be appealing to those who require large sample throughput.

Approaches to nonlinear curve fitting in laboratory medicine.

Nonlinear curve fitting is an important process in laboratory medicine, particularly with the increased use of highly sensitive antibody-based assays. Although the process is often automated in commercially available software, it is important that clinical scientists and physicians recognize the limitations of the various approaches used and are able to select the most appropriate model. This article summarizes the key nonlinear functions and demonstrates their application to common laboratory data. Following this, a basic overview of the statistical comparison of models is presented and then a discussion of important algorithms used in nonlinear curve fitting. An accompanying Microsoft Excel workbook is available that can be used to explore the content of this article.

The quantum mechanics of skincare: A context for the biochemistry curriculum.

Designing a relevant and engaging curriculum for biochemistry undergraduates can be challenging for topics which are at the periphery of the subject. We have used the framework of context-based learning as a means of assessing understanding of quantum theory in a group of students in their junior year. Our context, the role of retinol in skincare, provides a basis for the simple application of quantum mechanical principles to a biological context in an adaptation of the polyene in a box concept. As part of the learner journey, they gain experience of practical computational chemistry, which provided an in silico alternative to traditional laboratory work during the SARS-CoV-19 pandemic. Student feedback was overwhelmingly positive, and this approach is now firmly embedded in the undergraduate curriculum.

Physicochemical Properties of 4-(4-Hydroxyphenyl)-butan-2-one ("Raspberry Ketone") Evaluated Using a Computational Chemistry Approach.

Raspberry ketone (RK) is a product of the phenylpropanoid pathway in a variety of plants and is the second most expensive natural flavouring in the world. It is also widely used as a nutritional supplement due to its reported ability to promote lipolysis and fat oxidation in vivo. We have evaluated the thermodynamics of RK using the correlation consistent ccCA-CBS-2 approach which afforded calculation of (inter alia) the enthalpy of formation. To obtain pK a, log D, electrode potential, solubility, and reactivity indices, we used TPSS/def2-TZVP geometries followed by single-point energies obtained at the M06-2X/def2-TZVPP level of theory. We obtained Δf H o = -299.4 ± 0.17 kJ·mol-1; the pK a and logD were found to be 9.95 and 1.84, respectively, consistent with chemometric predictions. Using the enthalpy of fusion obtained from theory, we evaluated the aqueous solubility of RK to be in the region of 2.5 mg·mL-1 which is in agreement with limited literature reports. In terms of reactivity, we obtained a formal electrode potential of 1.29 V (vs SHE) at pH 7.4 and 298.15 K. The HOMO-LUMO energy separation in an aqueous environment was found to be ca. 7.8 eV, suggesting moderate chemical reactivity. Analysis of the frontier molecular orbitals using conceptual density functional theory supported this and revealed a reactivity pattern consistent with the metabolite profile obtained in mammals, namely, a propensity for nucleophilic attack at the carbonyl carbon and electrophilic addition of the benzene ring.

Mechanism for the enhanced peroxidation of linoleic acid by a titanium dioxide/hypochlorite system.

Nanosized titanium dioxide (TiO2) is a common component of sunscreen preparations and cosmetics as it reflects UV and visible light in accordance to Rayleigh's law. However, in aqueous environments, TiO2 is an efficient photocatalyst, producing superoxide O2⁻· and hydroxyl (HO·) radicals, which are highly damaging to biomolecules. We investigated the role of TiO2 in promoting the peroxidation of linoleic acid (LA) alone and in the presence of hypochlorous acid (HOCl). TiO2 significantly enhanced peroxidation of LA, which was further enhanced in the presence of HOCl. This latter finding involved the formation of singlet molecular oxygen in a Russell-type mechanism appearing to involve preformed lipid hydroperoxides (LOOH). In addition to lipid peroxidation, HOCl also mediated formation of 18:1 monochlorohydrins, which in the presence of TiO2 appeared to decompose to kinetic products which supplemented peroxidation of linoleic acid. We present a theoretical mechanism which fits the available experimental data and may partially explain the dichotomy associated with HOCls role in lipid modification.

Pourbiax Diagrams as an Aid to Understanding the Impact of Acid/Base Disturbance on Blood Glucose Point-of-Care Testing.

OBJECTIVE: Assays based on redox reactions that involve proton transfer are vulnerable to artifactual findings in metabolic acidosis/alkalosis. We evaluated the impact of pH on the measurement of blood glucose by the glucose dehydrogenase/pyrroloquinoline quinone system used in point-of-care-testing. METHODS: We applied a series of thermodynamic equations to adjust the Gibbs energy for the pyrroloquinoline quinone couple. This adjusts values taken under standard conditions to those more closely resembling the physiological state. RESULTS: Under standard conditions, the pyrroloquinoline quinone couple has Eo = -0.125 V whereas adjustment to the physiological state (pH 7.40, ionic strength 0.15 mol/L, and temperature 310.15°K) yields Eo' = -0.166 V. This corresponds to an uncertainty in blood glucose determination of approximately 0.13 mmol/L. CONCLUSION: We have demonstrated that the impact of pH on blood glucose determination by the glucose dehydrogenase/pyrroloquinoline quinone system (under physiologically relevant conditions of ionic strength and temperature) is not clinically significant.

Biphasic Effect of Curcuminoids on Oxidation of Postprandial Chylomicrons.

Chylomicrons are large, triglyceride-rich lipoproteins that transport dietary lipids and lipophilic micronutrients through the lymphatic system and into the venous circulation. They therefore represent a valuable means of investigating the bioavailability of a range of bioactive molecules. Curcuminoids have a complex reputation, with most in vitro studies suggesting a beneficial effect (e.g., as an antioxidant), while in vivo work is often, at best, equivocal. We have prepared a curcuminoid extract from fresh turmeric and investigated the transfer of curcuminoids to chylomicrons by rapid ultracentrifugation. We subsequently characterized the chylomicrons in terms of their susceptibility to oxidation. There was a dose-dependent relationship between the curcuminoid level in chylomicrons and decreased levels of preformed lipid hydroperoxides (P < .05), which was broadly mirrored by paraoxonase arylesterase activity. At lower dosages (1000-3000 mg), curcuminoids protected chylomicrons against copper(II)-mediated oxidation, but at higher levels (4000-5000 mg), a pro-oxidant effect was observed. In conclusion, we report a clear biphasic (hormetic) effect of curcuminoids on lipid oxidation, which supports the theory that low dosages of bioactive compounds can have beneficial effects, while higher dosages may have an equivocal or negative impact on the disease.

Paradoxical Hypercholesterolemia in an Otherwise Healthy Adult Man.

Hypercholesterolemia is characterized by serum cholesterol levels greater than 5 mmol per L. However, the distribution of cholesterol among lipoprotein classes has a significant bearing on diagnosis: high-low-density lipoprotein (LDL) cholesterol suggests familial hypercholesterolemia, whereas high-high-density lipoprotein (HDL) cholesterol is associated with hyperalphalipoproteinemia. On routine screening, a 23-year-old man presented with a total cholesterol level of 7.6 mmol per L but was subsequently found to have an HDL cholesterol level of 5.6 mmol per L. The clinical picture was confounded by his use of red yeast rice extract, a popular health supplement with hypolipidemic effects. In this case individual, the use of red yeast rice extract caused a hyperlipidemic state, ostensibly through downregulation of cholesteryl ester transfer protein. This case emphasizes the extended role of laboratory medicine in complex cases of hyperlipidemia.

A dual role for lecithin:cholesterol acyltransferase (EC 2.3.1.43) in lipoprotein oxidation.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme involved in lipoprotein metabolism. It mediates the transesterification of free cholesterol to cholesteryl ester in an apoprotein A-I-dependent process. We have isolated purified LCAT from human plasma using anion-exchange chromatography and characterized the extracted LCAT in terms of its molecular weight, molar absorption coefficient, and enzymatic activity. The participation of LCAT in the oxidation of very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) was examined by supplementing lipoproteins with exogenous LCAT over a range of protein concentrations. LCAT-depleted lipoproteins were also prepared and their oxidation kinetics examined. Our results provide evidence for a dual role for LCAT in lipoprotein oxidation, whereby it acts in a dose-responsive manner as a potent pro-oxidant during VLDL oxidation, but as an antioxidant during LDL oxidation. We believe this novel pro-oxidant effect may be attributable to the LCAT-mediated formation of oxidized cholesteryl ester in VLDL, whereas the antioxidant effect is similar to that of chain-breaking antioxidants. Thus, we have demonstrated that the high-density lipoprotein-associated enzyme LCAT may have a significant role to play in lipoprotein modification and hence atherogenesis.

Pioglitazone protects HDL(2&3) against oxidation in overweight and obese men.

BACKGROUND: The worldwide epidemic of obesity is a major public health concern and is persuasively linked to the rising prevalence of diabetes and cardiovascular disease. Obesity is often associated with an abnormal lipoprotein profile, which may be partly negated by pioglitazone intervention, as this can influence the composition and oxidation characteristics of low-density lipoprotein (LDL). However, as pioglitazone's impact on these parameters within high-density lipoprotein (HDL), specifically HDL(2&3), is absent from the literature, this study was performed to address this shortcoming. METHODS: Twenty men were randomized to placebo or pioglitazone (30 mg/day) for 12 weeks. HDL(2&3) were isolated by rapid-ultracentrifugation. HDL(2&3)-cholesterol and phospholipid content were assessed by enzymatic assays and apolipoprotein AI (apoAI) content by single-radial immunodiffusion. HDL(2&3) oxidation characteristics were assessed by monitoring conjugated diene production and paraoxonase-1 activity by spectrophotometric assays. RESULTS: Compared with the placebo group, pioglitazone influenced the composition and oxidation potential of HDL(2&3). Specifically, total cholesterol (P < 0.05), phospholipid (P < 0.001) and apoAI (P < 0.001) were enriched within HDL(2). Furthermore, the resistance of HDL(2&3) to oxidation (P < 0.05) and the activity of paroxonase-1 were also increased (P < 0.001). CONCLUSIONS: Overall, these findings indicate that pioglitazone treatment induced antiatherogenic changes within HDL(2&3), which may help reduce the incidence of premature cardiovascular disease linked with obesity.

A curvilinear approach to the kinetic analysis of linoleate peroxidation in aqueous liposomes by 2,2'azobis(2-amidoinopropane) dihydrochloride.

Lipid peroxidation is a common feature of many chemical and biological processes, and is governed by a complex kinetic scheme. A fundamental stage in kinetic investigations of lipid peroxidation is the accurate determination of the rate of peroxidation, which in many instances is heavily reliant on the method of finite differences. Such numerical approximations of the first derivative are commonly employed in commercially available software, despite suffering from considerable inaccuracy due to rounding and truncation errors. As a simple solution to this, we applied three empirical sigmoid functions (viz. the Prout-Tompkins, Richards & Gompertz functions) to data obtained from the AAPH-mediated peroxidation of aqueous linoleate liposomes in the presence of increasing concentrations of Trolox, evaluating the curve fitting parameters using the widely available Microsoft Excel Solver add-in. We have demonstrated that the five-parameter Richards' function provides an excellent model for this peroxidation, and when applied to the determination of fundamental rate constants, produces results in keeping with those available in the literature. Overall, we present a series of equations, derived from the Richards' function, which enables direct evaluation of the kinetic measures of peroxidation. This procedure has applicability not only to investigations of lipid peroxidation, but to any system exhibiting sigmoid kinetics.

High-content analysis of cancer-cell-specific apoptosis and inhibition of in vivo angiogenesis by synthetic (-)-pironetin and analogs.

The natural product (-)-pironetin is a structurally simple small molecule microtubule-perturbing agent whose biological activities appear to be exquisitely dependent on defined stereochemistry and the presence of an eletrophilic alpha,beta-unsaturated lactone moiety. We used alkaloid-catalyzed acyl halide-aldehyde cyclocondensation reactions in asymmetric total syntheses of (-)-pironetin and three synthetic analogs, and evaluated their biological activities by high-content analysis in cell culture and in a zebrafish model. Synthetic (-)-pironetin and 2,3-dihydro-3-hydroxypironetin caused mitotic arrest and programmed cell death in human lung cancer cells but not in normal lung fibroblasts, had nanomolar growth inhibitory activity in multi-drug resistant cells, and inhibited neovascularization in zebrafish embryos. Synthetic (-)-pironetin delayed the onset but increased the extent of tubulin assembly in vitro. The data illustrate the power of acyl halide-aldehyde cyclocondensation to generate biologically active synthetic analogs of stereochemically complex targets and suggest that (-)-pironetin and 2,3-dihydro-3-hydroxypironetin possess unique properties that may bestow them with advantages over existing microtubule-perturbing agents in the context of a whole organism or under conditions of multi-drug resistance.

Meayamycin inhibits pre-messenger RNA splicing and exhibits picomolar activity against multidrug-resistant cells.

FR901464 is a potent antitumor natural product that binds to the splicing factor 3b complex and inhibits pre-mRNA splicing. Its analogue, meayamycin, is two orders of magnitude more potent as an antiproliferative agent against human breast cancer MCF-7 cells. Here, we report the picomolar antiproliferative activity of meayamycin against various cancer cell lines and multidrug-resistant cells. Time-dependence studies implied that meayamycin may form a covalent bond with its target protein(s). Meayamycin inhibited pre-mRNA splicing in HEK-293 cells but not alternative splicing in a neuronal system. Meayamycin exhibited specificity toward human lung cancer cells compared with nontumorigenic human lung fibroblasts and retained picomolar growth-inhibitory activity against multidrug-resistant cells. These data suggest that meayamycin is a useful chemical probe to study pre-mRNA splicing in live cells and is a promising lead as an anticancer agent.

High density lipoprotein subfractions: isolation, composition, and their duplicitous role in oxidation.

The plasma HDLs represent a major class of cholesterol-transporting lipoprotein that can be divided into two distinct subfractions, HDL(2) and HDL(3), by ultracentrifugation. Existing methods for the subfractionation of HDL requires lengthy ultracentrifugations, making them unappealing for large-scale studies. We describe a method that subfractionates HDL from plasma in only 6 h, representing a substantial decrease in total isolation time. The subfractions so isolated were assessed for a variety of lipid and protein components, in addition to their susceptibility to oxidation, both alone and in combination with VLDL and LDL. We report for the first time a prooxidant role for HDL during VLDL oxidation, in which HDL donates preformed hydroperoxides to VLDL in a cholesteryl ester transfer protein (CETP)-dependent process. Examination of the participation of HDL in LDL oxidation has reinforced its classic role as a potent antioxidant. Furthermore, we have also implicated the second major HDL-associated enzyme, LCAT, in these processes, whereby it acts as a potent prooxidant during VLDL oxidation but as an antioxidant during LDL oxidation. Thus, we have identified a potentially duplicitous role for HDL in the pathogenesis of atherosclerosis, attributable to both CETP and LCAT.

Structure-activity and high-content imaging analyses of novel tubulysins.

The synthesis and biological evaluation of three tubulysin analogs provides the first structure-activity relationship in this family of potent cytotoxic myxobacteria metabolites. Most importantly, the labile N,O-acetal at N(14) is not essential for biological activity. Further, structural simplifications are possible without abolishing biological activities. The N-terminal amino acid can be replaced with N-methylsarcosine, and the configuration at the acetoxy-bearing stereocenter at C(11) is important but not critical for almost all aspects of the biological profile. Our data encourage further development of these compounds as potential therapeutic agents in cancer treatment.