Analysis of adipose tissue lipid using mass spectrometry.

Mass spectrometry technology has enabled significant advances in detailing the alterations of the lipidome in response to pathological conditions or experimental manipulations. Lipids comprise a wide range of compounds with functions that include structural, intracellular signaling, trafficking, and storage. Characterization of lipid species has evolved significantly over recent years due to the progress made in the area of mass spectrometry. This chapter details the methods used for the analysis of lipids tailored to the intrinsic characteristics of adipose tissue. Particular attention is given to the analysis of triglycerides, diacylglycerols, and phospholipid.

A surface-tethered spheroid model for functional evaluation of 3T3-L1 adipocytes.

In order to effectively treat obesity, it must be better understood at the cellular level with respect to metabolic state and environmental stress. However, current two-dimensional (2D) in vitro cell culture methods do not represent the in vivo adipose tissue appropriately due to the absence of complex architecture and cellular signaling. Conversely, 3D in vitro cultures have been reported to have optimal results mimicking the adipose tissue in vivo. The main aim of this study was to examine the efficacy of a novel conjugate of a genetically engineered polymer, elastin-like polypeptide (ELP) and a synthetic polymer, polyethyleneimine (PEI), toward creating a 3D preadipocyte culture system. We then used this 3D culture model to study the preadipocyte differentiation and adipocyte maintenance processes when subjected to various dosages of nutritionally relevant free fatty acids with respect to total DNA and protein content, cell viability, and intracellular triglyceride accumulation. Our results showed that 3T3-L1 preadipocytes cultured on the ELP-PEI surface formed 3D spheroids within 72 h, whereas the cells cultured on unmodified tissue culture polystyrene surfaces remained in monolayer configuration. Significant statistical differences were discovered between the 3D spheroid and 2D monolayer culture with respect to the DNA and protein content, fatty acid consumption, and triglyceride accumulation, indicating differences in cellular response. Results indicated that the 3D culture may be a more sensitive modeling technique for in vitro adipocyte culture and provides a platform for future evaluation of 3D in vitro adipocyte function.

Effect of processing temperature on the morphology and drug-release characteristics of elastin-like polypeptide-collagen composite coatings.

Elastin-like polypeptides (ELPs) exhibit an inverse phase transition temperature (Tt) in response to changes in their environment. We hypothesized that processing ELP-collagen composites at temperatures higher than the Tt of ELP (∼32 °C) will affect their microstructure and subsequently, achieve tunable release of model drugs. The composite coatings were prepared by formation of ELP-collagen hydrogels at 37 °C, incubation at 37, 45, or 55 °C, and finally air-drying at 37 °C. Scanning electron micrographs revealed that the fabrication process affected both the collagen and ELP microaggregate phases. A gradual time dependent bovine serum albumin (BSA) release that followed the power law and a burst antibiotic doxycycline release followed by a linear zero-order release were observed. Importantly, BSA and doxycycline releases were dependent on the ELP microaggregate size, which was governed by the processing temperatures. This study lays the foundation to achieve optimized composite microstructures by controlling processing conditions for drug delivery applications.

20-Hydroxyeicosatetraenoic acid inhibition attenuates balloon injury-induced neointima formation and vascular remodeling in rat carotid arteries.

20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling after balloon injury (BI). Sprague Dawley rats underwent BI of the common carotid artery and were treated with vehicle, 1-aminobenzotriazole (ABT, 50 mg/kg i.p. once daily), or HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (2 mg/kg s.c. twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography, and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61 and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries, and the levels were markedly suppressed in the groups treated with 1-ABT and HET0016 (P < 0.001). Immunostaining revealed that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries, and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation, resulting in a marked reduction in IH and vascular remodeling after endothelial injury.

Mass spectrometric detection of CYP450 adducts following oxidative desulfuration of methyl parathion.

Cytochrome P450 (CYP)-mediated desulfuration of methyl parathion results in mechanism-based inhibition of the enzyme. Although previous data suggest that reactive sulfur is released and binds to the apoprotein, the identities of neither the adduct(s) nor the affected amino acid(s) have been clearly determined. In this study, nanospray tandem mass spectroscopy was used to analyze peptide digests of CYP resolved by SDS-PAGE from liver microsomes of male rats following incubation in the absence or presence of methyl parathion. Oxidative desulfuration was confirmed by measurement of methyl paraoxon, and inhibition of specific CYP isozymes was determined by measurement of testosterone hydroxylation. Total CYP content was quantified spectrophotometrically. Incubation of microsomes with methyl parathion decreased CYP content by 58%. This effect was not associated with a comparable increase in absorbance at 420 nm, suggesting the displacement of heme from the apoprotein. Rates of testosterone 2ß- and 6ß-hydroxylation, respectively, were reduced to 8 and 2%, implicating CYP3A and CYP2C11 in the oxidative desulfuration of methyl parathion. Mass spectrometric analysis identified 96 amu adducts to cysteines 64 and 378 of CYP3A1. In addition, a peptide containing cysteine 433 that coordinates with heme was possibly modified as it was detected in control, but not methyl parathion samples. A comparison of rat CYP3A1 with human CYP3A4 suggests that cysteines 64 and 378 reside along the substrate channel, remote from the active site. Alteration of these residues might modulate substrate entry to the binding pocket of the enzyme.

Vitamin D Supplementation Suppresses Hypoxia-Stimulated Placental Cytokine Secretion, Hypertension and CD4+ T Cell Stimulation in Response to Placental Ischemia.

OBJECTIVE: To investigate a role of Vitamin D in the pathogenesis of preeclampsia (PE), and to discern any potential benefits of Vitamin D supplementation on hypertension in the RUPP rat model of PE. STUDY DESIGN: Blood and placentas from normal pregnancies (NP) and PE were collected following elective cesarean delivery without evidence of infection. Circulating Vitamin D was extracted by HPLC and measured via mass spectrometry. Media for placenta explants was supplemented with Vitamin D and exposed to hypoxic (1% O2) or normoxic (6% O2) conditions for 24 hours. ELISAs were performed on media and normalized to total protein to determine cytokine secretion. RUPP rats were supplemented with vitamin D by oral gavage, and blood pressure (MAP) and pup weights were measured in NP and RUPP rats with or without Vitamin D supplementation. Flow cytometry was used to evaluate CD4+ Tcells in control RUPP rats and RUPP rats treated with Vitamin D. RESULTS: Inflammatory cytokine secretion was higher (p<0.05) while the anti-inflammatory cytokine, IL-10, was significantly lower in the media of PE placentas compared to NP (p=0.005). Vitamin D supplementation decreased hypoxia stimulated pro-inflammatory cytokine secretion (p=0.003) in the media of PE placentas. Vitamin D decreased MAP and circulating CD4+ T cells in the RUPP rat model of PE (p<0.05). CONCLUSION: Vitamin D supplementation may be useful in the treatment or prevention of hypertensive disorders in pregnancy.

Determination of derivatized urea in exhaled breath condensate by LC-MS.

Elevation in one or more compounds in exhaled breath condensate (EBC) has been reported to be related to one or another lung disease. The increased concentration might be caused by increased chemicals in the airway surface liquid. However, it might also be due to an increased delivery of liquid samples into the airstream. Being evenly distributed throughout the body, urea is a likely candidate for a marker of such dilution. A liquid chromatography-tandem mass spectrometry method was developed for determination of EBC urea. Urea in EBC samples was converted to 2-hydroxypyrimidine (2-HPM) through a one step reaction, along with (15)N(2)-urea added as an internal standard. The product ion m/z 97/42 was selected for quantification with m/z 99/43 from (15)N(2)-2-HPM as a standard. Concentrations of urea in EBC from five lung cancer patients were found to be 35.1, 2.2, 103.5, 19.3, and 3.6 microM, respectively. The highest values were in patients dying of respiratory distress, whose lungs were filled with fluid. Lower values were seen in patients whose conditions were improving. Lately, one of the low EBC urea values was observed in a patient whose airway status did not contribute to his poor clinical condition.

Phorbol ester differentiates the levels of [3H]MK-801 binding in rats lines selected for differential sensitivity to the hypnotic effects of ethanol.

These studies addressed the possible involvement between sensitivity to the hypnotic action of ethanol and function of the NMDA receptor. The studies were carried out using high-alcohol sensitive (HAS) and low-alcohol sensitive (LAS) rats, two rats having differential sensitivity to the acute hypnotic action of ethanol. The animal models were developed by a selective breeding experiment. Using a quantitative autoradiograph technique, it was demonstrated that [3H]MK-801 binding to the NMDA receptor was highest in hippocampus in both HAS and LAS rats, but significant [3H]MK-801 binding was also detected in cortex, caudate-putamen, and thalamus of HAS and LAS rats. The density of [3H]MK-801 binding was lower only in cerebellar granule layers of untreated HAS rats as compared to the same brain area in untreated LAS rats. Activation of protein kinase C (PKC) by 100 nM PDBu, increased [3H]MK-801 binding in cortex, caudate-putamen, thalamus, central gray, and cerebellum of HAS rats but activation of PKC did not influence [3H]MK-801 binding in LAS rats. These activation of PKC differentiates between [3H]MK-801 binding of HAS and LAS rats in frontal cortex (layer II-IV and cingulate), caudate-putamen, and ventral lateral thalamic nuclei. The basal level of PKC-gamma mRNA was higher in HAS rats than that of LAS rats. These results suggest that the activation of PKC potentiates NMDA receptor function of the rat line which is more sensitive to alcohol (HAS) but does not affect [3H]MK-801 binding of alcohol resistant (LAS) rats.

Use of liquid chromatography-tandem mass spectrometry for the analysis of pentoxifylline and lisofylline in plasma.

A method was developed for the quantitation of pentoxifylline [1-(5-oxohexyl)-3,7-dimethylxanthine] and a primary active metabolite, lisofylline [1-(5-hydroxyhexyl)-3,7-dimethylxanthine], using high-performance liquid chromatography (HPLC)-tandem mass spectrometry. This method was developed in order to overcome problems encountered with HPLC-ultraviolet detection. The operating parameters of the electrospray interface (PE SCIEX, TurboIon Spray) and lens voltages of the triple-quadrupole detector (PE SCIEX 365) were optimized in positive ion mode to obtain the best sensitivity of the analytes. Collision-induced dissociation was used to produce fragment ions, and multiple reaction monitoring was used to quantitate pentoxifylline (m/z 279/181) and lisofylline (m/z 263/181). Dichloromethane was used to extract the drug, metabolite, and the internal standard (3-isobutyl-1-methylxanthine) from plasma. A reverse-phase C8(2) 150 x 1.0 mm HPLC column was used to resolve all three compounds in less than 6 min. Calibration curves were generated using peak area and were linear from 1 to 1000 ng/mL (R(2) > 0.99). The small sample volume, ease of extraction, and sensitivity provide advantages over more conventional methods of quantitation.

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle.

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.

Catecholamines and 5-hydroxytryptamine in tissues of the rabbit exocrine pancreas.

OBJECTIVES: Norepinephrine (NE), dopamine (DA), epinephrine (Epi), and 5-hydroxytryptamine (5-HT) all modulate pancreatic exocrine secretion, yet their concentrations in specific tissues of the exocrine pancreas are unknown. METHODS: Concentrations of catecholamines and 5-HT in rabbit pancreatic ganglia, acini, ducts and ampullae, and arteries and veins were measured using HPLC. RESULTS: Concentrations of NE in ganglia from the head/neck region were significantly higher than those from the body (1620 +/- 220 vs. 778 +/- 179 pmol/mg protein). Acini contained little NE, DA, or 5-HT (9 +/- 2, 0.9 +/- 0.2, 13 +/- 5 pmol/mg protein). Ducts and ampullae contained NE (314 +/- 74 and 156 +/- 24 pmol/mg protein), DA (43 +/- 14 and 13 +/- 4 pmol/mg protein), Epi (63 +/- 29 and 39 +/- 6 pmol/mg protein), and 5-HT (696 +/- 151 and 3563 +/- 288 pmol/mg protein). Arteries and veins contained the highest concentrations of NE (1962 +/- 463 and 736 +/- 80 pmol/mg protein, respectively). CONCLUSIONS: Pancreatic ganglia and blood vessels, rather than acini, are the main sites of noradrenergic sympathetic innervation of the rabbit exocrine pancreas. These nerves preferentially target ganglionic transmission in the head/neck versus the body. Serotonergic nerves provide little or no innervation of rabbit pancreatic ganglia or acini.

Arachidonoyl-phospholipid remodeling in proliferating murine T cells.

BACKGROUND: Previous studies have shown that the functional capacity of T cells may be modulated by the composition of fatty acids within, and the release of fatty acids from membrane phospholipids, particularly containing arachidonic acid (AA). The remodeling of AA within membrane phospholipids of resting and proliferating CD4+ and CD8+ T cells is examined in this study. RESULTS: Splenic T cells were cultured in the presence or absence of anti-CD3 mAb for 48 h then labeled with [3H]AA for 20 min. In unstimulated cells, labeled AA was preferentially incorporated into the phosphoglycerides, phosphatidylcholine (PC) followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). During a subsequent chase in unlabeled medium unstimulated CD4+ and CD8+ T cells demonstrated a significant and highly selective transfer of free, labeled AA into the PC pool. In contrast, proliferating CD4+ and CD8+ T cells distributed labeled [3H]AA predominantly into PI followed by PC and PE. Following a chase in AA-free medium, a decline in the content of [3H]AA-PC was observed in association with a comparable increase in [3H]AA-PE. Subsequent studies revealed that the cold AA content of all PE species was increased in proliferating T cells compared with that in non-cycling cells, but that enrichment in AA was observed only in the ether lipid fractions. Finally, proliferating T cells preincubated with [3H]AA exhibited a significant loss of labeled arachidonate in the PC fraction and an equivalent gain in labeled AA in 1-alk-1'-enyl-2-arachidonoyl-PE during a chase in unlabeled medium. CONCLUSION: This apparent unidirectional transfer of AA from PC to ether-containing PE suggests the existence of a CoA-independent transacylase system in T cells and supports the hypothesis that arachidonoyl phospholipid remodeling may play a role in the regulation of cellular proliferation.

Effects of chronic dermal exposure to nonlethal doses of methyl parathion on brain regional acetylcholinesterase and muscarinic cholinergic receptors in female rats.

The in vivo and in vitro effects of methyl parathion, a phosphorothionate insecticide, on cholinergic neurotransmitter systems in the brain of rats were investigated. Three groups of adult female rats received 0, 0.1, or 1.0 mg/kg methyl parathion via dermal exposure for 95 days. Exposure to 0.1 mg/kg methyl parathion produced inhibition of AChE in the caudate-putamen and thalamic nuclei, whereas 1.0 mg/kg resulted in inhibition of AChE in most brain regions. The same doses of methyl parathion had no effect on [(3)H]QNB binding to muscarinic receptors in the brain regions examined. The in vitro study demonstrated that methyl parathion causes preferential inhibition of AChE and [(3)H]QNB binding in specific brain regions. As an inhibitor of AChE, methyl paraoxon was 1,000-fold more potent than was methyl parathion. Similarly, methyl paraoxon showed brain region-specific inhibition of the enzyme. Generally, the brain stem was highly sensitive to organophosphate-induced inhibition of AChE activity and [(3)H]QNB binding. Because central respiratory neurons gather in the brain stem, preferential effects there and in other brain regions may underlie lethal toxicity of methyl parathion and other organophosphates.

Pharmacokinetics of methyl parathion: a comparison following single intravenous, oral or dermal administration.

Assessment of the risks posed by the residential use of methyl parathion requires an understanding of its pharmacokinetics after different routes of exposure. Thus, studies were performed using adult female rats to define the pharmacokinetic parameters for methyl parathion after intravenous injection and to apply the described model to an examination of its pharmacokinetics after single oral or dermal exposure. The pharmacokinetics of methyl parathion after intravenous administration (1.5 mg/kg) were best described by a three-compartment model; the apparent volume of the central compartment was 1.45 liters/kg, clearance was 1.85 liters/h/kg and the terminal half-life was 6.6 h with an elimination constant of 0.50 h(-1). The apparent oral absorption coefficient for methyl parathion (1.5 mg/kg) was 1.24 h(-1), and its oral bioavailability was approximately 20%. The latter likely includes a significant first pass effect. Concentrations of methyl parathion increased during the initial 10-60 min and then declined during the next 15-36 h. After dermal administration (6.25-25 mg/kg), methyl parathion concentrations peaked within 12-26 h and then declined dose dependently. The apparent dermal absorption coefficient was approximately 0.41 h(-1), and only two pharmacokinetic compartments could be distinguished. In conclusion, the pharmacokinetics of methyl parathion are complex and route dependent. Also, dermal exposure, because of sustained methyl parathion concentrations, may pose the greatest risk.

A comparison of cholinesterase activity after intravenous, oral or dermal administration of methyl parathion.

Time-dependent changes in blood cholinesterase activity caused by single intravenous, oral or dermal administration of methyl parathion to adult female rats were defined. Intravenous and oral administration of 2.5 mg/kg methyl parathion resulted in rapid (<60 min) decreases in cholinesterase activity which recovered fully in vivo within 30-48 h. In contrast, spontaneous reactivation of cholinesterase in vitro was complete within 6 h at 37 degrees C. Dermal administration of methyl parathion caused dose-dependent inhibition of cholinesterase activity which developed slowly (> or =6 h) and was prolonged (> or =48 h). Time- and route-dependent effects of methyl parathion on cholinesterase activity in brain and other tissues generally paralleled its effects on activity in blood. In conclusion, pharmacodynamics of methyl parathion differ substantially with route of exposure. Recovery of cholinesterase in vivo after intravenous or oral exposure may partially reflect spontaneous reactivation and suggests a rapid clearance of methyl parathion or its active metabolite methyl paraoxon. The more gradual and prolonged inhibition of cholinesterase caused by dermal administration is consistent with disposition of methyl parathion at a site from which it or methyl paraoxon is only slowly distributed. Thus, dermal exposure to methyl parathion may pose the greatest risk for long-term adverse effects.

Ethanol-enhanced cytotoxicity of alkylating agents.

BACKGROUND: Although ethanol itself is not genotoxic, chronic alcohol consumption increases the risk of neoplastic disease. The mechanism by which ethanol exerts a cocarcinogenic effect is not well established, and the aim of this study was to determine whether exposure to ethanol increased the cytotoxicity of known carcinogens. METHODS: To assess cell survival, the ability of Chinese hamster A10 cells, which express alcohol dehydrogenase, to form colonies was determined after exposure to ethanol and other substances, including both genotoxicants and non-DNA-reactive cytotoxic agents. RESULTS: 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) is an alkylating agent that forms covalent bonds with DNA. The cytotoxicity of MNNG at concentrations of 0.17 to 0.68 microM was markedly enhanced when cells were also treated with 50 mM ethanol. When combined with 0.34 microM MNNG, concentrations of ethanol as low as 2 mM exacerbated the toxicity of this alkylating agent. When these experiments were repeated in the presence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, pretreatment with ethanol did not affect the toxicity of MNNG. When ethanol treatment was combined with exposure to other carcinogens, as well as agents that do not directly damage DNA, the cytotoxicity of the DNA-reactive agents 4-nitroquinoline-N-oxide, mitomycin C, and 6-chloro-9-(3-[2-chloroethyl]aminopropylamino)-2-methoxyacridine was modestly enhanced, and that of a second alkylating agent, ethyl methanesulfonate, was markedly increased. CONCLUSIONS: The results are consistent with impairment of DNA repair processes, particularly base excision repair, by acetaldehyde, as a mechanism by which ethanol increases the genotoxicity of certain genotoxic agents.