scholarly journals Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations

Separations ◽  
2021 ◽  
Vol 8 (11) ◽  
pp. 204
Author(s):  
Rian Ka Praja ◽  
Wisitsak Phoksawat ◽  
Patcharaporn Tippayawat ◽  
Amonrat Jumnainsong ◽  
Chanvit Leelayuwat
Keyword(s):  
High Density Lipoprotein ◽  
Protein Pattern ◽  
Density Lipoprotein ◽  
Sample Volume ◽  
Molecular Diagnostic ◽  
Particle Sizes ◽  
Limited Sample ◽  
Modified Method ◽  
Diagnostic Approaches ◽  
Commercial Kits

High-density lipoprotein (HDL) and exosomes are promising sources of biomarkers. However, the limited sample volume and access to the ultracentrifuge equipment are still an issue during HDL and exosome isolation. This study aimed to isolate HDL and exosomes using an ultracentrifugation-free method with various small serum volumes. HDL was isolated from 200 µL (HDL200) and 500 µL (HDL500) of sera. Three different volumes: 50 µL (Exo50), 100 µL (Exo100), and 250 µL (Exo250) were used for exosome isolation. HDL and exosomes were isolated using commercial kits with the modified method and characterized by multiple approaches. The HDL levels of HDL200 and HDL500 were not significantly different (p > 0.05), with percent recoveries of >90%. HDL200 and HDL500 had the same protein pattern with a biochemical similarity of 99.60 ± 0.10%. The particle sizes of Exo50, Exo100, and Exo250 were in the expected range. All isolated exosomes exhibited a similar protein pattern with a biochemical similarity of >99%. In conclusion, two different serum volumes (200 and 500 µL) and three different serum volumes (50, 100, and 250 µL) can be employed for HDL and exosome isolation, respectively. The possibility of HDL and exosome isolation with small volumes will accelerate biomarker discoveries with various molecular diagnostic approaches.

Modification of the dextran-Mg2+ high-density lipoprotein cholesterol precipitation method for use with previously frozen plasma

1994 ◽  
Vol 40 (2) ◽  
pp. 233-239 ◽  
Author(s):  
J R McNamara ◽  
C Huang ◽  
T Massov ◽  
E T Leary ◽  
G R Warnick ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Density Lipoprotein ◽  
Hdl Cholesterol ◽  
Sample Volume ◽  
High Density ◽  
Precipitation Method ◽  
Modified Method ◽  
Fresh Plasma ◽  
Frozen Plasma ◽  
Clear Supernatant

Abstract Although dextran-Mg2+ precipitation produces accurate and precise results for high-density lipoprotein (HDL) cholesterol in fresh plasma and serum, precipitation of frozen specimens with triglycerides > 2.26 mmol/L (> 200 mg/dL) is difficult. We developed a modification that dilutes thawed samples by 35% and increases dextran-Mg2+ reagent to 15% of sample volume. Standard precipitations were performed on 62 fresh EDTA-treated plasma specimens; supernatant solutions were analyzed fresh and after freezing. Standard and modified methods were also performed on thawed, paired plasmas. In specimens with triglycerides < or = 2.26 mmol/L, HDL cholesterol results for all methods were similar. For triglycerides > 2.26 mmol/L, however, bias and precision were significantly affected by freezing, and 38.5% of samples with standard precipitation required additional procedures to produce clear supernatant solutions. HDL cholesterol concentrations for thawed samples with standard precipitation were significantly greater than for fresh samples (P < 0.02), but those for the modified method were not different from fresh samples, and only one specimen required additional steps to produce a clear supernate.

Multi-laboratory comparison of three heparin-Mn2+ precipitation procedures for estimating cholesterol in high-density lipoprotein.

1978 ◽  
Vol 24 (6) ◽  
pp. 853-856 ◽  
Author(s):  
J J Albers ◽  
G R Warnick ◽  
D Wiebe ◽  
P King ◽  
P Steiner ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Apolipoprotein B ◽  
Room Temperature ◽  
Density Lipoprotein ◽  
Sample Volume ◽  
Final Concentration ◽  
High Density ◽  
High Density Lipoproteins ◽  
Modification Method ◽  
Plasma High Density

Abstract Plasma high-density lipoprotein is commonly estimated by measuring the cholesterol remaining in plasma supernatant solutions after other lipoproteins, which contain apolipoprotein B, are precipitated with heparin and Mn2+. The method (method I) now in use by the Lipid Research Clinics, in which Mn2+ is at 46 mmol/liter final concentration, is reasonably accurate, but precipitation and sedimentation of lipoproteins other than high-density lipoproteins is often incomplete. We evaluated two modifications of method I. In method II, the Mn2+ concentration was doubled; the second modification (method III) included the increased Mn2+ concentration in a combined heparin Mn2+ reagent, decreased sample volume (2 ml), and a shorter incubation time (10 min at room temperature). The percentages of samples with turbid supernates (i.e., incomplete sedimentation) by methods I, II, and III were 9, 3, and 2%, respectively. Among non-turbid supernates, the percentages of samples containing measurable apolipoprotein B (incomplete precipitation) were 79, 19, and 16%, respectively. We conclude that method III is the most convenient and accurate of the three procedures.

High-density lipoproteins estimated by an enzymatic cholesterol procedure, with a centrifugal analyzer.

1978 ◽  
Vol 24 (12) ◽  
pp. 2180-2184 ◽  
Author(s):  
K O Ash ◽  
W M Hentschel
Keyword(s):  
High Density Lipoprotein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Sample Volume ◽  
High Density ◽  
High Density Lipoproteins ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Control Pool ◽  
Ambulatory Patients

Abstract We describe an assay for high-density lipoprotein cholesterol, adapted to a centrifugal analyzer, the GEMSAEC System 3, which includes use of an increased Mn2+concentration (91 mmol/liter) [J. Lipid Res. 19, 65 (1978)] and ethylenediaminetetraacetate [Clin. Chem. 22, 98 (1976)]. Modifications to the GEMSAEC system include reducing the mixing burst and preconditioning the sample tip. Accuracy of this procedure, as assessed by analysis of a control pool from the Center for Disease Control, was 99.2%. Day-to-day precision for two control pools was 320 +/- 13 and 506 +/- 17 mg/liter. Serum sample volume was decreased to 0.5 ml. In blanks with heparin/Mn2+ present, the pseudocholesterol concentrations resulting from a reaction of the enzymatic cholesterol reagent and the heparin/Mn2+ precipitating reagent depend on the source of the enzymatic reagent and appear to be enhanced slightly by the use of ethylenediaminetetraacetate. Pseudocholesterol concentrations reach a maximum at heparin/Mn2+ concentrations well below those needed to completely precipitate the low-density and very-low-density lipoprotein fractions. Population reference values were obtained from analyses done on 224 local physicians (mean: male, 500 mg/liter; female, 620 mg/liter) and 156 ambulatory patients (mean: male, 463 mg/liter; female, 553 mg/liter).

Triglyceride to high density lipoprotein cholesterol ratio, total cholesterol to high density lipoprotein cholesterol ratio and low ankle brachial index in an elderly population

VASA ◽  
2014 ◽  
Vol 43 (3) ◽  
pp. 189-197 ◽  
Author(s):  
Yiqiang Zhan ◽  
Jinming Yu ◽  
Rongjing Ding ◽  
Yihong Sun ◽  
Dayi Hu
Keyword(s):  
High Density Lipoprotein ◽  
Total Cholesterol ◽  
High Density Lipoprotein Cholesterol ◽  
Ankle Brachial Index ◽  
Density Lipoprotein ◽  
High Density ◽  
Lipoprotein Cholesterol ◽  
Lipid Lowering ◽  
Cholesterol Ratio ◽  
Potential Biomarker

Background: The associations of triglyceride (TG) to high-density lipoprotein cholesterol ratio (HDL‑C) and total cholesterol (TC) to HDL‑C ratio and low ankle brachial index (ABI) were seldom investigated. Patients and methods: A population based cross-sectional survey was conducted and 2982 participants 60 years and over were recruited. TG, TC, HDL‑C, and low-density lipoprotein cholesterol (LDL-C) were assessed in all participants. Low ABI was defined as ABI ≤ 0.9 in either leg. Multiple logistic regression models were applied to study the association between TG/HDL‑C ratio, TC/HDL‑C ratio and low ABI. Results: The TG/HDL‑C ratios for those with ABI > 0.9 and ABI ≤ 0.9 were 1.28 ± 1.20 and 1.48 ± 1.13 (P < 0.0001), while the TC/HDL‑C ratios were 3.96 ± 1.09 and 4.32 ± 1.15 (P < 0.0001), respectively. After adjusting for age, gender, body mass index, obesity, current drinking, physical activity, hypertension, diabetes, lipid-lowering drugs, and cardiovascular disease history, the odds ratios (ORs) with 95 % confidence intervals (CIs) of low ABI for TG/HDL‑C ratio and TC/HDL‑C ratio were 1.10 (0.96, 1.26) and 1.34 (1.14, 1.59) in non-smokers. When TC was further adjusted, the ORs (95 % CIs) were 1.40 (0.79, 2.52) and 1.53 (1.21, 1.93) for TG/HDL‑C ratio and TC/HDL‑C ratio, respectively. Non-linear relationships were detected between TG/HDL‑C ratio and TC/HDL‑C ratio and low ABI in both smokers and non-smokers. Conclusions: TC/HDL‑C ratio was significantly associated with low ABI in non-smokers and the association was independent of TC, TG, HDL‑C, and LDL-C. TC/HDL‑C might be considered as a potential biomarker for early peripheral arterial disease screening.

Studies on the Thromboplastic Effect of Human Plasma Lipoproteins

1976 ◽  
Vol 35 (01) ◽  
pp. 178-185 ◽  
Author(s):  
Helena Sandberg ◽  
Lars-Olov Andersson
Keyword(s):  
High Density Lipoprotein ◽  
Human Plasma ◽  
Platelet Rich Plasma ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasma Lipoproteins ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Free Plasma ◽  
Good Agreement

SummaryHuman plasma lipoprotein fractions were prepared by flotation in the ultracentrifuge. Addition of these fractions to platelet-rich, platelet-poor and platelet-free plasma affected the partial thromboplastin and Stypven clotting times to various degrees. Addition of high density lipoprotein (HDL) to platelet-poor and platelet-free plasma shortened both the partial thromboplastin and the Stypven time, whereas addition of low density lipoprotein and very low density lipoprotein (LDL + VLDL) fractions only shortened the Stypven time. The additions had little or no effect in platelet-rich plasma.Experiments involving the addition of anti-HDL antibodies to plasmas with different platelet contents and measuring of clotting times produced results that were in good agreement with those noted when lipoprotein was added. The relation between structure and the clot-promoting activity of various phospholipid components is discussed.

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