A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels.

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden.

Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 µM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste.

Automatic flow injection analysis (FIA) determination of total reducing capacity in serum and urine samples.

Automation of total antioxidant capacity assessment can substantially increase the determination throughput, allowing large scale studies and screening experiments. Total reducing capacity evaluation can be implemented under different chemistries, including the CUPRAC-Cupric Ion Reducing Antioxidant Capacity -assay. This assay is based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex by reducing (antioxidant) components of biological samples. In this chapter, we propose an automatic flow injection method for evaluation of total reducing capacity in serum and urine samples, attaining end-point data within 4 min using a kinetic matching strategy.

Insights on antioxidant assays for biological samples based on the reduction of copper complexes-the importance of analytical conditions.

Total antioxidant capacity assays are recognized as instrumental to establish antioxidant status of biological samples, however the varying experimental conditions result in conclusions that may not be transposable to other settings. After selection of the complexing agent, reagent addition order, buffer type and concentration, copper reducing assays were adapted to a high-throughput scheme and validated using model biological antioxidant compounds of ascorbic acid, Trolox (a soluble analogue of vitamin E), uric acid and glutathione. A critical comparison was made based on real samples including NIST-909c human serum certified sample, and five study samples. The validated method provided linear range up to 100 µM Trolox, (limit of detection 2.3 µM; limit of quantification 7.7 µM) with recovery results above 85% and precision <5%. The validated developed method with an increased sensitivity is a sound choice for assessment of TAC in serum samples.

Flow-injection spectrophotometric determination of bromate in bottled drinking water samples using chlorpromazine reagent and a liquid waveguide capillary cell.

In this work, aiming to develop a simple, inexpensive method for the determination of low bromate levels in water samples, a liquid waveguide capillary cell (LWCC) was coupled to a FIA system. The long optical path (100 cm) of the LWCC was used to improve the sensitivity and the limit of detection without resorting to any off-line or in-line preconcentration processes. The spectrophotometric determination was based on the oxidation of chlorpromazine by bromate in an acidic medium, resulting in the formation of a colored radical product. Sulfamic acid was added to the reagent for minimizing the interference of nitrite, and a chelating ion exchange resin was used to remove major cationic interferences. The developed system allowed the determination of bromate within the range between 1 - 20 µg L(-1) with a detection limit of 0.2 µg L(-1).

Review on recent applications of the liquid waveguide capillary cell in flow based analysis techniques to enhance the sensitivity of spectroscopic detection methods.

Incorporation of long path length liquid waveguide capillary cell (LWCC or LCW) into spectrometric detection systems can increase the sensitivity of these by orders of magnitude (up to 500 times), and consequently can reduce the detection limits. The combination of the long path length spectrophotometry with flow methodologies can provide analytical solutions for various challenges in the field of environmental, biochemical and food chemistry. In this present work, the analytical applications of the long capillary cells are summarised and critically discussed. A historical overview of the cell development is given; applications in different areas are presented and grouped by analyte type. Major improvements achieved based on the use of the LWCC in the analytical characteristics (like sensitivity and detection limit) are emphasised while some of the limitations are also discussed.

Determination of total protein content in white wines by solid phase spectrometry in a SI-LOV system.

Although present at low concentration in wine samples, proteins, have considerable technological importance, due to their capability of haze formation. The present work presents a methodology for the quantification of total protein in white wine in a sequential injection lab-on-valve system, exploiting the bead injection concept for solid phase extraction with spectrophotometric detection. The method is based on the retention of the proteins in the solid support, NTA (nitrilotriacetic acid) superflow beads, charged by Cu(2+). The change in the absorbance is monitored at 500nm at the surface of the beads after addition of the Folin-Ciocalteu's reagent (FCr). The developed method presented a sample consumption of 400µL per assay and a consumption of FCr and Cu(2+) solution of 25µL and 100µL per assay, respectively. It was possible to achieve a linear range up to 0.30g/L with a limit of detection and quantification of 0.03 and 0.10g/L, respectively. The proposed method was successfully applied to white wine samples.

Determination of glyphosate in water samples by multi-pumping flow system coupled to a liquid waveguide capillary cell.

A simple screening method was developed for the determination of glyphosate in water samples using a multi-pumping flow system. The proposed method is based on the reaction between glyphosate and p-dimethylaminocinnamaldehyde (p-DAC), in an acid medium where the reaction product can be measured spectrophotometrically at λ(max) = 495 nm. An experimental design methodology was used to optimize the measurement conditions. The proposed method was applied to the determination of glyphosate in water samples in a concentration range from 0.5 to 10 µg mL(-1). The limit of detection and quantification were 0.17 and 0.53 µg mL(-1), respectively. The results obtained (88.5 to 104.5%) in recovery studies for the determination of glyphosate in different water samples indicated good accuracy and no matrix effect for the developed method. Samples were also analyzed by a confirmatory HPLC method, and agreement within the two set of results was found.

Development of a flow method for the determination of phosphate in estuarine and freshwaters--comparison of flow cells in spectrophotometric sequential injection analysis.

A sequential injection system with dual analytical line was developed and applied in the comparison of two different detection systems viz; a conventional spectrophotometer with a commercial flow cell, and a multi-reflective flow cell coupled with a photometric detector under the same experimental conditions. The study was based on the spectrophotometric determination of phosphate using the molybdenum-blue chemistry. The two alternative flow cells were compared in terms of their response to variation of sample salinity, susceptibility to interferences and to refractive index changes. The developed method was applied to the determination of phosphate in natural waters (estuarine, river, well and ground waters). The achieved detection limit (0.007 µM PO(4)(3-)) is consistent with the requirement of the target water samples, and a wide quantification range (0.024-9.5 µM) was achieved using both detection systems.

Spectrophotometric determination of zinc and copper in a multi-syringe flow injection analysis system using a liquid waveguide capillary cell: application to natural waters.

This work exploits a multi-syringe injection analysis (MSFIA) system coupled with a long liquid waveguide capillary cell for the spectrophotometric determination of zinc and copper in waters. A liquid waveguide capillary cell (1.0m pathlength, 550 µm i.d. and 250 µL internal volume) was used to enhance the sensitivity of the detection. The determination for both ions is based on a colorimetric reaction with zincon at different pH values. The developed methodology compares favourably with other previously described procedures, as it allows to reach low detection limits for both cations (LODs of 0.1 and 2 µg L(-1), for copper and zinc, respectively), without the need for any pre-concentration step. The system also provided a linear response up to 100 µg L(-1) with a high throughput (43 h(-1)) and low reagent consumption and effluent production. The developed work was applied to natural waters and three certified reference water samples.