Detection of Francisella tularensis and analysis of bacterial growth in ticks in Japan.

UNLABELLED: Francisella tularensis is distributed in the Northern hemisphere and it is the bacterial agent responsible for tularaemia, a zoonotic disease. We collected 4 527 samples of DNA from ticks in Japan, which were then analysed by real-time PCR and nested PCR. Francisella DNA was detected by real-time PCR in 2·15% (45/2 093) of Ixodes ovatus, 0·66% (14/2 107) of I. persulcatus, 8·22% (6/73) of I. monospinosus and 0·72% (1/138) of Haemaphysalis flava specimens. Finally, Francisella DNA was detected by nested PCR in 42 and five samples I. ovatus and I. persulcatus, respectively, which were positive according to real-time PCR. Phylogenetic analysis showed that the sequence from I. ovatus and I. persulcatus were clustered with F. tularensis type B strains distributed in Eurasia. Microinjected live F. tularensis persisted in ticks, whereas heat-killed F. tularensis decreased. Microinjected F. tularensis hlyD mutant decreased in ticks significantly compared to parent strain, thereby suggesting that HlyD in F. tularensis contributes to the adaptation or survive of bacterial infection in ticks. SIGNIFICANCE AND IMPACTS OF THE STUDY: Francisella tularensis has been detected in ticks, suggesting that it is a tick-borne pathogen. However, F. tularensis has not been detected in ticks in Japan since 1991. In this study, we performed a large-scale analysis of DNA isolated from ticks in Japan and detected F. tularensis by real-time polymerase chain reaction (PCR) and nested PCR. We found that F. tularensis could survive in ticks based on an experimental tick-infection model. We also identified a bacterial factor that contributes to survival in ticks. Our results suggest that ticks are candidate vectors that mediate F. tularensis infection in Japan.

[Changes in the values of estrogen stimulated neurophysin, oxytocin and prostaglandin in the maternal blood after the administration of dehydroepiandrosterone-sulfate to the pregnant women near term].

UNLABELLED: In order to examine the effect of dehydroepiandrosterone-sulfate (DHA-S) on the production of uterus-contractive substances in perinatal women, the concentrations of estrogen stimulated neurophysin (ESN), oxytocin, 13, 14-dihydro-15-keto-Prostaglandin F2 alpha(PGF2 alpha) and DHA-S related steroids in the maternal blood of 12 cases of primigravida were serially estimated after intravenous administration of 200mg DHA-S twice a week from 38 weeks of gestation till the onset of labor. RESULTS: 1) The duration of labor was significantly shortened in the DHA-S group (mean; 9.3 +/- 2.8 hours) compared to the 11 control cases (13.5 +/- 5.2 hours). 2) i) The ESN concentration, which correlated well with that of free estradiol-17 beta, increased significantly at 1 week after the Ist administration. ii) The oxytocin concentration, which correlated well with that of ESN, increased significantly at 39 weeks of gestation, the first stage of labor and especially at delivery compared to the control. iii) The PGF2 alpha concentration, which increased in parallel with those of DHA-S, estrone-S and pregnenolone-S, increased significantly at delivery in comparison with the control and correlated well with estrone-S in the DHA-S group. CONCLUSION: These findings suggest that DHA-S administration to perinatal women may enhance the production of ESN, oxytocin and PGF2 alpha in the fetoplacento-maternal compartment, which may result in the shortening of labor.

[19-Hydroxyandrostenedione in human ovarian vein measured by GC-MS].

Nineteen-hydroxyandrostenedione (19-OHA) is considered to be an obligatory intermediate of estrogen synthesis. To clarify the role of 19-OHA in the human ovary, the following experiments were undertaken. Ovarian and peripheral vein blood in mid follicular and luteal phase were obtained from 14 women during gynecological surgery. The concentrations of androstenedione (A), testosterone (T), 19-OHA, estrone (E1) and estradiol (E2) were measured by gas chromatography mass spectrometry using deuterium-labeled steroids as internal standard. The effect of human chorionic gonadotropin (hCG) on those steroids was also studied in mid luteal phase. The concentrations of 19-OHA in ovarian vein were 1.20 +/- 0.16 ng/ml (mean +/- SE) in mid follicular and 0.78 +/- 0.18 ng/ml in mid luteal phase. The steroid levels measured in ovarian vein were significantly higher than those in peripheral vein. Though the administration of hCG in mid luteal phase enhanced A and E2 levels in ovarian vein, the levels of T, 19-OHA and E1 were not altered. Significant correlation between the levels of E2 and A was observed while none of the steroids correlated with 19-OHA. These results suggest, for the first time, that 19-OHA is produced and secreted from the human ovary. The question as to the physiological and biosynthetic role of ovarian 19-OHA has to be answered in the future.

[A study of non-aromatizing androgen C10-19lyase in adrenal tissue].

The conversion of androgen to estrogen is mediated by aromatase activity, and 19-hydroxylase and C10-19lyase are seen to be involved in C19-demethylation. The present study is to demonstrate C10-19lyase activity in adrenal tissue. Fetal bovine and pig adrenal tissue homogenates were incubated with 14C-androstenedione(A) and NADPH under air. The products 14C-19-OHA, 14C-19-norA, with added carrier standard 3H-19-OHA, 3H-19-norA were separated and purified by T.L.C.. The identification of 19-norA was established by recrystallization and that of 19-OHA by C.C.D.. The 3H/14C ratios of the products 19-norA were found to be constant in repeated crystallization. The amount of 19-OHA from A in fetal adrenal tissue was 30.0nmol/h/g protein, and that of 19-norA from A was 12.59pmol/h/g protein. C10-19lyase activity from 19-OHA, calculated by the amounts of 19-norA in fetal, bovine and pig adrenal tissue, was 49.7, 3.50, 10.3pmol/h/g protein, respectively. This is the first report to demonstrate androgen C10-19lyase in human fetal adrenal tissue, and it is assumed that fetal adrenal tissue forms 19-norA via 19-OHA.

Serum level of 19-hydroxyandrostenedione during pregnancy and at delivery determined by gas chromatography/mass spectrometry.

19-Hydroxyandrostenedione (19-OHA) is secreted from the adrenal glands in men and women and also from the placenta during pregnancy. It has been found to cause hypertension in animal models. We have synthesized [7,7-2H2]-19-OHA with high deuterium content and, together with [7,7-2H2]A and [9,11-2H2]estrone (E1), have developed a quantitative assay of serum level 19-OHA, A, and E1 using the gas chromatography/mass spectrometry-mass fragmentography method to monitor individual subjects throughout pregnancy. The labeled 19-OHA, used as internal standard, showed only 6.73% of unlabeled compound. Recovery of standard 19-OHA, A, and E1 (5,000 pg each) added to male plasma was 97.4 +/- 2.3%, 96.3 +/- 2.1%, and 100.1 +/- 4.1% (mean +/- SD), respectively; the intraassay coefficient of variation was 2.1%, 3.5%, and 3.8%, respectively. Ten pregnant subjects without complications and 10 pregnant subjects near term with hypertension were selected (with informed consent). The 19-OHA and E1 serum concentrations of maternal venous blood from uncomplicated pregnancies increased significantly as gestation progressed (19-OHA: first trimester, 225 +/- 72; second trimester, 656 +/- 325; third trimester, 1,518 +/- 544 pg/ml), reaching the highest level at delivery (19-OHA: 1,735 +/- 684 pg/ml). Whereas a positive correlation was found between the level of 19-OHA and E1, no apparent change of the A level was observed during pregnancy. Levels of the three steroid hormones in pregnancy complicated by hypertension in the second and third trimester were not found to be significantly different from those of normal pregnancy (19-OHA of hypertensive subjects: second trimester, 762 +/- 349; third trimester, 1,473 +/- 491 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of DHA-S on placental 3 beta-hydroxysteroid dehydrogenase activity, progesterone and 20 alpha-dihydroprogesterone concentrations in placenta and serum.

To study the effects of dehydroepiandrosterone sulfate (DHA-S) on placental steroid metabolism and maternal steroidal profiles at term, the following in vivo and in vitro experiments were performed. Two hundred mg of DHA-S was given to five pregnant women 30 minutes prior to delivery. After delivery, the placenta was collected and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and sulfatase activity was determined by measuring the rate of conversion of pregnenolone to progesterone and DHA-S to DHA. The amount of C21-delta 4-steroid in the placental tissue was measured by gas chromatography mass spectrometry (GC-MS) and compared with the control groups. The maternal serum concentration of several steroids was also measured by GC-MS before and after the administration of DHA-S. 3 beta-HSD activity in the placentae from the mothers who received DHA-S before delivery was significantly lower than in the controls. On the other hand, no significant change was observed in the activity of sulfatase. The serum concentration of progesterone (P) and 20 alpha-dihydro-P (20-P) before DHA-S loading decreased following the administration whereas estradiol (E), DHA, and androstenedione (A) levels increased. To study the direct effect of DHA-S and its related steroids on placental 3 beta-HSD activity, placental tissue samples were incubated with pregnenolone in vitro. Several other steroids were added simultaneously into the medium. It was observed that placental 3 beta-HSD activity was directly inhibited by DHA-S. These results indicate that DHA-S inhibits 3 beta-HSD activity in the placenta and subsequently causes a reduction in P and 20-P.

[Correlation between the levels of catecholamines (noradrenaline, adrenaline) and adrenal steroids (DHA-S, cortisol) in maternal and fetal blood during pregnancy and labor].

It is known that both catecholamines (CA) and cortisol (F) levels elevate during labor. To determine the correlation between adrenal steroids and medullary function, maternal blood was collected during pregnancy, first stage of labor (MVI) and at delivery (MVII). Umbilical arterial and venous blood (UA, UV) was also obtained at delivery. Further, ACTH or dexamethasone (Dx) was given during the first stage of labor, and maternal blood was collected before and 30 minutes after the administration. Plasma levels of CA[noradrenaline (NA), adrenaline (Ad)] were extracted by trihydroxyindole method and were measured by HPLC. DHA-S and F levels were determined by specific RIA. Results are as follows: 1. No apparent change was observed in maternal NA and Ad levels throughout pregnancy. DHA-S levels were high in first trimester and decreased as pregnancy advanced, while F levels showed an increase trend as pregnancy progressed. 2. All hormone levels in maternal blood increased remarkably during labor. A significant negative correlation between F and Ad levels at delivery was noted. When F levels were elevated by ACTH administration, Ad levels decreased. Ad levels elevated when F levels were suppressed by Dx administration. 3. NA, Ad and DHA-S levels in cord blood were higher than those in MVII. Levels of F in maternal blood were higher than those in cord blood. A significant correlation of F in MVII and UA was observed. These results indicate that the suppressive effect of F may be involved in the mechanism of Ad secretion, though the secretion of Ad increased with F in the course of labor. The response of fetal adrenal to the stress of labor may be different from that of maternal adrenal since a significant correlation was not noticed between the levels of Ad and cortisol in cord blood as was found in maternal blood.

[The newly developed method of 19-OH-A as intermediate of estrone biosynthesis by GC-MS].

To study the serum levels of 19-hydroxyandrostenedione (19-OH-A), known as an obligatory intermediate of estrogen biosynthesis and considered to be one of the hypertensinogens, a method using GC-MS with application of synthesized [7,7-d2]androstenedione (A), [7,7-d2] 19-OH-A and [9,11-d2]estrone(E1) as internal standards was newly developed. Normal pregnant women and pregnant women complicated with hypertension near term were selected for the study. The levels of 19-OH-A and E1 increased significantly as gestation progressed [19-OH-A; 224.7 +/- 72.1 (1st trimester), 655.5 +/- 325.4 (2nd trimester). 1517.8 +/- 543.6 (3rd trimester)pg/ml], and a positive correlation was found between the levels of the two steroids. No apparent change was observed in A levels during the course of pregnancy. The mean levels of 19-OH-A in pregnancy complicated with hypertension at 2nd and 3rd trimester were 761.7 +/- 348.9 and 1473.0 +/- 491.4 pg/ml, which were compatible with those in normal pregnancy. The levels of 19-OH-A at delivery in maternal vein (MV) were 1735.1 +/- 683.9 pg/ml. Significantly higher levels of 19-OH-A were found in umbilical vein (UV) (1977.2 +/- 564.9 pg/ml) than those in umbilical artery (109.7 +/- 49.1 pg/ml). 19-OH-A concentration in term placental tissue was 16.3 ng/g.w.w. tissue. This is the first report to demonstrate the serum 19-OH-A levels measured by GC-MS and also to demonstrate the levels in the cord blood. The results indicate that 19-OH-A may be the product of pregnancy and may be derived from the feto-placental compartment.

[Non-aromatizing androgen 19-hydroxylase in human fetal organs].

19-hydroxyandrostenedione (19-OH A), known for many years as an obligatory intermediate of estrogen biosynthesis, has recently been found to be an amplifier of aldosterone action and in itself a hypertensinogenic steroid (J. Steroid Biochem., 16; 329, 1982). To search the presence of a non-aromatizing andorgen 19-hydroxylase in fetal organs, metabolism of labeled androstenedione(A) in fetal adrenal, liver, lung, brain and kidney was studied. 19-hydroxylase activity was calculated by the amounts of 19-OH A formed from A. The tissue homogenate was incubated with [4-14C]A and NADPH for different periods of time under air. The product, [4-14C] 19-OH A with added carrier standard [6,7-3H] 19-OH A was separated and purified by TLC, acetylation and TLC to constant 3H/14C ratio. The identity of the product was established by C.C.D. and by GC-MS analysis of purified product 19-AcOA in a larger scale adrenal incubation of non-labeled A. 19-hydroxylase activity was 0.9 pmol/min/mg protein for adrenal, but there was a negligible quantity of 19-OH A in lung, brain and kidney. Estrogen production was also assayed by 3H-water method using [1 beta-3H, 4-14C] A (3H/14C = 69.9). No detectable amount of estrogen was found in incubation of any homogenate except for liver. These results indicate that the non-aromatizing 19-hydroxylase activity in fetal adrenal gland is much higher than that in other organs. This is the first report to demonstrate the presence of 19-hydroxylase in human fetal adrenal tissue. It is suggested that 19-OH A secreted from fetal adrenal may also be associated with serum concentration of fetal circulation.

[Serum concentrations of delta 5C21 steroids during pregnancy and at delivery].

In order to study the changes of C21-steroid levels which included Pregnenolone (P5), 20 alpha dihydropregnenolone (20P5), 16 alpha hydroxypregnenolone (16P5), progesterone (P4) and 20 alpha dihydroprogesterone (20P4) in maternal peripheral blood during pregnancy and at delivery, these steroids were measured by GC-MASS with application of deuterated steroids as internal standard. The accuracy of GC-MASS method of these steroids was satisfactory with C.V. value of less than 6%. Total delta 5C21 steroid concentrations in course of pregnancy and at delivery were as follows; P5 (mean +/- S.D. ng/ml): 66.6 +/- 36.2 (1st trimester), 80.9 +/- 24.6 (2nd trimester), 147.7 +/- 30.1 (3rd trimester) and 299.7 +/- 178.3 ng/ml (at delivery), 20Ps: 212.6 +/- 102.5, 143.4 +/- 53.9, 248.9 +/- 58.8, 563.4 +/- 198.2 ng/ml, 16P5: 8.6 +/- 8.6, 8.1 +/- 5.2, 124.3 +/- 40.3, 378.5 +/- 180.0 ng/ml, respectively. P4 (43.0 +/- 28.0 ng/ml) and 20P4 (8.0 +/- 4.0 ng/ml) in 1st trimester showed gradual increase to maximum level (P4: 138.2 +/- 30.1 ng/ml, 20P4: 105.4 +/- 21.6 ng/ml) at pre-pain period, afterward decreased rapidly (P4: 70.9 +/- 23.2 ng/ml, 20P4: 59.8 +/- 19.3 ng/ml) at delivery. P5, 20P5 and 16P5 levels were found to be significantly higher in umbilical artery (UA) as well as in umbilical vein (UV) than those in maternal vein (MV) regardless of labor pain. P4 and 20P4 did not show any differences in MV regardless of labor pain. P4 in UV (pain+) and 20P4 in UA (pain-), however, showed significantly higher than P4 in UV (pain-) and 20P4 in UA (pain+). P5, 20P5, 16P5 and 20P4 levels were significantly lower in the case of anencephalic pregnancy (ANC) at 3rd trimester than in normal pregnancy, especially 16P5 levels (22.2 +/- 5.0 ng/ml) showed 1/5 of those in normal pregnancy. From the results obtained above, it is suggested that these delta 5C21 steroids are actively produced in the feto-placental unit in the course of pregnancy. The levels of these steroids reached maximum at delivery, but the levels of P4, 20P4 decreased toward delivery after maximum levels were shown in the stage of pre-labor pain. No significant difference of P4 level in the case of ANC suggested that P4 production correlated with placenta as well as maternal and fetal precursor. Decreasing of 20P4 and P4 level after the stage of pre-labor pain suggested that activity of 3 beta hydroxysteroid dehydrogenase was reflected by uterine contraction during labor.

[Changes in maternal plasma levels of C21 and C19 steroid hormones during pregnancy].

To study the changes in circulating steroid levels during pregnancy, ten steroids which included Progesterone (P4), 16 alpha OH-Progesterone (16P4), free and conjugated Pregnenolone (P5), 16 alpha OH-Pregnenolone (16P5), Dehydroepiandrosterone (DHA) and 16 alpha OH-Dehydroepiandrosterone (16DHA) were measured simultaneously by RIA in 84 normal pregnant women from the 5th to 41st week of gestation. Ratios steroid levels were also calculated. The results were as follows; 1. P4 increased as gestation advanced and reached the maximum (126 +/- 18.24 ng/ml) in the 28th week. The concentration 16P4 increased sharply after the 32nd week and reached the maximum (16.3 +/- 1.67 ng/ml) in the 39th week. 2. Both free and conjugated P5 levels did not show an increasing trend as pregnancy progresses. 3. The concentration of free and conjugated 16P5 increased rapidly after the 30th week and reached the maximum at 39th week (free 2.5 +/- 0.3 ng/ml conjugated 42.4 +/- 5.1 ng/ml). 4. Both free and conjugated DHA decreased as pregnancy progressed. On the other hand, the concentration of free and conjugated 16DHA increased as pregnancy progressed. 5. The ratio of 16P4 to P4, 16P5 to P5 and 16DHA to DHA significantly increased after 30 weeks of gestation.

[Relation between size of fetal and neonatal adrenal glands and steroid levels in maternal and neonatal serum].

Fetal adrenal gland is known to develop till term and decrease in size after birth. In this study, the size of the adrenal glands was evaluated by real time ultrasonography and steroid levels in the maternal and neonatal peripheral veins were measured by Gas chromatography-Mass spectrometry with application of deutrated steroids as internal standards. One hundred normal pregnant women at 28-40 weeks of gestation and ten cases of cesarean section, not labor, infants were selected for this study. The size of the fetal adrenal glands increased as gestation advanced. Levels of maternal 16DHA-S, P5-S and E3 correlated well with the length of the fetal adrenal glands. No correlation between the size of fetal adrenal gland and maternal DHA-S levels was noticed, except at 36-40 weeks of gestation. On the 7th day after birth, the neonatal adrenal glands were about 20% longer than at delivery. Levels of DHA-S, 16DHA-S, P5-S and 16P5-S in neonatal blood were 85%, 49%, 71% and 50% lower than at delivery during this period. In the present study, the relation between the adrenal steroid levels in maternal/neonatal serum and the size of the fetal/neonatal glands was demonstrated for the first time.

[Metabolism of pregnenolone sulfate in feto-placental unit].

In order to study the metabolic fate of circulating pregnenolone sulfate (P5S) during pregnancy, 30 mg of newly synthesized tetra (2,2,4,6)-deuterated P5S was administered to the maternal vein 60 minutes prior to cesarean section at term. All pregnant women were volunteers and had been informed of nature of this study. The placenta, maternal urine and blood samples from maternal vein (MV), umbilical cord (U) were collected and deuterated metabolites were analysed by gas chromatography mass spectrometry with a multiple ion detector. Total amounts of metabolites were measured and the ratio of deuterated steroid to the total amounts were calculated (d%). d% of P5S, 16 alpha OH-P5S, 17 alpha OH-P5S, 20 alpha-dihydro-P5S (20P5S), 20P5 and progesterone (P4) in MV were 84.5, 51.6, 95.5, 85.1, 71.2 and 10.9%, respectively. In the placental tissue, 20P5, 20P4 and P4 were also found and d% of these steroids were calculated as 16.1, 3.2 and 3.1%. Only P4 was found with d% of 11.2% in U. In the urine collected for 2 hours after deuterated P5S administration, P5S (40.6%), 20P5S (56.6%) and pregnanediol (34.8%) were identified. Deuterated C19, C18 steroids were not detected in any of the samples studied. When 30 mg of non labeled P5S was also administered to MV at term, the levels of P5S, 20P5S, P4 and 20P4 in MV rose, but the levels of DHA-S were not changed. These results indicate that the circulating P5S in MV can be a precursor of 20P5S, 17P5S, 16P5S, 20P4 and P4, but can not be the precursor of C19, C18 steroid.

[Determination of serum 16 alpha-hydroxydehydroepiandrosterone, 16 alpha-hydroxyestrone and estriol by gas chromatography-mass spectrometry and their perinatal movement].

In order to study the mechanism of estriol (E3) formation in the fetoplacental unit, a method for determining E3 and its precursors, 16 alpha-hydroxydehydroepiandrosterone (16 alpha-OH DHA) and 16 alpha-hydroxyestrone (16 alpha-OH E1) in blood using Gas Chromatography-Mass Spectrometry (GC-MS) was newly developed. Deuterium labelled steroids, 3.4.4.-d3-16 alpha-OH DHA 2.4.17.-d3-E2 and 2.4.-d2-E3 were synthesized and used as the internal standards. After removal of free steroids with ether, the residue was solvolyzed and extracted with ethyl acetate as free form. Steroids were analysed after the formation of T.M.S. and T.F.A. derivative. The values for each steroid in maternal peripheral vein blood (M.V.), umbilical artery blood (U.A.) and vein blood (U.V.) were studied. Conjugated steroid values in M.V. obtained at normal vaginal delivery (13 cases) were as follows: 16 alpha-OH DHA 230.3 +/- 76.3 ng/ml, 16 alpha-OH E1 19.34 +/- 7.00 ng/ml and E3 158.8 +/- 50.2 ng/ml (mean +/- S.D.), respectively. The levels of these three steroids in cord blood were significantly higher than those in maternal blood. These results show that the steroids are actively produced in the fetoplacental unit. A significant increase in all the steroids was associated with labor. None of the values for E3 precursors in umbilical blood correlated with that of conjugated E3 in maternal blood. The significance of 16 alpha-OH DHA and 16 alpha-OH E1 as intermediates of E3 formation in the fetoplacental unit is discussed.

[Changes in plasma 2-hydroxyestrone levels in pubertal females and correlation with sex hormones].

Plasma levels of 2-hydroxyestrone (2-OHE1) were measured by specific radioimmunoassay during puberty to elucidate the physiological role of this hormone on female sexual development. Plasma levels of FSH, LH, PRL and estradiol (E2) were also measured with an RI-kit. 67 girls between aged 6 and 16 were selected for this study. Blood samples were collected into tubes containing 0.1W/V% EDTA and 0.1W/V% ascorbic acid and immediately centrifuged. 2-OHE1 in plasma was extracted and separated with a Sephadex LH-20 column. RIA was applied using anti 2-OHE1-17-CMO-BSA. Plasma 2-OHE1 levels before menarche were low (6-7 pg/ml) until 10 years of age. The levels started to increase from 11 and reached 11.2 +/- 5.8pg/ml at 16 years of age. The levels of 2-OHE1 in girls after menarche were significantly higher than that before menarche (p less than 0.01). There was a significant correlation between 2-OHE1 and E2 in girls before menarche (p less than 0.001, r = 0.5416). However, the 2-OHE1 to E2 ratio decreased significantly from 9 to 10 years of age. These results indicate that during this period, the E2 increase is more predominant than that of 2-OHE1. There was a significant negative correlation between 2-OHE1 and PRL after menarche while no correlation between 2-OHE1 and FSH, LH was noticed. These results suggested that 2-OHE1 may play a role in sexual development after menarche rather than in the initiation of menarche.

[Determination of free and conjugated 16 alpha-hydroxyestrone (16 alpha-OH E1) in serum by mass fragmentography using gas chromatography mass spectrometry].

A simple, rapid and highly specific method for the determination of maternal and cord serum 16 alpha-hydroxyestrone (16 alpha-OH E1) during pregnancy and at delivery was developed by mass fragmentographic technique using a Gas chromatography--Mass spectrometry (GC-MS) with application of deuterated estradiol as an internal standard. The equipment used was Shimadzu LKB9000 GC-MS (MID-PM). The T.F.A. derivative of the compounds was analyzed using the GC-MS system equipped with a 1 m glass coil, 1% OV-1 on chromosorb w 60 approximately 80 mesh. The flow rate of carrier gas (helium) was 25 ml/min. The temperatures of column oven, separator and ionization source were kept at 190 degrees C, 270 degrees C and 290 degrees C, respectively. The ionization energy and trap current were 70 eV and 60 microA, respectively. The mass spectrum showed molecular ions at m/ e467 and m/ e478 corresponding to the T.F.A. derivatives of d3-E2 and 16 alpha-OH E1 respectively. Evaluation of the method assessed by recovery experiments was 99.2 percent when 10 ng of 16 alpha-OH E1 was added to 1.0 ml of male serum treated with charcoal. The coefficient of variation was 4.2 percent. Serum conjugated 16 alpha-OH E1 levels during pregnancy increased with progressing gestation. The levels in early pregnancy (5th approximately 15th week), middle pregnancy (16th approximately 27th week) and late pregnancy (28th approximately 42th week) were 1.72 +/- 0.43 ng/ml, 5.35 +/- 2.60 ng/ml, 11.92 +/- 3.93 ng/ml (mean +/- S.D.), respectively. Mean levels of 16 alpha-OH E1 in the umbilical artery were 11.61 +/- 5.17 ng/ml which were statistically higher than these in the maternal peripheral vein (6.06 +/- 4.72 ng/ml) and umbilical vein (9.16 +/- 3.77 ng/ml). Serum 16 alpha-OH E1 levels in the samples obtained from anencephalic pregnancy were significantly lower than those in normal pregnancy, suggesting that fetal adrenal function may be involved in the formation of this steroid during pregnancy.

[Determination of serum unconjugated estetrol by selected ion monitoring with application of deuterated estetrol as an internal standard].

A rapid and highly specific method for determination of serum estetrol during pregnancy and at delivery is developed using a gas chromatography-mass spectrometry with application of newly synthesized deuterated estetrol as an internal standard. Evaluation of the method assessed by recovery experiments was 102.4 and 103.0 percent when 2 and 4ng of estetrol was added to 0.5ml of male serum. The coefficient of variation were 2.54 and 2.84 percent, respectively. The results obtained by the present method are correlated well with those obtained by conventional RIA. Serum unconjugated estetrol levels during pregnancy increased with progressing gestation. The levels from the 36th to the 40th week were 749.8 +/- 281.5pg/ml (mean +/- S.D.) The levels in umbilical cord blood at delivery were also measured: Umbilical vein were 8,064.0 +/- 6,595.4pg/ml which were statistically higher than in umbilical artery (3,515.9 +/- 1,553.8pg/ml) and in maternal peripheral vein (1,209.0 +/- 530.3pg/ml).

Determination of serum unconjugated estrone, estradiol-17 beta and estriol during pregnancy by selected ion monitoring.

A simple, rapid and highly specific method by selected ion monitoring (SIM), using 9 alpha, 11 alpha-[2H2]estrone, [2,4-2H2]estradiol-17 beta and 2,4-[2H2]estriol as internal standards, was developed for the determination of serum estrogens during pregnancy. Serum samples were submitted to a simple extraction procedure and were analysed after formation of the trifluoroacetic anhydride derivative. The inter-assay coefficients of variation for estrone, estradiol-17 beta and estriol were 3.73%, 3.42% and 3.49%, respectively. The results obtained by SIM were compared with analysis performed using radioimmunoassay.

[Changes of dehydroepiandrosterone (DHA), 16 alpha OH-DHA and estriol levels in maternal peripheral blood during late pregnancy and labor--measured by gas chromatography-mass spectrometry with application of deuterated steroids as internal standards (author's transl)].

Serum levels of DHA, 16 alpha OH-DHA and estriol (E3) were determined in maternal peripheral vein by gas chromatography-mass spectrometry (GC-MS) with application of deuterated steroids as internal standards during late pregnancy and labor. Deuterium labeled 3,4,4-d3-DHA, 3,4,4-d3-16 alpha OH-DHA and 2,4-d2-E3 were newly synthesized and highly specific method for determination of serum DHA, 16 alpha OH-DHA and E3 was developed by GC-MS. The mean total steroid concentrations in the third trimester (III), in the stage when patients had irregular contraction (P) and during the first stage of labor (1st) were as follows; DHA, 612.9 +/- 265.3, 877.5 +/- 365.6 and 1522.5 +/- 580.6, 16 alpha OH-DHA, 30.2 +/- 20.2, 49.4 +/- 21.9 and 161.3 +/- 43.6, E3, 50.9 +/0 25.7, 70.4 +/- 19.8 and 108.6 +/- 44.5 ng/ml (mean +/- S.D.). The values of these three steroids increased significantly from the III to the 1st. Correlation between the levels of total DHA and total E2 measured in ten patients individually in the III was 0.8665 (p less than 0.001) which changed to -0.6650 (p less than 0.01) and -0.5362 (p less than 0.05) in the stage of P and 1st. ACTH was given to eight pregnant women in the third trimester, and response of these steroids to ACTH was studied. In addition to these steroids, cortisol (F) levels were also measured by RIA. Following the administration, F increased significantly whereas no changes of DHA, 16 alpha OH-DHA and E3 levels were noticed suggesting that these steroids may not be influenced by maternal adrenal secretion. From the results obtained above, it is suggested that the endocrinological environment in maternal blood as indicated by correlations between DHA and E3 levels, changes prior to the onset of labor.