Beksac, M. S.

Cekan, S. Z.

Clinical Chemistry and Laboratory Medicine (CCLM)

Undetermined

Dokumenten-Publikationsserver der Humboldt-Universität zu Berlin

Beksac and Cekan: Measurements of progesterone in urine 183J. Clin. Chem. Clin. Biochem.Vol. 23, 1985, pp. 183-186Reliability of Progesterone Measurements in UrineBy M. S. Beksac*) and S. Z. CekanReproductive Endocrinology Research Unil, Karolinska Hospital, Stockholm, Sweden(Received September 10/November 2, 1984)Summary: A radioimmunoassay (RIA) of progesterone in urine is described. After the addition of labelledprogesterone, morning urine was extracted with -hexane and the residue was either directly subjected toRIA, or chromatographed on celite prior to RIA. The progesterone from celite chromatography wasradiochemically pure. RIA after chromatography was therefore considered valid. The non-chromatographedprocedure resulted in overestimations, the degree of which was inversely proportional to progesterone content.The results obtained by the two procedures were well correlated (r =%0.88 and 0.93, for 2 different groupsof samples).Zuverlässigkeit von Progesteronbestimmungen im HarnZusammenfassung: Ein Radioimmunassay zur Bestimmung von Progesteron im Harn wird beschrieben.Morgenharn wurde mit -Hexan extrahiert und der Rückstand entweder direkt oder nach Chromatographiean Celit dem Radioimmunassay unterzogen. Ein im Wanderungsbereich von Progesteron durchgeführter Testauf radiochemische Reinheit zeigte, daß radiochemisch reines Progesteron gemessen wurde. Der Radioimmun-assay nach Chromatographie wurde deshalb als gültig angesehen. Ohne vorangehende Chromatographiewurden zu hohe Werte erhalten, deren Abweichung vom Zielwert umgekehrt proportional zum Progesteronge-halt war. Beide Verfahren korrelierten gut miteinander (r = 0,88 und 0,93 für zwei Gruppen von Proben).IntroductionRadioimmunoassay (RIA) of progesterone in bloodplasma has beeil a major analytical tool for the as-sessment of the corpus luteum function in women(e.g. I.e. (1)). Blood letting is, however, an invasivemethod, frequently difficült to perform, especiallywhen serial samples are needed. Therefore, alternativeapproaches are of importance.Urine is a body fluid which is easy to obtain andwhich may be expected to yield Information equiv-alent to that provided by assays in plasma. Recentlytechniques were developed for the radioimmunoassayHolder pf a WHO Research Training Grant; present address:Department of Obstetrics and Gynaecology, School ofMediane, Hacettepe University, Ankara, Turkey.or chemijuminescence assay of a progesterone metab-olite — pregnanediol glucuronide (2, 3). These assaysrequire, however, special reagents (a labelledglucuronide and an antiserum to the glucuronide)which are not generally available. In addition, thepurification of the glucuronides, both labelled andnon-labelled, is laborious and difficült. From thispoint of view, a RIA progesterone in urine would bemuch more covenient.RIA of progesterone in urine has been describedearlier (4—7). In nöne of the publications, however,was a valid procedure presented, i.e. none of theprocedures was shown to produce accurate results.The aim of the present investigation was to develop,for progesterone in urine, a RIA procedure validatedby the test of radiochemical purity (8).J. Clin. Chem. Clin. Biochem. / Vol. 23, 1985 / No. 4184 Bcksac and Cekan: Measurements of progesteronc in urineMaterial and MethodsU r i n c samplesSamples of early morning urine were collected from 11 appar-ently healthy women (aged 24-32 years) with histories ofregulär menstrual cycles. The sampling was daily, during theentire menstrual cycle. A total of 316 samples were obtained.The samples were brought daily to the Department of Ob-stetrics and Gynaecology, Karolinska Hospital, Stockholm.They were frozen and kept at -20 °C until analysed.A pool of urine was prepared by mixing urine samples of 5normally menstruating women. The samples were collectedduring the follicular phase of the menstrual cycle. This poolwas kept frozen in appropriate aliquots and used for qualitycontrol and for the lest of radiochemical purity.Reagen t s[1,2,6,7-3H]Progesterone (3.66 TBq/mmol) was purchased fromthe Radiochemical Centre (Amersham, U K) and non-radio-active progesterone from Steraloids (Wilton, NH 03086, USA).Progesterone antiserum (Batch 82K) was a gift from the WorldHealth Organization, Matched Reagents Programme, Geneva,Switzerland. The cross-reactions exhibited by this antiserumwere äs follows: 17-hydroxyprogesterone 2.0%, 20oc-di-hydroprogesterone 2.6%, testosterone 0.2%, Cortisol <0.1%.All other reagerits were of analytical purity. The radioactivitywas measured in a scintillation fluid consisting of 5.5 g of"Permablend III" (91g of 2.5-diphenyloxazole and 9g of /?-bis(O-methylstyryl)benzene; Packard Instrument Co., DownersGrove, IL 60515, USA) in l litre of toluene. The compositionof the phosphate assay buffer was äs described earlier (9), butthe pH was 7.4.E x t r a c t i o n and ch romatographyUrine (0.5 ml) was equilibrated (25 °C, 15 min) with a solutionof labelled progesterone (approximately 40 Bq in 50 of assaybuffer) and extracted with /7-hexane (5 ml). The aqueous layerwas frozen in an ethanol: dry ice mixture, the hexane phasewas decanted and evaporated under nitrogen. The residue wasdissolved either in 0.5 ml of assay buffer (60 °C for 10 min)for RIA ("rapid" procedure), or in l ml of isooctane forchromatography.The chromatography was performed using celite/propyleneglycol columns (1 + 1, weight/volume) äs described earlier (9),except that a 3.5 ml progesterone zone (instead of 4 ml) wascollected. The eluent (isooctane) was evaporated and the residuewas dissolved in assay buffer (0.5 ml) for RIA("Chromatographie" procedure).Both procedures were run in duplicate for every urine sample.In both procedures a 0.2-ml aliquot of the 0.5 ml assay buffersolution was taken for the RIA proper and another 0.2-mlaliquot for a recovery measurement.Rad io immunoassay (RIA)The RIA was performed äs described in detail earlier (9).Briefly, the incubation of the Standard or unknown Solutionswith the antiserum and the tracer (total volume 0.3 ml) wascarried out at 60 °C for 10 min, followed by 30 °C for 30 min.The bound and free fractions were separated by charcoal at0°C. For the calculation of results, a logit-log transformationwas used. The lowest detectable concentration (sensitivity) was75 pmol/1.Test of radiochemical pur i ty (8)An aliquot (5.0 ml) of the urine pool was equilibrated withradioactive progesterone (approximately 500 Bq in 0.5 ml assaybuffer). Ten portions (0.5 ml) of this solution were separatelyextracted with hexane and chromatographed äs describedabove, except that 0.5 ml fractions were collected in the regionof the 3.5 ml progesterone zone. The corresponding fractionsfrom the 10 columns were combined (in order to accurriulatesufficient amounts of radioactivity and mass), the solution wasevaporated and the residue dissolved in 0.5 ml of assay buffer.Aliquots (0.2 ml) were used for the RIA on the one hand,and for the radioactivity measureme.r}ts on the other. In thecalculation of the RIA results, a correction for the mass of thetracer added was made (10). For each fraction, specific activity(Bq/pg) was calculated.ResultsIn order to test the validity of the progestef one assayin urine, radiochemical purity was tested by meas-uring the specific activities (Bq/pg) of progesteronein the first 5 consecutive half-milliliter fractions of theprogesterone Chromatographie zone. These fractionscontained 94—95% of the recovered radioactivity.(The last 2 fractions were not used for furthercalculations due tp a low content of radioactivity andmass). The test was repeated three times in order tomake possible a statistical evaluation. Since there wasno indication of an isotopic effect (11), the differencebetween the means of specific activities in individualfractions was tested by a one-way analysis of vari-ance. It was found that — at the 95% confidencelevel — the difference was not significarit; the F-value found was 2.53 (df:4,10). Consequently, theChromatographie procedure was considered valid andthe results yielded by this procedure were taken äsreference values for those obtained by the rapid pro-cedure. ' .The validity of the latter procedure was tested by acompariscm of the results with those obtained by theChromatographie method. For this cornparison, 316samples were assayed by both methods. The meas-urements were divided into 2 groups according to thevalues indicated by the Chromatographie procedure.In figure l a, values (n = 145) below l nmöl/1 areshown, and in figure Ib, measurements (n = 143)higher than l nmol/1 are depicted. The first grouprepresents the majority of measurements during thefollicular phase of the menstrual cycle, the secondgroup is typical for luteal phase values.Regression analysis indicated that the correlationcoefficients (r) were high in both gfoüps; they were0,88 arid 0.93, respectively. The slopes and their Stan-dard errors for the calculated best-fit straight lines(1.36 ± 0.06 and 1.15 ± 0.04, respectively) indicateda highly significant difference (P < 0.001 in bothcases) from the theoretical slope = 1. Comparisonof the calculated straight lines with the theoreticalJ. Clin. Chem. Clin, Biochem. / Vol. 23, 1985 / No. 4Beksac and Cekan: Measurements of progesterone in urine 185^ 2.0luIIs!o0.50.5 10Progesterone (chromatography)[nmol/l]Fig. 1. Scattcr diagrams of values obtained by the Chro-matographie (abscissa) and rapid (extraction only;Ordinate) procedures. The theoretical straight lines (y =Ix; r = 1) are represented by the broken lines, thebest-fit straight lines by the fully drawn lines. Scatterdiagrams a and b are drawn for regions of Chro-matographie values 0—1 nmol/1 and 1 — 10 nmol/1, re-spectively. The equations of the best-fit straight lines andthe correlation coefficients are äs follows:ya =0.137 (±0.036) + 1.360 (±0.061) xa; ra = 0.882,yb = 0.452 (±0.133) + 1.152 (±0.040) xb; rb = 0.925,where ± values in parentheses are Standard errors.Tab. 1. Degree of overestirnation of results by the rapid pro-cedure, äs calculated from the regression lines (cf.fig.1).Results (nmol/1)Chromatographie Rapidprocedure procedurePercentageof overestimation0.20.51.005.000.410.821.506.21105645024Tab. 2. Precision and recovery. Within-assay Variation was cal-culated äs an average from 316 true duplicates. Theexcessive error was expressed äs the percentage ofduplicates in which coefficients of Variation exceeded25% and which were excluded from the calculations.Between-assay Variation was calculated from qualitycontrol samples in 7 assays. Recovery is expressed äsmeans ± Standard errors of percentages (n = 626).Procedure Within- Excessive Between-assay Recoveryassay error in variatonVariation duplica-• lcs Mean(%) ( / ) (nmol/1) (%) (%)RapidChroma-tography8.5 5.19.3 5.20.540.3212.9 69.517.0 51.7± 0.4± 0.5one shows that the measurements yielded by the rapidmethod were, on average, higher than those obtainedby the Chromatographie method, and that the degreeof overestimation was inversely proportional to theprogesterone contefit of the sample (tab. 1). Theseobservations make the rapid procedure invalid inthe sense that it consistently overestimates the truecontent and therefore does not yield accurate results.The data for within-assay and between-assay vari-ations, and for recovery are summarized in table 2.It has to be pointed out that the assessment of thewithin-assay Variation was obtained by averagingindividüal, within-duplicäte coefficients of Variation(CV), after excluding all CV-values exceeding 25%.This percentage was considered to be a limitnecessitating the repetition of measurements. The be-tween-assay Variation was assessed by assaying a low-progesterone quality cöiitrol pool in 7 assays. Itfollows from the data of table 2 that the between-assay Variation was lower and recpveries higher inthe rapid procedure in comparison with theChromatographie method. Relatively low recoveriesfor both procedures (70% and 50%, respectively) areascribed to the use of hexane äs extractant.DiscussionIt is well established that extraction äs the only puri-fication step does not always provide valid estimatesin a RIA (12 — 13). This happens in those cases whenthe extraction does not completely separate the com-pound assayed from cross-reacting compounds pre-sent in plasma. Not even the inclusion of aChromatographie step, however, can guarantee thatthe method will invariably yield valid estimates. Sucha case was observed with the RIA of low levels ofprogesterone in the plasma of women in the follicularphase of the menstrual cycle (8).In the present case, however, chromatography didachieve radiochemical purity and thus validity of theprogesterone assay in urine. Because the chro-matography used in the present study was in principlethe same äs that for plasma, and the specificity ofthe antisera employed here and in the previous study(8) was apparently similar, the radiochemical purityachieved by a single chromatography of urinaryhexane extracts seems to be due to a lower contentof interfering cross-reacting compounds in urine thanin plasma, and/or to the use of hexane instead ofether äs a solvent.J. Clin. Chem. Clin. Biochem. / Vol. 23, 1985 / No. 4186 Beksac and Cekan: Measurements of progesterone in urineThe rapid procedure was found invalid, because itproduced a significant overestimation of the results.This fmding is not surprising in view of the datapublished by Johnson et al. (5). Their data showed amarked overestimation of progesterone in urine whenmeasured by RIA after hexane extraction, incomparison with the results obtained by gas chro-matography-mass spectrometry.The changing degree of overestimation by the rapidmethod in the present study is in accordance withour earlier observations that the specificity of a rapidassay decreases with decreasing plasma levels (13,14). The common factor causing overestimations inthe assays of plasma and urine samples seems, there-fore, to be a relatively large concentration of cross-reacting factors when the concentration of pro-gesterone is low.The present investigation shows that valid meas-urements of progesterone in urine can be achievedby radioimmunoassay, provided the progesterone issubjected to preliminary purification by celite chro-matography. This procedure is of a great value when-ever accurate measurements are needed.In the clinical practice, however, prompt Informationon significant — albeit relative — changes issometimes more important than accurate values. Itis conceivable in these cases that the inherentlyinvalid, but much more practicable procedurewithout chromatography (rapid method) is used. Thiscomment is made in view of the fact that there existsa good correlation of the results yielded by bothprocedures, and that the average overestimates re-sulting from the rapid procedure are moderate inabsolute terms (e. g., 0.4 nmol/1 vs. 0.2 nmol/1, 6.2nmol/1 vs. 5 nmol/1).A clinical study comparing measurements in urineof progesterone by the Chromatographie and rapidmethods on the one hand," and of pregnanediolglucuronide on the other hand is in progress. Thisstudy is expected to demonstrate whether or notdirect measurements of pregnanediol glucuronide canbe replaced by progesterone measurements using aslightly more laborious technique but achievingassays of known degree of validity with the use ofcommonly available reagents.Acknowledgement,We are indebted to Dr. B.-M. Landgren (Department of Ob-stetrics & Gynaecology, Karolinska Hospital, Stockholm) forprovidirig us with urine samples. This investigation receivedfinancial support from the Special Programme of Research,Development and Research Training in Human Reproduction,World Health Organisation.References1. Landgren, B.-M., Unden, A.-L. & Diczfalusy, E. (1980)Acta Endocrinol. (Copenhagen) 94, 89-98.2. Samarajeewa, R, Cooley, G. & Kellie, E. E. (1979) J.Steroid Biochem. //, 1165-1171.3. Eshhar, Z., Kim, J. B., Barnard, G., Collins, W. R, Gilad,S., Lindner, H. R. & Kohen, F. (1981) Steroids 38, 89-109.4. Chattoraj, S. C, Rankin, J. S., Turner, A. K. & Löwe, E.W. (1976) J. Clin. Endocrinol. Metab. 43, 1402-1405.5. Johnson, D. W., Phillipou, G., Ralph, M. M. & Seamark,R. F. (1979) Clin. Chim. Acta 94, 207-208.6. Schön, F., Hackenberg, K., Paar, D. & Reinwein, D. (1980)thisJ. 18, 355-361.7. Schöneshöfer, M. & Fenner, A. (1981) this J. 19, 71-74.8. Cekan, S. Z. & de Gomez, M. S. (1979) Anal. Leiters 12(B6), 589-602.9. Brenner, P. F., Guerrero, R., Cekan, Z. & Diczfalusy, E.(1973) Steroids 22, 775-794.10. Cekan, S. Z. (1982) Clin. Chem. 28, 1248-1249.11. Xing, S., Zheng, S. & Cekan, S. Z. (1981) Clin. Chem. 27,600-602.12. Kanluan, T., Masironi, B. & Cekan, S. Z. (1981) Clin.Chim. Acta 109, 169-174.13. De Gomez, M. Sr, Frei, A., Vitins, P. & Cekan, S. Z. (1981)Ann. Clin. Biochem. 18, 281-286.14. Xing, S„ Cekan, S. Z., Diczfalusy, U., Falk, O., Gustafsson,S. A., Äkerlöf, E. & Björkhem, I. (1983) Clin, Chim. Acta135, 189-201.S. Z. Cekan, Ph. D.Reproductive Endocrinology Research UnitKarolinska HospitalS-104 01 StockholmJ. Clin. Chem. Clin. Biochem. / Vol. 23, 1985 / No. 4

Reliability of Progesterone Measurements in Urine

Summary: A radioimmunoassay (RIA) of progesterone in urine is described. After the addition of labelled progesterone, morning urine was extracted with-hexane and the residue was either directly subjected to RIA, or chromatographed on celite prior to RIA. The progesterone from celite chromatography was radiochemically pure. RIA after chromatography was therefore considered valid. The non-chromatographed procedure resulted in overestimations, the degree of which was inversely proportional to progesterone content. The results obtained by the two procedures were well correlated (r =%0.88 and 0.93, for 2 different groups of samples). Zuverlässigkeit von Progesteronbestimmungen im Har

M. S. Beksac

S. Z. Cekan

CiteSeerX

http://edoc.hu-berlin.de/oa/degruyter/cclm.1985.23.4.183.pdf

Reliability of Progesterone Measurements in Urine

Abstract

Similar works

Full text

Available Versions

Dokumenten-Publikationsserver der Humboldt-Universität zu Berlin

CiteSeerX