Progesterone In Human Plasma Research Articles

Simultaneous quantification of endocannabinoids, oleoylethanolamide and steroid hormones in human plasma and saliva

Endogenous cannabinoids are an increasingly intriguing target for biological research, given the changing legal status of medicinal cannabinoid-based products throughout the world. However, studying the endogenous cannabinoid system is a relatively new field, with few research teams attempting to develop quantitative methods for these important modulatory analytes in human matrices, other than blood. Here we develop and validate simultaneous methods for quantifying arachidonoyl-ethanolamide, 2-arachidonoyl glycerol, oleoylethanolamide, cortisol and progesterone in human plasma and saliva using liquid–liquid extraction combined with ultra-high performance liquid chromatography coupled to tandem mass spectrometry. The method was fully validated over the linear concentration range 1–20 ng/mL for each analyte in plasma (R2 = 0.98–0.99) and saliva (R2 = 0.99). We find that salivary endogenous cannabinoids and cortisol are acutely responsive to exercise, suggesting that targeting the saliva system may present a convenient way for future research of endogenous cannabinoids. This finding also encourages a broader understanding of the endogenous cannabinoid system during stress responses, and our method may consequently lead to a better understanding of the role of endogenous cannabinoids in peripheral tissues.

Simultaneous quantitation of 17α-hydroxyprogesterone caproate, 17α-hydroxyprogesterone and progesterone in human plasma using high-performance liquid chromatography–mass spectrometry (HPLC–MS/MS)

A sensitive and specific assay method for the simultaneous quantitation of 17α-hydroxyprogesterone caproate (17-OHPC), 17α-hydroxyprogesterone (17-OHP), and progesterone (P) in human plasma using high-performance liquid chromatography and tandem mass spectrometry (LC–MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis ® HLB extraction cartridge prior to chromatography. Medroxyprogestrone acetate (MPA) was used as the internal standard. The compounds were separated using Waters C18 Symmetry analytical column (3.5 μm, 2.1 mm × 50 mm) using a gradient elusion with a mobile phase consisting of 5% methanol in water [A] and methanol [B], with ammonium acetate (2 mM) and formic acid (0.1%) being added to both [A] and [B], at a flow rate 0.3 ml/min. The retention times for 17-OHPC, 17-OHP, P and MPA were 4.5, 1.5, 2.5 and 2.2 min, respectively, with a total run time of 7 min. The analytes were detected by a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/ z 429.10 → 313.10 for 17-OHPC, m/ z 331.17 → 97.00 for 17-OHP, m/ z 315.15 → 109.00 for P and m/ z 387.15 → 327.25 for MPA (IS). The assay was linear over the range 1–200 ng/ml for 17-OHPC and 17-OHP, and 2–400 ng/ml for P, when 0.4 ml of plasma was used in the extraction. The overall intra- and inter-day assay variation was <15%. No significant variation in the concentration of 17-OHPC, 17-OHP or P was observed with different sample processing and/or storage conditions. This method is simple, allows easy, accurate and reproducible measurement of 17-OHPC, 17-OHP and P simultaneously in human plasma, and is used to evaluate the pharmacokinetics of 17-OHPC in pregnant subjects.

Neuroactive ring A-reduced metabolites of progesterone in human plasma during pregnancy: elevated levels of 5 alpha-dihydroprogesterone in depressed patients during the latter half of pregnancy.

Progesterone and its 5 alpha reduced metabolite, 5 alpha-dihydroprogesterone, rise greatly in pregnancy. Both are known to have anesthetic properties, as do a number of other ring A-reduced progesterone metabolites. The possible significance of these steroids with respect to the mood changes that are common in pregnancy and in the puerperium has not been explored. In this study, pregnenolone, progesterone, and five neuroactive progesterone metabolites: the 5 alpha and 5 beta dihydroprogesterones (DHP), and three tetrahydroprogesterones (THP)-3 alpha,5 alpha-THP, 3 beta,5 beta-THP, and 3 beta,5 alpha-THP-were studied at various stages of pregnancy and in the early postpartum period. Levels of all of the steroids rose greatly during pregnancy (P < 0.001), being highest for progesterone (562-fold the follicular level), 5 alpha-DHP (161-fold), 3 beta,5 alpha-THP (56-fold), 3 alpha,5 alpha-THP (37-fold), pregnenolone (30-fold), 5 beta-DHP (16-fold) and 3 beta,5 beta-THP (16-fold) at 37 wk of gestation. During the period 2-7 d postpartum, the level of progesterone fell precipitously, whereas those of pregnenolone and the metabolites fell more slowly and mean levels were still elevated compared with follicular levels 2 wk after delivery. By 7 wk postpartum, only 3 alpha,5 alpha-tetrahydroprogesterone and 3 beta,5 beta-tetrahydroprogesterone remained slightly elevated (P < or = 0.012 and 0.007, respectively). Mean levels of the progesterone metabolites tended to be higher in depressed patients compared with controls, and this difference reached significance for 5 alpha-dihydroprogesterone both at 27 wk (P = 0.04) and at 37 wk (P = 0.02) of gestation (combined, P = 0.003). These results show that all five of these metabolites rise markedly during pregnancy and suggest that alterations in progesterone metabolites may be involved in the mood changes of pregnancy and the puerperium.

Neuroactive Ring A-Reduced Metabolites of Progesterone in Human Plasma during Pregnancy: Elevated Levels of 5 -Dihydroprogesterone in Depressed Patients during the Latter Half of Pregnancy

Neuroactive Ring A-Reduced Metabolites of Progesterone in Human Plasma during Pregnancy: Elevated Levels of 5 -Dihydroprogesterone in Depressed Patients during the Latter Half of Pregnancy

Luminescent immunoassay (LIA) for progesterone in a heterogeneous system

An immunoassay procedure for determination of progesterone in human plasma is described which utilizes chemiluminescence as the endpoint. The assay utilizes progesterone-11-hemisuccinate-aminobutyl-ethyl-isoluminol (P-11-HS-ABEI) as the chemiluminescent marker conjugate and dextran coated charcoal for the separation of bound and free fractions of the ligand. An assay procedure for progesterone was established and validated in terms of sensitivity and precision, and assay results were compared with radioimmunoassay, using tritiated progesterone as the tracer. The two methods agreed well ( n = 35, r = 0.96). The most important advantage of this assay is the elimination of problems inherent in the use of radioactive materials.

Development of a solid-phase chemiluminescence immunoassay for plasma progesterone

A solid-phase immunoassay procedure for the determination of progesterone in human plasma is described, which utilizes chemiluminescence as the end-point. A progesterone-isoluminol conjugate serves as the chemiluminescent marker. An IgG fraction of antiserum to progesterone-11α-hemisuccinate bovine serum albumin is passively adsorbed to the walls of Lumacuvettea P polystyrene test tubes. After the binding reaction, the cuvettes are washed. The light yield of the bound conjugate upon oxidation with a H 2O 2-microperoxidase system at pH 13 decreased with increasing free progesterone concentration in a log-linear manner over the range 15–1000 pg/tube. The chemiluminescence immunoassay is comparable to radioimmunoassay with regard to sensitivity, specificity, precision and accuracy. The assay offers the advantages of speed and ease of automation. In addition, this method does not involve the use of radioisotopes or of a centrifugation step.

An improved enzyme immunoassay for progesterone in human plasma.

We describe an enzyme immunoassay for progesterone in which we use a progesterone-11alpha-hemisuccinyl-horseradish peroxidase conjugate as the "label" and an antiserum raised in rabbits to a progesterone-11alpha-hemisuccinyl-bovine serum albumin conjugate. In this assay, antibody-bound and free steroid are separated by using a second antibody precipitation procedure. The assay has a lower limit of sensitivity of 10 pg/assay tube and satisfies the usual criteria of specificity, precision, and accuracy. Results obtained with a comparison radioimmunoassay and our procedure agreed well (r = 0.98). Eighty samples can be assayed per day. It is not only well suited for surveys where accurate determination of progesterone concentrations in small plasma aliquots is required, but also for monitoring ovulation induction in patients attending infertility clinics.

A specific enzymeimmunoassay for progesterone in human plasma

An enzymeimmunoassay for plasma progesterone was established using progesterone covalently linked to the enzyme, horseradish peroxidase, as the ‘label’. Separation of free and bound steroid was effected by Sepharose-coupled anti-progesterone-11α-hemisuccinyl bovine serum albumin antiserum (Sepharose-antisera). The enzymeimmunoassay satisfied the normal criteria of specificity, precision and accuracy. Comparison of assay results obtained by radioimmunoassay (with and without thin-layer chromatography) and enzyme-immunoassay (with and without thin-layer chromatography) showed excellent agreement of results in all cases (r > 0.98). This enzymeimmunoassay is particularly applicable to the routine determination of plasma progesterone in the smaller clinical laboratory.

A quantitative method for the estimation of testosterone and progesterone in human plasma, using the gas chromatograph/mass spectrometer combination with single ion monitoring

This report describes the quantitative determination of plasma testosterone and progesterone by mass fragmentography of the molecular ion M + = 526 for the testosterone derivative and M + = 510 for the progesterone derivative. The total internal standards are added to the plasma before ether extraction and subsequent derivative formation. The lower limit of detection is 0.25 ng/ml plasma for testosterone and 0.19 ng/ml plasma for progesterone. Accuracy and precision of this method as well as the correlation with hormone concentrations measured by radioimmunoassay are discussed.

Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with 3H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.

The measurement of progesterone in human plasma by competitive protein-binding radioassay.

A method for the determination of progesterone in human plasma by competitive protein-binding is described. This method consists of extraction with petroleum ether, HTLC to purify the extracts and CBG-assay. The reliability criteria of the complete procedure are presented. The method is suitable for application to peripheral blood levels at all stages of the menstrual cycle and in men. The plasma values of progesterone in men and during the follicular and luteal phases of the menstrual cycle were 0.026 ± 0.014, 0.12 ± 0.02 and 0.83 ± 0.58 (S.D.) µg/100 ml, respectively.

A gas-chromatographic method for the quantitative determination of progesterone in human plasma

A method for the quantitative determination of progesterone in human plasma is described. This method consists of extraction with ether, solvent partition, thin-layer chromatography and finally gas-liquid chromatography for the quantitation of progesterone. The method is suitable for application to peripheral blood levels at all stages of the menstrual cycle and pregnancy. Data are presented on the sensitivity, accuracy, precision and reproducibility of the method.

QUANTITATIVE DETERMINATION OF PROGESTERONE IN HUMAN PLASMA BY THIN-LAYER AND GAS-LIQUID RADIOCHROMATOGRAPHY.

Quantitative Determination of Progesterone in Human Plasma by Thin-Layer and Gas-Liquid Radiochromatography

The fluorimetric determination of progesterone in human plasma during pregnancy and the menstrual cycle.

SUMMARY The fluorescence reaction for progesterone described by Touchstone &amp; Murawec (1960) has been used to determine the concentration of progesterone in nineteen samples of peripheral blood from pregnant women, and in seventeen samples of peripheral blood from women during the course of the menstrual cycle. There was good agreement between the ultraviolet and fluorescent estimates of progesterone in all the samples from pregnant women. The concentrations found during the follicular phase of the menstrual cycle were in general lower than those found during the luteal phase. In one woman who was sampled repeatedly during the course of a menstrual cycle, there was a well defined rise in the level of progesterone in the blood after the expected date of ovulation.