A high-throughput platform for detailed lipidomic analysis of a range of mouse and human tissues

Abstract

Lipidomics is of increasing interest in studies of biological systems. However, high-throughput data collection and processing remains non-trivial, making assessment of phenotypes difficult. We describe a platform for surveying the lipid fraction for a range of tissues. These techniques are demonstrated on a set of seven different tissues (serum, brain, heart, kidney, adipose, liver, and vastus lateralis muscle) from post-weaning mouse dams that were either obese (> 12 g fat mass) or lean (<5 g fat mass). This showed that the lipid metabolism in some tissues is affected more by obesity than others. Analysis of human serum (healthy nonpregnant women and pregnant women at 28 weeks' gestation) showed that the abundance of several phospholipids differed between groups. Human placenta from mothers with high and low BMI showed that lean placentae contain less polyunsaturated lipid. This platform offers a way to map lipid metabolism with immediate application in metabolic research and elsewhere.

Lipid extraction from dried blood spots and dried milk spots for untargeted high throughput lipidomics

“…Further checks could be made using orthogonal methods for determining molecular structure, for example through NMR. Dual spectroscopy 22 was designed to verify molecular structure using orthogonal spectroscopic methods, typically mass spectrometry and 31 P NMR, in lipidomics. Further work could include the use of 1 H NMR for this purpose, however this has not been attempted on DBS or DMS-based samples yet, to our knowledge.…”

“…This therefore adds to the work on extracting lipids from DMSs already reported. 19 Remarkably, the solvent system that proved to be best for extracting lipids from DMSs (xylene/ methanol/isopropanol) contrasts sharply with the mixture of dichloromethane, methanol and triethylammonium chloride that is superior for extracting lipids from wet biological samples both in high throughput 21,22,24 and large-sample-size 30 formats. This is based on calculations of the coefficient of variance that showed that the XMI method is generally more precise for extracting lipids from DMS.…”

“…As a visual appraisal of DMSs suggested that milk fat globules may aggregate on one face of the card, we also tested different solvent mixtures in order to determine which optimised isolation of the lipid fraction from this dense aggregation. We tested a novel extraction procedure against two methods reported for high throughput lipidomics, 'DMT', (dichloromethane/methanol/ triethylammonium chloride, 3 : 1 : 0.005) 21,22,24 and 'TBME' (tertiary-butylmethyl ether), 25 both of which used an aqueous wash as part of the extraction. The novel procedure was developed based on an established method that employed a mixture of chloroform, methanol and isopropanol (1 : 2 : 4).…”

“…The reference material used was designed to be as broad as possible. It comprised equal parts of an homogenate of mouse placenta, 22 pooled human blood serum and an infant-formula/animal milk preparation. The latter mixture comprised infant formula based on caprine, bovine and soya material, made up to a solution (15 mg mL À1 ) using Jersey milk described previously.…”

“…Treatment with this buffer has previously been found to facilitate preparation of biological samples for lipid extraction. 22 This led to considerable improvement in the extraction efficiency of DBSs but no improvement of that from DMSs, leading us to suppose that lipids were not inaccessible in DMSs and thus could be extracted by a standard or even non-aqueous method. We therefore tested several solvent systems for extracting lipids from DMSs in order to identify a method that was convenient for the simultaneous extraction of large numbers of small samples and for profiling as many of the lipid components present as possible.…”