Chapter 13 Miscellaneous applications and future prospects

CHAPTER 13

Miscellaneous applications and future prospects

From certain specific applications of liquid scintillation counting that have been made, there is potentially a much wider possible use of the technique in unsuspected fields. The principles involved in heterogenous scintillation counting provide a number of possible ways in which to exploit the system as a whole. Apart from the liquid-liquid systems used in colloidal counting there is also the solid-liquid system in which the scintillant may be in either phase. By fixing the scintillant in the solid phase, it is possible to measure the radioactivity of flowing liquids from chromatographic eluates or gases from gas chromatography. By incorporating an isotope into this solid scintillant phase, a convenient internal light source may be obtained, which can be used to determine the level of colour quenching brought about by a series of coloured solutions. A potentially most rewarding result of the fundamental study of those parameters which determine quenching, is the possibility of learning much more about the structural aspects of biochemical systems, especially those involving changing organelle structures. By locating soft beta-emitters within these structures by suitable selection of precursor molecules, much information could be obtained about the changing environment from the observed changes in quenching. There is also considerable scope for further developments in the field of controlled chemiluminescent reactions, especially in the ultrasensitive analysis of both trace metals and organic materials. Further bioluminescent systems employing different substrates could also 254

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MISCALLANEOUS APPLICATIONS A N D FUTURE PROSPECTS

255

provide powerful tools for ultra-sensitive assay of important biochemical substrates.

13.1. Flow cells The monitoring of a flowing stream of liquid or gas which contains a beta-emitter may be undertaken in two basic ways. The stream can be continuously monitored by passing it over an insoluble scintillant (heterogenous method) or by sampling the eluate either continuously or at intervals and assaying by conventional liquid scintillation methods (homogenous method). The maximum efficiency for the heterogenous method for tritium is about 1.0% using anthracene crystals and less than 2.0 % for diphenyl oxazole (PPO).However, for the more energetic isotopes, there is a significant advantage in being able to recover the sample easily with negligible running costs in scintillant. Thus for carbon- 14, sulphur-35 and similarly energetic beta-emitters there is an advantage in using the suspended scintillator method. Until relatively recently, the homogenous method was mechanically not very efficient but it is evident that the sensitivity of the method, particularly for tritium, is likely to be much superior to that of the heterogenous technique. The early aspects of flow monitoring have been reviewed by Rapkin (1963). A useful review of the methods and problems associated with the monitoring of aqueous solutions by means of the heterogenous flow methods is given by Schram (1970) and some aspects of the homogenous method by Hunt (1968). The two methods have been critically compared by Schutte (1972), and readers should consult these reviews for a more detailed appraisal of the different methods. Heterogenous flow methods. The use of plastic scintillator beads, europeum-activated fluorspar, cerium-activated lithium glass and similar scintillator beads have not proved as successful as anthracene for the monitoring of tritium-containing aqueous eluates. The use of diphenyloxazole (PPO) and butyl PBD have proved to be better solid scintillators from the point of view of counting efficiency for both 14C Sublei I inder p 309

256

SAMPLE PREPARATION FOR LIQUID SCINTILLATION COUNTING

and tritium, but they are slightly soluble in water, especially in acid pH conditions and could lead to artifacts and clogging of the cell. One of the problems is the fact that nucleotides appear to absorb on to most of the solid scintillants examined. From this point of view, cerium-activated lithium glass appears to be the least troublesome, and could be advantageously employed in these circumstances in spite of the relatively low efficiencies experienced. Anthracene crystals on the other hand seem to be useful in the monitoring of amino acid eluates from automatic analysers and this is still the heterogenous method of choice for this purpose. It is often inconvenient to use the usual counting instrument for flow-monitoring alone, and single sample coincidence counters are available commercially to undertake this. However, a cheaper single

+

-2mm c4.8mm

--c

Fig. 13.1. A flow cell constructed from a 2 1/4 x 2 1/4 inch block of Lucite by drilling three closely and evenly spaced holes, 0.48 cm in diameter and 3.7 cm deep. The interconnections are constructed of 20 guage stainless steel hypodermic needles fixed by means of an epoxy resin. The packing consists of glass wool plugs and the plastic scintillator NE 102 (Nuclear Enterprises) ground to 100 to 200 mesh powder (Tkachuk 1962). (Reproduced by kind permission of the National Research Council of Canada.)

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photomultiplier system can be used to obtain a qualitative record of the position of labelled peaks, and a simple device constructed from a suitably light-shielded perspex block and is often all that is required. Such devices have been available commercially (Nuclear Enterprises), but a simple apparatus (Fig. 13.1) which could probably be constructed in any reasonably equipped workshop was described by Tkachuk (1962). A useful pressure-resistant flow-cell (Fig. 13.2) for accelerated amino acid analysis (White and Mencken 1970) was intended to be used with the conventional coincidence counter. -BLACK

T E F L O N TLlBE LE

-FFE?HI

-

\-'ONNEC TOR

'-0

-FLOW

RING

CELL

Fig. 13.2. A flow cell constructed of Perspex and packed with detergent coatedanthracene is designed to fit into the well of a standard liquid scintillation counter. It is intended for use with accelerated amino acid analysis procedures (White and Mencken 1970). (Reproduced by kind permission of Academic Press.) Sublert mdexp. 309

258

SAMPLE PREPARATION FOR LIQUID SCINTILLATION COUNTING

Homogenous systems. The monitoring of tritium-containing eluates and low levels of carbon-14, requires a homogenous system where the sample is either continuously or intermittently monitored in the more conventional liquid scintillation counting system. A typical flow diagram was described by Schutte (1972) where a proportional pump was used to divert the flowing stream and to ensure mixing with a water-accepting liquid scintillation composition. A typical flow diagram is illustrated in Fig. 13.3a with a coiled tube inserted into a scintillation vial as shown in Fig. 13.3b, inserted into a scintillation counting well. The mixing spiral is a tube of internal diameter 2 mm and 70 cm long. Since mixing is assisted by a stream of air bubbles which periodically interrupts the flow, the resolution obtainable is maintained. The coil inside the vial has a total volume of 1.4 ml and the net flow rate is approx. 2.3 ml/min. In this system, a counting efficiency for tritium of 30% and for carbon-14 of 80% is recorded. With this system therefore, if only a sensitive record of label is required, the splitter can be dispensed with and a sensitivity of 5 nCi per peak can be obtained, In addition, for dual isotope experiments, good resolution of the isotopes can be made as pointed out by Hunt (1968). For those isotopes with a greater energy max than 0.5 MeV, the Cerenkov emission may be usefully employed since a simple coil of plastic immersed in a vial is all that is necessary for the monitoring of the label (Colomer et al. 1972).

13.2. Gas chromatography In the gas chromatographic technique, organic materials are heated and chromatographed over hot columns in the vapour state. O n many of these absorbant columns, there is also a stationary liquid phase, such as silicone oil. The gases are monitored in a variety of different ways, but it is often useful to be able to follow the level of radio isotope that may be used as a label in one of the components. However, to do so, the hot gases need first of all be concentrated either by

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259

uv

detect

a

Scintillator soln.

b

Fig. 13.3. A flow diagram (a) for the homogenous assay of chromatographic eluates using a Technicon Analyser proportionating pump by diverting a constant fraction of the stream with scintillant via a mixing spiral into a coil (b) placed in the scintillation vial. Adapted from Hunt (1968). The tubing and coil are made of a toluene resistant plastic (e.g. Acidflor, Technicon) and an interspacing of air bubbles prevents tailing of the peaks (For a more detailed description, see Schutte 1972.)

condensation on cooling or by absorption into some system which can be readily adapted for liquid scintillation counting. One of the most straight-forward, though not the most convenient ways to do this, is to lead the gases into an evacuated chamber. They are then passed from this chamber into a catalytic tube, where the carbon compounds are oxidised to carbon dioxide, which is then absorbed in a suitable absorbant scintillant, such as one based on Sublert mdexp. 309

260

SAMPLE PREPARATION FOR LIQUID SCINTILLATION COUNTING

ethanolamine (Bosshart and Young 1972). By using a weighed ethanolamine trap, the specific activity of the carbon dioxide collected can be calculated and together with the known mole fraction values normally determined in the gas chromatographic technique, the specific activity can be assayed for each of the peaks from the chromatogram. A detection limit of 0.04 dpm/ml can usually be obtained. An alternative procedure is to have a short section of gas chromatography tubing (2" long and 1/4 ins int dia.) at the exit port of the column, containing anthracene, coated with the liquid stationary phase, e.g. silicone oil ( 5 % w/w, oil/anthracene). The first two inches will trap the majority of the effluent if this tube is maintained at room temperature. It can then be assayed by counting inside a vial in a conventional liquid scintillation spectrometer (Karmen and Trich 1960).

13.3. Analytical applications There are a number of instances where the liquid scintillation spectrometer has been employed in an unconventional manner to assay light photons from sources other than a scintillator-isotope system. Mention has already been made of the extreme sensitivity of the bioluminescent system of the luciferin-luciferase (8 2.4). If the origin of chemiluminescence was better understood, there is clearly a potentially important field for the analysis of many inorganic and organic species. A useful chemiluminescent reaction which has already been exploited for a number of analyses (in most cases not using a liquid scintillation counter), is afforded by the peroxide-induction of light with luminol(5-amino-2,3-dihydro-l ,Cphthalazine dione). This reaction is catalysed by several inorganic ions, such as cadmium, cobalt, copper, iron, lead and vanadium, and the rate of catalysis is related to the concentration of the ion. Other organic molecules either catalyse or inhibit the reaction; in some instances, such as with the organophosphorus compounds, the chemiluminescent reaction has been used as an alternative to the cholinesterase reaction to determine the extent of anticholinesterase activity possessed by these molecules. Many

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26 I

other chemiluminescent reactions are now known, and it is likely that in this field the use of the liquid scintillation counter may develop. An alternative method of using the counter is to create a constant source of photons and measure the degree of quenching by changing colour reactions, particle density etc. An example of this application has already been described (# 8.3 and Fig. 1.10 for the assay of lipid mass. The method has also been modified for the assay of sulphydryl groups (Snyder and Moehl 1971). Sulphydryl group assay. The light source is immaterial, but the liquid source consists of a solution of 14C-tripalmitin in toluene containing approximately 98,000 cpm in 900 pl mixed with Tpp scintillant in the central tube of a glass vial of the type illustrated in Fig. 1.lf. The SH compound is dissolved in 0.1 m phosphate buffer (4 ml) pH 8.0 (reduced glutathione is usually employed as a reference in these determinations). An aliquot (200 p1) of a solution of 39.6 mg 5,5'-dithio-bis-2-nitrobenzoicacid in 10 ml 0.1 M potassium phosphate buffer (pH 7.0) is added in a standard vial. Either a liquid scintillator insert (e.g. Fig. 1.10 or scintillator beads are then added. A good linear correlation (Fig. 13.4) of the glutathione concentration with the degree of quenching is achieved. The method is a useful alternative to the spectrophotome:ric method, since, if several light sources of known light emission properties are made, the system may be automated. A plastic source may be more conveniently used as an alternative to a liquid one and these are compared in Fig. 13.4. Another use of an internal light source is that of counting particles in a suspension by addition of a Cerenkov emitter to the suspension. This procedure was described by Ashcroft (1969) for the measurement of Escherichia cofisuspensions as well as 0.5 nm diameter polystyrene latex beads. In this case, 0.5 pCi of 36C1is employed in 0.9% saline per vial as the source of Cerenkov light. An observation of Paus (1972) made during the assay of I4C labelled RNA, appears to be capable of considerable exploitation. The level of quenching increases as the molecule of labelled RNA associates with water molecules. These surround the beta-emitter and appear to reduce the degree of penetration of the beta particle into the scintillant. Sublert index p 309

262

SAMPLE PREPARATION FOR LIQUID SCINTILLATION COUNTING

Fig. 13.4. The effect of two different scintillation sources on the estimation of glutathione by colour quenching. Solid plastic light source 0- - -0,PPO-POPOP (Snyder and Moehl 1971). (Reproduced by kind permission liquid source 0-0 of Academic Press Inc.)

This association of water molecules appears to be a relatively loose one, since the quenching temporarily decreases on shaking the mixture. It would appear that further elaboration of the phenomenon could lead to a powerful method of assaying the rate and extent of water absorption to macromolecules of this type in relation to structure changes which may have important biological implications. The use of multi-isotope analyses have already been referred to but there are several ways in which use of such mixtures has been made, apart from the more obvious applications. Heidelberger et al. (1953) required to establish the identity of a metabolite of a labelled carcino-

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263

MISCEL.L.ANEOUS APPLICATIONS A N D FUTURE PROSPECTS

genic hydrocarbon following skin painting on an animal. Suspected metabolites were prepared unlabelled, and added in known amounts to the tissue digest prior to paper chromatography. Label associated with one of the added metabolite spots on the subsequent chromatogram was shown to be associated through repeated chromatographic runs and it was concluded that this was a true metabolite. The basic principle of employing isotope dilution is, of course, not new, but a combination of two isotopes may sometimes be employed to advantage. Prior to the widespread use of scintillation counting, Keston et al. (1950) described a procedure for the assay of amino acids on paper chromatograms by using labelled pipsyl chloride (in this case labelled with 13'I). The '311-pipsyl amino acids were then run with known quantities of 35S-pipsyl amino acid markers, and a constant ' 311/35Sratio could then be used to confirm both radiochemical purity and to determine the level of the original amino acid present. There appears to be no obvious reason why 14C or 3H compounds should not be used in a similar manner. Multiple isotope analysis has also been employed for a somewhat complex, but in certain cases a very convenient, way of assaying intracellular pH in whole animal tissue, in this case dogs (Schloerb and Grantham 1965). The method consists of the intravenous injection of a mixture of 100 pCi tritiated water, 10 pCi 14C, 5,5-dimethyl-2,4oxazolidine dione and 10 pCi 36Clin 10 ml of NaCl(0.9 %). After an equilibration time of not less than 3 hr, biopsy or tissue specimens, together with plasma samples are taken and the relative amounts of each isotope present are measured. The principles involved had previously been described by Wdddel and Butler (1959). The resulting intracellular pH, is then related to tissue (t) and plasma (p) radioactivity and to the extracellular pH, according to equation pHi

=

6 . 13

4Ct3H, + log 'I4C,3H, (I

{[

3

6

~

1

~

3

~

~

~

+

1 * 0536C1,3H1- 36Cl,3H,

SuhIeci index p . 309