Description

The 6-keto-PGF_1a/2,3-dinor-6-keto-PGF_1a [¹²⁵I] assay system provides the quantitative determination of 6-keto-prostaglandin F_1a (6-keto-PGF_1a) and 2,3-dinor-6-keto-prostaglandin F_1a (2,3-dinor-6-keto-PGF_1a) in biological fluids. 6-keto- and 2,3-dinor-6-keto-PGF_1a 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 (endoperoxide synthase). Endoperoxides are then converted to prostaglandins, thromboxane and prostacyclin (PGI₂) (1-5). Under in vitro circumstances prostacyclin has been demonstrated to have profound biological activities; it is a potent antiaggregatory and vasodilator agent (4-5). Platelet and vascular effects are generally accepted to underly the very potent pharmacological actions of prostacyclin in vivo.
Owing to its vinyl-ether moiety PGI₂ is rapidly converted in aqueous medium into 6-keto-PGF_1a, a chemically stable but biologically inactive hydration product. Because of very short half-life of the active species, prostacyclin synthesis of biological tissues can only be monitored by measuring 6-keto-PGF_1a, the primary breakdown product. Circulatory 6-keto-PGF_1a undergoes ß-oxidation, and appears in the urine as 2,3-dinor-6-keto-PGF_1a along with unmetabolized 6-keto-PGF_1a of renal origin.
Concentration of 6-keto-PGF_1a/2,3-dinor-6-keto-PGF_1a is very low in plasma (6), and rather low in urine (7). Therefore their quantitation require sensitive procedures. The combination of a high specific activity iodinated derivative of 6-keto-PGF_1a as tracer with rabbit anti-6-keto-PGF_1a antiplasma as specific antibody, provides an efficient tool for simple and sensitive determination of 6-keto-PGF_1a in biological fluids, and 2,3-dinor-6-keto-PGF_1a in urine.

Principle of the method

This assay is based on the competition between unlabelled 6-keto-PGF_1a/2,3-dinor-6-keto-PGF_1a and a fixed quantity of ¹²⁵I-labelled 6-keto-PGF_1a for a limited number of binding sites on 6-keto-PGF_1a 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 magnetizable 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 75 mm
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 6-keto-PGF_1a -[125I]TME in ethanolic solution. 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 4°C.

Antiserum

The antiserum was raised in rabbit against a bovine serum albumin conjugate of 6-keto-PGF_1a. 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 lyophilised 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 reconstitution, the solution contains 6-keto-PGF_1a 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.

6-keto- PGF_1a standard

Reconstitute the lyophilised 6-keto-PGF_1a standard by adding exactly 1.0 ml distilled water. Make ensure that the lyophilised material is in solution. The resulting solution contains 10 ng of 6-keto-PGF_1a 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 or re-use diluted standards.

2,3-dinor-6-keto-PGF_1a standard

Reconstitute the lyophilised 2,3-dinor-6-keto-PGF_1a standard by adding exactly 1.0 ml distilled water. Make ensure that the lyophilised material is in solution. The resulting solution contains 10 ng of 2,3-dinor-6-keto-PGF_1a 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 or re-use 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 azide and 0.05% Triton X-100. Stored at 4°C, it is stable until expiry date.

Sample handling

General comments

Due to special methodological pitfalls and low endogenous concentrations, it requires a special care and sophisticated procedures to reliably assay prostacyclin metabolites. Because of high variation with experimental procedures employed in full scale of biological studies, no universal methods can be declared. In the present leaflet, representative data and selected procedures applied to the assay of plasma and urine are dealt with for the sake of guidance and it remains the investigator's responsibility to evaluate the particular procedure employed during study. Users are kindly recommended to refer to selected publications (8-12) on methodology of eicosanoids analysis, with special emphasis on sample handling, extraction and purification methods, validation criteria, etc.

Concentration of 6-keto-PGF_1a in plasma

During the early stage of the application of prostanoids RIA a large number of studies appeared claiming plasma levels of primary prostanoids including 6-keto-PGF_1a in the range several hundred pg/ml up to a few ng/ml. These very high values far exceeded those expected theoretically, and were later demonstrated to be resulted from both ex vivo biosynthesis during processing samples and heterogeneous immunoreactivity. Determined by RIA under strictly controlled conditions, basal concentration of 6-keto-PGF_1a in human plasma was found to be as low as 1-2 pg/ml (6). This low level of 6-keto-PGF_1a suggests against either an antiaggregatory or an antihypertensive role for PGI as a circulating hormone and may cast some doubts about biological relevance of 6-keto-PGF_1a concentration in plasma. The extremely low plasma level of 6-keto-PGF_1a makes direct assay from untreated sample impossible. So as to have an amount enough for radio-immune determination, a high volume of plasma should be extracted and concentrated. By using even the present, highly sensitive, assay it is expected that approximately 25-50 ml of plasma will be required for the reliable determination of normal plasma concentration (i.e. to have an amount of 5-10 pg/tube fitting to optimal sensitivity range).

For the extraction of 6-keto-PGF_1a from human plasma solvent extraction proved to be unsuitable. By using solvent extraction followed by thin layer chromatography 6-keto-PGF_1a was reported to be decomposed into several degradation products, all of which was cross-reactive with 6-keto-PGF_1a antibody to varying degree (13).

Solid-phase extraction using Sep-Pak C₁₈ according to Powell (14), the most common procedure for isolating prostanoids from biological fluids, was demonstrated to result in heterogeneous immunoreactivity (6). Although in our laboratory a somewhat better profile was obtained on C₂-silica minicolumn (Fig. 2), the residual non-specific interference still remained too high. It is now generally agreed that solid-phase extraction followed by a chromatographic separation (preferably by reversed-phase HPLC) is the only method of choice to obtain reliable plasma concentration of 6-keto-PGF_1a by radioimmunoassay, but the biological relevance of even the most accurate concentration will still remain debated (8).

6-keto-PGF_1a immunoreactivity in urine

The majority of research studies has been aiming at quantitation of such an index compound that reflects prostacyclin production of the body, i.e. extrarenal biosynthesis. To achieve this, however, urinary 6-keto-PGF_1a is not the right choice. In spite of some conflicting reports (15), however, it is generally accepted that urinary 6-keto-PGF_1a represents renal prostacyclin biosynthesis. Prostacyclin of extrarenal origin is represented by urinary 2,3-dinor-6-keto-PGF_1a, the beta-oxidation product of 6-keto-PGF_1a. Since renal and extrarenal prostacyclin production are relevant parameters of different biological events, quantitation of 6-keto-PGF_1a and 2,3-dinor-6-keto-PGF_1a are of equal importance. Polyclonal antisera used originally as those specific for 6-keto-PGF_1a all turned out to have varying, but a considerable, cross-reaction with 2,3-dinor-6-keto-PGF_1a. As a consequence total 6-keto-PGF_1a immunoreactivity is always contributed by these two metabolites being present in unknown ratio in samples. (The presence of two immunoreactive fractions of a crude urine extract is illustrated in Fig. 3.) Apart from the high ratio of non-specific interference of unextracted urine, the dual nature, in itself, of specific immunoreactivity makes it impossible to quantitate urinary 6-keto-PGF_1a/2,3-dinor-6-keto-PGF_1a by direct assay. For the reliable determination, urine samples should be first extracted on solid phase, then separated by either thin layer chromatography or high-performance liquid chromatography, and metabolite fractions assayed individually.

The present assay system takes advantage of the dual nature of immunoreactivity of polyclonal rabbit antibody used. As described under Assay Procedure, this beneficial moiety enables one assay system to be utilized for the simultaneous determination of two metabolites; i.e. 6-keto-PGF_1a and 2,3-dinor-6-keto-PGF_1a. In addition to the capability of assaying two metabolite fractions obtained by HPLC, another, more practical option; to quantitate 2,3-dinor-6-keto-PGF_1a obtained by a selective solid-phase extraction procedure without HPLC (16) is also enabled by the current assay system.

Reported values of concentration/excretion of these metabolites are varying in a wide range. Recent study (based on authentic gas-chromatography/mass spectrometry technique (7) on reference intervals for daily excretion rates of urinary 6-keto-PGF_1a and 2,3-dinor-6-keto- PGF_1a in human, found 120 ng/24 h and 144 ng/24 h mean values, respectively. From these values, an average of 60-100 pg/ml for concentration range in normal human subjects can be expected. Values out of this range should be interpreted with cautiousness.

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 1mM indomethacin. At this concentration we have found no interference of indomethacin in the assay. If storage of plasma samples is necessary, -70°C or lower is recommended. Urine samples should be stored at -20°C after pooling the single samples collected from one patient. For tissue samples, storage at -70°C or lower until assay is recommended.

B) Preparation of samples prior to assay

SPE-1 Solid-phase extraction from human plasma. Co-extraction of 6-keto-PGF_1a and 2,3-dinor-6-keto-PGF_1a from human urine (16)

For the extraction of 6-keto-PGF_1a from plasma and urine Bond-Elut C₂ (Analytichem International) or Amprep C₂ (Amersham International plc) minicolumns have been applied successfully in our laboratory according to the following procedure.

SPE-2 Selective solid-phase extraction of 2,3-dinor-6-keto-PGF_1afrom human urine

For this procedure Spe-ed^TM C-1 Methyl silica 500 mg/6ml minicolumns (Applied Separations, PA, USA) are used according to the following procedure.

Preparation of prostanoid-free urine

Add 5% (w/v) activated charcoal to pooled normal human urine and stir it at room temperature for 1 hour. Centrifuge at about 3000xg for 15 minutes, and separate supernatant. The resulting liquid should be colourless and odorless. Filter if necessary, and store it at -20°C.

Remarks

The solid phase extraction procedure detailed above normally results in a recovery of > 90 and > 80% for SPE-1 and SPE-2, respectively, as checked by ³H-labelled 6-keto-PGF_1a/2,3-dinor-6-keto-PGF_1a, respectively, 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 these materials according to manufacturer's instruction, to check their radiochemical purity regularly, and to purify if needed, by chromatographic method.

Solvent residues, impurities as well as 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 and the lower the concentration the higher the relative error due to method blank. In order for 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.

Assay procedure

Day 1

Table 1. Dilution scheme (all volumes in microliters)

Note: 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)

Quantitation of urinary 6-keto-PGF_1a

Prepare the standard curve and quantitate 6-keto-PGF_1a fractions obtained in chromatographic separation according to the standard assay procedure as above. Concentrations will be obtained as pg/tube of 6-keto-PGF_1a.

Quantitation of urinary 2,3-dinor-6-keto-PGF_1a

Make the assay according to Assay Procedure, by using a dilution series of 2,3-dinor-6-keto-PGF_1a working standards according to the dilution scheme of Table 1. Use, as unknown samples, the 2,3-dinor-6-keto-PGF_1a fractions obtained in chromatographic separation.
Concentrations will be obtained as pg/tube of 2,3-dinor-6-keto-PGF_1a.

Remark

Cross-reaction value for 2,3-dinor-6-keto-PGF_1a as given later refers to the mean of 10 independent determinations. This value, however, as commonly referred to, only represents the ratio of effective doses at B / B₀ = 50%. Due to the non-parallelism of 2,3-dinor-6-keto-PGF_1a calibration curve with 6-keto-PGF_1a calibration curve, however, cross-reaction is varying throughout standard curve range (e.g 72% and 30% at B / B₀ = 80% and B / B₀ = 20%, respectively). Therefore the calculation of 2,3-dinor-6-keto-PGF_1a concentration by simply correcting the 6-keto-PGF_1a immunoreactivity for a constant per cent cross-reaction at B / B₀ = 50% (i.e. the Table value of 50.4%) would lead to an overestimation in the low and an underestimation in the high concentration range. So as to obtain a reliable value, the complete calibration curve constructed from serial dilution of 2,3-dinor-6-keto-PGF_1a should be prepared.

Calculation of results

The calculation is illustrated using representative data. The assay data collected should be similar to those shown in Table 3. and 4.

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 of the 6-keto-PGF_1a

6-keto-PGF_1a concentration pg/tube

Figure 1.
A typical standard curve
(Do not use to calculate sample values)
 

Table 4.    Typical Assay Data of the 2,3-dinor-6-keto-PGF_1a

2,3-dinor-6-keto-PGF_1a concentration pg/tube

Figure 2.
A typical standard curve
(Do not use to calculate sample values)

Characterization of the 6-keto-PGF_1a assay

Assay parameters

Specificity

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