Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington's disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner

Marullo, M., Valenza, M., Leoni, V., Caccia, C., Scarlatti, C., De Mario, A., et al. (2012). Pitfalls in the detection of cholesterol in Huntington's disease models. PLOS ONE [10.1371/505886e9a1968].

Pitfalls in the detection of cholesterol in Huntington's disease models

Leoni, V;
2012

Abstract

Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington's disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner
Articolo in rivista - Articolo scientifico
mass spectrometry, oxysterols, organic acids, fatty acids, metabolomics, cholesterol, neurodegenerative diseases
English
2012
e505886e9a1968
reserved
Marullo, M., Valenza, M., Leoni, V., Caccia, C., Scarlatti, C., De Mario, A., et al. (2012). Pitfalls in the detection of cholesterol in Huntington's disease models. PLOS ONE [10.1371/505886e9a1968].
File in questo prodotto:
File Dimensione Formato  
31 Marullo et al 2012_PLOSCurHuntDis_2012_4.pdf

Solo gestori archivio

Dimensione 398.26 kB
Formato Adobe PDF
398.26 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/221658
Citazioni
  • Scopus 18
  • ???jsp.display-item.citation.isi??? ND
Social impact