Accuracy of low-density lipoprotein cholesterol estimation at very low levels.

BACKGROUND: As the approach to low-density lipoprotein cholesterol (LDL-C) lowering becomes increasingly intensive, accurate assessment of LDL-C at very low levels warrants closer attention in individualized clinical efficacy and safety evaluation. We aimed to assess the accuracy of LDL-C estimation at very low levels by the Friedewald equation, the de facto clinical standard, and compare its accuracy with a novel, big data-derived LDL-C estimate. METHODS: In 191,333 individuals with Friedewald LDL-C < 70 mg/dL, we compared the accuracy of Friedewald and novel LDL-C values in relation to direct measurements by Vertical Auto Profile ultracentrifugation. We examined differences (estimate minus ultracentrifugation) and classification according to levels initiating additional safety precautions per clinical practice guidelines. RESULTS: Friedewald values were less than ultracentrifugation measurement, with a median difference (25th to 75th percentile) of -2.4 (-7.4 to 0.6) at 50-69 mg/dL, -7.0 (-16.2 to -1.2) at 25-39 mg/dL, and -29.0 (-37.4 to -19.6) at < 15 mg/dL. The respective values by novel estimation were -0.1 (-1.5 to 1.3), -1.1 (-2.5 to 0.3), and -2.7 (-4.9 to 0.0) mg/dL. Among those with Friedewald LDL-C < 15, 15 to < 25, and 25 to < 40 mg/dL, the classification was discordantly low in 94.9%, 82.6%, and 59.9% of individuals as compared with 48.3%, 42.4%, and 22.4% by novel estimation. CONCLUSIONS: Estimation of even lower LDL-C values (by Friedewald and novel methods) is even more inaccurate. More often than not, a Friedewald value < 40 mg/dL is underestimated, which translates into unnecessary safety alarms that could be reduced in half by estimation using our novel method.

A histogram approach to the quality of fit in sedimentation velocity analyses.

The quality of fit of sedimentation velocity data is critical to judge the veracity of the sedimentation model and accuracy of the derived macromolecular parameters. Absolute statistical measures are usually complicated by the presence of characteristic systematic errors and run-to-run variation in the stochastic noise of data acquisition. We present a new graphical approach to visualize systematic deviations between data and model in the form of a histogram of residuals. In comparison with the ideally expected Gaussian distribution, it can provide a robust measure of fit quality and be used to flag poor models.

Measurement of the temperature of the resting rotor in analytical ultracentrifugation.

Accurate measurements of rotor temperature are critical for the interpretation of hydrodynamic parameters in analytical ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor using iButton temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2°C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets.

Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references.

Sedimentation velocity (SV) is a method based on first principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton temperature logger to directly measure the temperature of a spinning rotor and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration that were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., Anal. Biochem., 437 (2013) 104-108), and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from 11 instruments displayed a significantly reduced standard deviation of approximately 0.7%. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard.

Review of Methodological Approaches to Human Milk Small Extracellular Vesicle Proteomics.

Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.

Quantitative Analysis of Protein Self-Association by Sedimentation Velocity.

Sedimentation velocity analytical ultracentrifugation is a powerful classical method to study protein self-association processes in solution based on the size-dependent macromolecular migration in the centrifugal field. This technique can elucidate the assembly scheme, measure affinities ranging from picomolar to millimolar Kd , and in favorable cases provide information on oligomer lifetimes and hydrodynamic shape. The present step-by-step protocols detail the essential steps of instrument calibration, experimental setup, and data analysis. Using a widely available commercial protein as a model system, the protocols invite replication and comparison with our results. A commentary discusses principles for modifications in the protocols that may be necessary to optimize application of sedimentation velocity analysis to other self-associating proteins. ©2020 Wiley Periodicals LLC. Basic Protocol 1: Measurement of external calibration factors Basic Protocol 2: Sedimentation velocity experiment for protein self-association Basic Protocol 3: Sedimentation coefficient distribution analysis in SEDFIT and isotherm analysis in SEDPHAT.

Determining the concentration and the absorptivity factor at 260 nm in sodium dodecyl sulfate of the adenovirus reference material using analytical ultracentrifugation.

A concentration of 6.4 +/- 0.2 (at 1 SD) x 10(11) virus particles (vp) per milliliter was determined for the adenovirus reference material (ARM) by averaging analytical ultracentrifugation concentration data obtained with refractometric detection with the completely independent concentration reported by the Adenoviral Reference Material Working Group (ARMWG). Using this concentration, the ARM absorptivity factor (in sodium dodecyl sulfate [SDS] at 260 nm) of 1.2+/-0.1 (at 1 SD) x 10(12) vp/ml per OD(260 nm) per centimeter was obtained.

A radial calibration window for analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first-principles based method for studying macromolecules and particles in solution by monitoring the evolution of their radial concentration distribution as a function of time in the presence of a high centrifugal field. In sedimentation velocity experiments, hydrodynamic properties relating to size, shape, density, and solvation of particles can be measured, at a high hydrodynamic resolution, on polydisperse samples. In a recent multilaboratory benchmark study including data from commercial analytical ultracentrifuges in 67 laboratories, the calibration accuracy of the radial dimension was found to be one of the dominant factors limiting the accuracy of AUC. In the present work, we develop an artifact consisting of an accurately calibrated reflective pattern lithographically deposited onto an AUC window. It serves as a reticle when scanned in AUC control experiments for absolute calibration of radial magnification. After analysis of the pitch between landmarks in scans using different optical systems, we estimate that the residual uncertainty in radial magnification after external calibration with the radial scale artifact is ≈0.2 %, of similar magnitude to other important contributions after external calibration such as the uncertainty in temperature and time. The previous multilaboratory study had found many instruments with errors in radial measurements of 1 % to 2 %, and a few instruments with errors in excess of 15 %, meaning that the use of the artifact developed here could reduce errors by 5-to 10-fold or more. Adoption of external radial calibration is thus an important factor for assuring accuracy in studies related to molecular hydrodynamics and particle size measurements by AUC.

Centrifugal ultrafiltration of human serum for improving immunoglobulin A quantification using attenuated total reflectance infrared spectroscopy.

Attenuated total reflectance infrared (ATR-IR) spectroscopy is a simple, rapid and cost-effective method for the analysis of serum. However, the complex nature of serum remains a limiting factor to the reliability of this method. We investigated the benefits of coupling the centrifugal ultrafiltration with ATR-IR spectroscopy for quantification of human serum IgA concentration. Human serum samples (n = 196) were analyzed for IgA using an immunoturbidimetric assay. ATR-IR spectra were acquired for whole serum samples and for the retentate (residue) reconstituted with saline following 300 kDa centrifugal ultrafiltration. IR-based analytical methods were developed for each of the two spectroscopic datasets, and the accuracy of each of the two methods compared. Analytical methods were based upon partial least squares regression (PLSR) calibration models - one with 5-PLS factors (for whole serum) and the second with 9-PLS factors (for the reconstituted retentate). Comparison of the two sets of IR-based analytical results to reference IgA values revealed improvements in the Pearson correlation coefficient (from 0.66 to 0.76), and the root mean squared error of prediction in IR-based IgA concentrations (from 102 to 79 mg/dL) for the ultrafiltration retentate-based method as compared to the method built upon whole serum spectra. Depleting human serum low molecular weight proteins using a 300 kDa centrifugal filter thus enhances the accuracy IgA quantification by ATR-IR spectroscopy. Further evaluation and optimization of this general approach may ultimately lead to routine analysis of a range of high molecular-weight analytical targets that are otherwise unsuitable for IR-based analysis.

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

HDL cholesterol performance using an ultracentrifugation reference measurement procedure and the designated comparison method.

BACKGROUND: Accurate high-density lipoprotein cholesterol (HDL-C) measurements are important for management of cardiovascular diseases. The US Centers for Disease Control and Prevention (CDC) and Cholesterol Reference Method Laboratory Network (CRMLN) perform ultracentrifugation (UC) reference measurement procedure (RMP) to value assign HDL-C. Japanese CRMLN laboratory (Osaka) concurrently runs UC procedure and the designated comparison method (DCM). Osaka performance of UC and DCM was examined and compared with CDC RMP. METHODS: CDC RMP involved UC, heparin-MnCl2 precipitation, and cholesterol analysis. CRMLN DCM for samples containing <200 mg/dl triglycerides involved 50-kDa dextran sulfate-MgCl2 precipitation and cholesterol determination. RESULTS: HDL-C regression equations obtained with CDC (x) and Osaka (y) were y=0.992x+0.542 (R(2)=0.996) for Osaka UC and y=1.004x-0.181 (R(2)=0.998) for DCM. Pass rates within ±1 mg/dl of the CDC target value were 91.9 and 92.1% for Osaka UC and DCM, respectively. Biases at 40 mg/dl HDL-C were +0.22 and -0.02 mg/dl for Osaka UC and DCM, respectively. CONCLUSIONS: Osaka UC and DCM were highly accurate, precise, and stable for many years, assisting manufacturers to calibrate products for clinical laboratories to accurately measure HDL-C for patients, calculate non-HDL-C, and estimate low-density lipoprotein cholesterol with the Friedewald equation.

Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors.

Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.

Comparison of an immunoprecipitation method for direct measurement of LDL-cholesterol with beta-quantification (ultracentrifugation).

A direct LDL cholesterol assay was evaluated using immunoprecipitation (Sigma Diagnostics, St. Louis, MO) with beta-quantification obtained by ultracentrifugation. Excellent intra- and interassay coefficients of variation were obtained (< 4.5%). There was a good correlation (r = 0.88, P < .0001) between the two methods for low-density lipoprotein cholesterol (LDL-C) in 249 samples with triglyceride levels ranging from 13 mg/dL to 2,236 mg/dL and LDL cholesterol levels ranging from 28 mg/dL to 290 mg/dL. Similar correlations were seen for patients with triglyceride levels < 400 mg/dL (r = 0.89, n = 174) and > or = 400 mg/dL (r = 0.89, n = 75). However, using the Friedewald equation, there was a good correlation only in samples with triglyceride levels < 400 mg/dL. No significant differences were found between LDL-C quantitated by the direct LDL assay and beta quantification for patients with dysbetalipoproteinemia (Type III disorder). However, calculated LDL values using the Friedewald equation were found to be significantly higher when compared to beta-quantification in patients with the Type III disorder. There was a slight but significant decrease in LDL-C determined by direct LDL cholesterol assay for non-fasting versus fasting serum (4.7%) despite a strong correlation between these samples (r = 0.98, P < .0001). In addition, freezing samples for 30 days resulted in a significant decrease in levels (15.1%). Thus, this direct LDL cholesterol assay is recommended in place of beta-quantification in hypertriglyceridemic samples (TG > or = 400 mg/dL) and to monitor LDL cholesterol levels in patients with Type III dyslipidemia, because it is less time consuming, more cost-effective and can be adapted to the clinical laboratory.

Selective adsorption: a new method for purification of protein A-gold complexes.

Protein gold complexes are prepared by adding gold colloids to cytochemically active proteins in solution. The gold particles of the colloid form complexes with the protein spontaneously, but some of the protein remains uncomplexed. Currently, when protein A-gold complexes are prepared, the uncomplexed protein. A is separated from the complex by ultracentrifugation, which is a lengthy procedure and requires special equipment. This report describes a simple and rapid method for removing uncomplexed protein A from freshly-prepared "crude" protein A-gold at the laboratory bench. In this method, larger gold particles of 15-nm diameter are added to a crude protein A-gold preparation made with smaller particles (e.g.,6nm diameter). The 15-nm particles adsorb uncomplexed protein A preferentially, but do not form complexes with already-formed 6-nm protein A-gold. The adsorbed protein A, attached to the 15-nm particles, can then be sedimented in a bench centrifuge, leaving the purified 6-nm protein A-gold complexes in the supernatant. The stability, immunocytochemical activity, and degree of aggregation of the protein A-gold complexes prepared by this method are comparable to protein A-gold complexes prepared by ultracentrifugation. The method is simple to perform, avoids lengthy purification procedures, and yields complexes with reproducible labelling characteristics.

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context.

We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how to measure the degree of chromatin assembly in the absence and presence of histone H1 using topological analysis and how to perform micrococcal nuclease digestion to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further, we describe the use of sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).

Beta-lactoglobulin B: a proposed standard for the study of reversible self-association reactions in the analytical ultracentrifuge?

Bovine beta-lactoglobulin B is proposed for use as a standard in the measurement of reversible self-association reactions in the analytical ultracentrifuge. The protein is well understood on a molecular level, is readily obtainable, and is stable under harsh conditions. Bovine beta-lactoglobulin B undergoes a simple monomer-dimer equilibrium which can be predictably controlled and consistently reproduced. In this investigation bovine beta-lactoglobulin B has been studied via sedimentation equilibrium experiments in the XL-A analytical ultracentrifuge at 5-30 degrees C in buffers of ionic strength 0.1-0.2 M and pH 2.0-3.0. Samples subjected to a number of different treatments and storage methods all yielded similar results. Molar equilibrium constants for the association reaction were determined by nonlinear regression fitting of a monomer-dimer model of association either to concentration versus radius data, using the programs NONLIN and ORIGIN, or to Omega versus concentration data using the program DUGOM. At 20 degrees C and pH 2.6, over the ionic strength range 0. 1-0.2 M, the equilibrium constant for the association reaction ranges between 1 x 10(4) and 5 x 10(4) M-1. The parameters of nonideal self-association behavior were found to be independent of the particular analysis strategy. Fitting to the concentration distribution, the apparent weight-average molecular weight, or the Omega function all returned identical parameters.

Evolution of methods for measurement of HDL-cholesterol: from ultracentrifugation to homogeneous assays.

BACKGROUND: Adoption of automated homogeneous assays for HDL-cholesterol (HDL-C) is increasing, driven by the need of clinical laboratories to cope with increasing workloads while containing costs. However, performance characteristics of homogeneous assays often differ in important aspects from those of the earlier precipitation methods. This review provides an overview of the new generation of homogeneous assays for HDL-C within the historical context of the evolution of methods and the efforts to standardize measurements of the lipoproteins. APPROACH: This is a narrative review based on method evaluations conducted in the laboratories of the authors as well as on relevant publications, especially comparative evaluation studies, from the literature. Publications considered here have been collected by the authors over the past 30 years of involvement as methods for HDL-C made the transition from their early use in lipid research laboratories to clinical laboratories and the recent emergence of homogeneous assays. CONTENT: The presentation includes descriptions of methodologies, including homogeneous, precipitation, electrophoresis, and ultracentrifugation assays. Reference methods and recommended approaches for assessing accuracy are described. Accuracy and imprecision are summarized in the context of the National Cholesterol Education Program (NCEP) standards for analytical performance. The effects of interfering substances and preanalytical sources of variation are presented. SUMMARY: Homogeneous assays have been shown to be reasonably well suited for use in routine clinical laboratories, generally meeting the NCEP criteria for precision, accuracy, and total error. However, discrepant results compared with the reference methods have been observed with some of the assays, and the sources of discrepancies are not well characterized. Some homogeneous reagents have not been thoroughly evaluated. At least three of the reagents have experienced successive adjustments in formulation; hence, the reagents may not yet be fully optimized. For these reasons, the homogeneous assays cannot be confidently recommended for use in long-term clinical trials and other research applications without thorough validation.

Optimization of beta-quantification methods for high-throughput applications.

BACKGROUND: Risk of cardiovascular disease is assessed, in part, by laboratory measurement of the concentrations of several lipoproteins. beta-Quantification is a method of lipoprotein measurement that uses ultracentrifugation to partially separate lipoprotein classes. Although beta-quantification is used largely in clinical and basic research, methods have not been described to allow the analysis of a large number of small-volume specimens with a short turnaround time. We report two variations of the traditional 5-mL method used by the Lipid Research Clinics Program that overcome these shortcomings. METHODS: Two lower-volume modifications of the traditional 5-mL beta-quantification method were developed. The methods used either 1 or 0.23 mL of specimen and required substantially less time for analysis (20 and 6 h, respectively) than the 5-mL method (2.5 days). The goal was to develop ultracentrifugation methods such that the concentration of cholesterol in the bottom fraction, from which LDL-cholesterol concentration is calculated, agreed with the 5-mL method. Fresh serum specimens (n = 45) were analyzed by the three methods to determine comparability of the methods based on the recovery of cholesterol in the bottom fraction after ultracentrifugation. To evaluate intrarun precision, replicate specimens (n = 17) were analyzed in a single run for each method. This experiment also evaluated how quickly the fractions would remix after separation by ultracentrifugation. For the 1-mL method, accuracy of the measurement of LDL- and HDL-cholesterol concentrations and the interrun precision were established by analysis of frozen serum specimens provided by the CDC, which established target values for the pools using reference methods. RESULTS: No clinically significant differences in cholesterol concentrations in the bottom fraction were observed for the 1- and 0.23-mL methods, which had mean biases of 0.8% and 1.5% relative to the 5-mL method, respectively. Intra- and interrun variability was acceptable for each method, e.g., <1.8% for cholesterol in the bottom fraction. Ultracentrifuged specimens were stable for at least 4 h with no evidence of contamination of cholesterol in the bottom fraction. For comparison specimens provided by the CDC, the 1-mL method met the accuracy and precision goals of the National Cholesterol Education Program for the measurement of HDL- and LDL-cholesterol concentrations (goals: total error <13% and <12%, respectively), with total errors of 6.45% and 5.43%, respectively. CONCLUSIONS: Both the 1- and 0.23-mL beta-quantification methods are suitable substitutes for the traditional 5-mL method for use in clinical and basic research for the determination of LDL-cholesterol concentration. Both methods provide much higher throughput and require substantially less specimen volume. The 0.23-mL method can be performed in 1 day, but it is slightly less precise than the 1-mL method. In our laboratory setting, as many as 80 specimens are routinely processed per day using the 1-mL method.