The measurement of nanogram amounts of acetylcholine in tissues by pyrolysis gas chromatography

Abstract— A method previously described for measuring ACh in biological effluents has been simplified and extended for use with tissues. The tissue is homogenized in acetonitrile containing propionylcholine as the internal standard and after centrifugation the acetonitrile is removed by shaking with toluene. To the aqueous solution is added a solution of KI-I₂ to precipitate the quaternary compounds. The precipitate is dissolved in aqueous acetonitrile and then drawn through a small column of ion-exchange resin to convert the periodides of the quaternary compounds to chlorides which are then simultaneously pyrolysed and gas chromatographed. On the column the pyrolytic product of choline has a slower retention time than that of acetylcholine; under these circumstances the choline present in tissues does not obscure the measurement of acetylcholine. Specificity was demonstrated by several procedures including mass spectroscopy. The method can measure 25 ng (171 pmoles) of acetylcholine in extracts of brain, simply, and with high reproducibility. With the usual gas chromatograph, 16 samples can be run in a working day. The content of acetylcholine in rat brain was 26.4 nmol/g or almost precisely the values found with other gas chromatographic methods. The pyrolytic method was shown to be applicable to the detection of biologically interesting substances other than choline esters, including betaine, carnitine and the non- quaternary compound, ?-aminobutyric acid, which is readily converted to a volatile compound (probably its methyl ester) when pyrolysed in the presence of tetramethylammonium hydroxide. Of additional general interest is the demonstration of the advantages of acetonitrile as a solvent for extracting water-soluble compounds from tissues.     

Quantitative analysis of hyaluronate in nanogram amounts.

A radiosorbent technique allowing the specific analysis of 10 ng of hyaluronate has been developed.     

Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues

Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w/v, in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng.microliter-1, respectively.     

The viroimmunotest for mass determinations of nanogram amounts of antigens

Simple highly sensitive screening variant of viral immunoassay for detection of nanogram quantity of antigen is proposed. The antigen linked with antibodies adsorbed on polystyrene 96-well plates was revealed by conjugate of Fab' fragments of antibodies with phage phi X174. The concentration of the antigen could be detected qualitatively and quantitatively (from 0.3 ng/ml to 1000 ng/ml).     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

Quantitative analysis of volatile halothane metabolites in biological tissues by gas chromatography

A simple and sensitive gas chromatographic method for the determination of 2-chloro-1, 1-difluoroethylene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE), two highly volatile metabolites of halothane, in blood, liver and isolated hepatic microscomes is described. The entire head-space in equilibrium with a known volume or weight of the sample is injected into the gas chromatograph equipped with a flame ionization detector. Quantification is accomplished with standards prepared by fortifying blank samples with known concentrations of CDE and CTE which are treated under the same conditions as the samples. Detection limits for CDE and CTE were 2 pmole/ml in blood and 10 pmole/g in liver and the mean relative standard deviations are no greater than ± 6% except for CTE in hepatic microsomes (± 9%). A preliminary study of blood CDE and CTE levels in humans anesthetized with halothane is reported.     

Determination of hydrogen sulfide and acid-labile sulfur in animal tissues by gas chromatography and ion chromatography

A sensitive and reliable method was developed for the determination of hydrogen sulfide and acid-labile sulfur (ALS) in animal tissues using gas chromatography with flame photometric detector (GC-FPD) and ion chromatography (IC). Hydrogen sulfide trapped in alkaline solution was determined by GC-FPD as hydrogen sulfide or by IC as sulfate after oxidation with hydrogen peroxide. Sodium sulfide used as a source of hydrogen sulfide was standardized by IC. Fresh rat liver and heart tissues contained 112.2±23.0 and 274.1±34.6 nmol/g of ALS respectively. Free hydrogen sulfide was not detected.     

A silver-binding assay for measuring nanogram amounts of protein in solution

We recently reported a highly sensitive assay for measuring protein in solution based on the capacity of glutaraldehyde-treated protein to bind silver. This assay has now been made more sensitive, with a lower limit of detection of 5 ng, and more reproducible by supplementing protein samples with sodium dodecyl sulfate (SDS) to reduce protein loss to glassware. Two procedures have been developed. In one, protein samples are supplemented with both SDS and Tween 20 to yield very steep protein dose-response curves, which allow for more precise protein determinations, and very stable color formation, permitting OD measurements to be made several hours after the assay has been completed. In the second procedure, protein samples are supplemented with SDS alone which results in a less steep dose-response curve and less stable color formation but makes the assay substantially more tolerant of interfering substances. Thus, proteins in most commonly used buffers can be assayed directly with the second procedure without the need for buffer exchange. The procedure of choice, therefore, depends on the type and concentration of interfering substance. Proteins in buffers totally incompatible with either assay procedure (e.g., those containing reducing agents) can be easily buffer exchanged by centrifugation through 0.2% SDS equilibrated, drained Bio-Gel P-2 beads. The clinical utility of this improved assay is demonstrated by the accurate quantitation of protein in 0.5 μl of samples of human cerebral spinal fluid. This assay should therefore prove especially useful when a limited amount of protein is available for quantitation.     

Quantitation of nanogram amounts of nucleic acids in the presence of proteins by the ethidium bromide staining technique.

Modification of the method for determining low amounts of RNA and DNA is proposed. It consists in nucleic acid staining in solution with EtBr (1 microgram/ml) followed by photography of 10 microliters drops on a UV-transparent plate under UV illumination. Densitometric measurements of the Polaroid negatives were used to construct standard concentration curves in the range of 1-16 micrograms/ml of DNA or RNA. This permitted to determine nucleic acid in amounts as little as 10 micrograms. The measurements were not influenced by the presence of proteins such as bovine serum albumin, DNase, RNase or proteinase K, thus the method proposed may be useful in determining the nucleic acid content of very small samples or of scarce biological material.     

Determination of lincomycin residues in salmon tissues by gas chromatography with nitrogen-phosphorus detection

A sensitive method for the determination of lincomycin residues in fish tissues is described. Lincomycin was extracted from fish tissues with phosphate buffer (pH 4.5). The extract was concentrated with a C₁₈ solid-phase extraction cartridge and further cleaned up by solvent extraction. Lincomycin was derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide to form a trimethylsilyl derivative before being analyzed by gas chromatography with nitrogen-phosphorus detection. Coumaphos was used as the internal standard. Assays showed good linearity in the range 25–250 ppb (ng/g) (r = 0.9994). Recoveries of fortified lincomycin at 50, 100 and 200 ppb were>80% with relative standard deviation <6%. The limit of detection of the method was 1.7 ppb and the limit of quantitation was 3.8 ppb.     

Efficient extraction and quantitative determination of nanogram amounts of cellular RNA

An assay for quantitating nanogram amounts of cellular RNA is described. RNA is efficiently extracted from cells, using RNA-free DNA as carrier, by conventional chloroform: phenol procedures and the nucleic acids are precipitated with ethanol. Isolated RNA is hydrolyzed by RNase T2 to ribonucleoside 3′-monophosphates which in turn are converted to 5′-³²P-labeled ribonucleoside 3′,5′-diphosphates in the presence of T4 polynucleotide kinase and [γ-³²P]ATP. Radiolabeled products are separated from remaining [γ-³²P]ATP by chromatography on polyethyleneimine-cellulose, located by autoradiography, excised from the chromatogram, and subjected to liquid scintillation counting to quantitate the amount of RNA. Using mouse liver ribosomal RNA as a standard, the assay is linear over a range of 0 to 64 ng of RNA. The assay has been used to determine the amount of RNA in fully grown mouse oocytes arrested at the dietyate stage of first meiotic prophase. Each oocyte contains 0.61 ± 0.05 ng of RNA and only 25 oocytes have been used for such assays.     

Analysis of physiological variations of piperidine levels in tissues by mass fragmentography

By use of a mass fragmentographic technique the distribution of piperidine in tissues and changes in levels under physiological conditions were examined. Only trace amounts of piperidine existed in the brain of several animal species, although higher concentrations were detected in other species. In the brain of rats and rabbits, some regional differences were seen and high concentrations were found in the striatum. Piperidine levels in the brain and adrenal gland similarly showed the daily and seasonal rhythmic fluctuations.