Measurement of Lecithin–Cholesterol Acyltransferase Activity with the Use of a Peptide-Proteoliposome Substrate

Vaisman, Boris; Remaley, Alan T.

doi:10.1007/978-1-60327-369-5_16

Cited by 14 publications

(16 citation statements)

References 13 publications

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“…The peptide forms stable complexes with phosphatidylcholine and sterol that react suitably well with LCAT [24]. Several advantages are obtained by employing the peptide.…”

Section: Resultsmentioning

confidence: 99%

“…A further advantage is obtained in terms of substrate preparation and cost by substituting a synthetic, LCAT-activating peptide for ApoA-I. The peptide, which has previously been studied under various identifications for its anti-atherosclerotic properties [22;23], forms stable lipid complexes that are reported to react with LCAT at least as well as reconstituted HDL preparations formed from ApoA-I, PC and cholesterol [24]. …”

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confidence: 99%

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A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

Homan

Esmaeil

Mendelsohn

et al. 2013

Analytical Biochemistry

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We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate and measure scheme. This is possible by using the fluorescent sterol, dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE non-fluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of non-radiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.

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“…The peptide forms stable complexes with phosphatidylcholine and sterol that react suitably well with LCAT [24]. Several advantages are obtained by employing the peptide.…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

Homan

Esmaeil

Mendelsohn

et al. 2013

Analytical Biochemistry

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“…LCAT activity in plasma was assayed as described previously ( 50 ) with modifi cations ( 51 ). Briefl y, artifi cial substrate for LCAT, [1,[2][3] H(N)]cholesterol (Perkin Elmer, NET139001MC) labeled liposomes were prepared as described ( 51 ).…”

Section: Plasma Lcat Activity Assaymentioning

confidence: 99%

“…Briefl y, artifi cial substrate for LCAT, [1,[2][3] H(N)]cholesterol (Perkin Elmer, NET139001MC) labeled liposomes were prepared as described ( 51 ). Each plasma sample was analyzed in triplicate using 2 l per measurement.…”

Section: Plasma Lcat Activity Assaymentioning

confidence: 99%

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

Stukas

Freeman

Lee

et al. 2014

Journal of Lipid Research

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Journal of Lipid Research Volume 55, 20141721 of the apoE4 allele increases risk and decreases the age of onset of late-onset AD ( 2 ), the putative roles of apoE in AD pathogenesis have been a topic of intensive focus. apoE is the primary apolipoprotein secreted by astrocytes and microglia in the CNS, and lipidation of nascent apoEcontaining discoidal particles eventually leads to the formation of mature CNS HDL-like particles in cerebrospinal fl uid (CSF) ( 1, 3 ). It is well-established that amyloid burden in humans follows an isoform-dependent pattern of apoE4>apoE3>apoE2 ( 4-6 ). In contrast to human apoE, murine apoE exists in only one isoform with limited homology to human apoE. Amyloid burden is robust in AD mice expressing murine apoE and nearly abrogated in apoE-defi cient mice ( 7-11 ). Reconstitution of human apoE isoforms into AD mice greatly delays amyloid deposition relative to murine apoE, but retains the human isoform-specifi c infl uence on amyloid accumulation (12)(13)(14)(15)(16)(17). Taken together, the relative amyloid burden across murine and human apoE is: murine apoE>>apoE4>apoE3>apoE2>>apoE Ϫ / Ϫ . These observations clearly show that apoE affects amyloid burden in vivo. In vivo, neither apoE isoform nor gene dose significantly affects the rate of ␤ -amyloid (A ␤ ) production from amyloid precursor protein ( 16,17 ), leading to the consensus that apoE is largely involved in modulating the clearance of A ␤ peptides from the brain. apoE participates in each of the three known pathways of A ␤ clearance, including proteolytic degradation, egress across the blood-brain barrier, and interstitial fl uid drainage, although many details remain poorly understood ( 18,19 ). It is important to note, however, that apoE isoforms, levels, and lipidation status are all important variables.Abstract A key step in plasma HDL maturation from discoidal to spherical particles is the esterifi cation of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fl uid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer's disease, including facilitating ␤ -amyloid (A ␤ ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for A ␤ clearance is unknown. Here we characterized the impact of LCAT defi ciency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT defi ciency did not increase A ␤ or amyloid in APP/PS1 LCAT ؊ / ؊ mice. Finally, LCAT expression and plasma activity were unaffected by age or the ons...

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“…However, these findings must be interpreted with caution. The authors do not provide free cholesterol and cholesterol ester data before and after therapy, which are more precise measures of LCAT activity(Vaisman and Remaley, 2013). Moreover, the small sample size, lack of a randomized placebo controlled design, and inability to determine which specific treatment improved HDL function all remain to be addressed.…”

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confidence: 99%

High-Density Lipoprotein Cholesterol Function Improves after Successful Treatment of Psoriasis: A Step Forward in the Right Direction

Mehta

Gelfand

2014

Journal of Investigative Dermatology

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Measurement of Lecithin–Cholesterol Acyltransferase Activity with the Use of a Peptide-Proteoliposome Substrate

Cited by 14 publications

References 13 publications

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

High-Density Lipoprotein Cholesterol Function Improves after Successful Treatment of Psoriasis: A Step Forward in the Right Direction

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