A Simple and Improved Method for Extraction of Phospholipids from Hemoglobin Solutions

Abstract

This study introduced a liquid-liquid extraction method designed for complete recovery of phospholipids from protein-rich samples, such as hemoglobin (Hb) solutions. In order to minimize protein denaturation and maximize lipid extraction from protein-rich samples, isopropanol was used as the major organic extraction solvent. In a wide range of the volume ratio of isopropanol to Hb solution at low temperatures, such extraction system resulted in limited protein precipitation and did not cause heme pigment contamination. The efficiency of phospholipid extraction from Hb solutions was pH-dependent, with the lowest extraction yield around neural pH and the highest extraction yield around pH 5.0. At pH 5.0, salt with concentrations up to 400 mmol/L in the samples did not increase extraction efficiency. Compared to other available methods, this method is simpler, needs significantly less organic solvent and, most importantly, consumed much less Hb, which is very expensive to make for the purpose of production of therapeutic products, hemoglobin-based oxygen carriers (HBOCs). This method is suitable not only for Hb solutions but also for other protein-rich biological samples, such as erythrocytes, plasma and liver.

Validation and application of a protocol for the extraction and quantitative analysis of sphingomyelin in erythrocyte membranes of patients with non-alcoholic fatty liver disease

“…Broekhuyse [14] proposed a system with multiple solvents. The inconvenience of multiple solvents and steps, led other researchers to develop methods requiring only a single step, involving either isopropanol or chloroform/isopropanol at a ratio of 1:1.5 [15][16][17]. Recently, Zhao and Xu [18], developed a rapid and efficient method for the analysis of phospholipids and lysophospholipids, which has not been evaluated for the analysis of erythrocyte sphingomyelin.…”

Highly Efficient Microscale Purification of Glycerophospholipids by Microfluidic Cell Lysis and Lipid Extraction for Lipidomics Profiling

“…4–6 Low detection limits are important to lipid studies because some lipids occur in trace (picomolar (10 −12 ) – nanomolar (10 −9 )) and even ultra-trace (femtomolar (10 −15 ) and below) concentrations in samples. 7–9 Two branches of chemical analysis play an important role in `omics': separation science and mass spectrometry. In lipidomics, distinct subclasses of lipids, obtained by liquid extraction from a cell or tissue mass, are isolated by solid phase extraction, liquid chromatography, or thin-layer chromatography and then analyzed by a further dimension (or two) of liquid chromatography and two or more dimensions of mass spectrometry.…”

“…Lipids have various biochemical functions including energy storage (fats and sterols), cell structure (lipid bilayers and micelles), and cell signaling. − The quantitative study of lipids, or “lipidomics”, is a burgeoning field in bioanalytical chemistry. − Low detection limits are important to lipid studies because some lipids occur in trace (picomolar (10 –12 ) to nanomolar (10 –9 )) and even ultratrace (femtomolar (10 –15 ) and below) concentrations in samples. − Two branches of chemical analysis play an important role in “omics”: separation science and mass spectrometry. In lipidomics, distinct subclasses of lipids, obtained by liquid extraction from a cell or tissue mass, are isolated by solid phase extraction, liquid chromatography, or thin-layer chromatography and then analyzed by a further dimension (or two) of liquid chromatography and two or more dimensions of mass spectrometry. , One difficulty in profiling the chemical population of a small sample is that the lipidome exists over a wide range of concentrations.…”