Quantification Of Cholesterol Research Articles

Soraphen A enhances macrophage cholesterol efflux via indirect LXR activation and ABCA1 upregulation

Increased cholesterol efflux from macrophage foam cells in the subendothelial space confers protection against atherosclerosis. Soraphen A, a myxobacterial macrolactone, is an inhibitor of acetyl coenzyme A carboxylases (ACC), which control fatty acid synthesis and oxidation. To assess a potential direct link between macrophage cholesterol efflux and ACC inhibition, we examined [3H]-cholesterol efflux from human THP-1-derived foam cells in the presence of soraphen A. We dissected underlying molecular events by western blot analyses, RT-qPCR, reporter gene and coactivator recruitment assays as well as relative quantification of free and total cholesterol. Soraphen A increased cholesterol efflux from macrophage foam cells via upregulation of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1). Soraphen A enhanced transcription of ABCA1 in an LXR-dependent manner, however, without direct binding to the ligand-binding domain of this nuclear receptor. Soraphen A elevated the cellular level of free cholesterol, and failed to activate LXR upon exogenous supplementation with fatty acids or inhibition of cholesterol synthesis. Thus, impeded conversion from acetyl- to malonyl-CoA by soraphen A may lead to more unesterified cholesterol and thus potential LXR agonists. The present study reveals ACC inhibition as a previously unrecognized mechanism to regulate macrophage cholesterol efflux via indirect LXR activation and ABCA1 upregulation.

Stevia: limiting cholesterol synthesis in Hep-G2 cells

As of today, no literature has been reported on the efficacy of stevia on lipid regulations conducted in vitro. Thus, the current study was focusing on the potential of Stevia rebaudiana bertoni as an anti-hypercholesterolemia substitute in limiting the de novo cholesterol synthesis in Hep-G2 cell line. The cytotoxicity and lipid internalization effects of stevia on Hep-G2 cells were assessed quantitatively and qualitatively in this study. As evaluated by MTT assay, commercialized stevia (0.5-20.0 mg/ml) and stevioside (1.0-10 µM) inhibited Hep-G2 cells viability in a dose-dependent manner for 24 hours. IC50 was detected at 8.68 mg/ml (commercialized stevia) and 10.91 µM (stevioside). From the assay, suitable concentrations were chosen to study the effect of stevia on cholesterol internalization in Hep-G2 cells supplemented with exogenous lipids. Cholesterol quantification assay revealed that high concentration commercialized stevia and stevioside promoted significant cholesterol internalized in Hep-G2 cells as compared to simvastatin. Finally, immunofluorescent microscopy assessment was done to qualitatively observe the formation of lipid droplets and low-density lipoprotein receptor in relation to total cholesterol extracted. The microphotographs of immunofluorescent microscopy were in parallel to results obtained from the cholesterol quantification assay which further revealed the effect of stevia as a potential anti-hypercholesterolemia agent.

Loss of HNF1α Function Contributes to Hepatocyte Proliferation and Abnormal Cholesterol Metabolism via Downregulating miR-122: A Novel Mechanism of MODY3.

PurposeMutations in hepatocyte nuclear factor 1α (HNF1α) are the cause of maturity-onset diabetes of the young type 3 (MODY3) and involved in the development of hepatocellular adenoma and abnormal lipid metabolism. Previously, we have found that the serum microRNA (miR)-122 levels in MODY3 patients were lower than those in type 2 diabetes mellitus and healthy controls. This study aimed to investigate the mechanism of decreased miR-122 levels in patients with MODY3 and whether low levels of miR-122 mediate tumorigenesis and abnormal lipid metabolism associated with HNF1α deficiency in human hepatocytes.MethodsThe expression of miR-122 was examined by real-time PCR. Dual-luciferase reporter assay was performed to confirm the transcriptional regulation of miR-122 by HNF1α. HepG2 cells were transfected with siRNA or miRNA mimic to downregulate or upregulate the expression of HNF1α or miR-122, respectively. CCK-8 and colony formation assay were used to determine cell proliferation. Lipid accumulation was examined by Oil Red O staining and intracellular triglyceride and cholesterol quantification assays.ResultsHNF1α regulated the expression of miR-122 by directly binding to its promoter. Knockdown of HNF1α in HepG2 cells reduced the expression of miR-122, increased proliferation and promoted intracellular cholesterol accumulation. Overexpression of miR-122 partially rescued the phenotypes associated with HNF1α deficiency in human hepatocytes. Mechanistically, HNF1α modulated cholesterol homeostasis via miR-122-dependent activation of sterol regulatory element-binding protein-2 (SREBP-2) and regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). Moreover, circulating miR-122 levels were associated with serum cholesterol levels.ConclusionLoss of HNF1α function led to hepatocyte proliferation and abnormal cholesterol metabolism by downregulating miR-122. Our findings revealed a novel mechanism that low levels of miR-122 mediate tumorigenesis and abnormal lipid metabolism associated with MODY3. MiR-122 may be a potential therapeutic target for the treatment of MODY3.

Development of Non-enzymatic Cholesterol Electrochemical Sensor Based on Polyindole/Tungsten Carbide Nanocomposite

Non-enzymatic electrochemical sensor was developed for estimation of low-level cholesterol. Polyindole/tungsten carbide (PIN/WC) nanocomposite was synthesized and used as an electroactive material to develop low-cost modified stainless steel plate electrode (SSPE). Surface morphology of developed electrode was characterized by scanning electron microscopy. Electrochemical behavior of cholesterol was investigated through electron impedance spectroscopy, potentiodynamic polarization and cyclic voltammetry in 1-M KOH electrolytic solution. The quantification of cholesterol was studied by square wave voltammetry and differential pulse voltammetry. The calibration plots between the cholesterol concentration and peak current were in linear relation with limit of detection of 1.23 × 10−6 mol L−1. The overall result reveals that developed PIN/WC/SSPE electrode has excellent performance for trace-level cholesterol estimation and can be further employed for cholesterol monitoring in blood serum samples. • Synthesis of polyindole/tungsten carbide nanocomposite as electroactive sensing material. • Development and optimization of polyindole/tungsten carbide modified working electrode. • Non enzymatic cholesterol sensing at developed electrode by different electrochemical methods. • Monitoring of cholesterol in blood serum samples with developed electrode.

Simultaneous Quantification of DPPG, DEPC and Cholesterol in Propofol Liposome by HPLC-ELSD Using Alkaline Hydrolysis.

A high-performance liquid chromatography method with evaporative light-scattering detection (ELSD) was performed for simultaneous determination of dipalmitoyl phosphatidylglycerol (DPPG), dierucoyl phosphatidylcholine (DEPC) and cholesterol in propofol liposome by the pretreatment of alkaline hydrolysis (temperature, concentration of KOH anhydrous ethanol solution and reaction time were 90°C, 1mol · L-1 and 10min, respectively). The analysis was carried out on an Agilent TC-C18 column (4.6mm×250mm, 5μm) with isocratic elution of methanol and 0.1% acetic acid aqueous solution (95:5, v/v) at a flow rate of 1.0mL· min-1. The column temperature was 30°C. The drift tube temperature of the ELSD system was set at 30°C, and the pressure of carrier gas was 350KPa. The regression equation revealed a good linear relationship (r=0.9990-0.9993) during the test ranges. The RSD of stability and repeatability (n=6) was found to be less than 1.96 and 1.46%, respectively. The average recoveries ranged from 97.90 to 101.00%. The proposed method was validated and showed good precision, stability, repeatability and recovery, which indicated that the method could be readily utilized as a quality evaluation method for the determination of DPPG, DEPC and cholesterol in propofol liposome.

A Validated Method for Cholesterol Determination in Turkey Meat Products Using Relative Response Factors.

The objective of this study was to develop a precise and accurate method to quantify cholesterol in turkey meat products using relative response factors, based on a modification of a previously published method for plant sterols determination. Validation was performed using neat solutions to determine linearity, precision, and accuracy. The method was linear in the concentration range considered (1–20 µg/mL, r2 ≥ 0.991). Precision and accuracy were within the acceptability guidelines of the U.S. Food and Drug Administration (FDA) for method validation (<20% relative standard deviation (RSD) at the lower limit of quantification (LLOQ) and <15% RSD for other standards). Turkey meat was spiked with cholesterol at two levels (low = 3 µg/mL and high = 18 µg/mL), either before or after saponification, to establish the recovery and matrix effects. Recovery ranged from 94% to 105%, with a mean value of 105% at the low spike level and 95% at the high spike level. No significant matrix effects were found (90% to 112% recovery). This method is reliable for the quantification of cholesterol in turkey meat products in the range 0.4–8 mg/g.

Validation of Analytical Method for Quantification of Egg Cholesterol Using Reversed Phase-High Performance Liquid Chromatography-Multiwavelength Detector

In this research, analytical method of cholesterol content in eggs by Reversed Phase-High Performance Liquid Chromatography-Multiwavelength Detector (RP-HPLC-MWD) was validated. Our experiment validated the modified method of AOAC 994.10:2012 to get a more simple and efficient analytical method of cholesterol content. The sample was saponified using 10% KOH concentration for 15 min at 80 °C, then this analytical method was validated. RP-HPLC-MWD condition was at 100% MeOH as a mobile phase, flow rate of 1.0 mL/min, detection UV at 205 nm, cholesterol was detected at 10.38±0.13 min. As a result, the coefficients of determination for instrument and method linearities reached 0.9991 and 0.9912, respectively. The limits of detection and quantification of RP-HPLC-MWD instrument were found at 5 and 10 μg/mL, respectively, while the method-detection limit and quantification limit were 250 and 500 μg/g sample, respectively. Recovery values for the cholesterol analysis ranged from 98.62% to 112.26%, with a precision of 1.05%‒3.90%. Additionally, intralab reproducibility was known to reach 3.27%. This validated method can be applied for the analysis of cholesterol in various eggs available in the market.

UV-VIS-NIR spectroscopy and artificial neural networks for the cholesterol quantification in egg yolk

Artificial neural networks (ANN) are non-linear calibration methods that try to simulate the human nervous system with neurons, layers and transfer functions. In this study the input neurons were the UV-VIS-NIR spectra of egg yolks and the outputs were cholesterol content. Therefore, the aim of this work was to develop and assess a UV-VIS-NIR method coupled to chemometrics and ANN for cholesterol determination in egg yolks. A total of 57 samples of egg yolks were obtained from fresh shell eggs and pasteurised egg yolks. A total of 2311 variables (wavelengths) with four variable selection methods were analysed. An enzymatic method was used as reference method for cholesterol content. The effect of the solvent was also evaluated. The best results were obtained based on selected absorbance peaks. Calibration results showed r2cal over 0.90 and RMSEC below 0.65. Validation results showed r2val close to 0.7. The ANN model developed could be useful to determine cholesterol in egg yolk for routine quality control.

Doxycycline reduces osteopenia in female rats

Doxycycline, a member of the tetracycline family, is a drug used as an antibiotic (dosage of 100 mg/day) and as an anti-inflammatory drug on the dosage of 20 mg twice a day, this use has Matrix Metalloproteinases (MMP) inhibitor action. Doxycycline is a calcium chelator and therefore interferes in bone remodeling. The main objective of this study was to evaluate the action of the drug doxycycline in the control of osteopenia. Sixty three Wistars rats were divided into 9 groups with n = 7 each, as follow: the control group with doxycycline 10 mg/kg/day (C10), control with doxycycline 30 mg/kg/day (C30) and control (C), ovariectomized group with doxycycline 10 mg/kg/day (OVX10), ovariectomized with doxycycline 30 mg/kg/day (OVX30), and ovariectomized with water (OVX), sedentary group with 10 mg/kg/day (Se10), sedentary with doxycycline 30 mg/kg/day (Se30), and sedentary group with water (Se). Left femoral bone was used for bone densitometry, right femoral bone for histological analysis. The right tibia was intended for chemical quantifications, the total serum was used for cholesterol and calcium quantification. The length of the left femoral bone was measured after the densitometry analysis. Statistical analysis was performed using multivariate general linear model (ANOVA two factors with Bonferroni adjustment) and the TRAP analysis was subjected to normality test and then were subjected to nonparametric test, both with p < 0.05 significance. Statistically significant differences were found, with better results for the groups exposed to the medication (10 and 30 mg/kg/day): Se vs. Se10 and Se vs. Se30 for BMC, quantification of magnesium, amount of cancellous bone in the distal portion; OVX vs. OVX10 for BMC, BMD and calcium in serum; OVX vs. OVX10 and OVX30 for quantification in proximal and distal portion of cancellous bone; Se vs. Se30 and OVX vs. OVX30 for immunostaining for TRAP, all results with minimum of p ≤ 0.05. Doxycycline had a deleterious effect on control groups and positive action for bone organization on female rats affected by bilateral ovariectomy-induced osteopenia and sedentary lifestyle.

Biallelic pathogenic variants in the lanosterol synthase gene LSS involved in the cholesterol biosynthesis cause alopecia with intellectual disability, a rare recessive neuroectodermal syndrome

PurposeLanosterol synthase (LSS) gene was initially described in families with extensive congenital cataracts. Recently, a study has highlighted LSS associated with hypotrichosis simplex. We expanded the phenotypic spectrum of LSS to a recessive neuroectodermal syndrome formerly named alopecia with mental retardation (APMR) syndrome. It is a rare autosomal recessive condition characterized by hypotrichosis and intellectual disability (ID) or developmental delay (DD), frequently associated with early-onset epilepsy and other dermatological features. MethodsThrough a multicenter international collaborative study, we identified LSS pathogenic variants in APMR individuals either by exome sequencing or LSS Sanger sequencing. Splicing defects were assessed by transcript analysis and minigene assay. ResultsWe reported ten APMR individuals from six unrelated families with biallelic variants in LSS. We additionally identified one affected individual with a single rare variant in LSS and an allelic imbalance suggesting a second event. Among the identified variants, two were truncating, seven were missense, and two were splicing variants. Quantification of cholesterol and its precursors did not reveal noticeable imbalance. ConclusionIn the cholesterol biosynthesis pathway, lanosterol synthase leads to the cyclization of (S)-2,3-oxidosqualene into lanosterol. Our data suggest LSS as a major gene causing a rare recessive neuroectodermal syndrome.

Update on low-density lipoprotein cholesterol quantification.

β-Quantification is considered the reference measurement procedure for low-density lipoprotein cholesterol (LDL-C). However, this technique is time-consuming and thus is inappropriate for routine clinical practice. Therefore, the Friedewald equation or homogeneous assays have been widely utilized. As several pitfalls exist with these two methods, a novel method for estimating LDL-C was developed by Martin et al. RECENT FINDINGS: Martin's method uses a strata-specific median for the triglycerides/very low-density lipoprotein cholesterol (VLDL-C) ratio on the basis of triglycerides and non-HDL-C concentrations. Recent studies show that Martin's method better correlates with β-quantification or homogeneous assays compared with the Friedewald equation, especially with values of triglycerides at least 150 mg/dl and/or LDL-CD less than 70 mg/dl. Such findings have also been demonstrated in other ethnic groups (Japanese and Korean) and disease populations, including diabetes and cardiovascular disease, in which the triglycerides/VLDL-C ratio can be affected. For the current therapeutic goal of LDL-C values below 70 mg/dl in high-risk patients, accurate assessment of LDL-C levels at very low levels is required. Martin's method could overcome pitfalls such as underestimation of the Friedewald equation at this level. Further evaluation of the triglycerides/VLDL-C ratio in participants with diverse ethnic backgrounds or clinical conditions would expand the implementation of this novel method.

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography–Urgent need for harmonisation of analytical methods

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols).Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Quantification of Cholesterol and Cholesteryl Ester by Direct Flow Injection High-Resolution Fourier Transform Mass Spectrometry Utilizing Species-Specific Response Factors.

The quantification of free cholesterol (FC) and cholesteryl ester (CE) in mammalian samples is of great interest for basic science and clinical lipidomics. Here, we evaluated the feasibility of direct flow injection analysis (FIA) coupled to electrospray ionization high-resolution mass spectrometry (ESI-HRMS) to quantify FC and CE in lipid extracts from human serum, cultured cells, and mouse liver. Despite poor ionization efficiency of FC, the limit of quantitation was sufficient for precise and accurate quantification of FC by multiplexed HRMS (MSX) analysis without using a derivatization step. However, it was demonstrated that, upon full scan Fourier transform MS (FTMS) quantification, CE species show substantial differences in their analytical responses depending on number of double bonds, length of the acyl chain, infused lipid concentration, and other lipid components. A major determinant for these response differences is their susceptibility to in-source fragmentation. In particular, introduction of double bonds lowers the degree of in-source fragmentation. Therefore, CE species-specific response factors need to be applied for CE quantification by FTMS to achieve accurate concentrations. Method validation demonstrated that FIA-ESI-HRMS (MSX and FTMS) is applicable for quantification of FC and CE in samples used in basic science as well as clinical studies such as cultured cells, tissue homogenates, and serum.

Cholesterol determination in egg yolk by UV-VIS-NIR spectroscopy

A quick and cheap method for cholesterol quantification in egg yolk by UV-VIS-NIR spectroscopy was developed. Cholesterol was saponified and extracted from shell egg yolks or pasteurised egg yolks. Spectral data (144 spectra) were obtained from 38 samples. Different transformations of the spectra (smoothing Savitzky-Golay, standard normal variate, multiplicative scatter correction, detrending, baseline correction and first and second derivative) and modelling strategies such as Partial Least Squares (PLS) regression or Principal Component Regression (PCR) were evaluated. An enzymatic method was selected as reference procedure. Cholesterol content quantified was significantly lower in pasteurised egg yolks than in shell egg yolks. Best results were obtained using PLS and first derivative Savitzky-Golay combined with baseline correction, achieving a model with a determination coefficient of 0.93. Total cholesterol quantification was increased with saponification conditions improvements. Results obtained demonstrate that UV-VIS-NIR spectroscopy can be used to determine cholesterol from shell egg yolks or pasteurised egg yolks.

Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4.

Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at the plasma membrane and inside cells. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to GFP (GFP-D4) and the polyene macrolide filipin. First, we report a robust method for quantifying plasma membrane cholesterol by flow cytometry using the GFP-D4 probe. Second, to optically distinguish and quantify intracellular cholesterol accumulation, we have adapted the classical filipin cholesterol staining protocol. Indeed, we observed that treatment of living cells with methyl-β-cyclodextrin, a chemical known to extract cholesterol from the plasma membrane, improves the visualization of the intracellular cholesterol pool with filipin. To complement these staining procedures, we developed an image analysis protocol based on image segmentation to quantify, in a robust manner, intracellular cholesterol stained with filipin. Thus, this chapter is a guideline for cellular cholesterol staining and signal quantification.

Identification of lipids of the stratum corneum by high performance thin layer chromatography and mass spectrometry

The stratum corneum, the outermost layer of the epidermis, is the most important skin barrier against exogenous physical and chemical effects, in addition to protecting against dehydration. Ceramides are integral parts of the intercellular lipid lamellae of the stratum corneum and play an important role in the barrier function of mammalian skin. Ceramides are sphingolipids consisting of sphingoid bases linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine, sphingosine, phytosphingosine, and 6-hydroxysphingosine, and the fatty acid acyl chains are composed of non-hydroxy fatty acid, α-hydroxy fatty acid, ω-hydroxy fatty acid, and esterified ω-hydroxy fatty acid. Analytical methods, such as gas chromatography/mass spectrometry, high performance thin layer chromatography with UV detection, and liquid chromatography/mass spectrometry, have been developed for the identification and quantification of ceramides in the stratum corneum. However, only a few publications relate to the mass fragmentation patterns specific to ceramide types to determine the structure of skin ceramides. Moreover, these studies provide very limited structural information and only for some ceramides. Therefore, the aim of our study was to develop a quick and easy method of quantification of ceramides, cholesterol, and free fatty acids by high performance thin layer chromatography with ultraviolet detection. High performance thin layer chromatography with ultraviolet detection was also coupled with mass spectrometry using negative ionization by electrospray and tandem mass spectrometry (MS/MS) for identification of ceramides' structure.

Convenient Colorimetric Detection of Cholesterol Using Multi-Enzyme Co-Incorporated Organic-Inorganic Hybrid Nanoflowers.

We have developed a microscale well-plate colorimetric assay for the multiplexed detection of cholesterol in clinical human blood samples. This system utilizes a novel multi-enzyme incorporated organic-inorganic hybrid nanoflower which entrap both cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) as the organic components with copper phosphate as the inorganic component to detect cholesterol levels in blood samples. The hybrid nanoflowers, synthesized via an extremely simple but rapid sonication-mediated method within 5 min at room temperature, enable an efficient one-pot two-enzyme cascade reaction. The ChOx in the nanoflowers catalyze the generation of H2O2 only in the presence of cholesterol in the sample. This subsequently activates the HRP co-entrapped in the nanoflowers, thereby leading to the conversion of the employed chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB), into a blue-colored product. This strategy can be used to detect target cholesterol concentrations as low as 8 μM, with a linear range from 10 to 70 μM, which is suitable to diagnose high levels of cholesterol (hypercholesterolemia) with excellent stability over three weeks at room temperature. The biosensor also exhibited an excellent selectivity to detect target cholesterol even in the presence of common interfering biomolecules in human blood and showed a high degree of precision when employing human blood serum samples. Therefore, this hybrid nanoflower-based assay can be used in clinical practice for the multiplexed and reliable quantification of cholesterol, and readily extended to other enzymes to prepare multi-step cascade enzymatic reactions for various biotechnological applications.

Determination of Egg Number Added to Special Pasta by Means of Cholesterol Contained in Extracted Fat Using GC-FID.

Pasta with eggs added (generally termed “special pasta” for Italian legislation) is made by adding no less than 4 eggs without shells (or no less than 200 g of liquid or lyophilized egg product) per kilogram of semolina, as provided by law. In this work, to determine the final content of eggs added to dough, an analytical procedure was developed for the rapid analysis of the cholesterol content in the finished pastas. The proposed procedure was simpler, faster, and more accurate than that of official methods of analysis based on the gravimetric determination of sterols. Moreover, the determination of the quality of fat content in the special pasta (egg pasta in this case) allowed the evaluation of its origin, avoiding possible fraud resulting from the addition of foreign fat as an alternative to fat derived from eggs. In this new gas chromatographic procedure, the internal standard squalene for the quantification of cholesterol was used because a more polar GC capillary column was used (RTX 65 TG-HT) for the separation of sterols, rather than 5% phenyl methylsilicone. The ratio between cholesterol and squalene allowed for the determination of the number of eggs added, while from analysis of the same gas chromatogram, it was also possible to evaluate the composition of triglycerides in the fat contained in the pasta, allowing discrimination of foreign fats with respect to fats contained in eggs and therefore avoiding adulteration of pasta. The same analytical procedure was applied to the determination of cholesterol content in lyophilized yolk.

Quantifying Atherogenic Lipoproteins: Current and Future Challenges in the Era of Personalized Medicine and Very Low Concentrations of LDL Cholesterol. A Consensus Statement from EAS and EFLM.

The European Atherosclerosis Society-European Federation of Clinical Chemistry and Laboratory Medicine Consensus Panel aims to provide recommendations to optimize atherogenic lipoprotein quantification for cardiovascular risk management. We critically examined LDL cholesterol, non-HDL cholesterol, apolipoprotein B (apoB), and LDL particle number assays based on key criteria for medical application of biomarkers. (a) Analytical performance: Discordant LDL cholesterol quantification occurs when LDL cholesterol is measured or calculated with different assays, especially in patients with hypertriglyceridemia >175 mg/dL (2 mmol/L) and low LDL cholesterol concentrations <70 mg/dL (1.8 mmol/L). Increased lipoprotein(a) should be excluded in patients not achieving LDL cholesterol goals with treatment. Non-HDL cholesterol includes the atherogenic risk component of remnant cholesterol and can be calculated in a standard nonfasting lipid panel without additional expense. ApoB more accurately reflects LDL particle number. (b) Clinical performance: LDL cholesterol, non-HDL cholesterol, and apoB are comparable predictors of cardiovascular events in prospective population studies and clinical trials; however, discordance analysis of the markers improves risk prediction by adding remnant cholesterol (included in non-HDL cholesterol) and LDL particle number (with apoB) risk components to LDL cholesterol testing. (c) Clinical and cost-effectiveness: There is no consistent evidence yet that non-HDL cholesterol-, apoB-, or LDL particle-targeted treatment reduces the number of cardiovascular events and healthcare-related costs than treatment targeted to LDL cholesterol. Follow-up of pre- and on-treatment (measured or calculated) LDL cholesterol concentration in a patient should ideally be performed with the same documented test method. Non-HDL cholesterol (or apoB) should be the secondary treatment target in patients with mild to moderate hypertriglyceridemia, in whom LDL cholesterol measurement or calculation is less accurate and often less predictive of cardiovascular risk. Laboratories should report non-HDL cholesterol in all standard lipid panels.

Colourimetric Determination of High-Density Lipoprotein (HDL) Cholesterol Using Red–Green–Blue Digital Colour Imaging

ABSTRACTA rapid, low-cost, and sensitive method was developed for the quantification of high-density lipoprotein cholesterol based on enzymatic colorimetric reactions and digital image analysis. The proposed method was adapted to a 96-microwell enzyme-linked immunosorbent assay plate and imaging acquisition was performed using a conventional desktop scanner. The images were recorded using the red–green–blue color system in which the resolved absorbance for each color channel was used for multiple linear regression. The regression model presented a root mean squared error of calibration and a correlation coefficient (R2) value of 1.53 mg dL−1 and 0.995, respectively. Prediction was obtained with a root mean square error of prediction of 2.42 mg dL−1 and R2 of 0.993, thereby showing accurate prediction response. A limit of detection of 0.43 mg dL−1 and precision better than 1.72% reinforced these results. This method was compared with a reference methodology using ultraviolet–visible spectroscopic measurements at 500 nm and no statistical differences were observed at the 95% confidence level, showing the potential for future clinical applications.