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Direct observation of lipoprotein cholesterol ester degradation in lysosomes.

ABSTRACT: We have investigated whether pyrene-labelled cholesterol esters (PyrnCEs) (n indicates the number of aliphatic carbons in the pyrene-chain) can be used to observe the degradation of low-density lipoprotein (LDL)-derived cholesterol esters (CEs) in the lysosomes of living cells. To select the optimal substrates, hydrolysis of the PyrnCE species by lysosomal acid lipase (LAL) in detergent/phospholipid micelles was compared. The rate of hydrolysis varied markedly depending on the length of the pyrenyl chain. Pyr10CE was clearly the best substrate, while Pyr4CE was practically unhydrolysed. Pyr10CE and [3H]cholesteryl linoleate, the major CE species in LDL, were hydrolysed equally by LAL when incorporated together into reconstituted LDL (rLDL) particles, thus indicating that Pyr10CE is a reliable reporter of the lysosomal degradation of native CEs. When rLDL particles containing Pyr4CE or Pyr10CE were incubated with fibroblasts, the accumulation of bright intracellular vesicular fluorescence was observed with the former fluorescent derivative, but not with the latter. However, when the cells were treated with chloroquine, an inhibitor of lysosomal hydrolysis, or when cells with defective LAL were employed, Pyr10CE also accumulated in vesicular structures. HPLC analysis of cellular lipid extracts fully supported these imaging results. It is concluded that PyrnCEs can be used to observe degradation of CEs directly in living cells. This should be particularly useful when exploring the mechanisms responsible for the accumulation of lipoprotein-derived CEs in complex systems such as the arterial intima.

SUBMITTER: Lusa S

PROVIDER: S-EPMC1219500 | biostudies-other | 1998 Jun

REPOSITORIES: biostudies-other

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Project description:AimsLow-density lipoprotein (LDL-C) lowering is imperative in cardiovascular disease prevention. We aimed to compare accuracy of three clinically-implemented LDL-C equations in a clinical trial of cholesterol ester transfer protein (CETP) inhibition.Methods and resultsMen and women aged 18-75 years with dyslipidaemia were recruited from 17 sites in the Netherlands and Denmark. Patients were randomly assigned to one of nine groups using various combinations of the CETP inhibitor TA-8995 (obicetrapib), statin therapy, and placebo. In pooled measurements over 12 weeks, we calculated LDL-C by the Friedewald, Martin/Hopkins, and Sampson equations, and compared values with preparative ultracentrifugation (PUC) LDL-C overall and with a special interest in the low LDL-C/high triglycerides subgroup. There were 242 patients contributing 921 observations. Overall median LDL-C differences between estimates and PUC were small: Friedewald, 0.00 (25th, 75th: -0.10, 0.08) mmol/L [0 (-4, 3) mg/dL]; Martin/Hopkins, 0.02 (-0.08, 0.10) mmol/L [1 (-3, 4) mg/dL]; and Sampson, 0.05 (-0.03, 0.15) mmol/L [2 (-1, 6) mg/dL]. In the subgroup with estimated LDL-C <1.8 mmol/L (<70 mg/dL) and triglycerides 1.7-4.5 mmol/L (150-399 mg/dL), the Friedewald equation underestimated LDL-C with a median difference versus PUC of -0.25 (-0.33, -0.10) mmol/L [-10 (-13, -4) mg/dL], whereas the median difference by Martin/Hopkins was 0.00 (-0.08, 0.10) mmol/L [0 (-3, 4) mg/dL] and by Sampson was -0.06 (-0.13, 0.00) mmol/L [-2 (-5, 0) mg/dL]. In this subgroup, the proportion of LDL-C observations <1.8 mmol/L (<70 mg/dL) that were correctly classified compared with PUC was 71.4% by Friedewald vs. 100.0% by Martin/Hopkins and 93.1% by Sampson.ConclusionIn European patients with dyslipidaemia receiving a CETP inhibitor, we found improved LDL-C accuracy using contemporary equations vs. the Friedewald equation, and the greatest accuracy was observed with the Martin/Hopkins equation.RegistrationClinicalTrials.gov, NCT01970215.

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Direct observation of lipoprotein cholesterol ester degradation in lysosomes.

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