Novel corrective equations for complete estimation of human tissue lipids after their partial destruction by perchloric acid pre‐treatment: high‐resolution ¹H‐NMR‐based study

Abstract

Owing to the small quantity of tissue available in human biopsy specimens, aqueous and lipid components often have to be determined in the same tissue sample. Perchloric acid (PCA) used for the extraction of aqueous metabolites has a deleterious effect on lipid components; the severity of the damage is not known. In this study, human muscle tissue was first treated with PCA to extract aqueous metabolites, and the residue was then used for lipid extraction by conventional methods, i.e. the methods of Folch and Bligh & Dyer and a standardised one using methanol/chloroform (1:3, v/v) used in our laboratory. A (1)H-NMR spectrum was obtained for each lipid extract. Lipid was quantified by measuring the integral area of N(+)-(CH(3))(3) signals of phospholipids (PLs). Triacylglycerol (TG) and cholesterol (CHOL) were quantified using the -CH(2)- signals of glycerol and the C18 methyl signal, respectively. This study shows that prior use of PCA caused marked attenuation of TG, PL, and CHOL. This was confirmed by recovery experiments and observation of the direct effect of PCA on the standard lipid components. On the basis of the quantity of lipid lost in each case, three novel equations (with respect to TG, PL, and CHOL) were derived. Application of these equations to lipid quantities estimated in different pathological tissues after PCA pre-treatment produced values equivalent to those estimated without PCA use. This study conclusively shows that PCA pre-treatment damages all three lipid moieties, TG, PL, and CHOL. When PCA is used in a fixed ratio to the tissue, the lipid damage is also proportional and correctable by statistically derived equations. These equations will be useful in human biopsy specimens where aqueous and lipid components have to be studied using the same tissue sample because of the small quantity available.

Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS

“…The extraction with PCA showed low matrix effects similar to those of the CHCl 3 :MeOH method. Its similar behavior to that of the CHCl 3 :MeOH method may be due to PCA's low extraction capacity and the deleterious effects of the lipid components [45].…”

Metabolic fingerprinting of joint tissue of collagen-induced arthritis (CIA) rat: in vitro, high resolution NMR (nuclear magnetic resonance) spectroscopy based analysis

“…Aqueous metabolites were extracted from joint tissue of the CIA and control rats by the method of perchloric acid extraction, as described previously (Srivastava et al, 2008[ 50 ]). After completing the extraction, the sample was lyophilized.…”

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study

“…Using literature references (15)(16)(17)(18)(19)(20)(21) and our own additional 2D correlation spectroscopy ( 1 H 1 H-TOCSY) measurements, an overall of 18 metabolites and 11 lipid resonances were assigned. For chemometric analysis of the muscle biopsies for the reproducibility study, a total of 63 buckets (between 0.95 and 8.62 ppm) were selected, with a variable size according to the peak width (see supplementary material, figure S1).…”