Description

The TXB₂/2,3-dinor-TXB₂ [¹²⁵I] assay system enables the quantitative determination of thromboxane B2 (TXB₂) and 2,3-dinor-thromboxane B₂ (2,3-dinor-TXB₂) in biological fluids. TXB₂ and 2,3-dinor-TXB₂ can be assayed in the range 1.2-100 pg/tube. Each kit contains materials sufficient for 100 assay tubes, permitting the construction of one standard curve and assay of 25 unknowns in triplicate.

Introduction

Arachidonic acid, released from the cell wall by phospholipase A₂, is converted to prostaglandin endoperoxides on the effect of cyclo-oxygenase (endoperoxyde synthase). Endoperoxydes are then converted to prostaglandins, prostacyclin (PGl₂) and thromboxane A₂ (TXA₂) (1-5). Being a potent platelet-aggregating and very efficient vasoconstrictor agent, TXA₂ is an antagonistic to prostacyclin. It is believed that physiological balance between the two compounds plays an important regulatory role in the maintainance of normal vascular tone and in pathogenesis of various cardiovascular disorders (6).

Since TXA₂ is rapidly converted to thromboxane B₂ (TXB₂), a chemically stable but biologically inactive hydration product, thromboxane synthesis of biological tissues has been monitored by measuring TXB₂.

One of the futher metabolic processes which the circulatory TXB₂ undergoes is the common ß-oxidation that leads to the appearance of 2,3-dinor-TXB₂ in urine. Urinary 2,3-dinor-TXB₂ has been used as an index of the extrarenal thromboxane production. The extremely low concentration of thromboxane B₂ in different biological media necessitates sensitive procedures to be used for quantitation.The current assay system works as a twin-analyte tool – it enables the simple and sensitive determination of both TXB₂ and 2,3-dinor-TXB₂, by using the very same assay reagents, and a single-analyte calibration.

Principle of the method

This assay is based on the competition between unlabelled TXB₂/2,3-dinor-TXB₂ and a fixed quantity of ¹²⁵I-labelled TXB₂ for a limited number of binding sites on TXB₂ specific antibody. Allowing to react a fixed amount of tracer and antibody with different amounts of unlabelled ligand the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand. Upon addition of magnetiz-able immunosorbent the antigen-antibody complex is bound on solid particles which are then separated by either magnetic sedimentation or centrifugation. Counting the radioactivity of solid phase enables a standard curve to be constructed and samples to be quantitated.

Contents of the kit

Materials and equipment required

Round bottom polystyrene or polypropylene assay tubes, about 12 x 75mm
Plastic film to cover tubes
Precision pipettes (100 µl and 500 µl)
Vortex mixer
Magnetic separator, or, alternatively, centrifuge
Decanting racks
Gamma counter

Recommended tools and equipment

orbital shaker
repeating pipettes

Preparation of reagents

Tracer

One vial of the tracer concentrate contains approximately 75 kBq of thromboxane B₂-[¹²⁵I]TME in organic solvent. For use in the assay dilute the tracer with 10 ml assay buffer. The resulting solution contains 75 kBq of the tracer in 50 mM phosphate buffer, pH 7.3, with 0.1% gelatin and 0.01% thimerosal. The diluted solution is stable until expiry date, if stored at -20°C.

Antiserum

The antiserum was raised in rabbit againts a bovine serum albumin conjugate of thromboxane B₂. For use in the assay, reconstitute the antiserum by adding 10 ml of distilled water with gentle mixing to avoid foaming. Make ensure that the lyophilized material is in solution. So as to make complete solution faster, the material can be equilibrated in water bath of 35°C for a few minutes. After resonstitution, the solution contains thromboxane B₂ antiserum of appropriate binding ability in 50 mM phosphate buffer, pH 7.3, with 0.1% gelatin and 0.01% thimerosal. This solution should be stored at 4°C. Under these conditions the solution is stable until expiry date.

Standard

Reconstitute the lyophilized thromboxane B₂ standard by adding exactly 1.0 ml distilled water. Make ensure that the lyophilized material is in solution. The resulting solution contains 10 ng of thromboxane B₂ per ml in 50 mM phosphate buffer, pH 7.3 with 0.1% gelatin and 0.01% thimerosal. Immediately before use in the assay dilute an appropriate aliquot of the standard stock solution to prepare standards. A suggested dilution scheme is shown later. Store the remaining standard stock solution at -20°C. Do not store diluted standards.

Assay buffer

To prepare assay buffer for use in the assay, warm the bottle containing buffer concentrate to room temperature and add 80 ml water. The assay buffer thus obtained contains 50 mM phosphate buffer, pH 7.3 with 0.1% gelatin and 0.01% thimerosal. Stored at 4°C, it is stable until expiry date.

Magnetic immunosorbent

This reagent contains paramagnetic particles coated with anti-rabbit immunoglobulin suspended in 50 mM phosphate buffer, pH 7.3 with 0.1% sodium-azid and 0.05% Triton X-100. Stored at 4°C, it is stable until expiry date.

Sample handling

General comments

Prostanoids and thromboxanes are found in a great variety of tissues at extremely low concentrations. It requíres a special care and sophisticated procedures to reliably assay an analyte of such a fow level. Because of diversity of potential research projects as well as of experimental procedures employed, no universal methods can be declared. In the present leaffet, representatíve data and selected procedures applied to the assay of more common biologica! media (i.e. plasma and urine) are dealt with for the sake of guidance only. Any modification of standard assay procedure or sample preparation makes investigator responsible to evaluate the particular procedure employed. Users are kindly recommended to refer to selected publications (6-10) on methodology of eicosanoids analysis, with special emphasis on sample handling, extraction and purification methods, validation criteria, etc.

Concentration of thromboxane B₂ in plasma

During the early stage of the application of prostanoids RIA a large number of studies appeared claíming plasma levels of primary prostanoids including thromboxane B₂ in the range several hundred pg/ml up to a few ng/ml. These very high values far exceeded those expected theoretícally, and were later proved to be resulted from both ex vivo biosynthesis during processing samples and heterogeneous immunoreactivity. Upon theoretical consideration TXB₂ concentration in plasma cannot be expected to exceed a few pg/ml, which is in contrast to most frequently reported plasma concentrations being about 100 pg/ml (7). Based on theoretical as well as on methodological reasons, reliability and bíological relevance of plasma thromboxane B₂ levels have been a subject to serious criticism (7-8).

The low plasma level of thromboxane B₂ makes it impossible to use direct assay from unextracted sample. So as to have an amount enough for radioimmunological determination, an high volume of plasma should be extracted and concentrated. By using even the present, highly sensitive, assay it is expected that approximately 10-50 ml of plasma will be required for the reliable quantitation (i.e. to have a sample concentration of 5-10 pg/tube fitting to optimal sensitivity range). Although no limitation with sensitivity is expected for values most frequently obtained (i.e. in range of 100 pg/ml), they should be critically interpreted on theoretical reasons.

Thromboxane B₂ in human serum

In contrast to extremely low TXB₂ concentration in plasma, an exceptionally high amount of thromboxane B₂ is produced ex vivo in blood after clotting. Serum thromboxane B₂ thus produced can be monitored as an index of platelet cyclo-oxygenase activity (11). The high concentrations obtained after clotting (sometimes over 100 ng/ml!) enables serum level of thromboxane B₂ to be measured directly, as detailed later.

Thromboxane metabolites in human urine

In spite of some conflicting reports (12), it is generally accepted that urinary thromboxane B₂ represents renal thromboxane biosynthesis (13). Studies aimed at finding a suitable index metabolite whích would really reflect extrarenal thromboxane production demonstrated that thromboxane produced in vivo is excreted into urine as 2,3-dinor-thromboxane B₂, the beta-oxidation product of thromboxane B₂ (14). Based on this finding urinary immunoreactive thromboxane B₂ was regarded, for long time, as a parameter entirely irrelevant to in vivo thromboxane biosynthesis.

Recently, however, thromboxane B₂-like immunoreactivity of normal human urine turned out to be contributed by 2 compounds; thromboxane B₂ and 2,3-dinor-thromboxane B₂ (15). Although their actual ratios are reported to vary from one individual to other, total thromboxane B₂-like immunoreactivity is always dominated by 2,3- dinor-thromboxane B₂, whereas percentage that could be attributed to thromboxane B₂ itself did not usually exceed 25% (15). In good agreement with these data, immunoreactivity profile determined in pooled human urine of healthy males (Fig. 3) showed the majority of immunoreactivity corresponding to 2,3-dinor-thromboxane B₂, with only a negligible contribution of thromboxane B₂. As a consequence, urinary thromboxane B₂ immunoreactivity is an index of extrarenal rather than renal thromboxane production. These experimental evidence offer two alternatives for monitoring thromboxane metabolites in urine:

– Urine samples after common solid-phase extraction (e.g. the one suggested in present manual), are assayed for thromboxane B₂ and the values obtained are used as the measure of 2,3-dinor-thromboxane B₂ concentration. The contribution of thromboxane B₂, as being in the range of the common between-assay error, is thus ignored. It should be emphasized that the values thus obtained can be regarded as the rough estimate rather than an exact quantity, of 2,3-dinor-thromboxane B₂.

– In majority of studies simultaneous quantitation of thromboxane B₂ and 2,3-dinor-thromboxane B₂ can be expected. Since the composition, in the actual sample, of the two compounds must be unknown, precise quantitation can only be carried out by separating thromboxane B₂ from 2,3-dinor-thromboxane B₂. This is more efficiently done by high-performance liquid chromatography. Once separated, both compounds can be determined by radioimmunoassay.

The present assay kit has the exceptional feature to enable any of above alternatives carried out as simple as possible. The antibody used does not differentiate 2,3-dinor-thromboxane B₂ from thromboxane B₂; i.e. it has 100% cross reaction with 2,3-dinor-thromboxane B₂. This offers a particularly simple way of determination; unless cross-contaminated, both thromboxane B₂ and 2,3-dinor-thromboxane B₂ can be measured by using exactly identical assay procedure. There is no need to use any correction factor for cross-reactivity, nor to standardize with 2 different compounds!
Details of all these options are described under Assay Procedure.

Reference intervals as determined by authentic gas-chromatography/mass spectrometry technique (16), revealed 120 ng/24 h and 432 ng/24 h mean value for daily excretion rates of urinary thromboxane B₂ and 2,3-dinor-thromboxane B₂, respectively. From this value, an average of 60-100 pg/ml (thromboxane B₂) and 20-400 pg/ml (2,3-dinor-thromboxane B₂) for concentration ranges in normal human subjects can be expected. Values out of this range should be interpreted with cautiousness.

2,3-dinor-thromboxane B₂ is not the only suitable metabolite to monitor in vivo thromboxane production. Based on detailed metabolic studies, 11-dehydrothromboxane B₂ rather than 2,3-dinor-thromboxane B₂ has been suggested as a more convenient major index compound (17). Determination of this metabolite by the use of our 11-dehydrothromboxane B₂ RIA kit (Code RK-67) can be preferred on several reasons; urinary concentration of 11-dehydrothromboxane B₂ is much higher than that of 2,3-dinor-thromboxane B₂, and the elaboration of a selective solid-phase extraction procedure (18,19) eliminates the expensive and time-consuming chromatographic separation. In conclusion, a great variety of research studies is enabled by RIA systems suitable for monitoring thromboxane metabolites; thromboxane B₂, 2,3-dinor-thromboxane B₂ and 11-dehydro-
thromboxane B₂.

A) Collection and storage

Blood samples should be collected in pre-chilled plastic or siliconized glass tubes containing anti coagulant and cyclo-oxygenase inhibitor. In our laboratories blood is drawn in polypropylene tube containing 10% (v/v) of 2% EDTA buffer (pH 7.3) with 1 mM indomethacin. At this concentration we have found no interference of indomethacin in the assay. If storage of plasma samples is necessary, -70oC or lower is recommended. Urine samples should be stored at -20oC after pooling the single samples collected from one patient. For tissue samples, storage at -70oC or lower until assay is recommended.

B) Preparation of samples prior to assay

Direct assay of serum thromboxane B₂

Concentration of thromboxane B₂ in serum is usually high enough to enable samples assayed in 500-fold up to 1000-fold dilutions. These highly diluted samples can be added directly to incubation mixture, and quantitated by using standard assay procedure. However, due to considerable interference of plasma proteins with assay, thromboxane-free serum of a dilution identical to unknowns should be added into standard curve, if samples are diluted in the range 100-500-fold. Serum samples diluted less than 100-fold should not be assayed directly!

Solid-phase extraction of human plasma and urine

For the extraction of prostanoids from biological tissues, solid-phase extraction carried out according to Powell (20) has become the most popular method of choice. In our laboratory, Bond-Elut C₂ (Analytichem International) minicolumns have been applied successfully according to the following procedure.

Remarks

This solid phase extraction procedure detailed above usually results in a recovery of > 90%, as checked by ³H-labelled thromboxane B₂ as the recovery marker. So as to eliminate random error with individual samples, it is suggested that extraction efficiency is determined for each sample throughout whole procedure. Quality requirements for tritiated prostanoids being suitable as recovery marker are high; specific activity, chemical and radiochemical purity should be as high as possible. Depending on the chemical structure of prostanoid, tritium is quickly exchanged by solvent hydrogen, a phenomenon resulting in serious underestimation of extraction efficiency. It is recommended to store tritiated thromboxane B₂ according to manufacturer's instruction, to check its radiochemical purity regularly, and to purify it, if needed, by chromatographic method.

Solvent residues, impurities as well as the biological matrix itself may often introduce an astonishingly high non-specific immunoreactivity whose degree is a function of analyte, assay medium, quality of antibody and solvents, etc. This may give rise to a considerable overestimation of real concentration. The lower the concentration the higher the relative error due to this method blank. In order for the blank contribution to be corrected, prostaglandin-free sample (to estimate matrix contribution and method blank simultaneously) and/or buffer (to determine method-blank only) should be subjected to the procedure strictly identical to that used for unknowns. Concentration of unknowns should be corrected accordingly.

³H-labelled TXB₂ can be used as the marker of both.

Assay procedure

Day 1

Table 1. Dilution scheme (all volumes in microliters)

Notes: vial "s" is prepared by reconstituting the lyophilised standard with 1.0 ml distilled water
To prepare standard dilution and to dissolve or dilute assay buffer must be used.

Day 2

Table 2. Assay Protocol, Pipetting Guide (all volumes in microliters)

Estimation of urinary 2,3-dinor-thromboxane B₂ without chromatography

Extract urine samples according to the suggested procedure and make determinations according to the standard assay procedure. Amounts measured are expressed as pg/tube of
2,3-dinor-thromboxane B₂. (Refer to General comments for theoretical limitations.)

Quantitation of urinary thromboxane B₂ and 2,3-dinor-thromboxane B₂

Extract urine samples according to the suggested procedure, make chromatographic separation and collect the fractions of thromboxane B₂ and 2,3-dinor-thromboxane B₂. Make determinations according to the standard assay procedure as above. Concentrations will be obtained directly as pg/tube of thromboxane B₂ and 2,3-dinor-thromboxane B₂ of respective fractions.

Calculation of results

The calculation is illustrated using representative data. The assay data collected should be similar to those shown in Table 3.

Figure 1 shows a typical standard curve. Determine the TXB₂ or 2,3-dinor-TXB₂ levels in the unknown samples by interpolation from the standard curve. Values can be read directly as pg TXB₂/12,3-dinor-TXB₂ per assay tube from the standard curve. Never extrapolate values beyond the standard range.

Calculation by computing data using various fitting programs may also be applied but is not dealt with here. In our laboratories, smoothed cubic spline is routinely used for both calculation of unknowns and for regular Quality Control of the present assay system.

Table 3.    Typical Assay Data

TXB₂ concentration pg/tube
Figure 1.
A typical standard curve
(Do not use to calculate sample values)

Characterization of the assay

Assay parameters

Specificity

Cross reactivity was defined by weight at the 50% displacement level in per cent.

* 97.6 ± 3.4% as determined from 6 independent assays.

Reproducibility

To determine inter-assay precision 3 control samples were measured in triplicates in 12 independent assays by 2 operators using different kit batches. Values obtained are shown below.

Evaluation of immunoreactive purity by immuno-chromatography

Figure 2.  Immunoreactivity profile obtained with plasma after solid-phase extraction
 

Pooled normal human plasma containing tritiated thromboxane B₂ was extracted on Bond Elut C₂ according to suggested procedure and the extract separated by reversed-phase HPLC using a complex línear gradient elution with water : acetonitrile (0.1 % CH₃COOH) as the mobile phase on Spheri-5 C Microbore column at a flow rate of 0.4 ml/min. Fractions eluted were measured in thromboxane B₂ radioimmunoassay. The main immunoreactive peak co-migrated with tritiated thromboxane B₂ (marked by asterisks).

Figure 3.  Immunoreactivity profile obtained with urine after solid-phase extraction
 

24-hour pooled urine collected from healthy male volunteers was subjected to solid-phase extraction on Bond Elut C₂ according to suggested procedure and the extracts separated by reversed phase HPLC using a complex linear gradient elution with water:acetonitrile (0.1% CH₃COOH) as the mobile phase on Spheri-5 C₁₈ Microbore column at a flow rate of 0.4 ml/min. Fractions eluted were measured in thromboxane B₂ radioimmunoassay. The majority of immunoreactivity co-migrated with authentic 2,3-dinor thromboxane B₂ (peak-A) and a negligible ratio of immunoreactivity was seen in thromboxane B₂ fraction (peak-B).

Additional information

Storage

This kit is shipped ambient. Upont receipt store the individual components as detailed in this leaflet.
Pay special attention to preventing magnetic immunosorbent suspension from freezing.

Availability

From stock.

Shelf life

The shelf life of kit reagents is 8 weeks from the date of manufacturing. To make maximum benefit of long-term stability it is recommended to adjust the date of ordering to labelling calendar issued each year. The actual expiry date is given on package label and in the quality certificate. Components from various lots or from kits of different manufacturers should not be mixed or interchanged.

Precautions and warnings

This kit should only be used for in vitro research purposes.

Radioactivity

This kit contains radioactive material. Receipt, acquisition, possession, or use of radioactive materials are subject to regulations, and a licence of (inter)national authorizing bodies. It is the responsibility of the user to ensure that local regulations or codes of practice are satisfied.

Chemical and other hazard

Magnetic immunosorbent contains sodium azide (0.1% w/v) as an antimicrobial agent. Dispose the waste by flushing it with copious amounts of water to avoid build up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 55 mg.

References