Quantitation of submandibular proteins resolved from normal individuals and children with cystic fibrosis

Submandibular secretions collected from children with cystic fibrosis (CF) showed increased protein concentration (milligrams/milliliter) and increased amylase specific activity (units/milligram of protein) relative to normal secretions. These differences between normal (N) and CF secretions were as follows: protein, 1.25 ± 0.51 (N), 1.75 ± 0.35 (CF) (P < 0.02); and amylase, 58 ± 18 (N), 80 ± 19 (CF) (P < 0.001). To determine the basis for elevated protein in CF saliva, several major proteins resolved by polyacrylamide disc gel electrophoresis were quantitated by densitometry. These included four phosphoproteins (PP), serum albumin, an acid phosphatase-containing fraction, amylase, and an unidentified protein referred to as PI-7.1. Together, these proteins comprise greater than 75% of the total protein in the secretion. Differences in individual protein concentrations (milligrams/milliliter) resolved from normal and CF secretions, respectively, were as follows: PP₂, 0.02 ± 0.01, 0.03 ± 0.02 (NS, not significant); PP₃, 0.06 ± 0.04, 0.05 ± 0.03 (NS); acid phosphatase fraction, 0.06 ± 0.04, 0.12 ± 0.07 (P < 0.05); amylase, 0.09 ± 0.04, 0.27 ± 0.16 (P < 0.01); and pI-7.1, 0.04 ± 0.02, 0.13 ± 0.08 (P < 0.02). Amylase, the most significant contributor to the elevated protein, comprised 26% of the total protein of normal secretions and 38% of the total protein of CF secretions. Thus, our results do not support the concept of a generalized increase in all organic components in CF submandibular secretions but, rather, increases in specific proteins, namely amylase, component pI-7.1, and an acid phosphatase-containing fraction.     

Detection of cystic fibrosis homozygotes and heterozygotes by serum isoelectrofocusing

Summary It is suggested that the majority of individuals with the cystic fibrosis (CF) gene have a unique serum protein (CFP) which can be demonstrated by means of isoelectrofocusing (IEF) in thin layer polyacrylamide gels. We have found the CFP in 90% of patients with cystic fibrosis, in approximately 80% of individuals heterozygous for the CF gene, and in 8% of normal control individuals. We conclude that CFP is a useful marker for the CF gene.     

The first report on immunoglobulins A,E, G and M levels in cystic fibrosis patients in Saudi Arabia

BackgroundPrevious reports have shown that pulmonary and systemic hypergamma-globulinemia in CF patients is a reflection of chronic pulmonary infection. Infection with Pseudomonas aeruginosa is known to have major prognostic significance in patients CF. This study aims to identify the incidence of immunoglobulins (especially: IgG, and IgE) in a cohort of CF patients.MethodsA total of 297 patients recruited all over the country’s region for this study were a as part of the CF registry data from 1st January 1984 to 1st June 2016. All patients had their immunoglobulin levels measured by enzyme link immunosorbent assay (ELISA) in 3 stages, at presentation and two follow-ups.ResultsOf the 297 patients recruited, 139 (46.8%) were males while 158 (53.2%) were females. IgA and IgM levels were found not to have risen above the previously reported levels in healthy individuals in all stages. On the contrary, IgE level increased from 209.51 ± 32.30 KU/L to 303.58 ± 37.11 KU/L from baseline to stage 3 while IgG level rose from 12.26 ± 0.43 mg/mL to 17.17 ± 1.68 mg/mL for baseline and stage 3 respectively all above previously reported levels in healthy individuals.ConclusionThis study establishes a potential for the use of IgE and IgG in disease diagnosis as well as the prognostic implications. However, further study is needed to identify the role of infection or medications in relation to the rise of both IgE and IgG with advancement of age and progression of disease severity which may inherently confound the observed results.     

Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy

Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-¹³C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13±0.02 μmol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21±0.02 μmol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59±0.10 μmol/h per ml erythrocytes, compared to normal erythrocytes, 1.64±0.10 μmol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16±0.16 μmol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to simulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.     

Involvement of serum vascular endothelial growth factor family members in the development of obesity in mice and humans

Adipose tissue is highly vascularized implying that angiogenesis takes place in its expansion. The aim of this study was to compare the concentrations of members of the vascular endothelial growth factor (VEGF) family in obesity. Serum concentrations of VEGFs were analyzed in 15 lean (BMI 20.3±2.5 kg/m²) and 24 obese (BMI 47.6±5.9 kg/m²) volunteers. Obese patients showed significantly increased circulating VEGF-A (150±104 vs. 296±160 pg/ml; P<.05), VEGF-B (2788±1038 vs. 4609±2202 arbitrary units; P<.05) and VEGF-C (13 453±5750 vs. 17 635±5117 pg/ml; P<.05) concentrations. Interestingly, levels of VEGF-D were reduced in obese individuals (538±301 vs. 270±122 pg/ml; P<.01). In addition, VEGF-A significantly decreased after weight loss following Roux-en-Y gastric bypass (BMI from 46.0±8.0 to 28.9±4.2 kg/m² P<.0001 vs. initial) from 345±229 to 290±216 pg/ml (P<.01). Moreover, in order to corroborate the human findings VEGF-A levels were analyzed during the expansion of adipose tissue in two dynamic models of murine obesity. Serum VEGF-A was significantly increased after 12 weeks on a high-fat diet (43.3±9.0 vs. 29.7±9.1 pg/ml; P<.01) or in ob/ob mice (52.2±18.0 vs. 29.2±7.7 pg/ml; P<.01) and was normalized after leptin replacement in the latter (32.4±14.0 pg/ml; P<.01 vs. untreated ob/ob). Our data indicates the involvement of these factors in the expansion of adipose tissue that takes place in obesity in relation to the need for increased vascularization, suggesting that manipulation of the VEGF system may represent a potential target for the pharmacological treatment of obesity.     

Pseudomembranous Type of Oral Candidiasis is Associated with Decreased Salivary Flow Rate and Secretory Immunoglobulin A Levels

Saliva plays an important role in maintaining microbial homeostasis in the oral cavity, while salivary gland hypofunction predisposes the oral mucosa to pathologic alteration and increases the risk for oral candidiasis. This study sought to determine the salivary flow rate (SFR) and secretory immunoglobulin A (SIgA) levels in HIV-positive and HIV-negative individuals and evaluate their relationship with the determinants of oral candidiasis. Sixty HIV-positive (30 with and 30 without oral candidiasis) and 30 healthy HIV-negative individuals were enrolled. Cotton pellet was weighed pre- and post-saliva collection for the assessment of SFR, while SIgA levels were estimated by commercial ELISA (Diametra, Italy) kit. The mean ± SD, SFR and SIgA levels in HIV-positive individuals with candidiasis, without candidiasis and HIV-negative controls were 0.396 ± 0.290, 0.546 ± 0.355 and 0.534 ± 0.214 ml/min and 115.891 ± 37.621, 136.024 ± 51.075 and 149.418 ± 31.765 µg/ml, respectively. A positive correlation between low CD4 counts (indicator of immunodeficiency) and SIgA was observed in HIV-positive individuals with candidiasis (r = 0.373, p = 0.045). We also report here for the first time the significant decrease in SFR and SIgA levels in individuals presenting with pseudomembranous type of oral candidiasis and Candida albicans infection.     

Use of porcine follicle stimulating hormone after chromatographic purification in superovulation of cattle

Luteinizing hormone was removed from a commercial follicle stimulating hormone preparation (FSH-P) by QAE-A50 column chromatography. Dose rate of the FSH-rich fraction (FSH-W) significantly affected the number of transferable embryos recovered (P = 0.003), increasing from 4.5 ± 3.5 (x ± S.D.) at 2.7 units to 7.0 ± 6.5 at 5.4 units and then declining to 3.1 ± 3.9 at 10.8 units. These differences were the result of changes in the number of embryos fertilized (P = 0.004) (5.9 ± 4.4, 10.1 ± 9.1, and 5.1 ± 5.8 at the three dose rates) and in the total embryos recovered (8.0 ± 6.2, 13.8 ± 14.6, 11.2 ± 8.8). The percent transferable embryos declined with increasing dose of FSH-W from 66 ± 35% to 57 ± 31% and then 27 ± 25% (P < 0.001). In 130 Brahman crossbred cows, 5.4 units of FSH-W produced significantly more transferable embryos (6.3 ± 6.7) than 28-mg equivalents (Armour units) FSH-P (2.9 ± 4.0, P < 0.001). The number of fertilized embryos increased from 5.8 ± 6.7 to 9.0 ± 8.2 (P = 0.019). Adding LH to the FSH-W reversed the advantages of FSH-W. Fertilized embryo production declined from 12.8 ± 8.1 to 5.3 ± 3.6 (P < 0.001), resulting in a decline in the number of transferable embryos from 7.8 ± 6.6 to 4.4 ± 5.0 (P = 0.052). The blood progesterone levels at estrus in cows superovulated with FSH-P were higher (0.88 ng/ml) than in cows superovulated with FSH-W (0.45 ng/ml, P = 0.016). LH had a deleterious effect, due to reduction in fertilization rates on the number of transferable embryos recovered from donor cows stimulated with FSH.     

Establishment of Radioimmunoassay for Human Macrophage Colony-stimulating Factor Using Recombinant Human Macrophage Colony-stimulating Factor as Tracer and Immunogen

A sensitive and reliable radioimmunoassay (RIA) for human macrophage colony-stimulating factor (M-CSF) was developed using recombinant human M-CSF (rhM-CSF) as tracer and immunogen. The assay was quantitative over the range of 50 pg/ml and 5.0 ng/ml for M-CSF in human urine and serum, and more sensitive and specific than the murine bone marrow assay. The average level of human M-CSF in urine from normal males (N = 71) and females (N = 46) was 3.94 ± 1.78 ng/ml (2.85 ± 1.15 μg/g creatinine), and 3.53 ± 1.70ng/ml (3.31 ± 1.12 μg/g creatinine), respectively. The serum levels were 1.95 ± 0.38ng/ml for males (N = 117), and 1.93 ± 0.49 ng/ml for females, (N = 16). The results with the urine and sera showed that there was no difference in the M-CSF levels due to age or gender.     

A mutation in the second nucleotide binding fold of the cystic fibrosis gene.

The discovery last year of the deletion of a phenylalanine residue at amino acid position 508 of the cystic fibrosis (CF) gene has meant that approximately 70% of mutant chromosomes associated with CF can be accounted for. We report the finding of a substitution at nucleotide position 4041 of the CF gene, resulting in a change from asparagine to lysine at amino acid position 1303. We believe that this is a disease-causing mutation, as it involves a nonconservative amino acid change and has only been found on CF chromosomes with a consistent haplotype background. The mutation was detected using direct sequencing of PCR-amplified genomic DNA and was confirmed by dot hybridization to both normal and mutant allele-specific oligonucleotides. The mutation was detected on three chromosomes from four individuals but not on any normal chromosome. Its presence in the heterozygous state is not correlated with the clinical status of the individual patients.     

Sex differences in the circadian profiles of melatonin and cortisol in plasma and urine matrices under constant routine conditions

Conflicting evidence exists as to whether there are differences between males and females in circadian timing. The aim of the current study was to assess whether sex differences are present in the circadian regulation of melatonin and cortisol in plasma and urine matrices during a constant routine protocol. Thirty-two healthy individuals (16 females taking the oral contraceptive pill (OCP)), aged 23.8 ± 3.7 (mean ± SD) years, participated. Blood (hourly) and urine (4-hourly) samples were collected for measurement of plasma melatonin and cortisol, and urinary 6-sulfatoxymelatonin (aMT6s) and cortisol, respectively. Data from 28 individuals (14 females) showed no significant differences in the timing of plasma and urinary circadian phase markers between sexes. Females, however, exhibited significantly greater levels of plasma melatonin and cortisol than males (AUC melatonin: 937 ± 104 (mean ± SEM) vs. 642 ± 47 pg/ml.h; AUC cortisol: 13581 ± 1313 vs. 7340 ± 368 mmol/L.h). Females also exhibited a significantly higher amplitude rhythm in both hormones (melatonin: 43.8 ± 5.8 vs. 29.9 ± 2.3 pg/ml; cortisol: 241.7 ± 23.1 vs. 161.8 ± 15.9 mmol/L). Males excreted significantly more urinary cortisol than females during the CR (519.5 ± 63.8 vs. 349.2 ± 39.3 mol) but aMT6s levels did not differ between sexes. It was not possible to distinguish whether the elevated plasma melatonin and cortisol levels observed in females resulted from innate sex differences or the OCP affecting the synthetic and metabolic pathways of these hormones. The fact that the sex differences observed in total plasma concentrations for melatonin and cortisol were not reproduced in the urinary markers challenges their use as a proxy for plasma levels in circadian research, especially in OCP users.     

Increased superoxide anion production is associated with early atherosclerosis and cardiovascular dysfunctions in a rabbit model

Objective Hypercholesterolemia (HC) has been associated with impairment of vascular and myocardial functions. As HC could generate an alteration in the oxidative status, we studied the effects of a 1-month cholesterol diet on cardiovascular oxidative stress. Methods and Results New Zealand rabbits received cholesterol (1%) or normal chow for 1 month. At 30 days, superoxide anion levels, assessed by ESR spectroscopy, NAD(P)H oxidase (NOX) activity, and dihydroethidium (DHE) staining of aortas were higher in the cholesterol-fed (CF) group compared with control (respectively, 4.0 ± 0.6 Arbitrary Units/mg (AU/mg) vs. 2.6 ± 0.3, p < 0.05; 4231 ± 433 vs. 2931 ± 373 AU/mg, p < 0.05; 21.4 ± 1.2 vs. 12.9 ± 1.7% fluorescence/mm², p < 0.001). NOX gp91phox and p67phox expression in the aortas were higher in the CF group vs. control (1.5 ± 0.2 vs. 0.5 ± 0.2, p < 0.001; 0.9 ± 0.2 vs. 0.3 ± 0.2, p < 0.05). The endothelium-dependent relaxation evaluated on the iliac arteries was higher in control than in the CF group (64.8 ± 10.1 vs. 13.1 ± 3.70%, p < 0.001). The cardiac diastolic pressure estimated on isolated hearts was higher in the CF group than in control (21.1 ± 4.1 vs. 10.3 ± 1.4 mmHg, p < 0.05) after 60 min of ischemia. Conclusions Hypercholesterolemia induced increased levels of superoxide in the aortas and a higher expression of NOX subunits, associated with altered vasorelaxation. The increased diastolic pressure observed in hearts, consistent with a post-ischemic contractile dysfunction might be mediated by the production of superoxide.     

Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man

Levels of norepinephrine (NE) in human plasma have been determined by a radioenzymatic technique sufficiently sensitive to measure 0.014 ng NE per ml plasma. Several procedures which raise plasma NE levels have been compared and a standard procedure developed to evaluate sympathetic neuronal function based on the increments in plasma NE produced by postural change and a standard amount of exertion. The mean basal level of NE in plasma of 74 resting, supine, normal subjects ranging in age from 10 to 70 (mean 32.7 years) was 0.292 ± 0.016 (± SEM) ng/ml and ranged from 0.112 to 0.738 ng/ml. There was a significant correlation between age and basal levels of NE (L.R. = 0.33, p < 0.01). In 44 subjects who stood for 5 minutes after the basal sample of blood was obtained, the mean plasma level of NE increased to 0.538 ± 0.044 ng/ml and further increased to 0.778 ± 0.080 ng/ml after a subsequent isometric hand grip for 5 minutes.     

Developmental patterns of plasma dehydroepiandrosterone sulfate and testosterone in male pigs

The relationship between plasma levels of dehydroepiandrosterone sulfate (DHAS) and testosterone (T) was determined by radioimmunoassays in growing and adult pigs. Seven young males were bled at 2-weekly intervals between 1 and 47 weeks of age and two adult boars were cannulated for short-term studies. Plasma samples were extracted with methylene chloride and T was isolated by Celite chromatography. DHAS was assayed directly in the aqueous phase.Dehydroepiandrosterone occurred predominantly (89.7 ± 10.6%) as the sulfoconjugate in boar plasma (n = 50). Plasma DHAS was undetectable in castrated males (n = 2). At 1 week of age, mean levels (± S.D.) of DHAS and T were 5.0 ± 3.0 ng/ml and 0.15 ± 0.10 ng/ml, respectively; and they rose to small peaks of 16.0 ± 2.0 ng/ml and 0.63 ± 0.10 ng/ml at 3 weeks. At 7 weeks, the levels of DHAS and T increased gradually from 10.0 ± 6.7 and 0.11 ± 0.10 ng/ml to 27.0 ± 6.6 and 1.84 ± 0.61 ng/ml at 19 weeks. There followed a marked increase to 4.90 ± 3.30 ng/ml at 21 weeks for T and a less abrupt rise to 44.0 ± 9.3 ng/ml at 23 weeks for DHAS. The mean levels remained high from then onwards, fluctuating between 24.0 ± 8.7 and 54.5 ± 5.0 ng/ml for DHAS and between 1.73 ± 0.86 and 4.43 ± 1.26 ng/ml for T. Episodic fluctuations were noted in two boars during hourly collection for 24 h, with mean levels of 9.0 ± 4.9 and 50.0 ± 10.4 ng/ml for DHAS, and 1.76 ± 0.83 and 3.26 ± 0.63 ng/ml for T, respectively.For all ages of males, plasma DHAS and T levels were highly correlated (r = 0.95) with greater concentrations of DHAS in all samples. Although individual differences in steroid profiles were noted, concentrations for DHAS and T showed almost parallel increases at puberty and corresponding fluctuations in adult boars. It is suggested that plasma DHAS determinations provide a simple, sensitive assessment of androgen production in the male pig.     

A Substrate for Direct Measurement of L-iduronic Acid 2-sulfate sulfatase

Commercially available sodium heparinate has been sequentially treated with methanolic 0.06M hydrogen chloride and nitrous acid. The nondegraded material was separated by gel filtration from the nonsulfated and monosulfated disaccharides produced. The latter ones, obtained in 10% yield, have been used as a substrate for the direct measurement of the enzyme L-iduronic acid 2-sulfate sulfatase present in human plasma and fibroblast homogenates. Studies of the kinetics and pH optimum of the enzyme, by use of plasma of a patient with mucolipidosis II, indicated an apparent K_m of 2.5mM and a pH optimum of 4.6-4.8. The levels of activity in normal plasma and plasma of a patient with Hunter's disease were found to be 20.4 ± 1.22 units (μmol sulfate/24 h/g protein) and 3.25 ± 0.35 units, respectively. In homogenates of cultured skin fibroblasts, the levels were 137.6 ± 10.7 units for normal controls and 6.4 ± 5.1 for patients with Hunter's disease. The plasma of two obligated heterozygotes gave intermediate levels of activity, whereas the plasma of two possible heterozygotes gave either intermediate levels or entirely normal levels of activity.     

Serum luteinizing hormone levels in cattle under various reproductive states

Serum lutinizing hormone (LH) levels in cattle during various reproductive states were measured by radioimmunoassay. A sharp LH peak observed at estrus (22.72 ± 5.68 ng/ml) was about 26 times higher than at other stages of the cycle (0.87 ± 0.06 ng/ml). LH levels during the first 90 days of pregnancy (0.75 ± 0.15 ng/ml) were similar to those of the estrous cycle, except during estrus, while those during the second (0.22 ± 0.07 ng/ml) and third trimesters of pregnancy (0.22 ± 0.08 ng/ml) were significantly lower. Higher levels than those of the cycling cows, except during estrus, were seen in ovariectomized cows (2.21 ± 0.56 ng/ml). Levels of LH in cows with cystic follicles (2.00 ± 0.49 ng/ml) were higher than the levels in the cycle. LH levels in bulls (1.29 ± 0.39 ng/ml) were comparable to that of estrous cows. Serum LH of calves increased with age from 1.00 ± 0.32 ng/ml (less than 30 days of age), to 2.30 ± 0.83 ng/ml (181 to 210 days of age), and the level after 151 days was significantly higher than that of the cyclic cows, except during estrus.     

Low frequency of the deltaAF508 mutation of the CFTR gene in a highly admixed population in Bahia, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease in the European (Caucasian) population, with an incidence of 1:2000 to 1:8000. The deltaF508 mutation (66%) is predominant among more than 1300 different mutations of the CFTR gene. The population of the state of Bahia, in northeastern Brazil, is highly admixed (mainly African and Portuguese descendants), and so far, no study has been carried out to assess the molecular basis of CF in this population. We determined the deltaF508 mutation frequency in 503 individuals from the general population of Salvador, the capital of the state of Bahia, and in 144 CF patients from several cities in Bahia. In the general population samples we found 4 individuals heterozygous for the deltaF508 mutation (allele frequency of 0.4%). This frequency was lower than that found in the state of Rio de Janeiro, in southeastern Brazil, and similar to that reported for the state of Paraná, in the far south. In the CF patients we found 9 heterozygous individuals and 8 homozygous individuals (allele frequency of 8.68%) for the deltaF508 mutation. This frequency is considerably lower than the average frequency of CF in the world population and in the Brazilian CF population of European ancestry (47%). These data could be explained by the intense admixture among the population in Bahia, and they suggest a heterogeneous molecular basis for CF in this area of Brazil.     

Diurnal variations of plasma testosterone in men

Plasma testosterone (T) levels were assayed by a Competitive Protein Binding (CPB) technique in a group of 31 healthy males. In 22 subjects a single blood sample was taken between 8:00 and 9:00 A.M. and the mean T concentration was 6.84 ± 2.11 ng/ml. In the other 9 normal men, blood samples were taken every 4 hours. The existence of temporal variations for testosterone was confirmed by finding the highest mean plasma levels at 4:00 A.M. (9.28 ± 1.17 ng/ml) and lowest mean levels at 8:00 P.M. (2.66 ± 0.52 ng/ml).     

Passage of certain penicillins into the uterine tubal fluid of estrous and pregnant rabbits

Four penicillins, procaine penicillin G (PPG), potassium penicillin G (KPG), sodium ampicillin (SA), and benzathine penicillin G (BPG) were administered intramuscularly at the rate of 300,000 units per rabbit. Estrous uterine tubal fluids were collected from rabbits during estrous and the concentrations of PPG, KPG, SA and BPG found in estrous rabbit uterine tubal fluid following two, 300,000 units intramuscular injections at 24 hour intervals were 0.20± .0017 U/ml and 0.44± .010 U/ml for PPG; 0.038± 0.006 and 0.033± 0.004 for KPG and 0.019± 0.004 and 0.18± 0.001 for SA and from 0.027± 0.002 to a low of 0.002± 0.002 U/ml after 96 hours post injection for BPG. Probenecid reduced the concentration of PPG and SA in uterine tubal fluid 50% when compared with nonprobenecid treated rabbits. A close correlation between the plasma and uterine tubal fluid concentrations of PPG, SA, and KPG was also found. The concentrations of PPG, KPG, and SA were reduced by about 50% during day 2, 3 and 4 of pregnancy.     

Simultaneous radioimmunoassay of testosterone and dihydrotestosterone

A radioimmunoassay, which simultaneously measures both testosterone (T) and dihydrotestosterone (DHT) in the same serum sample, is presented. Celite column chromatography is employed to separate T from DHT, and these two steroids from other potentially cross-reacting and interfering steroids. The normal values for men, women in the follicular phase, women in the luteal phase, ovariectomized and adrenal ectomized women, post-menopausal women and ovariectomized women for T are 5, 140 ± 1190 pg/ml, 307 ± 97 pg/ml, 285 ± 46 pg/ml, undetectable (<5 pg/ml), 262 ±47 pg/ml and 199 ±44 pg/ml; and for DHT 470 ± 165 pg/ml, 160 ±45 pg/ml, 147 ±44 pg/ml, undetectable (<5 pg/ml), 168 ± 27 pg/ml, 94 ± 15 pg/ml. The maximum sensitivity of the method was 10 pg/ml for T and 14.3 pg/ml for DHT when 1 ml was extracted. The blank in most assays was undetectable, but rarely exceeded 10 pg.