Reverse Isotope Dilution Technique Research Articles

Enhanced dehydroepiandrosterone synthesis by amnion compared to chorion: A comparative study using the reverse-isotope dilution technique

With a view to establishing whether the term human fetal membranes possess the enzymic ability to synthesize dehydroepiandrosterone (DHEA) from pregnenolone, homogenates of amnion and chorion obtained from women (n = 5, age 27-34 years) after spontaneous labor at term (37-42 weeks gestation) from uncomplicated pregnancies were incubated with [7n-3H]pregnenolone as substrate. Reverse-isotope dilution analysis gave positive identification of [3H]DHEA acetate in all incubations of viable tissues. No such metabolite was evident in control incubations with heat-denatured tissues. Virtually radiochemically pure esters under three recrystallizations were obtained with mean concentrations of between 15787 and 30137 dpm mol(-1) for amnion which was considerably higher than that of chorionic tissues at 4316-5528 dpm mol(-1). The magnitude of elevation in DHEA production by amnion was noted to be between 3.6- and 5.5-fold higher than the corresponding chorion. This study provides evidence that the fetal membranes possess 17-alpha hydroxylase and C-17, 20 lyase activities capable of synthesis of DHEA, an important androgen necessary for aromatization to estrogens in need by the developing fetus.

Isotope ratio measurements of magnesium and determination of magnesium concentration by reverse isotope dilution technique on small amounts of 26Mg-spiked nutrient solutions with inductively coupled plasma mass spectrometry

In the present work, an analytical method has been developed for the measurement of magnesium isotope ratios and the determination of magnesium concentration in 26Mg-spiked plant nutrient solutions by the reverse isotope dilution technique. The experimental parameters of the mass spectrometric measurements using quadrupole-based inductively coupled plasma mass spectrometry (ICP-MS) were optimized with respect to the lowest relative standard deviation (RSD) in the magnesium isotopic ratio ( 25Mg/ 24Mg and 26Mg/ 24Mg). The RSD of the measured isotopic ratios was determined in different concentrations of isotopic standard solution (NIST SRM 980T 26Mg-enriched isotopic standard) of natural isotopic abundances (from 1 to 50 μg/L with 1.9% to 0.22% RSD, respectively). Long-term isotopic ratio measurements using 50 μg/L isotopic standard solution (NIST SRM 980) yielded 0.14% RSD for 25Mg/ 24Mg and 0.23% RSD for 26Mg/ 24Mg. The accuracy of measured isotopic ratios was 0.1% to 0.3% [ 25Mg/ 24Mg 0.12684 ± 0.00018 (measured) versus 0.12663 ± 0.00013 (certified) and 26Mg/ 24Mg 0.13958 ± 0.00036 (measured) versus 0.13932 ± 0.00026 (certified)]. Synthetic isotopic mixtures of NIST SRM 980 and highly enriched 26Mg solutions were prepared and the measured isotopic ratios were compared with the calculated ones. The RSD of the isotope ratios measurements ranged between 0.14% to 0.07% for the 25Mg/ 24Mg ratio and 0.2-0.04% for the 26Mg/ 24Mg ratio. The developed method has been successfully applied for the determination of the magnesium isotope ratios and the concentration measurements in 26Mg-enriched plant nutrient solution samples with reverse isotope dilution technique. The nutrient solutions investigated contained high amounts of calcium and potassium (20–35 μg/ml and 9.7–30 μg/ml, respectively). Magnesium concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) and ICP-MS with external calibration and the measured values are in agreement with the results obtained by the reverse isotope dilution technique.

Evidence for Progesterone Synthesis by Human Umbilical Cord Blood Erythrocytes

In order to determine whether human umbilical cord blood erythrocytes are capable of progesterone biosynthesis, sonicated preparations of plasma-free erythrocytes at the range of total cell numbers between 18.4 × 10⁹ and 62.9 × 10⁹ cells obtained from umbilical cord arterial and venous blood collected from normal pregnant women (n = 6, age 28–39 years) following spontaneous vaginal delivery were incubated with [7n-³H]-pregnenolone as substrate. The leucocyte content of the incubates was negligible (<0.005%). Controls (n = 4, age 29–36 years; 27.6 × 10⁹ to 42.7 × 10⁹ erythrocytes) obtained from cord blood of normal pregnant women were heat-denatured preparations of cells. Using the reverse-isotope dilution technique, [³H]-progesterone was isolated and characterized yielding an overall enzymic conversion which ranged between 0.27 and 0.46%. The results indicate for the first time that cord blood erythrocytes possess the 3β-hydroxysteroid dehydrogenase-5,4-en isomerase activity and are a source of progesterone in human pregnancy.

Evidence for de novo Cholesterol Synthesis by Term Human Fetal Amnion and Chorion: A Comparative Study Using the Reverse-Isotope Dilution Technique

The cholesterol biosynthetic activity was assessed using [2-¹⁴C]-acetate as substrate in the homogenates of amnion and chorion obtained from women (n = 6, age 26–39 years) after spontaneous labour at term (37–40 weeks of gestation) having uncomplicated pregnancies. Reverse-isotope dilution analysis gave positive identification of [¹⁴C]-cholesterol acetate in all incubations of viable tissues. This metabolite was not evident in heat-denatured homogenates which served as controls. The extent of enzymic conversion for amnion at 2.6 × 10^–3 to 0.19% was persistently higher than that of the chorion at 1.7 × 10^–3 to 9.0 × 10^–3%. The results indicate that human term fetal membranes possess the full complement of enzymes to catalyze the transformation of acetate to cholesterol. This study provides evidence that fetal membranes possess the capacity for de novo cholesterol biosynthesis, the sterol being essential for steroidogenesis as well as in embryo viability during pregnancy.

Expression of C-20, 22 desmolase activity by the human fallopian tube in vitro: evidence for steroidogenesis.

With a view to establishing whether cells of the human Fallopian tubes possess the cholesterol side-chain cleavage activity, homogenates of the tubes, obtained from 6 women (39-45 years) following abdominal hysterectomy for benign conditions, were incubated with (7n-3H)-cholesterol as substrate. Controls (n=6, age 40-44 years) were homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution technique, (3H)-pregnenolone was isolated and characterized. No such metabolite was evident in control incubations of heat-denatured enzymes. The extent of enzymic conversion varied from 1.9 x 10(-3) to 1.3 x 10(-2)%. The results reveal for the first time the existence of an active cholesterol-specific C-20, 22 desmolase system in the viable tissues. It is suggested that there exists a potential for substantial pregnenolone synthesis in vivo. This rate-limiting steroid biosynthetic conversion provides a new dimension to the functional capacity of the Fallopian tubes in the synthesis of steroids, which may be necessary for modulating ciliary beat frequency and in maintenance of hormonal milieu essential for embryogenesis.

A delta 4-3-keto pathway for testosterone synthesis in the human spermatozoa.

The ability of human spermatozoa to metabolize pregnenolone to progesterone and progesterone to testosterone was assessed. Sonicated specimens of freshly ejaculated sperm from two groups of husbands (n = 6, age 32-38 years; n = 6, age 30-51 years) of infertile couples in the range of sperm concentration between 237.5 and 568.5, 100.1 and 248.8 millions per ejaculate, were separately incubated with [7n-3H]pregnenolone and [1,2,6,7,16,17-3H]progesterone. Using the classical reverse-isotope dilution technique the desired products [3H]progesterone and [3H]testosterone formed from the respective substrates were isolated and characterized, yielding 1.4 to 12.2% and 3.1 x 10(-2) to 2.0 x 10(-1)%. Such metabolites were not evident in the controls. The results indicate that the human spermatozoa contain the enzymes necessary for the transformation of pregnenolone to testosterone via the delta 4-3-keto route.

Characterization of a delta 5-3 beta-hydroxy pathway of testosterone synthesis in the human spermatozoa.

Sonicated specimens of freshly ejaculated sperm from two groups of husbands (n = 6, age 32-38 years; n = 7, age 27-38 years) of infertile couples in the range of sperm concentration between 237.5 and 568.5; 131.1 and 256.7 millions per ejaculate were separately incubated with [7n-3H]pregnenolone and [1,2,6,7(-3)H]dehydroepiandrosterone. Using the reverse-isotope dilution technique [3H]dehydroepiandrosterone and [3H]testosterone formed from the respective substrates were isolated and characterized, yielding 1.2 x 10(-2) to 4.6 x 10(-20/0) and 7.1 x 10(-2) to 2.5 x 10(-10/0)/ Such metabolites were not evident in the controls. These results provide evidence for metabolic transformation of pregnenolone to testosterone via the delta 5-3 beta-hydroxy route.

In vitro formation of testosterone via the Δ 5-pathway in the human umbilical cord

Homogenates of two groups of term umbilical cord ( n = 6, 37–40 weeks; n = 6, 38–40 weeks) were separately incubated with [7n- 3H]pregnenolone and [1,2,6, 7- 3H]dehydroepiandrosterone. Using the reverse-isotope dilution technique, [ 3H]dehydroepiandrosterone and [ 3H] testosterone formed from the respective substrates were isolated and characterized. The extent of enzymic conversions were 0.015-0.28% and 0.044-2.2%. These results provide evidence for the metabolic transformation of pregnenolone to testosterone via the Δ 5-3β-hydroxy route.

Enhanced Uptake and Metabolism of Riboflavin in Erythrocytes Infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [14C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5-8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0-3 h at 37 degrees C in phosphate buffered saline containing MgCl2, glucose, and [14C]riboflavin (2.5-7.5 microM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.

Human Umbilical Cord as a Possible Source of Progesterone

Homogenates of human term umbilical cord were incubated with 7n-3H-pregnenolone. Using a reverse-isotope dilution technique, 3H-progesterone was isolated and characterized. The extent of enzymic conversion ranged from 2.5 to 55.4%. The results indicate that the cord can be a source of progesterone during human pregnancy.

Development of a dual isotopes technique and two-stage column chromatography to characterize the prostaglandin F 2α, E 2 and thromboxane B 2 formed by the guinea Pig lung

A sensitive dual isotopes ([ 14C]-arachidonic acid, ([ 3H]-PGF 2α, and [ 3H]-PGF 2) technique coupled with two-stage column chromatographic systems was developed to characterize the prostaglandins synthesized by guinea pig lung. The assay consisted of incubating 600 g supernatant fraction of lung homogenate (75–600 μg protein) with 7.88 μM of [ 14C]-arachidonic acid, 1–2 nM [ 3H]-PGE 2 and [ 3H]-PGF 2α as internal standards, 5 mM glutathione, 5 mM epinephrine and 100 mM Tris HCl buffer (pH 8.2) in a total volume of 0.5 ml. The reaction was terminated by 0.25 ml of 1 N HCl and the mixtures extracted with 1.5 ml ethyl acetate. PGF 2α and PGE 2-like materials (“PGE 2”) in the ethyl acetate extract were separated from arachidonic acid by chromatography on two-stage silicic acid columns. Reverse isotope dilution technique revealed that [ 14C]-PCF 2α synthesized from [ 14C]-arachidonic acid by the lung enzyme was 100% pure. Moreover, the amount of PGF 2α synthesis as measured by this method was in accord with the amount obtained by radioimmunoassay. In contrast, only 15% of the [ 14C]-“PGE 2” was PGE 2; 85% of it was a KOH resistant and NaBH 4-reducible product that has been tentatively identified as thromboxane B 2. The kinetics of PGF 2α and “PGE 2” synthesis by guinea pig lung enzyme have been studied under various experimental conditions employing this method. The two stage open silicic acid column chromatography as described in the present report offers several advantages such as economy of time and expense and capacity to process multiple samples on a routine basis. Furthermore, this method also offers the advantage of correction for procedural losses of prostaglandins using internal recovery standards. This method should have broad applicability to study the prostaglandin synthesizing ability of various tissues especially in those laboratories that cannot afford to employ sophisticated instruments needed for prostaglandin analysis.

Corticosteroids in human blood—VI. Isolation, characterization and quantitation of sulfate conjugated metabolites of cortisol in human plasma

Evidence has been obtained for the presence of a total spectrum of sulfate-conjugated metabolites of cortisol in human plasma: 19 monosulfates, two disulfates and six glucuronosulfates. Two hours and 15 min following i.v. administration of a tracer dose of [4- 14C]-cortisol to 11 normal subjects, large samples of heparinized blood were obtained. From the separated, deproteinized and defatted plasma, all conjugated steroids were extracted by means of an XAD-2 amberlite column. Mono- and di-sulfate conjugated steroids were then separated from other free and conjugated metabolites of cortisol by means of high voltage paper electrophoresis. Individual monosulfate conjugated metabolites and glucuronosulfates were subsequently separated (as seven sub-groups) by paper chromatography. These conjugates were eluted and subjected to cleavage by solvolysis (monosulfates and disulfates) or consecutive solvolysis and β-glucuronidase hydrolysis (glucuronosulfates). The liberated steroid moieties were separated by multiple successive paper chromatographies, and identified by reverse isotope dilution technique. The individual steroids were then quantitated. Sulfate-to-steroid molar ratios were also determined on major conjugates, prior to their cleavage. The steroid metabolites found in plasma are identical with those isolated by us previously from human urine. The total sulfate fraction constituted 5.3 ± 1.9% of all conjugated metabolites of cortisol in plasma. Of those, 76% were monosulfates, 19% were glucuronosulfates, and 5% were disulfates. A large proportion of monosulfates consisted of steroids conjugated at C-21 (27%), which contrasts with glucuronide conjugates. The steroids found in largest concentrations were: 20β-cortolone-3-sulfate (17%), 5α-tetrahydrocortisol-3-sulfate (14%), 6β-hydroxycortisol-21-sulfate (9%), cortisol-21-sulfate (8%), tetrahydrocortisol-3-sulfate (7%), and 20β-dihydrocortisol-21 -sulfate (6%). From the correlation of plasma and urinary concentration of individual steroid sulfates, the following conclusions are drawn: (1) sulfoconjugation is a consistent, though quantitatively minor, pathway in the peripheral metabolism of cortisol; (2) C-21 sulfates of 4-ene-3-oxo-steroids are formed (and excreted) considerably faster than C-3 sulfates, the formation of which requires a prior reduction of ring-A; (3) C-3 sulfates of steroids with dihydroxyacetone side-chain are formed (and excreted) more slowly than the corresponding sulfates of steroids with glycerol side-chain, ergo, the reduction of 20-ketone appears to facilitate sulfoconjugation.

Androgen synthesis in vitro by adrenal tissue from a newborn anencephalic infant.

SUMMARY Incubation of slices of adrenal tissue from a newborn anencephalic infant with [14C]pregnenolone and [3H]progesterone yielded radioactive testosterone, androst-4-ene-3,17-dione and 11β-hydroxyandrost-4-ene-3,17-dione which were characterized by reverse isotope dilution techniques. These findings indicate the presence of C(17)—C(20) desmolase, steroid 11β-hydroxylase, 3β-hydroxysteroid dehydrogenase-isomerase and 17β-hydroxysteroid dehydrogenase enzymes in this tissue. Therefore, despite the deficient production of C19 3β-hydroxy-unsaturated steroids by the anencephalic foetus, as indicated by cord blood levels, a potential for synthesis of C19 3-oxo-4-unsaturated steroids has been demonstrated in vitro.

Are C 19 androgens formed by cleavage of A C 8 fragment from cholesterol?

Attempts have been made to confirm the suggestion that cholesterol can be metabolized to C 19 steroids by cleavage of the C 8 side-chain between C-17 and C-20. The evidence previously adduced for the existence of this pathway has come from in vitro incubation experiments in which [26- 14C] cholesterol was used as the substrate for the formation of [ 14C]6-methylheptan-2-one . The identification of the C 8 product was established by the reverse-isotope dilution technique. In this study, the procedures previously reported have been repeated and have been extended by the addition of several purification steps designed to validate the association of radio-carbon with this C 8 moiety. In none of our experiments was it possible to prove unequivocally that [ 14C]6-methylheptan-2-one is formed from [26- 14C]-cholesterol during incubation with tissues from a variety of steroid-producing glands. If the pathway, C 27 → C 19 + C 8 exists, corroboration will have to come from other kinds of evidence.

Transformation of cortisol in rheumatoid synovial tissue in vitro.

ABSTRACT 11β-Hydroxyandrostenedione, cortisone, 20α- and 20β-dihydrocortisol and cortisol were identified, by means of paper and thin layer chromatography and reverse isotope dilution technique, as transformation products of cortisol-4-14C in rheumatoid synovial membrane. Non-enzymatic conversion of cortisol to 11β-hydroxyandrostenedione and other unknown compounds has been shown in control experiments. The major metabolic pathways of cortisol in human rheumatoid synovial membrane involve oxidation of the 11β-hydroxyl to 11-ketone, reduction of the 20-ketone to 20α-ol and 20β-ol. No ring A reduced metabolites could be detected.

In vitro metabolism of pregnenolone-7-alpha-3H, progesterone-4-14C and androstenedione-4-14C by rat placental tissue.

The in vitro metabolism of radioactive androstenedione, pregnenolone and progesterone by rat placental homogenates has been studied. No estrogens were isolated but there was extensive ring A reduction of each compound. From the androstenedione incubations, 3α-hydroxy-5α-androstan-17-one, 3β-hydroxy- 5α-androstan-17-one, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol were isolated by paper chromatography and identified by thin layer chromatography and the reverse isotope dilution technique. The metabolites of pregnenolone and progesterone were quantitatively and qualitatively similar. From these incubations 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3α,20β-diol were isolated and identified. In the presence of a progesterone trap, progesterone- 7α-3H was found following the incubation of pregnenolone-7α-3H, demonstrating the presence of a 3β-ol-dehydrogenase Δ4–5-isomerase enzyme system. (Endocrinology 87: 151, 1970) ATTEMPTS to evaluate possible endocrine functions of the rat placenta during pre...

Patterns of steroid biosynthesis by human adrenals incubated in vitro with [7alpha-3H) pregnenolone: changes with (a) gestational age, (b) incubation period and (c) weight of incubated tissue.

SUMMARY Human adrenal tissue was sliced and incubated with 25 μc [7α-3H]-pregnenolone. The percentage conversion of radioactive precursor to dehydroepiandrosterone (DHA), DHA sulphate, androstenedione, 11β-hydroxyandrostenedione, cortisol and 11-deoxycortisol was estimated by the reverse isotope dilution technique. Incubations of both adult and foetal adrenal slices gave reproducible results. The duration of the incubation period was an important factor affecting the pattern of radioactive steroids synthesized, the adult adrenal pattern being dissimilar to that of the foetal adrenal. A direct proportional relationship between the weight of the incubated tissue and the biosynthesis of cortisol from exogenous [7α-3H]-pregnenolone was not established. The production of DHA sulphate relative to free DHA increased markedly from 12 to 27 weeks gestation. When incubated for 2 hr. the combined production of DHA and DHA sulphate relative to cortisol and androstenedione decreased with gestational age. There was evidence for considerable 3β-hydroxysteroid dehydrogenase activity in foetal adrenals at 12 to 27 weeks gestation.

Transformation of labeled cholesterol, 20α-hydroxycholesterol, (22R)-22-hydroxycholesterol, and (22R)-20α,22-dihydroxycholesterol by adrenal acetone-dried preparations from guinea pigs, cattle and man: I. Establishment of radiochemical purity of products

The preparation of radiochemically pure tritiated 20α-hydroxy-cholesterol, (22R)-22-hydroxycholesterol, (22S)-22-hydroxycholesterol and (22R)-20α,22-dihydroxycholesterol has been described. The enzymatic transformation of cholesterol to 20α-hydroxycholesterol, (22R)-22-hydroxycholesterol, (22S)-22-hydroxycholesterol, (22R)-20α,22-dihydroxy-cholesterol and pregnenolone has been studied using adrenal acetone-dried powder preparations from guineapigs, cattle and man. No significant conversion of cholesterol to (22S)-22-hydroxycholesterol took place. The amount of radioactivity appearing in 20α-hydroxy-cholesterol was exceedingly small and the radiochemical purity of this product was not firmly established. Using reverse isotope dilution techniques, involving chromatographic separations, derivative formation, specific reactions and crystallization, the conversion of cholesterol to (22R)-22-hydroxycholesterol and (22R)-20α,22-dihydroxycholesterol has been established for the first time without the use of “trapping” procedures. The further conversion of 20α-hydroxycholesterol and (22R)-22-hydroxycholesterol to (22R)-20α,22-dihydroxycholesterol and pregnenolone and that of (22R)-20α,22-dihydroxycholesterol to pregnenolone also has been investigated. The transformation of (22S)-22-hydroxycholesterol to (22S)-20α,22-dihydroxycholesterol and pregnenolone was insignificant in comparison to the transformation of the (22R) isomer.

Studies on the metabolism of steroid hormones and their precursors by the human placenta at various stages of gestation. II. In vitro metabolism of 3 beta-hydroxyandrost-5-en-17-one.

ABSTRACT The in vitro metabolism of 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) by human placentae of various gestational ages was studied by incubation of 7α-3H-dehydroepiandrosterone with a total of 39 samples of placentae obtained at from 4 to 24 weeks of gestation, and extraction and identification of the resulting radiolabeled metabolites by reverse isotope dilution techniques. Metabolites obtained under the in vitro conditions employed include estra-1,3,5(10)-trien-3,17β-diol, 3-hydroxyestra-1,3,5(10)-trien-17-one, 2,3-dihydroxyestra-1,3,5(10)-trien-17-one, androst-4-ene-3,17-dione, 17β-hydroxyandrost-4-en-3-one, 5α-androstane-3,17-dione, 5α-androstane-3β,17β-diol, androst-5-ene-3β,17β-diol, 30-hydroxy-5α-androstan-17-one, and unaltered dehydroepiandrosterone. When the relative capacities of the various placental samples to carry out the observed metabolic transformations of the steroid substrate under the in vitro conditions employed are compared, it is seen that the differences in the r...

Fate of exogenous marinobufagin- 3H in Bufo marinus

After marinobufagin- 3H was injected into toads, 80 per cent of the radioactivity could be extracted from various tissues 2–3 months later. One-half of the tissue radioactivity was found in the ovarian tissues, of which 80–90 per cent could be identified as unchanged marinobufagin by thin-layer chromatography and reverse isotope dilution techniques. Of the other tissues, 27.2 per cent was found in the gastrointestinal tract. Skin and parotoid glands, where these steroids presumably are synthesized and stored, had together only 1 per cent activity. Other tissues had a total of less than 0.25 per cent of the injected radioactivity. Little was excreted. The major conversion product of marinobufagin is tentatively identified as telocinobufagin. Thus the formation of 14β-hydroxyl group of cardiotonic steroids, at least in toads, would be by reduction of a 14β, 15β-epoxide.