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.

Validation of a high-performance liquid chromatographic assay for lysylpyridinoline in urine: a potential biomarker of bone resorption.

We developed and validated a high-performance liquid chromatographic (HPLC) method for quantifying the bone-specific collagen crosslink, lysylpyridinoline (LP), in urine, LP was purified from cortical bone and characterized by spectrophotometry, HPLC, and 1H NMR spectroscopy. Our HPLC detected urinary LP independently of the sample volume and the range of quantification was between 63 nM and 1 microM. Average recovery of added LP standard to human urine samples was 106 +/- 21%. Mean inter- and intraassay CVs, respectively, for urines containing low, medium, and high concentrations of the crosslink LP were 7.4 and 6.3%. This analytical method is more efficient than previously published HPLC assays for LP because of the significant 24-h reduction in urinary sample preparation time. There was agreement between urinary LP concentrations measured with this method and the Metra Pyrilinks-D enzyme immunoassay (r2 = 0.714). These results emphasize the importance of using a thoroughly standardized HPLC assay as the "gold standard" for comparison of results with newly developed immunoassays.

Clinical evaluation of near-continuous oral micronized progesterone therapy in estrogenized postmenopausal women.

In an open non-comparative prospective trial of 12 months' duration, we investigated the role of a novel hormone replacement therapy regimen in 40 post-menopausal women who sought hormone replacement therapy. The regimen consisted of continuous administration of 0.625 mg of conjugated equine estrogen coupled with a fixed low-dose of micronized oral progesterone administered for 23 days every calendar month. The regimen was well-tolerated, producing no major side-effects and was effective in relieving menopausal symptoms. The study showed that 40% of the women experienced side-effects and 20% withdrew from the study. Half of the 20% of the women who dropped out did so for reasons not related to treatment. All symptomatic women experienced improvement after the 1st month, and virtually all were asymptomatic by the 3rd month of treatment, persisting until the end of the trial with the average number of hot flushes per day declining from the pretreatment levels by 96%. Amenorrhea was observed in 47% of patients, amenorrhea and minimal vaginal bleeding in 78% but acyclic bleeding was present in 28% of those in whom bleeding was re-established. Endometrial atrophy was induced in the majority of patients and no atypical endometrial hyperplasia was encountered. No significant changes were observed in blood glucose or liver enzymes. The mean percentage changes from baseline for serum cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoproteins (LDL) and LDL/HDL ratio were -6%, +32% (p < 0.001), -16% (p < 0.05), +15% (p < 0.05) and -23% (p < 0.05), respectively. The regimen was clinically effective and its apparent lack of major side-effects, the protective effect on the endometrium, the added advantage of minimal vaginal bleeding and the beneficial effect on lipid/lipoprotein levels, offer an attractive therapy and improved compliance with postmenopausal hormone replacement therapy.

Trends in HIV-1 and HIV-2 prevalence and risk factors in pregnant women in Harare, Zimbabwe.

The spread of the human immunodeficiency virus (HIV) and the resulting morbidity and mortality are major public health challenges with adverse social and economic implications. The pregnant population serves as an important marker of the extent of the problem in the sexually active low risk categorized population. Furthermore, a high HIV prevalence among women of childbearing age contributes to neonatal and infant mortality through perinatal transmission and also a large number of uninfected children become orphans. The objective of the present study was to determine the HIV prevalence rate and the risk factors in pregnant women attending antenatal care clinics in the Greater Harare area of Zimbabwe. Women presenting for antenatal care in four maternity clinics between May 1994 and June 1995 were tested for HIV-1 and HIV-2 antibodies following informed consent, counselling and completion of a questionnaire. Of the 1.168 women, 30.4 pc tested HIV-1 positive, with prevalence rates ranging from 23.6 pc at a lower density clinic, 28.6 pc in a medium density clinic, 30.7 pc in a higher density clinic and 33.2 pc at the referral maternity hospital. HIV-2 was present in 7.6 pc of the women. The 20 to 29 years age group had the highest HIV prevalence of 35 pc, (Odds Ratio (OR) = 2.4; 95 pc CI-1.33 to 4.32). Single pregnant women were more likely to test positive, (OR = 2.1; 95 pc CI = 1.2 to 3.7). Thirty five pc of the women reported previous use of condoms and in those where condom use was reported in casual relationships, there was a higher risk of HIV (OR 6.1; 95 pc CI = 2.1 to 17.3). Reported use of intravaginal herbs was associated with HIV risk (OR 1.4; 95 pc CI = 1.1 to 1.8; p < 0.03). History of genital ulcer (OR = 2.3), discharge (OR = 2.4), rash (OR = 2.7), genital ulcer with PID (OR = 5.8) was significantly associated with HIV infection. Present findings indicate a 30.4 pc HIV prevalence rate for a sample of 1,168 pregnant women in Harare. This rate is much higher than the 18 pc HIV prevalence rate reported for 1,008 pregnant women in the same Greater Harare area in 1990. We conclude that there is need for further innovative and aggressive community based as well as institutional interventions aimed at reducing HIV risk. Prevention strategies should include a wide range of socially contextualized initiatives.

PIP: During May 1994-June 1995 in Zimbabwe, after informed consent and HIV counseling, 1168 pregnant women attending their first prenatal care visit at Harare Maternity Hospital and three Harare-based maternity clinics had blood taken to test for HIV-1 and HIV-2. The study aim was to determine the HIV prevalence and risk factors for HIV infection among pregnant women. 30.4% tested positive for HIV-1 (compared to 18% for a similar group in 1990). The HIV-1 prevalence ranged from 23.6% at the lowest density clinic to 33.2% at the referral maternal clinic of the hospital. 7.6% tested positive for HIV-2. Risk factors for HIV-1 infections included being 20-29 years old (odds ratio [OR] = 2.1), unmarried (OR = 2.1), unemployed (OR = 2.1); use of condoms in casual relationships as opposed to with husband or boyfriend (OR = 6.1); having a partner who frequented the bar everyday, every weekend, or twice per month (OR = 2); and history of sexually transmitted disease (STD) (OR = 2.3). Medical conditions associated with HIV-1 infection were palpable lymph glands (OR = 10.8), weight loss (OR = 4.5), and chronic diarrhea (OR = 2.2). STDs associated with HIV-1 infection included genital ulcer (OR = 2.3), genital discharge (OR =2.4), genital rash (OR = 2.7), and genital ulcer with pelvic inflammatory disease (OR = 5.8). The researchers recommend multisectoral and multilevel interventions to prevent HIV infections at all socioeconomic levels.

Follicular and luteal phase salivary progesterone profiles in women with endometriosis and infertility.

Twenty-three women with laparoscopically diagnosed early-stage endometriosis collected daily saliva samples at home and brought them to the laboratory at the completion of each menstrual cycle. Levels of progesterone in the saliva were determined by enzymeimmunoassay. Samples were collected for three cycles by one patient, for two cycles by 15 patients and for one cycle be seven women, resulting in a total of 40 cycles for analysis. Only 20 (50%) cycles were classified as normal. In 18 cycles (45%) progesterone levels exceeded the 95th percentile of the normal range. These high levels occurred in the follicular phase only in seven (18%) cycles, in the luteal phase only in eight (20%) and in both follicular and luteal phases in three (7.5%) cycles. Three cycles were anovulatory. One of these cycles also demonstrated elevated progesterone levels in the follicular phase and is therefore included in the 18 cycles mentioned above. We conclude that ovarian function is altered in a significant number of infertile women with endometriosis. However, these alterations are often subtle and only detected by detailed investigation.

Development and validation of a monoclonal antibody enzyme immunoassay for measuring progesterone in saliva.

A nonextraction, competitive, solid-phase enzyme immunoassay with a monoclonal antibody was developed and validated for measuring progesterone in saliva. The antibody was raised against 11 alpha-hydroxyprogesterone hemisuccinate-bovine serum albumin conjugate and was indirectly immobilized to the walls of microtiter wells. The labeled analyte (progesterone-horseradish peroxidase conjugate) was homologous with the immunogen. The lower detection limit (concentration equivalent to B0-3 SD) was 38 pmol/L of saliva sample. We validated the assay with studies to establish the independence of the concentration determined from the volume of saliva assayed, quantitative recovery of progesterone added to saliva, interference from possible cross-reactants, and agreement with a similar assay that incorporated an extraction step. In addition, we determined luteal-phase concentrations of salivary progesterone in normal women and compared the results with those of an older assay involving a polyclonal antibody.

Radioimmunoassay of 2-hydroxyestrone in urine.

Catechol estrogens such as 2-OH estrone are interesting estrogen metabolites formed in several human tissues and excreted in urine. We developed and thoroughly validated a radioimmunoassay for urinary 2-OH estrone that has several advantages over published RIAs. Because we developed a relatively specific antiserum, we did not include a preliminary chromatographic step to eliminate cross-reacting urinary steroids. We hydrolyzed urinary steroid conjugates with beta-glucuronidase from Helix pomatia because recoveries after acid hydrolysis were only 49.6% compared with 73.8% after enzyme hydrolysis. Published RIAs for urinary 2-OH estrone use acid hydrolysis. Our RIA measured 2-OH estrone independently of the volume of sample, and the detection limit was between 100 and 240 ng/L (10-24 pg per tube). The ED50 was 370 ng/L, and inter- and intraassay CVs for low, medium, and high concentrations were 22.5%, 22.8%, and 19.9%, and 17.4%, 14.3%, and 10.8%, respectively. Median concentrations measured in 14 controls and 33 postmenopausal patients with breast cancer were 0.96 and 1.55 micrograms/g creatinine, respectively, but there was considerable overlap between values from controls and patients.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin.

We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

Assessment of bioavailability of oral micronized progesterone using a salivary progesterone enzymeimmunoassay.

Salivary progesterone was measured sequentially by enzymeimmunoassay following 1 month and 6 months of oral therapy with 100 mg of micronized progesterone (MOP) in 40 healthy estrogenized postmenopausal women (aged 40-68 years). MOP was administered for 23 days every month. There were striking differences in the absorption of MOP between various subjects. Significant increases occurred in salivary progesterone concentrations over baseline and pretreatment levels and persisted for at least 10 h. Levels of salivary progesterone remained higher than pretreatment levels for at least 24 h after administration of MOP. Maximum mean concentrations of salivary progesterone of 827.2 and 888 pmol/l in the 1st and 6th months of therapy, respectively, were achieved within 2 h of administration and were above the 95th percentile of a control corridor which corresponds to the range found in the luteal phase. The areas under the salivary progesterone curve (AUC0-24 h, pmol/l) were 7177.75 and 7388.20 respectively, in the 1st and 6th months of therapy but the difference was not statistically significant. Serum and salivary progesterone peaked simultaneously and there was a significant correlation between the concentrations measured concurrently (y = 233.08 + 35.575x; r = 0.89, p < 0.001) thus supporting the current concept of a relatively rapid diffusion of steroids from plasma to saliva. Results of this study confirm those of previous investigations which monitored the bioavailability of MOP with the use of serum progesterone measurements and showed that luteal phase progesterone concentrations can be attained easily. The use of non-invasive salivary sampling and a cost-effective, direct enzymeimmunoassay showed a considerable advantage in the present study, compared with previous ones. We conclude that 100 mg MOP should be given at least twice-daily to maintain a stable physiological luteal phase level of progesterone during clinical hormone replacement therapy.

Low-dose progesterone therapy in oestrogenised postmenopausal women: effects on plasma lipids, lipoproteins and liver function parameters.

BACKGROUND: Cardiovascular disease among older women is a major health problem and is the leading cause of death in this group in developed countries. The risk is reduced in oestrogen users secondary to favourable lipid changes, but the beneficial effect of oestrogen may be counteracted when concomitant progestogens are administered. OBJECTIVE: To study the effects of a novel hormone replacement therapy regimen on liver enzymes, lipids and lipoproteins in postmenopausal women. DESIGN: Prospective open, non-comparative trial for 12 months. METHODS: 40 healthy postmenopausal women, (mean age +/- S.D.), 53.5 +/- 3 years received 0.625 mg of conjugated equine oestrogen daily and 100 mg of micronised oral progesterone (P) for the first 23 days every calendar month for 12 months without interruption. MAIN OUTCOME MEASURE: Gonadotrophins, liver function parameters and lipoproteins were measured before treatment and at the 6th, 9th and 12th months of treatment. RESULTS: Compliance with treatment was confirmed by a 33% decrease in mean serum level of follicle stimulating hormone at the end of 1 year of treatment. In the same period, the mean serum cholesterol, LDL and LDL/HDL ratio decreased by 6%, 16% and 23% of the base line levels, respectively. The percentage changes in triglycerides and HDL from the basal levels were +32% (P < 0.001) and +15% (P < 0.05), respectively. CONCLUSION: These results indicate that near continuous administration of fixed low-dose of P has no adverse effects on the lipid milieu of postmenopausal women when combined with long-term continuous oestrogen replacement therapy provided women with borderline triglyceridaemia are excluded.

The frequency of salivary progesterone sampling and the diagnosis of luteal phase insufficiency.

A profile of salivary progesterone concentrations, based on daily samples taken over a full menstrual cycle, provides a detailed picture of changes in luteal function, at the expense of analyzing a large number of samples. Strain can be placed on analytical services by assaying daily samples instead of one or a few serum (or saliva) samples. This study sought to determine the minimum number of salivary progesterone determinations which adequately describe luteal function. Daily salivary progesterone levels from 215 cycles, of which 29 cycles had progesterone profiles indicative of luteal phase insufficiency, were analyzed to ascertain the efficiencies of various sampling patterns of reduced frequency. A single mid-luteal salivary progesterone estimation or the mid-luteal Lenton progesterone index (n = 4) satisfactorily reflected the normal luteal phase, but a frequency of one sample every 3 days over the luteal phase (n = 5-6) was necessary to allow recognition of a short luteal phase or poor progesterone surge.

The return of postpartum fertility monitored by enzyme-immunoassay for salivary progesterone.

A longitudinal study in which daily salivary progesterone and estrone were measured by solid-phase enzyme-immunoassays was performed in 30 postpartum women to monitor the return of ovarian activity. Ovulation was inferred from a sustained rise in salivary progesterone over 251 pmol/l, but salivary estrone measurements were not as informative as progesterone in this regard. Recovery of ovarian activity was slower in lactating women compared with non-lactators; the mean delivery-menstruation interval were 123 (+/- 10) and 57 (+/- 7) days, respectively. An abnormal luteal phase was noted in 35% of the first ovulatory cycles, 20% had short luteal phases and 15% were less than the 5th percentile of a normal control corridor. The pregnancy rate in this study of 3.3% was lower than the anticipated rate of 8.8%. We conclude that salivary progesterone measurements are useful for monitoring the return of ovarian activity postnatally.

Osteocalcin concentrations in plasma prepared with different anticoagulants.

We investigated the effects on plasma osteocalcin concentrations of different anticoagulants used to collect the blood samples. Plasma osteocalcin concentrations measured by enzyme immunoassay and radioimmunoassay are influenced by the nature of the anticoagulants used. The most significant difference between concentrations found in plasma and serum was seen with oxalate/fluoride anticoagulant, which reduced osteocalcin concentrations to 37.3% of serum values. This is probably related to increased hemolysis with this anticoagulant compared with osteocalcin concentrations in plasma prepared with other anticoagulants. Samples prepared with sodium citrate (0.105 mol/L) or lithium heparin gave values 92.4% and 83.6% of those obtained with matched serum samples. Osteocalcin concentrations were relatively stable in plasma and serum at -20 degrees C for two freeze/thaw cycles. In blood from 100 patients there was a good correlation between osteocalcin concentrations in serum and plasma (lithium heparin) (r2 = 0.831); the slope and intercept (+/- SE) were 0.924 +/- 0.04 and 4.92 +/- 1.25 micrograms/L, respectively. However, in 10 patients, serum osteocalcin concentrations were two- to threefold higher than those in matched plasma samples.

Osteocalcin: diagnostic methods and clinical applications.

Osteocalcin is a small (Mr 5800), very interesting bone specific protein, synthesized by osteoblasts and measured in plasma as a biochemical indicator of bone formation. Many immunoassays for osteocalcin have been developed, including radio- and enzymoimmunoassays, with the use of monoclonal and polyclonal antibodies. These are used in many different clinical settings, including bone, kidney, and liver diseases. However, there is a wide range of published values for plasma osteocalcin concentrations in control and patient samples and this has hindered a more widespread adoption of osteocalcin measurement by clinicians. This review discusses how various immunoassays for osteocalcin may contribute to the wide variation of published values and suggests approaches for the development of standardized assays. For example, epitope specificity and immunoreactivity with multiple forms of osteocalcin and osteocalcin peptides in plasma are discussed. It also includes a recent update on interesting clinical applications of osteocalcin.

Follicular growth and corpus luteum function in women with unexplained infertility, monitored by ultrasonography and measurement of daily salivary progesterone.

Ovarian function was evaluated over a minimum of 3 consecutive menstrual cycles from each of 41 women with unexplained infertility. Follicular development and ovulation were monitored using real time ultrasonography and luteal function was evaluated by daily salivary progesterone measurement. In 129 spontaneous cycles, normal single ovulations were detected in 121 (93.8%). Luteal phase insufficiency was identified in 21 (17.4%) of these 121 cycles and this was a recurrent phenomenon in the cycles of 5 of the 41 women (12.2%). A successful pregnancy was seen only in association with consistently normal salivary progesterone profiles or where the empirical use of clomiphene citrate therapy had corrected previously diagnosed luteal phase insufficiency. Basal body temperature records or mid-luteal serum progesterone measurements were less satisfactory indices of luteal function than a salivary progesterone profile.

Direct solid-phase enzymoimmunoassay of testosterone in saliva.

This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.