Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma

We have developed and validated a sensitive, simple and direct (i.e. without extraction) enzyme immunoassay (EIA) system for the measurement of progesterone in bovine milk and blood plasma. Progesterone (P) has been analysed by a microtiterplate EIA, employing polyclonal antibodies against P-7α-carboxyethylthioether-BSA as the antigen. The enzyme used as a label is horseradish peroxidase (HRP) and the chromogen is tetramethylbenzidine (TMB). Sensitivity of the EIA has been greatly improved by introduction of a heterologous tracer, in which progesterone is coupled to HRP at the 6β position. Compared to the radioimmunoassay (RIA) in which the same antiserum has been used, the sensitivity is 20 times greater. The detection limit of the assay is 0.4 pg per well. The working range of the standard curve is 0–20 pg per well (i.e. 0–40 ng per ml), and 50% reduction of the initial binding is obtained with 2.5-5.0 pg. Results can be obtained either by spectrophotometric measurement at 450 nm, or by naked eye. Total time needed for the assay of 40 replicate samples is approximately 3 h. Comparison of the EIA system with a previously validated RIA system gave a regression line EIA = 0.85 RIA + 2.11 (r = 0.93, n = 400 milk samples). Application of the milk-progesterone EIA to pregnancy testing (n = 66) gave an accuracy of 79.6% for positive diagnoses and 100% for negative diagnoses.     

Correlation between homologous and heterologous enzyme immunoassays for progesterone determinations in milk from cows

Two enzymeimmunoassays, homologous and heterologous, have been used for measuring progesterone in unextracted bovine milk using HRP as the enzyme label. Antibody raised by immunization of the rabbit against 11 alpha-hemisuccinate-BSA was used for the homologous system (EIA-11 alpha) and 7 alpha-carboxyethylthioether-BSA (EIA-7 alpha) for the heterologous. The progesterone derivatives used for the enzyme-hormone conjugates were 11 alpha-hemisuccinate and 6 beta-OH-hemisuccinate respectively. Milk progesterone in 60 samples measured by EIA-11 alpha and EIA-7 alpha were highly correlated (r = 0.93). Both systems were further compared with a conventional direct progesterone radioimmunoassay (RIA) in regular use for the same samples showing a good correlation. The sensitivity estimated was much higher in the EIA-7 alpha (0.5 pg/well) than in the EIA-11 alpha (32 pg/well).     

Oestrus control and early pregnancy diagnosis in the swamp buffalo: comparison of enzymeimmunoassay and radioummunoassay for plasma progesterone

A preliminary study has been undertaken, in order to investigate the suitability of a progesterone assay in blood plasma for oestrus control and pregnancy diagnosis in the swamp buffalo (Bubalus bubalis ). Progesterone was determined both by radioimmunoassay (RIA) and by enzymeimmunoassay (EIA). Values obtained by EIA were considerably lower than values obtained by RIA. This may be partly due to the fact that only RIA values were corrected for procedural losses. Blood samples were taken on day 1 (= day of insemination) and on days 24, 27 and 30 after insemination (p.i.). Additional samples from pregnant animals were taken around day 170 p.i.. Normal progesterone values during oestrus were lower than 0.5 ng/ml, and generally the same low values were found in case of non-pregnancy at days 24, 27 and 30 p.i.. Pregnant animals showed in all cases progesterone concentrations higher than 0.5 ng/ml at days 24, 27 and 30, as well as around day 170 p.i.. These preliminary results indicate that the analysis of progesterone in plasma may be suitable for fertility control in the swamp buffalo. Furthermore we suggest that a modified EIA method can be used as a simple and rapid oestrus detection test under field conditions.     

Use of progesterone 11-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre-plate enzymeimmunoassay of progesterone in milk and its application to pregnancy testing in dairy cattle

A simple direct-addition microtitre plate enzymeimmunoassay (EIA) for progesterone in whole milk is described. The assay used antiserum raised against 11 alpha-hydroxyprogesterone 11-hemisuccinate (progesterone 11-hemisuccinate) and a heterologous label prepared by conjugation of 11 alpha-hydroxyprogesterone 11-glucuronide (progesterone 11-glucuronide) with alkaline phosphatase using an active ester procedure. The sensitivity, analytical recovery, linearity of response and precision of the assay compared favourably with radioimmunoassay (RIA). Results from EIA of milk samples were compared with determinations made after isolation of progesterone by HPLC (r = 0.910). Milk samples (200) were assayed by RIA at both the Milk Marketing Board and the Cattle Breeding Centre and the results were correlated with EIA performed at the Cattle Breeding Centre (r = 0.890 and r = 0.833 respectively). Calving data were obtained from a further 110 cows for which the milk progesterone EIA had provided a pregnancy test 24 days after AI; 46 cows were correctly identified as non-pregnant and 58 as pregnant and there were 4 false positive and 2 inconclusive results.     

Development of a sensitive enzymeimmunoassay (EIA) for FSH determination in bovine plasma.

A highly sensitive enzymeimmunoassay (EIA) procedure for FSH determination in bovine plasma on microtiterplates using the biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to FSH and used to bridge between streptavidin-peroxidase and the immobilized antiserum in the competitive assay. The EIA was carried out directly in 50 microl of bovine plasma and compared with an established radioimmunoassay (RIA) employing 100 microl plasma. Same FSH standards and FSH specific antiserum were used in both procedures. FSH standards prepared in hormone free plasma were used. The sensitivity of the EIA procedure was 6.25 pg/well FSH which corresponded to 125 pg/ml plasma; the 50% relative binding sensitivity was seen at 200 pg/well. In comparison to RIA, the EIA was at least four times more sensitive besides requiring 6 times less FSH specific antiserum. Plasma volumes for the EIA ranging from 12.5 to 50 microl did not influence the shape of the standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. When both EIA and RIA methods were used to measure FSH in cows, the levels were detectable only by the EIA procedure. The assay detects high and low plasma FSH levels within the physiological variation as well as changes in plasma FSH after stimulation with a GnRH analog. In conclusion, in addition to being non-radioactive and low cost in nature, the method offers several advantages over the conventional FSH RIA procedure; these are (a) higher sensitivity, (b) less labour and time saving, (c) more economical use of precious FSH antiserum and (d) long shelf-life of the biotinyl-FSH label (in contrast to the short half life of iodinated FSH in RIA).     

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Development of a direct enzyme immunoassay of milk progesterone and its application to pregnancy diagnosis in cows

The purposes of this study were to develop an enzyme immunoassay (EIA) for determination of progesterone in unextracted whole milk and to apply it to pregnancy diagnosis. Paper fibers covalently coupled to antibody specifically and competitively bound ³H-progesterone and 11α-hydroxyprogesterone hemisucccinate-horseradish peroxidase and were stable for 9 mo at ?20°C. The sensitivity, recovery, precision, and cross reactivity of the EIA and a radioimmunoassay (RIA) of milk progesterone were evaluated, and showed that this EIA was comparable to the RIA. Milk samples were collected once daily for one estrous cycle from ten lactating Holstein cows and the progesterone levels were determined by RIA. Milk progesterone in 70 samples measured by EIA were highly correlated (r = 0.90) with the values of RIA for the same samples. Milk samples for pregnancy diagnosis by EIA of milk progesterone were obtained daily from days 20 to 24 after 115 artificial inseminations of 85 lactating Holstein cows. Pregnancy diagnosis by EIA was confirmed by rectal palpation at 30 to 60 days after insemination or return to estrus. The accuracy based on single, two, three, four, and five consecutive samples was from 67.2 to 80.0%, 77.3 to 84.0%, 79.2 to 87.5%, 82.0 to 85.4%, and 85.4%, respectively, for pregnancy diagnosis; and from 95.0 to 98.3% for nonpregnancy.     

Elisa for salivary and plasma estriol in pregnancy

A competitive, sensitive, and rapid enzyme-linked immunoadsorbent assay (ELISA) was developed for the determination of estriol in saliva and in plasma. Horseradish peroxidase (HRP) was used as the label enzyme; separation between free and bound steroid was carried out by insolubilized antibody prepared by adsorbing purified IgG of rabbit anti-6-oxoestriol-6-(0-carboxymethyl)oxime-BSA on polystyrene balls. The enzyme activity was measured by a colorimetric reaction using o-phenylenediamine dihydrochloride and hydrogen peroxide as substrate. The sensitivity of the assay was 12 pg/tube.In order to compare ELISA to RIA estriol estimations in different biological fluids, we selected six women during normal pregnancy, from the 30th to the 40th week of gestation. Salivary estriol was assayed by direct and extraction methods, while the corresponding plasma samples of the same subjects were analyzed only for unconjugated estriol by an extraction method.A good agreement was found between the results obtained by RIA and ELISA: r=0.897, p <0.001 between direct RIA and direct ELISA in saliva; r=0.909, p < 0.001 between extraction RIA and direct ELISA in saliva; and r=0.916, p < 0.001 between extraction RIA and extraction ELISA in plasma. A good correlation (r=0.793, p<0.001) was present between plasma samples by RIA and saliva samples by ELISA (direct method).These results indicate that: 1. ELISA is a reliable method for the determination of estriol in plasma and saliva. 2. Saliva samples can be used for the assay of estriol and therefore for the assessment of fetal conditions during pregnancy.     

Bioluminescent enzyme immunoassay for progesterone using monoclonal antibodies and glucose-6-phosphate dehydrogenase labels

Bacterial luciferase, NAD(P): FMN oxidoreductase and anti-mouse immunoglobulin were co-immobilized on Sepharose 4B. This reagent together with a progesterone glucose-6-phosphate dehydrogenase conjugate and various anti-progesterone monoclonal antibodies was used to develop a non-separation bioluminescent immunoassay for progesterone. This monoclonal antibody based assay was sensitive and reliable and using the tracer progesterone-11-acetate-glucose-6-phosphate dehydrogenase, the majority of the monoclonal antibodies give a better sensitivity with this enzymatic tracer than that obtained with an iodinated tracer. In a second assay design progesterone-glutathione was co-immobilized with bacterial luciferase and NAD(P): FMN oxidoreductase on Sepharose 4B and three monoclonal antibodies were labelled with glucose-6-phosphate dehydrogenase. With aqueous progester-one standards, this assay gave comparable sensitivity to the bioluminescent enzyme immunoassay using the second antibody immunoadsorbant and to an RIA but was unsuitable for plasma samples.     

The danger of hepatitis B virus infection from transfusions of plasma in which the HBs-antigen was detected retrospectively by highly sensitive methods

The prospective dynamic laboratory and clinical study of premature children was carried out: 55 children who received plasma transfusion during the first weeks of their life and were retrospectively (i. e. after plasma transfusion) found to have HBsAg detected by the passive hemagglutination (PHA) test, the enzyme immunoassay (EIA) and the radioimmunoassay (RIA) and 127 children who received the transfusion of plasma found to be HBsAg-negative according to the results of EIA and RIA. As revealed in this study, the risk of hepatitis B virus infection in such children after the transfusion of plasma containing HBsAg at low concentrations, determined only in the PHA test or in EIA and RIA, was, respectively, 7.5 and 6.3 times greater than in children receiving plasma found to be HBsAg-negative according to the results of EIA and RIA. The results of this investigation showed the tendency towards a decrease not only in the total contamination of plasma recipients with hepatitis B virus, but also in morbidity rate in icteric forms of acute posttransfusion hepatitis B, depending on the concentration of HBsAg in plasma used for transfusion.     

Evaluation of an on-farm blood progesterone test for predicting the day of parturition in cattle

An on-farm blood progesterone enzymeimmunoassay (EIA) was evaluated as a diagnostic test to predict the time of calving within a 24-hour period in near-term dairy cows. Blood samples were taken daily from 45 cows beginning 5 days prior to their expected due dates until calving, and plasma was stored at -20 degrees C until all cows had calved. The EIA test was performed on frozen-thawed plasma samples, and progesterone concentrations were determined to be low (positive test for calving within 24 hours) or high (negative test for calving within 24 hours). Sensitivity, specificity and predictive value of the EIA to accurately determine parturition within 24 hours were 86.7, 90.8 and 75.0%, respectively. The EIA correctly predicted the day of parturition in 168 of 187 (89.8%) plasma samples. Ten additional cows were similarly monitored except the EIA was performed on whole blood immediately after collection, and the sensitivity, specificity and predictive value of the test were 80.0, 97.6 and 88.9%, respectively. The day of parturition was correctly predicted in 49 of 52 (94.2%) whole blood samples. More than 95% of the cows calved within 24 hours when their plasma progesterone reached < 1.3 ng/ml. When results of the EIA were compared with those of a radioimmunoassay (RIA), the EIA findings were used to correctly classify 190 of 232 (81.9%) plasma samples as having low (< 2.0 ng/ml) or high (>/= 2.0 ng/ml) concentrations of progesterone. The EIA test was found to be a quick, practical means of estimating progesterone concentrations in bovine plasma or whole blood and was a useful test for predicting the day of parturition in cows.     

A comparison of enzyme-immunoassay and radioimmunoassay for detection of hepatitis A virus and antibodies against hepatitis A virus

A direct comparison has been made of tracers labelled with an enzyme and with 125I in solid phase enzyme-immunoassay (EIA) and solid phase radioimmunoassay (RIA) for the detection of hepatitis A virus (HAV) antigen and antibodies to HAV. By comparing the binding capacity of peroxidase-labelled anti-HAV-IgG and anti-HAV-F(ab)2 fragments tracers, anti-HAV-IgG was found to have a higher binding capacity than anti-HAV-F(ab)2 fragments in both EIA and RIA. For EIA 16.25-fold more anti-HAV-IgG was needed for one test probe compared to RIA and 32.5-fold more anti-HAV-F(ab)2 fragments. For the detection of HAV antigen from stool preparations and IgG and IgM antibodies against HAV, there were only minor quantitative differences in titre. EIA was as sensitive as RIA.     

Predictive value of palpation per rectum vs milk and serum progesterone levels for the diagnosis of bovine follicular and luteal cysts

Diagnosis of bovine follicular and luteal cysts by palpation per rectum was compared with diagnosis by progesterone status, as determined by an enzymeimmunoassay (EIA). A 2x2 contingency table analysis allowed the calculation of predictive value, sensitivity and specificity for the palpation diagnosis of both cyst types. The results were 75.0, 61.9 and 50.0% for follicular cysts and 35.1, 50.0 and 61.9% for luteal cysts, respectively. The EIA results were compared to radioimmunoassay (RIA). The predictive value for a low progesterone determination (<5ng/ml) was 91.8% and for high progesterone (5ng/ml or >) it was 83.3%. The sensitivity and specificity for the EIA were both 88.2%. Within the EIA system, milk and serum samples agreed a total of 85 out of 87 times (97.7%). Definitive diagnosis of follicular or luteal cysts should not be based upon palpation per rectum alone. The EIA was found to be the better definitive diagnostic technique.     

Radioimmunoassay and enzymeimmunoassay of plasma progesterone as monitors of progesterone sponge treatment in ewes

The daily plasma progesterone (P) concentrations achieved during insertion of P (750 mg) sponges into two groups of ewes were examined. Group I received prostaglandin (PG) treatment, which was required to suppress the P production (to levels of < 0.3 ng hormone/ml plasma) from the corpora lutea (CL) of a previous superovulation treatment, following which these Group I ewes and the anestrous Group II ewes were sponge treated. Radioimmunoassay (RIA) and enzymeimmunoassay (EIA) were used to measure the P levels in both groups. Progesterone (750 mg) sponges with and without citric acid impregnation were inserted into all the ewes for 12 days (d). Citric acid lowered the P levels reaching the plasma from the sponges, but it did not mask the characteristic profile (during the treatments) determined by the states of the ewes (single PG and double PG injected, Group I or in the anestrous Group II). The plasma P levels in Group I and II ewes rose to at least 7.0 ng/ml at intervals during treatment. The duration and magnitude of the P concentrations in the plasma were higher in the single PG compared with the double PG ewes during sponge insertion in Group I. The anestrous Group II ewes showed two major peaks (Day 1, P<0.01 and Days 11 to 12, P<0.05) during sponge treatment. A P level > 2.0 ng/ml was maintained over the entire treatment in the single PG and in the anestrous hormone-treated ewes, and was of shorter duration (7 d) in the double PG-treated animals. These endogenous patterns in P profiles of the ewes indicate that the hormone level during sponge insertion varies in magnitude and duration, parameters determined by the physiological/endocrinological state of the ewes at the start of the treatment. The EIA correlated significantly (P<0.001) with the RIA for the measurement of P concentration, when analyzed daily on an individual animal basis.     

Analysis of 6-keto PGF1 alpha, 5-HETE, and LTC4 in rat lung: comparison of GM/MS, RIA, and EIA

The recent availability of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the lung extract assayed for 6-keto-PGF1 alpha, 5-HETE and LTC4. By EIA lung tissue was found to contain large quantities of 6-keto-PGF1 alpha after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF1 alpha amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF1 alpha, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Pak) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher values obtained by RIA. LTC4 was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC4 levels were identical in unpurified lung extract and after Sep-Pak purification, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproducible results in lung tissue, although LTC4 and 5-HETE must be purified prior to analysis.     

A highly specific radioimmunoassay for progesterone using antibodies covalently linked to arylamine-glass particles

Antibody was produced in sheep following the administration of the antigen, progesterone-11α-succinyl-BSA. After treatment with Rivanol, the protein of the albumin-free antiserum was covalently bound to arylamine glass particles using glutar-aldehyde as the coupling agent. This antibody-glass preparation was used for measurement of plasma progesterone by radioimmunoassay. Progesterone was extracted from human plasma with petroleum ether. Of those Steroids which are petroleum ether extractable, only pregnenolone exhibited minimal cross-reactivity. Immobilization of the antibody by attachment to an insoluble support allows the separation of free from antibody-bound steroid without the use of absorbents, such as florisil and charcoal.     

Analysis of 6-Keto PGF1a, 5-hete and LTC4 in rat lung: Comparison of GC/MS,RIA, nad EIA

The recent avaibility of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the ling extract assayed for 6-keto-PGF_1a, 5-HETE and LTC₄.By EIA lung tissue was found to contain large quantities of 6-keto-PGF_1a after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF_1a amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF_1a, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Park) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher obtained by RIA. LTC₄ was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC₄ levels were identical in unpurified lung extract and after Sep-Pak purifucation, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproductive results in lung tissue, although LTC₄ and 5-HETE must be purified prior to analysis.     

Development and testing of radio and enzyme immunoassays for acidic fibroblast growth factor (aFGF)

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor from bovine brain stimulate growth in a variety of tissues in several species. Despite the 55% amino acid sequence homology of the two forms of FGF, a specific immunoassay of aFGF has been developed using a polyclonal antibody raised in a rabbit. Two immunoassays were compared: a radioimmunoassay (RIA) using 125I aFGF and an enzyme immunoassay (EIA) using aFGF coupled to the tetrameric form of acetylcholinesterase (aFGF-AchE) as tracer. With EIA, the detection limit was 1.5 ng/ml, versus 2.2 ng/ml with RIA, while the dose at 50% was 5.9 ng/ml for EIA and 9.6 ng/ml for RIA. Using a modified EIA procedure where aFGF-AchE was added 2 h after the other reagents, the dose at 50% binding was 1.5 ng/ml. Examples of the performance of both immunoassays are presented for various brain extracts of different species including human. The aFGF content obtained by these methods correlates (CR = 0.987) with the values obtained by biological assay.     

Use of group-specific antibodies to detect fecal progesterone metabolites during the estrous cycle of cows

Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P < 0.001) was 0.76 (y = 155.2 × + 37.2). Fecal 20-oxo-pregnane concentrations during the follicular and luteal phase were 39.5 ± 2.2 and 341 ± 15.2 ng/g feces, respectively. In conclusion, fecal 20-oxo-pregnanes are significantly correlated to milk progesterone concentrations. They consist of several metabolites and compared to a 6HS-progesterone antibody, their evaluation was improved using antibodies against 5-reduced pregnanes.     

Progesterone fate in rabbit cornea

Excised cornea from adult New Zealand rabbits were incubated with progesterone-4-14C in Eagle's media for 96 hr. Samples were inactivated at intervals of 24 hr incubation periods. The following metabolites of progesterone were isolated: 20 alpha-Hydroxy-4-pregnen-3-one, 20-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione; 5 beta-pregnane-3,20-dione and 6 beta-hydroxy-4-pregnen-3,20-dione. 20 alpha-Hydroxy-pregnen-3-one was the predominant metabolite of progesterone-4-14C. A linear increase was observed throughout 96 hr. The opposite was found for 5 alpha and 5 beta pregnane-3,20-dione. Compounds remaining at the origin of the paper chromatograms contained 6 beta-hydroxy-4-pregnen-3,20-dione and other still unidentified metabolites of progesterone-4-14C. Presence of 20 alpha and 20 beta-reductase; 5 alpha and 5 beta-reductase and 6 beta-hydroxylase enzyme systems are involved in corneal progesterone metabolism. No fungal neither bacterial enzymatic biotransformation occurred in the culture media.