2nd-trimester maternal serum marker results are not altered by delayed analysis

OBJECTIVE: The goal of the study was to evaluate the significance of delayed laboratory analysis of maternal serum alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, and unconjugated estriol for prenatal screening. METHODS: Biochemical analysis of 30 consecutive biochemical screening specimens of maternal serum alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, and unconjugated estriol was performed immediately upon arrival to the laboratory, 7 days later, and again 14 days after maternal blood was drawn. Differences among the results of the three sets of biochemical studies were evaluated by one-way analysis of variance for repeated measures. RESULTS: No significant differences were found among the results of immediate assays as compared with those at a 7- or a 14-day delay for all three biochemical markers. CONCLUSIONS: Our data suggest that up to a 14-day delay in the performance of the 2nd-trimester maternal serum biochemical screening assays will not alter the results significantly. The results of maternal serum screening are, thus, clinically valid even if the laboratory assays were performed several days after maternal blood was drawn.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-3H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5alpha-androstane-3alpha,17beta-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5alpha-androstane-3,17-dione and 3beta-hydroxy-5alpha-androstan-17-one were isolated only from MCF-7. The metabolism of 3H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15--0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Undetectable maternal serum unconjugated estriol levels in the second trimester: risk of perinatal complications associated with placental sulfatase deficiency

OBJECTIVE: Our purpose was to determine the prevalence of undetectably low second-trimester maternal serum unconjugated estriol levels and the association with increased perinatal morbidity or mortality in pregnancies at risk for placental sulfatase deficiency. STUDY DESIGN: Nine centers in New England identified singleton pregnancies with undetectably low unconjugated estriol levels. Each unexplained case was matched with four controls; pregnancy outcome information was sought. RESULTS: Among 130,295 pregnancies surveyed, undetectably low unconjugated estriol levels were identified in 167 (13/10,000). Explanations included fetal death (53), overestimated gestational age (50), nonpregnancy (12), and chromosome abnormalities (5). The 41 unexplained cases were compared with 163 matched controls. Male offspring were more frequent (85%) among cases than among controls (55%). Although rates of perinatal complications were not significantly different, primary cesarean sections occurred about twice as often among cases. No perinatal deaths occurred. CONCLUSIONS: Neither severity of symptoms nor perinatal morbidity or mortality currently warrant routine interpretation of unexplained undetectably low unconjugated estriol levels as a marker for placental sulfatase deficiency.     

Rapid radioimmunoassay of total urinary estriol

We describe a radioimmunoassay for total urinary estriol in pregnancy. A 25-mul aliquot of the urine specimen is acid hydrolyzed, neutralized, and diluted before assay. We use rabbit antisera against estriol-6-(O-carboxymethyl)oxime/bovine serum albumin and ammonium sulfate precipitation at room temperature. Results are unaffected by glucose or methenamine mandelate, a urinary tract antiseptic. Using semi-automatic pipetting equipment, one laboratory technologist can complete 50 assays within 8 h. This technique is both reliable and convenient and should decrease the expense of routine estriol assays.     

Primary alcohols and phosphatidylcholine metabolism in rat brain synaptosomal membranes via phospholipase D

The prognostic value of maternal serum triple analyte screening with AFP, hCG and uE3 (unconjugated estriol) was studied early in the second trimester of pregnancy. In this case-control study of 38 women and 76 matched controls derived from a consecutive screened population of 28,897, case selection was based upon elevated MSAFP and MShCG (> or = 2 MOM) and low MSuE3 (< or = 0.6 MOM). Adverse pregnancy outcome was found in 65.8% of cases and 2.6% of controls (RR 25, 95% CI 6.3-100.0). When increased odds (> or = 1 in 270) for Down's syndrome were considered with the abnormal analyte screen, fetal/congenital defects, fetal neonatal loss or low birth weight were noted in 17/26 cases (65.4%). Elevated MSAFP and MShCG with low values for estriol, with or without increased odds for Down's syndrome, imply an unfavorable prognosis for both the fetus and the child.     

Intestinal metabolism of estrogens

Estrogen metabolism in the human intestine was studied in two ways. Firstly, by measuring the excretion of 12 estrogens in pooled human late pregnancy feces before and during the administration of ampicillin (2 g/day). Secondly, by administering 5.4 and 20 mg of 16alpha-hydroxyestrone orally to two postmenopausal women and analyzing the estrogens in simultaneously drawn portal and peripheral venous blood samples at time intervals from 0 to 150 min after steroid administration. The majority of the estrogens in normal pregnancy feces were unconjugated. The amounts of estradiol, estreon and 16-epiestriol excreted, relative to the principal estrogen estriol, were greater than in pregnancy bile or urine and 16alpha-hydroxyestrone, an important biliary estrogen, was only present in trace amounts. Considerable quantities of 15alpha-hydroxyestradiol-17beta were also found. Ampicillin administration, which decreases intestinal bacterial steroid metabolism, caused a huge increase in the fecal excretion of conjugated estrogens. In particular it caused very striking increases in the excretion of both unconjugated and conjugated, estriol, 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol and 2-methoxyestrone. These findings emphasize the active role played by the intestinal microflora in estrogen metabolism under normal conditions. Administration of 16alpha-hydroxyestrone resulted in increases in portal venous unconjugated and conjugated 16alpha-hydroxyestrone, 16-oxoestradiol-17beta, 15alpha-hydroxyestrone, 16-epiestriol and conjugated estriol levels. The most significant finding in both subjects was the large increase in portal venous unconjugated 15alpha-hydroxyestrone. This would suggest that the human intestine (or intestinal contents) has the ability to carry out the transformation, 16alpha-hydroxyestrone leads to 15alpha-hydroxyestrone. Increases in the same estrogens were found in peripheral plasma, with the increase in conjugated estriol occurring in peripheral blood before it was seen in portal blood. The largest elevations in peripheral plasma values were seen in unconjugated estriol and conjugated 16alpha-hydroxyestrone in the subject who received the 20 mg dose and in unconjugated 16alpha-hydroxyestrone and 16-oxoestradiol-17beta in the subject who had the 5.4 mg dose. The intestinal and enterohepatic metabolism of estrogens is discussed in relation to these findings.     

Determination of 17 beta-estradiol and its metabolites in sewage effluent by solid-phase extraction and gas chromatography/mass spectrometry

The paper describes a simple and quantitative method for monitoring non-conjugated 17 beta-estradiol (E2) and its metabolites estrone (E1) and estriol (E3) as environmental contaminants in municipal sewage effluents. Estrogens were preconcentrated and cleaned up by solid-phase extraction using a reversed-phase C18 cartridge. They were derivatized with pentafluoropropionic acid anhydride, and the products were analyzed by gas chromatography/mass spectrometry. Recoveries from spiked distilled water and sewage were better than 87% at fortification levels of 100 and 20 ng/L. For a 1 L sewage sample and a concentration factor of 5000, detection limits were 5 ng/L for E1 and E2 and 10 ng/L for E3. In a brief survey of Canadian wastewater, these estrogens were detected in many raw sewage and effluent samples at concentrations ranging from 6 to 109 ng/L for E1, from < 5 to 15 ng/L for E2, and from < 10 to 250 ng/L for E3.     

On being a medical student in the 1930s

The obstetrical management of women with renal disease is complicated and associated with increased fetal and maternal morbidity. However, maternal serum screening is an integral part of obstetrical care and should be offered to all women. We found that maternal serum levels of a-fetoprotein and human chorionic gonadotropin did not significantly change as a result of hemodialysis, whereas levels of unconjugated estriol were markedly decreased following hemodialysis. Maternal serum screening should be limited to alpha-fetoprotein analysis in women undergoing hemodialysis until the effects of hemodialysis on all serum analytes are better delineated.     

Study of human isoursodeoxycholic acid metabolism

BACKGROUND/AIMS: The aim of this study was to examine the metabolism of isoursodeoxycholic acid (isoUDCA) in humans. METHODS: IsoUDCA was synthesized of >99% purity and administered orally for 1 week, 3 x 250 mg/day, to six healthy male subjects. Bile acids were extracted from duodenal bile, serum, and 24-h urine samples collected before and at the end of the study period, separated into groups of conjugates, and analyzed by gas chromatography-mass spectrometry and fast atom bombardment mass spectrometry. RESULTS: IsoUDCA was tolerated without any side effect. Liver function tests did not change. Bile acid concentrations (mean+/-SEM) increased from 11.9+/-1.87 to 15.3+/-1.37 mmol/l in bile (n.s.), and from 3.4+/-0.10 to 6.8+/-0.43 micromol/l in serum (p<0.05). Urinary excretion of bile acids increased from 5.3+/-0.29 to 82.2+/-7.84 micromol/24 h (p<0.01). All changes were due to significant increases of isoUDCA and UDCA in bile, serum and urine, and of 3-dehydro-UDCA, the 3-oxo intermediate of isomerization, in bile and in serum. The relative enrichments of isoUDCA, UDCA, and 3-dehydro-UDCA, were: in bile, 2.2%, 25.7%, and 0.7%; in serum, 24.7%, 23.5%, and 6.1%; and in urine, 83.7%, 2.0%, and 2.4%. Whereas 78% of serum isoUDCA was unconjugated, 93-94% of biliary and urinary isoUDCA was conjugated with N-acetylglucosamine. CONCLUSIONS: This study indicates good tolerance and significant intestinal absorption of orally administered isoUDCA. IsoUDCA is extensively isomerized, probably both by intestinal and hepatic enzymes to yield UDCA which became the major biliary compound. In vitro, using the human hepatoblastoma cell line Hep G2, isoUDCA was found to be cytoprotective towards ethanol-induced cell injuries.     

Screening of maternal serum for fetal Down's syndrome in the first trimester

BACKGROUND: Screening of maternal serum to identify fetuses with Down's syndrome is now routinely offered during the second trimester of pregnancy. Prenatal screening by means of serum assays or ultrasonographic measurements, either alone or in combination, may also be possible in the first trimester. METHODS: We measured serum alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin (hCG), the free beta subunit of hCG, and pregnancy-associated protein A in 4412 women (82 percent of whom were 35 years of age or older) who came to 16 prenatal diagnostic centers for chorionic-villus sampling or early amniocentesis at 9 to 15 weeks of gestation. Ultrasound measurements of fetal nuchal translucency were also reported. Fetal chromosomal analysis was performed in all pregnancies. Altogether, there were 61 fetuses with Down's syndrome. RESULTS: A total of 48 pregnancies affected by Down's syndrome and 3169 unaffected pregnancies were identified before 14 weeks of gestation; the rates of detection of Down's syndrome for the five serum markers were as follows: 17 percent for alpha-fetoprotein, 4 percent for unconjugated estriol, 29 percent for hCG, 25 percent for the free beta subunit of hCG, and 42 percent for pregnancy-associated protein A, at false positive rates of 5 percent. The results of the measurements of serum hCG and its free beta subunit were highly correlated. When used in combination with the serum concentration of pregnancy-associated protein A and maternal age, the detection rate was 63 percent for hCG (95 percent confidence interval, 47 to 76 percent) and 60 percent for its free beta subunit (95 percent confidence interval, 45 to 74 percent). Measurements of nuchal translucency varied considerably between centers and could not be reliably incorporated into our calculations. CONCLUSIONS: Screening for Down's syndrome in the first trimester is feasible, with use of measurements of pregnancy-associated protein A and either hCG or its free beta subunit in maternal serum.     

Direct colorimetric monoclonal antibody enzyme immunoassay for estradiol-17 beta in saliva

We developed a direct microtiter plate enzyme immunoassay to measure estradiol-17 beta in saliva. The assay has a commercially available monoclonal antibody, raised against estradiol-17 beta-6-carboxymethyloximebovine serum albumin, and a homologous horseradish peroxidase conjugate measured colorimetrically. The detection limit (equivalent to B0-3 SD) is 365 amol/well or 7.3 pmol/L when 50-microL samples are assayed. Cross-reactivity with estrone and estriol, testosterone, or progesterone is < 0.2%. Estradiol-17 beta was measured in daily samples over five natural menstrual cycles and eight cycles stimulated as a preliminary to in vitro fertilization, and the concentrations and fluctuations found agreed with previously published data. This method gives results in approximately 3 h and may be useful for fertility monitoring and management.     

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).     

Correlation between morning urine estriol concentration and 24-hour estriol excretion

In an attempt to circumvent the need for 24-hour urine collections for estriol analyses in assessment of fetal status, the possibility of using morning urine samples was investigated. Results indicate 1) good correlation between 24-hour estriol excretion and morning estriol concentration, and between corresponding E/C ratios, 2) similar morning and 24-hour estriol concentrations, 3) high dependence of estriol and creatinine excretion on 24-hour urine volume but not on morning volume, 4) larger variations in 24-hour than in morning urine volume, and 5) better consistency of values in morning than in 24-hour samples in pathologic pregnancy. The use of serial morning urine concentrations at least as an outpatient monitoring procedure is suggested.     

Surgical repair of coronary sinus type partial anomalous pulmonary venous drainage with intact atrial septum

A method to prevent co-elution of steroid sulfates with proteins in serum from the pre-column in column-switching HPLC was developed. The pre-column, a polymer-coated mixed function column, was used for ion-pair chromatography with 5 mM tetra-n-butylammonium (TBA) ion. As steroid sulfates, estriol 3-sulfate, dehydroepiandrosterone 3-sulfate and pregnenolone 3-sulfate were used. Human serum (25 microl) was diluted with mobile phases including 5, 100 and 500 mM TBA ion, and then injected directly into the pre-column. The peak areas of the steroid sulfates in serum samples were compared with those of the steroid standards without serum. When 25/microl of serum was diluted with mobile phase including 100 or 500 mM TBA ion, the steroid sulfates in serum were retained in the pre-column; however, the steroid sulfates from the same sample diluted with mobile phase containing 5 mM TBA ion were not retained in the pre-column. Addition of an excess amount of counter ion (TBA ion) into the serum sample made it possible to retain the steroid sulfates in the pre-column. This method was applied to column-switching HPLC for measurement of steroid sulfates in serum using a semi-microcolumn as the analytical column.     

Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6-150 pg injected. The calibration curves for each amino acid in the range of 0.02-2 micrograms were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83-112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3-8.9% (n = 3) and 1.9-15.8% (n = 3), respectively.     

Analysis of estrone, estradiol and estriol in serum and urine by mass fragmentography using GC-MS (author's transl)

The first successful analysis of estrogens in serum and urine was performed by mass fragmentography using a GC-MS combined system. The equipment used was Shimadzu LKB 9000 GC-MS (MID-PM). The TMSi derivative of the compounds were analyzed using the GC-MS system equipped with a 3 ft x 3mm column packed with 1.5% OV-1 and the temperature was programed from 200 degrees to 260 degrees C at 10 degrees C/min increments. The mass spectrum showed molecular ions at m/e 342, 416 and 504 which correspond to the TMSi derivatives of Estrone (E-1), Estradiol (E-2) and Estriol (E-3) respectively. Molecular ion peak of each compound was applied to establish the precise quantitative evaluation of E-1, E-2 and E-3. 1) The minimum detectable limits of the compounds injected into the column were ca. 2 pg for E-1, E-2 and 5 pg for E-3 respectively. 2) The sensitivity was of the order of ng or pg which enables quantitation with 2 ml of human serum and urine samples obtained from normally ovulating women. 3) This method was very convenient for extracting the compounds from biological fluids, and the procedure can be carried out easily in a short time. 4) Mass fragmentography makes possible the simultaneous measurements of E-1, E-2 and E-3 in samples obtained from pregnant women.     

Blood flow rate during orthostatic pressure changes in the pulp skin of the first toe

BACKGROUND: The development of simple and standardised methods to measure airborne levels of workplace biological allergens is an important step in reducing the incidence of occupational asthma. Such a method would be useful for measuring wheat flour allergens which cause asthma in bakers. Measurement of allergen per se rather than total dust enables exposure to be better defined. METHODS: Monoclonal antibodies were produced, their specificity analysed by immunoblotting and then used to affinity-purify a putative flour allergen. The importance of this protein as an allergen was tested by RAST using sera from allergic bakers and it was identified by N-terminal sequencing. Suitable monoclonal antibodies were chosen to develop an enzyme-linked immunosorbent assay. Commercial baking flours and personal airborne dust samples were analysed using the immunoassay. RESULTS: A sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay was developed to measure a wheat alpha-amylase inhibitor. The wheat alpha-amylase inhibitor content of bulk wheat flours was 0.124% (95% confidence limits 0.083-0.164%) and airborne levels in bakeries had a geometric mean of 744 ng/m3 (95% confidence limits 371-1,496 ng/m3). CONCLUSION: This assay is suitable for widespread use as the monoclonal antibodies and standards are well defined and potentially infinitely available. The assay therefore offers distinct advantages over those exposure assessment methods currently in use. Comparable results would be obtained by different investigators over a prolonged time period. The assessment of flour allergen exposure and the relationship with clinical response could then be investigated using a multi-centered approach.     

Solitary vertebral plasmacytoma

This study compares fetal corticoid response from conventional dose (12.0 mg) intramuscular betamethasone to large dose (1,000 mg) intravenous cortisol administered to women in premature labor for acceleration of fetal lung maturity. To compare these two regimens, 14 women selected at random were treated in groups of seven with either cortisol or betamethasone. Peripheral levels of unconjugated estriol were measured by specific radioimmunoassay prior to the cortisol dose and at 1, 4, 8, and 12 hours following the dose. The rate of corticoid delivery to the fetal hypothalamic-adrenal axis was estimated by the per cent suppression of unconjugated estriol at each post-treatment interval. Least-squares regression lines fitted (P less than 0.01) for each regimen were compared for time saved (delta t) when cortisol was used. Mean delta t (1, 4, 8, and 12 hours) was 9.0 +/- 0.2 S.E.M. hours. It is concluded that: (1) Intravenous cortisol delivers a fetal corticoid effect that is significantly more rapid in onset and more profound in magnitude than does intramuscular betamethasone and that (2) the cortisol regimen is probably better suited to the acceleration of fetal lung maturation in premature labor when time is short and rapid action is essential.     

First trimester sonographic diagnosis of acrania

Anencephaly, a well-known lethal fetal malformation, was long considered to result from primary nonclosure of the neural tube. In the past few years other pathogenic mechanisms, such as reopening or degeneration of a closed neural tube have been suggested. High-resolution transvaginal ultrasonography, which provides fine visualization of the different stages in embryogenesis, allowed us to detect fetal acrania as early as the 12th week of gestation. Very high levels of alpha-fetoprotein, almost undetectable levels of unconjugated estriol (E3), and postabortion histology were consistent with anencephaly, suggesting that anencephaly is the end result of fetal acrania.