Quantitative Analysis Of Molecular Species Research Articles

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.

Atmospheric trace gas analysis with cavity ring‐down spectroscopy

AbstractCavity ring‐down spectroscopy (CRDS) is a highly sensitive laser absorption method. It can be used for quantitative analysis of molecular species at the sub‐ppb level. The absorption cell (cavity) is sealed by two high‐reflective mirrors on each side, which results in an effective absorption path‐length of some kilometers. Our experiments for atmospheric gas analysis have been carried out so far with an Excimer pumped dye laser in the UV‐VIS and a CO overtone sideband laser in the wavelength region around 3 μm. Experiments with an all solid‐state difference frequency laser system will follow. In the UV‐VIS region, we measured trace gas molecules like SO2, NO2, and CH2O. In the mid‐infrared, around 3 μm, we measured hydrocarbons like CH4, C2H6, and C2H4 with a detection limit of less than 1 ppb. The noise equivalent absorption coefficients in the MIR are in the order of 1.7·10−9 cm−1. Due to the high data acquisition rate and the high sensitivity, CRDS enables real‐time detection of trace gases in ambient air.

Determination of lysophosphatidic acids by capillary electrophoresis with indirect ultraviolet detection

Lysophosphatidic acid (LPA) is the simplest form of lysophospholipid. Molecular species of LPA have been identified as the potent components in the ovarian cancer activation factor. The elevated plasma LPAs may be used as potential biomarkers for the early detection of ovarian cancer. This paper is the first report on the quantitative analysis of molecular species of LPA using capillary electrophoresis. In this work, the separation of LPAs was achieved within 14 min in an adenosine monophosphate-borate–methanol–water solution, and the measurement was accomplished by indirect UV detection. With LPA (D) as internal standard, the method had linear calibration ranges for LPAs from 2.8 to 75 μ M. The detection limits for various molecular species of LPA were from 1.2 to 2.3 μ M by the pressure injection at 3.45 kPa for 5 s. The method had been applied to serum fortified with LPA (S), LPA (O), LPA (P), and LPA (M) and the recoveries ranged from 83 to 112%.

Quantitative analysis of ceramide molecular species by high performance liquid chromatography

A method was developed for quantitative analysis of molecular species of ceramide (N-acyl-sphingosine) and dihydroceramide (N-acyl sphinganine) by high performance liquid chromatography (HPLC). Various N-acyl chain-containing ceramides or dihydroceramides were semi-synthesized as standard materials and allowed to react with anthroyl cyanide, a fluorescent reagent. Anthroyl derivatives of ceramide and dihydroceramide containing C16, C18, C20, C22, and C24 saturated N-acyl chain could be completely separated to each molecular species by reversed-phase HPLC equipped with fluorescence detector, although some ceramide molecular species containing monoenoic acyl chain were eluted together with saturated dihydroceramide species. Ceramide molecular species could be quantified using N-heptadecanoyl or N-tricosanoyl sphingosine as an internal standard, and the lower detection limit was below 1 pmol. This method was applied to the analysis of sphingomyelin and free ceramide in U937 cells. The analysis of the ceramide obtained by hydrolysis of sphingomyelin of U937 cells revealed that the ceramide moiety was mainly composed of N-palmitoyl sphingosine, N-ner vonoyl sphingosine, and N-lignoceroyl sphingosine, representing 50.0, 27.4, and 6.7% of sphingomyelin, respectively. The total free ceramide and dihydroceramide of U937 cells was determined to be 254 ± 5 pmol/106 cells. Major molecular species of the free ceramide fraction were N-lignoceroyl, N-palmitoyl, and N-nerovonoyl sphingosine, representing 27.6%, 26.6%, and 13.6% of this fraction, respectively. Different distribution of free ceramide molecular species from sphingomyelin species may suggest that selective metabolism of molecular species occurs in the synthesis or degradation of sphingomyelin. These results indicate that the picomole level of molecular species of ceramide and dihydroceramide is successfully determined by fluorescence HPLC and that this newly developed method may be useful to reveal the metabolism and function of ceramide and related compounds in cultured cells.—Yano, M., E. Kishida, Y. Muneyuki, and Y. Masuzawa. Quantitative analysis of ceramide molecular species by high performance liquid chromatography. J. Lipid Res. 1998. 39: 2091–2098.

Quantitative Analysis of Changes in the Molecular Species of Glycerolipids in Cultured Cells during Signal Transduction

A method for the quantitative analysis of the molecular species of glycerolipids present In biological samples has been described. 1,2-Diacyl- sn-glycerol, either isolated from biological samples or enzymatically generated from phosphoglycerides, is benzoylated at the sn-3 position and then subjected to reverse-phase (C 18-silica) HPLC to separate the molecular species of different hydrophobicitles. An internal standard (1,2-distearoyl- sn-glycerol) is used to identify and quantify the various species eluted from the reverse-phase column. Examples are given for the quantitative analysis of molecular species and precursor-product relationships of glycerolipids generated In SK-N-SH neuroblastoma cells after stimulation of the cell-surface muscarinic acetylcholine receptors.

Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells.

Quantitative changes in the total mass and the molecular species of 1,2-diacyl-sn-glycerol (DAG) and phosphatidic acid (PA) formed upon muscarinic receptor activation were studied in cultured human SK-N-SH neuroblastoma cells. DAG was isolated from the total lipid extracts of carbachol (CCh)-stimulated and unstimulated cells and after benzoylation, was subjected to reverse phase high performance liquid chromatography to separate the component species. The molecular species of DAG were identified by analyzing the fatty acid composition of each separated fraction by gas chromatography, and their total and individual masses were quantified from the known amount of an internal standard, 1,2-distearoyl-sn-glycerol, added during the extraction of the lipid. Relatively high basal levels of DAG (1.5 nmol/mg protein) are present in these cells, and addition of CCh elicited a 50-60% increase in the total amounts of DAG within 5 min. The increase was biphasic: an initial major peak at 5 min was followed by a sustained increase that persisted for at least 30 min. An increase in DAG was elicited by both full and partial muscarinic agonists and was blocked by atropine. The presence of extracellular Ca2+ was necessary for muscarinic receptor-activated formation of DAG. To determine the source of the DAG, the molecular species of the major phospholipids present in SK-N-SH cells were also analyzed. The phospholipids were first enzymatically hydrolyzed to DAGs which were then analyzed as described above. A number of unusual fatty acids, the major one being 20:3 (n-9), were present in these lipids especially in the phosphoinositides and also in the DAG formed after CCh stimulation. Within 5 s of CCh stimulation there were transient increases in the DAG species representative of phosphoinositides. By 5 min the newly formed molecular species of DAG resembled a mixture of phosphoinositides and phosphatidylcholine (PC). Quantitative comparison of the molecular species compositions of phosphoinositides, PC, and newly formed DAGs indicated that at time periods up to 10 min, approximately 30% of the DAG originated from the phosphoinositides and the rest from PC. At longer intervals (greater than 20 min), most (85%) of DAGs originated from PC. Activation of muscarinic receptors in SK-N-SH cells also elicited an increase in PA (200% in 5 min). A quantitative molecular species analysis, using 1,2-distearoyl-sn-glycerol-3-P as internal standard, was performed by enzymatic (alkaline phosphatase) hydrolysis of PA to DAG and subsequent analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

A method for the quantitative analysis of molecular species of alkylacylglycerol and diacylglycerol

We describe a method for the quantitative analysis of molecular species of diacylglycerol and alkylacylglycerol as their diradylglycerobenzoate derivatives. Synthetic internal standards were used to provide quantitative determinations of the low levels of diacylglycerol and alkylacylglycerol and their individual molecular species in cultured cells. Diradylglycerols were isolated by thin-layer chromatography (TLC), converted to their benzoate derivatives and separated into subclasses by TLC. The molecular species of each subclass were analyzed by reversed-phase high performance liquid chromatography. Thirty-six species of diglyceride-type molecules were identified in Madin-Darby canine kidney cells. These cells were shown to contain 7.88 nmoles of diacylglycerol and 3.97 nmoles of alkylacylglycerol per mumole of phospholipid. Both subclasses contain predominantly monoenoic and saturated species. This technique should be valuable for studies examining the origin and metabolism of these important intracellular mediators.

Quantitative analysis of retinal glycerolipid molecular species acetylated by acetolysis.

A method for the quantitative analysis of molecular species of 1,2-diacylglycerol acetates (1,2-DGAC) containing polyunsaturated fatty acids is described. Phosphatidylethanolamine (PE) isolated from frog retina was used to test the method. PE was converted to 1,2-DGAC by acetolysis. The molecular species of the 1,2-DGAC were resolved by reverse-phase high performance liquid chromatography (HPLC), detected by UV absorption spectroscopy at 210 nm, and identified by gas-liquid chromatography (GLC) of the fatty acid methyl esters (FAME). Molar response curves were generated for each DGAC molecular species that eluted as a single entity from HPLC by determining the moles of fatty acids in the molecular species collected and the response (peak area unit) of the UV detector. Each molecular species response curve was linear from about 10 pmoles to 4-8 nmoles, allowing the slope of each curve to be used as a molar absorptivity. This method provides a means for quantification of most of the molecular species of all glycerolipid classes.

Analysis of molecular species of ether analogues of phosphatidylcholines from biological samples

A method is described for quantitative analysis of molecular species of ether analogues of phosphatidylcholines. The choline-containing phospholipids are isolated by chromatography on a lipophilic weak anion exchanger and are hydrolyzed with phospholipase C. The neutral lipids formed are separated by straight-phase chromatography on Lipidex®-5000 into three classes: 1-alky 1-2-acyl-, 1-alk-1'-enyl-2-acyl- and 1,2-diacylglycerols. The trimethylsilyl ethers of the ether lipids are then separated into molecular species by reversed-phase chromatography on Lipidex® -5000 and the species are analyzed by gas chromatography-mass spectrometry. For a more detailed analysis, the ester bond is hydrolyzed and the fatty acids are separated from the 1-alkyl- and 1-alk-1'-enylglycerols on a lipophilic strong anion exchanger. The glyceryl ethers are then analyzed as trimethylsilyl ethers by gas chromatography-mass spectrometry. The method can be used for analysis of 50–1000 gmg of ether analogues present in samples containing a 100–1000-fold excess of 1,2-diacylglycerophosphocholines. When ether analogues of phosphatidylcholines in beef heart and rat liver were analyzed, 10–20 molecular species were identified. In beef heart, the composition of molecular species was similar for 1-alkyl-2-acyl- and 1-alk-l'-enyl-2-acylglycerophosphocholines, 1-hexadecyl-2-octadecadienoyl- and 1-hexadec-1'-enyl-2-octadecadienoylglycerophosphocholine constituting 42% and 38% of the respective classes. In rat liver, the three major molecular species of 1-alkyl-2-acylglycerophosphocholines were 1-hexadecyl-2-palmitoyl- (28%), 1-octadecenyl-2-palmitoyl- (26%) and 1-hexadecyl-2-eicosatetraenoylglycero-phosphocholine (19%). In contrast, the vinyl ether analogue of 1-octadecenyl-2-palmitoylglycerophosphocholine was almost absent from the l-alk-1'-enyl-2-acylgly cerophosphocholines.