The impact of high-dose acetylcholine on bovine corneal epithelium.

PURPOSE: Ion channels are formed when acetylcholine (ACh) combines with specific receptors on the postsynaptic membrane. We are testing whether this occurs in vitro in corneal epithelial cells when exposed to ACh, using intracellular ascorbic acid (AA) as a tracer. METHODS: The corneal epithelium was separated from Bowman's membrane as an intact sheet with thermolysin (TL). These cells were incubated in a medium containing phospholine iodide (Phi) to block acetylcholinesterase (AChE) activity, and then, five different groups of specimens were examined: the basic level of ACh and AA in the cells was tested in groups I and II, respectively. In groups III-V, the culture medium was supplemented with AA, ACh and ACh+AA, respectively, and following 2 hr of incubation the cells were tested for AA. AA was determined by high-performance liquid chromatography (HPLC) with UV detection, and ACh by LC-MS/MS. RESULTS: The ACh concentration in the corneal epithelial cells was 23.7 ng/mg wet weight. AA values were as follows: preexposure 0.17 mg/g. After exposure to AA, ACh and ACh+AA, the values were 0.30, 0.12 and 0.21 mg/g, respectively. The AA concentration mechanism of the corneal epithelium was intact despite exposure to Phi and ACh. CONCLUSION: The main observation is that the AA content of the corneal epithelium drops in response to ACh exposure. Various explanations are discussed, in particular the possibility that ACh exposure may cause cell membrane leakage through pores (nicotinic receptors). This would be similar to the moderate membrane leakage (depolarization) in neural synapses in response to ACh release. Interesting in vivo consequences may occur when considering that AChE is extremely UV sensitive, as this substance could be a target at which actinic radiation may influence the ACh-AChE complex in this epithelium.

Absolute ProGRP quantification in a clinical relevant concentration range using LC-MS/MS and a comprehensive internal standard.

The objective of this study was to develop a method using liquid chromatography with tandem mass spectrometric detection for the absolute quantification of the small cell lung cancer biomarker ProGRP in human serum, using its tryptic signature peptide NLLGLIEAK. The samples were precipitated for most of its proteins using acetonitrile prior to tryptic digestion. Further sample clean-up and enrichment was achieved by the use of an on-line restricted access media column, followed by separation on a BioBasic C8 column. Detection and quantification was carried out by operating a triple quadrupole MS in the selected reaction monitoring mode. This setup allowed analysis of realistic samples and detections limits in human serum of 150 pg ProGRP on column. Using an internal standard derived from the parent ProGRP after acetylation of the lysine side chain allowed better quantification through variation correction in all sample pretreatment steps, trypsination included.

Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples.

Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.

Targeted determination of the early stage SCLC specific biomarker pro-gastrin-releasing peptide (ProGRP) at clinical concentration levels in human serum using LC-MS.

Pro-gastrin-releasing peptide (ProGRP) is used as a specific diagnostic marker for small cell lung cancer (SCLC), a rapidly growing neoplasm with high mortality. Our object was to develop an LC-MS method for the detection and quantification of ProGRP in human serum using the specific tryptic digestion product NLLGLIEAK (m/z 485.8 for [M + 2H](2+)). For this purpose the sample pretreatment, clean-up, enrichment, and LC-MS conditions were evaluated. Sample pretreatment was carried out using ACN precipitation to decrease the sample complexity. Although ProGRP (31-98) standards were soluble in 99% ACN, it showed that optimal signal intensities were obtained by adding ACN to the serum in a 1:1 ratio v/v. A simplified tryptic digest protocol was carried out using 100 mM triethanolamine buffer to ensure pH stability during the whole procedure. The simplified protocol also includes omission of reducing and alkylating reagents. Necessary additional sample clean-up was achieved by trapping NLLGLIEAK on a RAM column (ADS-C8) which was back-flushed onto the analytical BioBasic C8 column. Volume of injection, sample enrichment, and column capacity are among the factors optimized to reach a mass LOD of 150 pg on column (OC) ProGRP (31-98). Detection of ProGRP in the serum sample of a patient suffering from SCLC with a clinically relevant concentration shows the potential of the method in diagnosis, prognosis, and monitoring of the disease.

Acetylcholine in the corneal epithelium of diurnal and nocturnal mammals.

PURPOSE: To analyze the concentration of acetylcholine (ACh) in the corneal epithelium and aqueous humor of various diurnal and nocturnal mammals. METHODS: The following species were examined: roe deer, cattle, horse, human, moose, sheep, reindeer, wild boar, polecat, lynx, cat, and rat. ACh was determined by liquid chromatography-tandem mass spectrometry. RESULTS: ACh was present in significant amounts in the corneal epithelium of all diurnal mammals, the concentrations varying from one species to the next. By contrast, it was not detected in any of the nocturnal tissue samples. None of the species showed detectable amounts of ACh in the aqueous humor. CONCLUSIONS: The amount of ACh in the corneal epithelium varies from one species to the next, with diurnal species showing significant concentrations and nocturnal species no detectable concentrations.

Ion-pair mediated transport of angiotensin, neurotensin, and their metabolites in liquid phase microextraction under acidic conditions.

This paper discusses the behaviour of angiotensin 1 and neurotensin together with their metabolites in a three-phase liquid phase microextraction under acidic conditions. Variations in donor phase, organic phase, and acceptor phase are studied with extraction recovery as response variable. It is proved that for all peptides the transport across the organic phase is mediated by heptane-1-sulphonic acid. n-Octanol gave overall best results as organic phase. A donor phase volume of 1.0 mL was chosen as a compromise between optimal recovery and robustness of the LPME device. The optimal pH of the donor phase (using acceptor phase of pH 2) was found to be different for the peptides, which opens opportunities for selective sample preparation. Decreasing the acceptor phase pH to 1.0 resulted in increased extraction recoveries. On using 1.0 mL of donor phase containing 50 mM heptane-1-sulphonic acid pH 3, n-octanol as organic phase immobilized in the pores of the fibre, and 20 microL of acceptor phase containing 0.1 mol/L HCl, extraction recoveries up to 82% (enrichment factor = 41) were achieved. To our knowledge this is the first report on liquid phase microextraction of angiotensins and neurotensins.

Application of supplementary flow in comprehensive 2D liquid chromatography combining SEC and RPC.

A comprehensive two-dimensional HPLC system (SEC x RPC) was evaluated. Various model compounds with differing hydrophobicity (log D: -0.08 to 2.22) and size (MW: 194 to 66.0 x 10(3)) were used. In order to reduce the run time of the second dimension, and thereby optimize the number of runs per unit volume from the first dimension, short RPC columns were used (7.5 mm). This column size demanded a low concentration of methanol in the mobile phase from the first dimension, in order to avoid severe band broadening and solute loss. Secondary interactions on SEC make a high methanol concentration in the mobile phase a necessity. Up to 40% methanol was required to diminish non-ideal SEC behavior. These conditions were non-compatible with trapping of hydrophilic compounds on RPC. The use of a supplementary flow (0.1% TFA) mixed after the first dimension led to better peak shape and trapping of hydrophilic compounds in the second dimension. This demanded flow adjustment in SEC, which in turn caused performance improvement and an increase in analysis time, making more RPC separations possible. These factors contributed to a larger peak capacity.

Ion-pair mediated transport of small model peptides in liquid phase micro extraction under acidic conditions.

This paper discusses the behaviour of five small model peptides in a three phase (aqueous donor-organic-aqueous acceptor) liquid phase micro extraction system in relation to their physico-chemical properties (charge, hydrophobicity). It is proved that for all peptides transport over the organic phase is mediated by aliphatic sulphonic acids. Heptane-1-sulphonic acid gave the best overall recoveries. It appeared that peptides with hydrophobic properties (IPI) and a high number of positive charges (KYK) show good recoveries and are enriched in the acceptor phase. Variation in the pH (1.6-4.4) of the donor phase shows that there are peptide-dependent optimal pH-values for their recovery. Increasing pH in the acceptor phase shows that in most cases the recovery decreases due to decreased ion-pair mediated membrane transport. For KYK the partition between the organic phase and the aqueous acceptor-phase is also driven by the solubility in the aqueous acceptor phase. Increase of the ion strength of the acceptor phase did not affect the recovery of the peptides. Except for KYK, which showed decreased recovery when the ion strength increased. Another finding is that delocalisation of positive charge causes bad recovery, probably due to incomplete ion-pair-peptide complex formation.