On-line Radioactivity Detection Research Articles

Site specific radioiodination of recombinant hirudin

Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated −125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.

Profile of phosphatidylinositol metabolism stimulated by carbachol and glutamate in primary cultures of rat cerebellar neurons

The formation of inositol phosphates, after stimulation of primary cultures of cerebellar neurons of the neonatal rat, in the presence of lithium chloride, by glutamate, carbachol, norepinephrine, histamine and Mg 2+-free conditions, was measured by anion exchange high-pressure liquid chromatography (HPLC) with on-line radioactivity detection. All of the above agents caused a persistent, dose-dependent and calcium-sensitive preferential accumulation of inositol-4-phosphate, while the levels of inositol-1-phosphate were virtually unaffected. Agonist stimulation produced also a transient increase of a second peak which co-eluted with the standard for inositol 1,4-bisphosphate. However, no significant accumulation of inositol-1,4,5-trisphosphate and inositol-1,3,4,5-tetrakisphosphate was detected, possibly due to the fast kinetics of the metabolism of inositol phosphate. The results indicate that receptor-stimulated metabolism of inositol phosphate, in cultures of cerebellar granule cells, is due to a preferential hydrolysis of polyphosphoinositides and leads to the formation of inositol-4-phosphate through several calcium- and lithium-sensitive enzymatic steps.

Determination of 75Se-labelled selenite and metabolites in plasma and urine by high-performance liquid chromatography with on-line radioactivity detection.

A reversed-phase ion-pair high-performance liquid chromatographic method with on-line radioactivity detection for the simultaneous determination of 75Se-labelled selenite and metabolites has been developed. With this system a good resolution of various radiolabelled selenium complexes can be achieved. The detection limit of the radioactivity detector (signal-to-noise ratio = 3) is 49 pg of selenium (specific activity of 3 GBq/mg selenium) per millilitre of urine or plasma ultrafiltrate. The detector response is independent of both the chemical structure of the selenium complexes and the matrix composition of the samples. This method may serve as a reference system for other high-performance liquid chromatographic systems with less specific and sensitive detectors.

Determination of cisplatin and related platinum complexes in plasma ultrafiltrate and urine by high-performance liquid chromatography with on-line radioactivity detection

A reversed-phase ion-pair chromatographic method with on-line radioactivity detection for the simultaneous determination of 195mPt-labelled cisplatin and related platinum complexes has been developed. With this system a good resolution of various radiolabelled platinum complexes can be achieved. The detection limit of the radioactivity detector is 10 ng of cisplatin (specific activity of 15 MBq/mg cisplatin) per millilitre of urine or plasma ultrafiltrate. The detector response is independent of both the chemical structure of the platinum complexes and the matrix composition of the samples. This method may serve as a reference system for other high-performance liquid chromatographic systems with less specific and sensitive detectors.

Sensitive radiometric assay for chloramphenicol acetyltransferase using automated HPLC.

Chloramphenicol acetyltransferase (CAT) is widely used as a reporter element in studies of eukaryotic gene expression. We report a new technique for measurement of CAT activity that incorporates several advantages over the existing procedures from which it is derived. The combination of direct reverse-phase HPLC analysis, substitution of butyryl for acetyl coenzyme A substrate, automated sample loading and continuous on-line radioactivity detection significantly improves the assay. This technique eliminates the need for organic extraction; improves chromatographic resolution of radioactive substrate and products; provides low background with high sensitivity, linearity, and precision; and yields rapid, quantitative results, even at low levels of cellular CAT expression.

HPLC Analysis of [3H]-Arachidonic Acid Metabolites Produced by Smooth Muscle and Endothelial Cells in Response to Leukotriene D4

Abstract Clonally derived murine and rat smooth muscle cells and bovine endothelial cells were incubated with [3H]-arachidonic acid for 16 hours (10 μCi/ml). The cells were then rinsed twice with saline and then treated with leukotriene D4 (LTD4) (1 μM) for 10 min. The supernatants from these cells were acidified with phosphoric acid (0.1% vol:vol), and then extracted with two volumes of ethylacetate. The radioactivity in the organic phase was analyzed by HPLC using an on-line radioactivity detector equipped with a solid scintillating flow cell. This procedure allowed us to monitor the arachidonic acid metabolites produced in response to LTD4. From data obtained by this method we conclude that LTD4 increases both lipoxygenase and cyclooxygenase product formation in these cells and that this method may be useful in obtaining qualitative information concerning eicosanoid metabolism in other biological systems.

Enzymatic synthesis of 3H-labeled Ring-A reduced metabolites of aldosterone and their separation by high pressure liquid chromatography

Specific methods are described for the enzymatic synthesis of each of the six possible 3H-labeled Ring-A reduced metabolites of aldosterone (5α- and 5β-DHAldo; 3α,5α-THAldo; 3β,5α-THAldo; 3α,5β-THAldo; and 3β,5β-THAldo; see footnote 1 for full names). Use of heated jacketed columns (C 8-reverse phase) and two HPLC solvent systems, with isocratic aqueous methanol or acetonitrile, respectively, have been developed which resolve all six Ring-A reduced metabolites of aldosterone. The relative retention times and elution order of each reduced metabolite are different with each solvent system and hence help confirm the identities of Ring-A reduced metabolites made in vivo from physiological quantities of [ 3H]aldosterone. The use of an on-line β -radioactivity detector (Berthold LB-504) enhanced the sensitivity of detection and markedly improved the resolution of these metabolites, compared with that obtained by off-line scintillation counting. Thus, the use of increased temperature with these two solvent systems, together with an on-line radioactivity detector, provide a useful and efficient analytical tool for the separation and identification of each reduced metabolite of aldosterone.

Identification of Pea Gibberellins by Studying [14C]GA12-Aldehyde Metabolism

Experiments were designed to test the hypothesis that the labeled products recovered from plant tissue incubated with [(14)C]GA(12)-7-aldehyde ([(14)C]GA(12)ald) would serve as appropriate [(14)C]markers for the recovery of naturally-occurring gibberellins (GAs). The [(14)C]GA(12)ald (about 200 millicuries per millimole) was synthesized from pumpkin endosperm using [4,5-(14)C]mevalonic acid. It was added to the adaxial surface of isolated pea cotyledons at 22 days after flowering. Products recovered after 0.5 and 4.0 hour incubations yielded four major peaks which were separated by high performance liquid chromatography (HPLC). These products were purified by multiple-column HPLC using on-line radioactivity detection. They were then added as [(14)C]markers to two unlabeled pea extracts. In general, preparative HPLC followed by further HPLC purification resulted in a single UV-absorbing peak co-eluting with each [(14)C]marker. These [(14)C] and UV-absorbing peaks were shown to contain GA(53), GA(44), GA(20), GA(19), and GA(17) by GC-MS. The finding of GA(53) is novel; all others have previously been found in pea. Endogenous GAs of pea were thus readily detected using [(14)C]GA(12)ald metabolites as [(14)C]markers to recover naturally occurring GAs suggesting that the method may be applicable in detecting naturally occurring GAs in other species.

Bimodal column switching liquid chromatographic assay of six metabolites of [ 14C] felodipine in rat urine

A liquid chromatographic method using bimodal column switching is presented which allows for the separation of six urinary metabolites of [ 14C] felodipine and their quantification using an on-line radioactivity detector. Evaluation of the chromatographic conditions was performed with non-labelled reference compounds and UV detection. Pre-separation of the metabolites into two groups, one consisting of carboxylic acid metabolites and the other of the hydroxylated analogues, was performed on underivatized silica. The mobile phase used was optimized with respect to pH and the character of quaternary ammonium ion, and was 0.01 M tetrapropylammonium in 5% (v/v) methanol in 0.05 M phosphate buffer (ph 5.0). Each group was introduced and separated, after band compression, by a gradient of increasing methanol concentration on an octyl-bonded column. The analysis time was 70 min. The method was applied to urine collected from rats ( n  4, 0–24 h) after oral dosing of [ 14C]felodipine (5 μmol/kg). The urine was analysed with no pre-treatment other than slight dilution. The six metabolites accounted for 58% of the excreted amount (13% of the dose).

Monitoring radioactive compounds in high-performance liquid chromatographic eluates: Fraction collection versus on-line detection

This study compared two methods of monitoring radioisotopes in high-performance liquid chromatographic eluates (on-line radioactivity detector versus fraction collection and counting). Testing was accomplished by pumping solutions of tritiated water in acetonitrilewater mixtures through the detector or to the fraction collector. At most solvent compositions, the detector's counting efficiency and detection limits were poorer than those of the scintillation counter. However, the reproducibility of the detector data was superior at acetonitrile concentrations of less than 50%. This was attributed to the difficulty in collecting fractions of small equal volumes at the lower organic solvent concentrations in short time intervals. We conclude that on-line monitoring with homogeneous detection is the preferred method for detecting radiolabeled compounds in high-performance liquid chromatographic eluates.