Measurement of 14CO2 evolution during tissue oxidation in vitro; an alternative to the Kontes well

1. An alternative method to the use of the disposable Kontes well for trapping 14CO2 produced in the course of biological oxidations is described. 2. A polyethylene miniature scintillation vial was used to contain the hyamine hydroxide-impregnated filter paper wick. 3. The two methods are compared in their abilities to trap 14CO2 produced directly by acidification of sodium [14C]bicarbonate and during beta-oxidation of 1[14C] palmitic acid. 4. The miniature vial and Kontes well methods showed similar efficiencies in the trapping of 14CO2 (97% and 95%, respectively, on average) the radioactivity of which was determined in the miniature vial using 5 ml only of scintillation fluid compared with a minimum of 10 ml required by the standard scintillation vial used to accommodate the Kontes well. 5. The technical advantages of the suggested miniature vial system, during both incubation and counting stages, are discussed.     

A device for the liberation and determination of 14CO2

A simple closed system for the serial determination of 14CO2 in small volumes of fluid samples, is described. The device consists of commercially available scintillation vials and silicone tube seals. 14CO2 is selectively liberated by citric acid and absorbed in a scintillation vial by Hyamine. Experiments on the effect of dichloroacetate on pyruvate dehydrogenase activity in rat hindlimbs perfused with [1-14C]pyruvate demonstrate the applicability of the method.     

Determination of evolved 14CO2 in decarboxylase reactions with application to measurement of [14C]oxalic acid.

An assay is described for the determination of the radioactive purity of [14C]oxalic acid preparations and the quantity of [14C]oxalic acid in biological samples. In this method oxalate decarboxylase is used to convert oxalate to formate and CO2. The entire procedure is carried out in a scintillation vial. The 14CO2 released in the enzymic reaction is allowed to diffuse off in a fume hood following acidification. Scintillation fluid is added to reacted and unreacted vials and the radioactivity measured. The loss of radioactivity from the reacted versus the unreacted vials provides the quantity of evolved 14CO2. This value is equal to 50% of the [14C]-oxalate (dpm) present. The radioactive purity of four preparations of [U-14C]oxalic acid was 99.0% while a fifth batch had a purity of 88%. A single batch of [U-14C]oxalic acid had a radioactive purity of 99.0% following storage of an aqueous solution, at -20 degrees C for 7 years. Recovery of [14C]oxalic acid from rat fecal extracts was 101.3%. Eight replicate analyses of a [U-14C]oxalic acid preparation gave a coefficient of variation of 0.3%. Following subcutaneous infusion of [U-14C]oxalic acid to rats, 100.2 +/- 2.9%, mean +/- SD, of the 14C in fecal extracts was present as [14C]oxalic acid (n = 10). The procedure provides a rapid, sensitive, and specific method to determine [14C]oxalic acid. It avoids the time consuming and inconvenient procedure for trapping and counting the evolved 14CO2. The approach used to determine the evolved 14CO2 may find application in other radiochemical methods that require its measurement.     

体外~(14)C-尿素呼气试验检测胃内幽门螺杆菌感染研究

目的　建立诊断胃内幽门螺杆菌感染 (Hp)的体外 1 4 C-尿素呼气试验 (1 4 C- U BT)。方法　 47例 Hp阳性和 32例 Hp阴性患者接受测试 ,用口服微量胃液采集胶囊的办法收集胃液标本于一 10 m l无菌试管内 ,加入生理盐水 0 .5 m l和 18.5 k Bq1 4 C-尿素后立即加橡皮塞密封试管 ,室温放置反应 3h,注射器经橡皮塞注入 2 M H2 SO41.0 ml,使 1 4 CO2 释出。同一注射器回抽气体并立即注入装有 6 .5 ml的 1 4 CO2 搜集闪烁剂液闪瓶内搜集 1 4 CO2 ,最后在液体闪烁计数仪上作 1 4 C放射性测定。结果　 47例 Hp阳性病人 1 4 C放射性几何均数为 5 30 dpm,而 32例 Hp阴性者结果为 2 1dpm,二者相差 2 3倍 (Wilcoxon秩和检验 ,u=5 .5 976 ,P<0 .0 1)。以受试者工作特征曲线分析法得出判别阈值为 75 dpm ,对 Hp诊断的敏感性和特异性为 92 %(4 3/ 47)和 91% (2 9/ 32 )。结论　体外 1 4 C- UBT诊断 Hp感染具有高度的准确性 ,无放射性损伤之虞 ,可适用于临床诊断。     

A simple fast micromethod for measuring enzyme activities which release tritium as tritium water

The entire procedure is carried out in a counting vial by mixing the reagents as a 20- to 30-microliters drop in the cap of a counting vial, incubating, quenching the reaction, and then distilling the tritium water produced into the chilled vial, in which it is assayed after the addition of scintillation solvent and a clean cap. The application of this technique to the analysis of serum transaminases is described.     

Liquid Scintillation Vial for Cumulative and Continuous Radiometric Measurement of In Vitro Metabolism

A two-compartment vial is described in which suspensions of bacteria, cells, or tissues may be cultured and their growth and metabolism measured radiometrically by using a liquid scintillation counter. The device consists of a scintillation vial lined with a cylinder of scintillating paper into which is placed a sterilized inner culture vial containing a carbon-14 substrate. The assembled device can be carried by the sample transport systems of conventional liquid scintillation counters. Evolved (14)CO(2) is collected and measured cumulatively and continuously. The device can be constructed simply and economically from readily available reagents and glassware. Data are given on relative sensitivity and on the effect of the color and transparency of the inner vial. A pilot experiment with bacteria (Escherichia coli) is described.     

Scintillation counting of 14C-labeled soluble and insoluble compounds in plant tissue

A method is described for the liquid scintillation counting of 14C in plant tissues. Samples are fixed, in the scintillation vial, in a solution of ethanol and acetic acid (3:1) and decolorized with commercial bleach before the addition of scintillation liquid. The method was compared to other techniques of tissue oxidation or digestion and found to be equally effective at least with thin tissue samples. The technique is simple, rapid, and inexpensive and does not result in loss of 14C.     

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.     

Method for rapid screening of pesticide mineralization in soil

A method has been developed for the analysis of (14)CO(2) evolution from the mineralization of (14)C-labelled organic compounds in soil samples. The new method is less space demanding and substantially cuts down laborious manual work compared to the traditional incubation bottle method used. Furthermore, the use of scintillation cocktail is largely reduced with the new method. In the new method, (14)CO(2) is trapped in filter paper held in the lid of a 20 ml glass vial by surface tension. The trapping solution used is Ca(OH)(2), which fixates CO(2) in the filter paper and the analysis of trapped (14)CO(2) is done using the Cyclone trade mark Storage Phosphor system. The lids are placed in a 32 well holder and exposed to a phosphor screen prior to scanning in a Cyclone trade mark scanner. The new filter method has been tested and compared to results obtained using the traditional method. The results show good agreement but due to a smaller capacity for CO(2) with the filter method compared to the traditional method, the interval between sampling has to be shorter using the filter method when the CO(2) development is high. The detection limits for the filter method is higher compared to the traditional method. With the filter method, the level of radioactivity has to exceed 300 dpm before detection is possible, while the same limit for the traditional method is around 30 dpm. On the other hand, the gas trapping faster and the efficiency is higher with the filter method.     

Development of a sensitive radiorespiration method for detecting microbial activity at subzero temperatures

We have developed a simple and sensitive method to detect microbial respiration at subzero temperatures. Microbial activity was detected by measuring (14)CO(2) evolved during the microbial-mediated mineralization of [1-(14)C] acetic acid or [2-(14)C] glucose in microcosm assays using modified (14)CO(2) traps. Various (14)CO(2) traps, designed to withstand freezing at subzero temperatures, were tested for their quench characteristics during liquid scintillation spectrometry and their ability to trap (14)CO(2). Solutions consisting of 1 M KOH supplemented with 20% or 30% v/v ethylene glycol did not freeze at temperatures above -20 degrees C and had a minor quenching effect on liquid scintillation spectrometry. Addition of ethylene glycol did have an effect on the efficiency of (14)CO(2) trapping, as the cumulative recovery of (14)CO(2) was reduced by 14% and 32% in the 1 M KOH+20% ethylene glycol and 1 M KOH+30% ethylene glycol solutions, respectively. Using the modified (14)CO(2) traps, microbial activity in representative Canadian high Arctic environmental samples was detected at temperatures as low as -15 degrees C. This simple method allows for sensitive, specific, and reliable detection of microbial activity occurring at subzero temperatures and is readily adaptable for studies in other cryoenvironments.     

A microassay for the measurement of androgen receptors in human prostatic tissue.

A microassay utilizing R 1881 (methyltrienolone) has been developed for the measurement of androgen receptor sites in the cytosol and nuclear extract of human prostatic tissue. Binding of R 1881 to the progesterone binding molecule in cytosol was eliminated by the addition of triamcinolone acetonide. Utilizing a six tube, single point assay, the number of binding sites estimated in nuclear extract averaged 95% of the number measured by a full 7 point Scatchard analysis; the number estimated by the microassay in cytosol averaged 91%. When the single point assay was applied to needle biopsy specimens (200 mg of tissue), the estimated number of binding sites in nuclei averageed 83% of the number measured in bulk tissue (2 grams) utilizing a 7 point Scatchard analysis; the number in cytosol estimated by the microassay on needle biopsy specimens averaged 73%. It is hoped that this technique may be useful in correlating receptor content with hormonal responsiveness in men with metastatic carcinoma of the prostate.     

A radioisotopic assay for polyamine oxidase

A new radioisotopic assay for polyamine oxidase with N1-acetylspermine as substrate is presented. A modified method for the chemical synthesis of radioactive N1-acetylspermine, which gave a good yield, is also described. The reaction mixture, containing N1-[14C]acetylspermine and tissue homogenate, was incubated for the enzyme reaction and applied to a minicolumn of Amberlite CG-50. The reaction product 3-[14C]-acetamidopropanal did not adsorb to the column, but passed through it; thus the eluate could be directly subjected to liquid scintillation counting. The blank levels were low and relatively constant even with crude tissue homogenates. The detection limit obtained was 0.05 nmol per tube. This method is simple, highly sensitive, and highly specific.     

Direct counting of tissues containing radioactive cholesterol by a two-phase liquid scintillation method

A rapid and simple method for counting radioactivity in tissue samples containing [³H]- or [¹⁴C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems.     

Apolipoprotein C-II synthesis as studied in explants of rat liver in culture

Adult male Sprague-Dawley rats (200-250 g) were used to study apolipoprotein C-II synthesis and secretion. Liver slices were prepared and incubated in RPMI 1629-medium (tissue amount and incubation time studies) and in Minimum Essential Medium (Eagle) with Earle's salts (hormone experiments). Incubation was performed in scintillation vials in a 95% O2-5% CO2 atmosphere, at 37 degrees C from 1 to 21 hr (2 and 4 hr with hormones). The hormones used and their amounts per millilitre medium were: oestradiol-17 beta 0.1 microgram, progesterone 3.0 micrograms and dexamethasone 1.5 micrograms. Apolipoprotein C-II was determined by specific double immunoprecipitation technique and TCA-insoluble protein fraction represented total protein. Optimal tissue amount was 100 mg/vial and the results show that liver slices quickly secrete the newly synthesized apo C-II (also total protein) into the surrounding medium. There were only minor differences between apo C-II values with the hormones used. The portion of apo C-II synthesis from total protein synthesis was 0.47-1.50%. After 4 hr incubation the [3H]leucine incorporation was almost equal for controls and hormone treated slices.     

Adsorbed soluble [3H]elastin as substrate for proteinase activity. A new microassay technique.

A microassay for elastase activity has been designed. Microtubes are coated with soluble elastin substrate by passive adsorption to the tube walls. On contact with enzymes in test samples, radioactivity is released into the sample buffer, which is then transferred to vials containing scintillation fluid without further processing. Porcine pancreatic elastase (EC 3.4.21.11) is easily detected in concentrations down to 8 micrograms/litre. The interassay coefficient of variation (CV) was below 10% for values above 50 micrograms/litre. The assay is rapid, precise, economical and sensitive.     

A radiochemical assay for beta-ureidopropionase using radiolabeled N-carbamyl-beta-alanine obtained via hydrolysis of [2-(14)C]5, 6-dihydrouracil.

A radiochemical assay was developed to measure the activity of beta-ureidopropionase in human liver homogenates which is based on the detection of the reaction product (14)CO(2) by liquid scintillation counting. Radiolabeled N-carbamyl-beta-alanine was prepared within 15 min by a simple hydrolysis of [2-(14)C]5, 6-dihydrouracil under alkaline conditions at 37 degrees C. The enzymatic reaction proved to be linear with time up to at least 3.5 h and protein concentrations up to at least 1 mg/ml. Human beta-ureidopropionase obeyed Michaelis-Menten kinetics with an apparent Km for N-carbamyl-beta-alanine of 15.5 +/- 1.9 microM. The assay proved to be very accurate and sensitive with an intraassay coefficient of variation of 2% and a detection limit of 28 pmol for the product CO(2).     

Metabolism of [1-(14)C]arachidonic acid in ruminant tissues during the pre- and postnatal periods of development under in vitro conditions]

After in vitro incubation of liver, skeletal muscle and adipose tissues from the fetuses and adult cattle as well as of placenta tissue with [1-14C]arachidonic acid, about 90% of the radioactive label was found in the lipids, 10%-in the prostaglandins, and 0.1%-in 14CO2. Arachidonic acid was utilized for the synthesis of lipids and prostaglandins in the majority of fetal tissues in a much greater degree, whereas that in energy-linked process--in a smaller degree compared with adult cattle tissues. [1-14C]arachidonic acid metabolism in the placenta and liver of the given species proceeds much more intensely than that in the skeletal muscles and adipose tissue. In tissues of adult animals [1-14C]arachidonic acid is predominantly utilized for the synthesis of phospholipids, whereas that in fetal tissues is utilized for the synthesis of phospholipids, cholesterol, esters and triglycerides.     

Highly specific micromethod for the enzymatic determination of radioactive [14C]lactate

By collecting released 14CO2 following the enzymatic decarboxylation of radiolabeled lactate, picomoles of the latter can be precisely, easily, and reproducibly measured in small biological fluids. This radioactive [14C]lactate microassay does not require a neutralization step, nor does it require chemical extractions and partioning procedures, ion exchange, or pyruvate derivatization. Under our specified conditions this simple reaction goes to completion in 90 min. Using this assay in porous adipose cells, with the cell number logarithmically less than that found in other literature methods, the measured glycolytic flux rates were consistent with those previously reported. In these studies, glycolysis was initiated with [U-14C]glucose 6-phosphate. This microradioactive lactate assay is useful when dealing with minute tissue samples and/or microliter aliquots of biological fluids.     

Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.

Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.     

Comparison of reactors for oxygen-sensitive reactions: reductive dechlorination of chlorophenols by vitamin b(12s)

Serum bottles are frequently used for studies of reductive dechlorination by vitamin B(12), but reducing conditions can be maintained only for several days. This time period is inadequate for evaluating the reductive dechlorination of some slow-reacting aromatic compounds. Sealed glass ampoules maintain reducing conditions for many months, but this method has the disadvantage of disallowing subsampling of the reaction mixture. A glass serum tube was modified for these experiments which not only maintained anoxic conditions for several days but also allowed subsamples to be removed during experiments. The modification was a restriction placed in the middle of the tube by heating in a flame, creating two chambers separated by a narrow neck. The lower chamber contained the oxygen-sensitive reaction mixture. The upper chamber, sealed with a septum and screw cap, was purged with purified nitrogen or argon introduced and vented through fused silica capillaries. Reductive dechlorination of chlorophenols by vitamin B(12) reduced with Ti(III) citrate was monitored in all three reactor types. Sealed ampoules maintained reducing conditions for up to 12 months. The two-chambered reactor maintained reducing conditions longer than the serum vials when frequent samples were taken.