Keith L. Clay

On the metabolic acidosis of ethylene glycol intoxication.

AND MURPHY, R. C. (1977). Toxicol. Appl. Phnrmacol. 39, 39-49. Administration of ethylene glycol to pigtail monkeys (Macaca nemesrrina) or to dogs produced a severe metabolic acidosis. An assay for glycolic acid was developed and used to show that the decrease in blood bicarbonate concentrations was matched by increased blood glycolate concentrations. Treatment of the monkeys with 4-methylpyrazole prior to administration of ethylene glycol prevented metabolic acidosis. Treatment with 4-methylpyrazole after an acidosis had been produced with ethylene glycol resulted in restoration of normal acid-base balance as blood glycolate concentrations decreased. The contribution of other possible ethylene glycol metabolites to the metabolic acidosis was determined to be negligible.

Experimental methanol toxicity in the primate: Analysis of metabolic acidosis

Abstract Methanol was administered to rats, rhesus monkeys ( Macaca mulatta ), and pigtail monkeys ( Macaca nemestrina ). Of these animals, only the pigtail monkey reliably developed a severe metabolic organic acidosis resembling that observed in humans. Blood and urine specimens drawn from methanol-treated pigtail monkeys were analyzed for organic acid content by gas chromatography-mass spectroscopy and specific assays were performed for formate. The anion gap resulting during methanol acidosis was accounted for in full by increased blood concentrations of formate. Systemic formate concentrations remained low in rats and monkeys which failed to become acidotic following methanol administration.

Phosphatidylethanolamine Has an Essential Role inSaccharomyces cerevisiae That Is Independent of Its Ability to Form Hexagonal Phase Structures

Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn). Mitochondrial Psd1p provides ∼90% of total cellular phosphatidylserine decarboxylase activity. When thePSD1 gene is deleted, the resultant strain(psd1Δ) grows normally at 30u2009°C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37u2009°C) or on the nonfermentable carbon source lactate, the growth of psd1Δ strains is minimal without ethanolamine supplementation. The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid. These results suggest that there is a critical level of PtdEtn required to support growth. This theory is supported by growth data revealing that a psd1Δ psd2Δ dpl1Δ strain can only grow in the presence of ethanolamine. In contrast, a psd1Δ psd2Δstrain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Δ psd2Δ cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn). PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn. The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content. The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by31P NMR.

Biosynthesis of platelet activating factor and 1-O-acyl analogues by endothelial cells

Mass spectrometric procedures have been used to measure 1-O-alkyl-2-O-acetyl-glycero-3-phosphocholine (PAF) and a structural analogue, 1-O-acyl-2-O-acetyl-glycero-3-phosphocholine, biosynthesis in stimulated human umbilical vein endothelial cells (HUVEC). The primary species of acetylated glycerophosphocholine detected were of the 1-O-acyl type, rather than PAF. The amounts of PAF synthesized were much less than reported in earlier studies. In addition, mass spectrometric procedures were used to profile the molecular species of glycerophosphocholine in HUVEC and to determine those species which have arachidonate in the sn-2 position. The pattern of the occurrence of arachidonate in the sn-2 position is very similar to the pattern of synthesis of the acetylated glycerophosphocholines. These results suggest that 1-O-acyl-2-O-acetyl-glycero-3-phosphocholines may have significant but unappreciated biological activities. These results also support the view that glycerophosphocholine molecular species which have arachidonate esterified at the sn-2 position are the immediate precursors for the hydrolysis-acetylation steps which result in the synthesis of sn-2 acetylated glycerophosphocholines, including PAF.

Acute keratinocyte damage stimulates platelet-activating factor production.

Abstract Recent evidence suggests that the phosphocholine-derived lipid mediator platelet-activating factor (PAF) is involved in keratinocyte function and cutaneous inflammation. PAF is found in various inflammatory skin diseases, and intradermal injection of PAF directly results in cutaneous inflammation. Keratinocytes also synthesize PAF and related 1-acyl species in response to ionophores, cytokines and growth factors, and in response to activation of the epidermal PAF receptor. Since keratinocytes are routinely exposed to potential damage by thermal or oxidative stressors with resultant induction of cutaneous inflammation, the objective of these studies was to assess whether exogenous thermal or oxidative damage can induce the production of PAF and related 1-acyl species. Cells of the immortalized human keratinocyte cell line HaCaT were subjected to acute heat or cold, or treatment with the pro-oxidant lipid tertiary butyl hydroperoxide, and PAF and 1-palmitoyl-2-acetyl-GPC were measured by gas chromatography/mass spectrometry. We report that these diverse toxic stimuli resulted in the accumulation of these biologically active lipids. These studies suggest that the PAF system is involved in the inflammatory response seen following acute epidermal damage.

Compounds biologically similar to platelet activating factor are present in stored blood components

Agents which prime the neutrophil NADPH oxidase develop during routine storae of whole blood and packed red blood cells. This plasma priming activity can be inhibited by bepafant (WEB 2170), a specific platelet activating factor (PAF) receptor antagonist. Quantitation of the priming agent(s), by a commercially available radioimmunoassay for PAF, reproducibly demonstrated high levels of PAF activity. However, analysis of these plasma samples from stored blood components by gas chromatography/mass spectroscopy did not reveal any 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. We conclude that the polyclonal antibody to PAF used in these studies may have recognized different epitopes of a family of heterogeneous, biologically active lipids that manifest their effects through the PFA receptor.

Binding of platelet activating factor to albumin

Binding of platelet activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) to albumin is an important facet of the biological activity of this phospholipid. Measurement of that binding has been hampered by the physical nature of the lipid, which made estimation of the free and bound concentrations difficult. With the use of ultracentrifugation to generate an albumin gradient and to produce a region free of protein, the successful measurement of free PAF and PAF bound to albumin was accomplished. This study has demonstrated that PAF binds to albumin at four binding sites and that the average equilibrium dissociation constant for this binding is 1.10(-7) M. Consideration of these data has led to the hypothesis that the receptor active form of PAF is the albumin-PAF complex, rather than free PAF.

Copper-Catalyzed Oxidation Mediates PAF Formation in Human LDL Subspecies: Protective Role of PAF:Acetylhydrolase in Dense LDL

Free radical-mediated oxidation of cholesterol-rich LDL plays a key role in atherogenesis and involves the formation of oxidized phospholipids with proinflammatory biological activity. We evaluated the production of platelet-activating factor (PAF), a potent inflammatory mediator, in human LDL subspecies on copper-initiated oxidation (4 mumol/L CuCl2, 80 micrograms/mL for hours at 37 degrees C). PAF formation was determined by biological assay of HPLC-purified lipid extracts of copper-oxidized lipoproteins; chemical identity was confirmed by gas chromatographic and mass spectrometric analyses. PAF, characterized as the C16:0 molecular species, was preferentially produced in intermediate LDL (d = 1.029 to 1.039 g/mL) (8.6 +/- 5.7 pmol PAF/3 h per mg LDL protein) and light LDL (d = 1.019 to 1.029 g/mL), but was absent from dense LDL particles (d = 1.050 to 1.063 g/mL). As PAF:acetylhydrolase inactivates PAF and oxidized forms of phosphatidylcholine, we evaluated the relationship of lipoprotein-associated PAF:acetylhydrolase to PAF formation. We confirmed that PAF:acetylhydrolase activity was elevated in native, dense LDL (41.5 +/- 9.5 nmol/min per mg protein) but low in LDL subspecies of light and intermediate density (d 1.020 to 1.039 g/mL) (3.5 +/- 1.6 nmol/min per mg protein) [Tselepis et al, Arterioscler Thromb Vasc Biol. 1995;15:1764-1773]. On copper-mediated oxidation for 3 hours at 37 degrees C, dense LDL particles conserved 20 +/- 14% of their initial enzymatic activity; in contrast, PAF:acetylhydrolase activity was abolished in light and intermediate LDL subspecies. Clearly, the elevated PAF:acetylhydrolase activity of dense LDL efficiently diminishes the potential inflammatory role of endogenously formed PAF; nonetheless, formation of proatherogenic lysophospholipids results. In contrast, LDL particles of the light and intermediate subclasses can accumulate PAF on oxidative modification.

Preparation of labeled molecules by exchange with oxygen-18 water.

Publisher Summary This chapter discusses the synthesis of 18 O-labeled molecules to equilibrium exchange reactions with H 2 18 0. Several functional groups containing oxygen have the property of undergoing exchange of their oxygen atoms with water and these reactions can be catalyzed by both chemical and enzymatic means. Examples of such moieties are carboxylic acids, ketones, phenols, and phosphate esters, and it should be noted that the rates of 18 O exchange differ for each of the functional groups. The combined use of stable isotope-labeled molecules and mass spectrometry has been a powerful means to address numerous problems in chemistry and biochemistry which had been difficult or impossible to probe by other techniques. Molecules labeled with 18 O have been used in numerous mass spectrometric studies from the classic determination of the fate of the carboxyl oxygen atoms in ester hydrolysis, mechanistic studies of gas-phase ion decompositions, internal standards for quantitative mass spectrometric analysis, and detailed studies of chemical and biochemical reaction mechanisms.

Tandem mass spectrometry of negative ions from choline phospholipid molecular species related to platelet activating factor

Fast atom bombardment mass spectrometry of choline phospholipids produces negative ions characteristic of the intact molecule and tandem mass spectrometry of collision-induced decomposition of M-15 anions characterizes both the identity and substituent position of radyl groups. Certain choline phospholipid molecular species which may be of special interest in the generation of platelet activating factor contain a highly unsaturated fatty acyl substituent atsn-2 and an ether radyl group atsn-1; other choline phospholipid molecular species which contain esterified arachidonic acid are of interest as potential sources of arachidonate for eicosanoid biosynthesis. Collisional activated decomposition of 1-hexadecanoyl-2-arachidonoyl-sn-glycerophosphocholine produce abundant carboxylate anions atm/z 303 (arachidonate) andm/z 255 (hexadecanoate) in a ratio of 3∶1, diagnostic for thesn-2 arachidonoyl position. The ether analog, 1-O-hexadecyl-2-arachidonoyl glycerophosphocholine, produces only one collision-induced dissociation ion atm/z 303 and no product ions corresponding to the ether substituent atsn-1. Molecular weight information from the M-15 ion combined with the CID generated carboxylate anions completely characterize these important phospholipids.Precursor ion studies of M-15 anions from glycerophosphocholine lipids indicate that this ion is derived directly from a unique adduct ion formed by attachment of the molecular species to a matrix alkoxide ion, neutralizing the positive charge of the quaternary choline nitrogen. Decomposition of this adduct ion yields a methylated matrix molecule and the nominal M-15 ion.