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Summary. Background: Microparticle size measurements are often calibrated on flow cytometers using polystyrene microspheres that forward scatter more light vs. particle diameter than cellular microparticles. Methods: We compared theoretical with measured forward angle light scattering on the LSRII, FC500 and Apogee A40 using polystyrene and silica microspheres vs. synthetic lipid vesicles and platelets, then compared plasma microparticle counts using different calibration strategies. Results: Polystyrene and silica microspheres with higher refractive indices forward scattered more light with a wavelength of 488 nm for a given size microparticle than did lipid vesicles or platelets. The LSRII and FC500 did not count many, and were unable to separate by size, polystyrene microspheres

1. Investigations of the role of cAMP in the stimulation of the steroidogenesis of zona glomerulosa (ZG) cells by increased extracellular K+ concentration are reviewed. 2. Possible reasons for discrepancies in the results of different investigators on whether K+ increases the cAMP content or output of ZG tissue or dispersed cells are discussed. 3. The concentration of cAMP in the incubating media of ZG tissue or cells, rather than their cAMP content, seems to respond more sensitively to stimulation by extracellular K+, as was also found for adrenocorticotropic hormone stimulation of zona fasciculata-reticularis cells. 4. The addition of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (IBMX) to incubations with the aim of increasing the sensitivity of the response in cAMP to extracellular K+ in ZG cells may give rise to effects, probably nonspecific, which actually inhibit the measured response. 5. The immediate stimulation in the steroidogenesis of ZG cells with raised extracellular K+ is probably mostly due to the direct effect of increases in cytoplasmic Ca2+ (arising from increases in Ca2+ influx) on mitochondrial processes. However, increases in cAMP may prolong the stimulation of steroidogenesis by increased extracellular K+. This increased cAMP is probably due to stimulation of adenylyl cyclase activity. 6. It has been concluded that the increase in Ca2+ influx output after rises in the extracellular K+ concentration of ZG cells is responsible for most of the increase in cAMP. 7. According to one group of investigators, there is weak stimulation of phospholipase C (PLC) activity after increasing the extracellular K+ concentration of rat ZG cells. 8. If there is such a stimulation of PLC activity, it seems that the action of increased extracellular K+ can potentially involve all known mechanisms for the stimulation of steroidogenesis in endocrine cells. The common primary event is probably the increase in Ca2+ influx. The relative importance of these various potential mechanisms may depend on the particular in vitro conditions used.

Increased exposure of phosphatidylserine (PS) in erythrocytes has been postulated to contribute to the pathophysiology of sickle cell disease because of its possible effects on blood coagulation, cell adhesion, and cell clearance. We developed a flow-cytometric assay to measure exposure of PS on the outer face of the erythrocyte membrane based on addition of fluorescein-annexin V to whole-blood specimens. The assay correlated linearly with binding of 125I-annexin V (r2 = .95, n = 125 samples). Normal donors (n = 30) showed virtually no annexin-positive cells (0.34% +/- 0.18% for 24-hour old samples). In contrast, annexin V binding was above the upper limit of normal in 96% of 205 specimens from 17 adult sickle cell and 2 beta-thalassemia patients; the mean percentage of annexin-positive cells was 2.86% +/- 2.00% (range, 0.4% to 12.0%). Values varied substantially over time for some patients, and mean values varied between patients. The percentage of annexin-positive cells always decreased after transfusion (11 events in 6 patients), and out of proportion to the amount of blood transfused. In conclusion, increased exposure of PS on a subpopulation of erythrocytes in vivo is a virtually universal feature of sickle cell disease, and its measurement may be useful to evaluate clinical status and response to therapeutic measures such as blood transfusion.

Earlier views indicated that globulin (corticosteroid-binding globulin (CBG) or sex hormone-binding globulin (SBG)) but not albumin binding in plasma, protects steroids from splanchnic metabolism in man. Also, the splanchnic extraction (HE) of a steroid seemed to be highly dependent on the rate of disassociation of the steroid–protein complex. However, the faster rate of disassociation (τ½ = 0·9 s) of cortisol–CBG, as determined by later accurate fluorescence methods, intuitively meant that this complex must disassociate completely in a single 9 s passage through the liver. The low HE of total cortisol was then a puzzling anomaly. Using a differential equation solver (TUTSIM) and a model with unbound, albumin- and globulin-bound pools of steroid (with metabolism of unbound and also possibly albumin-bound steroid), the mechanism of splanchnic metabolism has been studied. The 'complex', probably most realistic, model includes 13 steroids, which can simultaneously bind to plasma albumin, CBG and SBG. The steroid concentration and numbers of occupied binding sites of the globulins decrease during the time of metabolism. The experimental data used are the in-vitro binding characteristics of the steroid–protein complexes, including the equilibrium constants and rates of disassociation and the in-vivo HE of nine steroids, usually measured by direct analysis of hepatic venous blood. However, the HE of cortisol had to be calculated from the metabolic clearance rate/splanchnic blood flow, giving a maximum value of 12%. The fractional metabolic rate of unbound steroid is generally represented by e. A certain value of e (RE) is required to give a remaining steroid concentration after 9 s of metabolism, which is made equal to (1–HE) in the model to simulate splanchnic extraction. If the fractional rate of metabolism of albumin-bound steroid is h (f = h/e), then RE will depend on the value of f. The maximum RE for cortisol is RE0 = 0·42 and RE1 = 0·16 for f = 0 and 1 respectively. For either value of RE, there will be the appreciable reassociation of cortisol to CBG after disassociation of the cortisol–CBG complex. With such reassociation, the total cortisol remaining after 9 s metabolism is fairly independent of the rate of disassociation of the cortisol–CBG complex. This explains the low total HE of cortisol in spite of the high rate of disassociation of cortisol–CBG. Generally, for all nine steroids studied, HE estimations in vivo in humans indicate that the steroid–globulin disassociation rate will only be markedly rate-limiting for dihydrotestosterone–SBG and, to a lesser extent, testosterone–SBG. These results are examples of the principle that the divergence in hormone concentration with the disassociation rate of the hormone-protein complex after metabolism depends on the value of e. The RE0 and RE1 values of the nine steroids: aldosterone, progesterone, testosterone, dihydrotestosterone, androstenedione, androstanediol, oestradiol, oestrone and cortisol have also been calculated from all in-vivo HE estimates. If it is assumed that the steroid in the albumin-bound pool is not metabolized (f = 0), then the RE0 values of the nine steroids considered have a large variation (s.d./mean = 115%). There is a correlation between the RE0 values and their albumin-binding index (BIA). However, if f = 1 then the variation of RE is smaller (s.d./mean = 36%) and there is no correlation between RE1 and BIA. The ratio of the RE0 and RE1 values for a particular steroid is nearly equal to (1 + BIA), which is determined solely by the dynamics of the situation. However, the appropriate value of f depends on the mechanisms involved. With the F1 hypothesis (f = 1), albumin binding does not protect steroids from splanchnic extraction because of metabolism of albumin-bound steroid. With the FO hypothesis (f = 0), this seeming lack of protection of albumin binding is essentially artefactual. Albumin-bound steroid would not be metabolized directly but the fractional rate of metabolism of unbound steroid would be increased with the decreasing polarity of the steroid (i.e. with increasing BIA). With the available data, the choice between the hypotheses can only be on an intuitive basis. Journal of Endocrinology (1991) 131, 339–357

The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells.Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant.Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts.These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.

Intramedullary apoptosis of hematopoietic tissue is believed to play a major role in the pathophysiology of myelodysplastic syndrome. Annexin V, a specific marker of the early to intermediate phases of apoptosis, has been applied to the in vitro study of bone marrow aspirates. A noninvasive measure of intramedullary apoptosis in vivo that could serially monitor the clinical progression of myelodysplastic syndrome may be helpful.We used 99mTc-radiolabeled annexin V and radionuclide gamma camera imaging to serially study the sites, extent, and severity of intramedullary apoptosis induced by cyclophosphamide treatment.Intravenously administered radiolabeled annexin V localized preferentially in the femur, pelvis, vertebrae, and spleen; increased uptake in these organs was easily visualized as early as 8 h after injection of 100 mg/kg cyclophosphamide in 8- to 10-wk-old animals. Higher doses of cyclophosphamide (150 mg/kg) in animals of the same age increased annexin V uptake in the bone marrow and splenic tissue and delayed recovery of these organs as seen histologically compared with lower doses. Older animals, 5-6 mo old, showed a slower response to cyclophosphamide treatment and delayed recovery of bone marrow and splenic tissues.Radiolabeled annexin V can be used to detect and directly quantify the degree of intramedullary and splenic apoptosis in a noninvasive fashion using current clinical radionuclide imaging equipment. Annexin V imaging may be useful clinically in the diagnosis and management of myelodysplastic syndrome.

A 13-year-old girl presented with a two year history of declining school performance, loss of coordination, and increased difficulty with sports. The family history was positive for Huntington's disease (HD). An MRI was suggestive for bilateral atrophy of the caudate. HD is autosomal dominant and the HD gene contains a polymorphic trinucleotide (CAG) repeat, which is expanded beyond 36 CAG repeats in HD. This patient had one normal-sized allele and one abnormally expanded allele with 68 CAG repeats, confirming the clinical diagnosis of HD. Juvenile onset of HD is uncommon, and is associated with unusually large CAG repeat numbers as was observed in this patient.

Whereas unperturbed endothelial cells provide potent anticoagulant properties, exposure to inflammatory and atherogenic stimuli can rapidly lead to a procoagulant behavior. Because recent studies provide evidence that apoptosis of vascular cells may occur under conditions such as atherosclerosis and inflammation, we investigated whether apoptotic endothelial cells may contribute to the development of a prothrombotic state. In this report, it is shown that both adherent and detached apoptotic human umbilical vein endothelial cells (HUVECs) become procoagulant. Apoptosis was induced by staurosporine, a nonspecific protein kinase inhibitor, or by culture in suspension with serum deprivation. Both methods resulted in similar findings. As assessed by flow cytometric determination of annexin V binding, HUVECs undergoing cell death exhibited typically a more rapid exposure of membrane phosphatidylserine (PS) than DNA fragmentation. Depending on the stage of apoptosis, this redistribution of phospholipids was found to induce an increase of the activity of the intrinsic tenase complex by 25% to 60%. Although apoptotic cells did not show antigenic or functional tissue factor (TF) activity, when preactivated with lipopolysaccharide, TF procoagulant activity increased by 50% to 70%. At 8 hours after apoptosis induction, antigenic thrombomodulin, heparan sulfates, and TF pathway inhibitor decreased by about 83%, 80%, and 59%, respectively. The functional activity of these components was reduced by about 36%, 52%, and 39%, respectively. Moreover, the presence of apoptotic HUVECs led to a significant increase of thrombin formation in recalcified citrated plasma. In conclusion, apoptotic HUVECs, either adherent or in suspension, become procoagulant by increased expression of PS and the loss of anticoagulant membrane components.

Annexin V is a human protein that binds with high affinity to the abundant phosphatidylserine molecules exposed on activated platelets and accumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis. We designed two chimeras that use annexin V as a means to target thrombolytic agents to platelet-containing thrombi: prourokinase (1-411)-annexin V (1-320); and prourokinase (144-411)-annexin V (1-320) (amino acid numbers of parent proteins given in parentheses). Chimeras were produced by cytoplasmic expression in Escherichia coli, refolded, and purified in single-chain form. Both chimeras had the same specific activity as annexin V in binding to cell membranes containing exposed phosphatidylserine. After activation with plasmin, both chimeras had specific amidolytic activity similar to that of urokinase. Both chimeras activated plasminogen in vitro with kinetic parameters similar to those for urokinase, and both showed full activity compared to urokinase in an assay of clot lysis in vitro. This study shows the feasibility of producing chimeric plasminogen activators in which annexin V provides the thrombus-targeting component; although not yet tested in vivo, such chimeras may have advantages over antibody-based targeting agents.

We investigated the use of annexin V (placental anticoagulant protein I), a calcium-dependent protein that binds tightly to phosphatidylserine-containing membranes, as a means to measure membrane phospholipid asymmetry in human erythrocytes. Iodine 125-labeled annexin V bound to erythrocytes in a specific, reversible, calcium-dependent reaction (dissociation constant = 25 +/- 4 nmol/L at 37 degrees C and 2.5 mmol/L calcium). Lysed erythrocytes contained 1.2 x 10(6) binding sites for annexin V. Treatment of erythrocytes with 1 mumol/L A23187 in the presence of 2.5 mmol/L calcium at 37 degrees C caused a gradual but marked increase in annexin V binding sites, reaching a level of approximately 300,000 sites per cell after 8 hours of incubation. We also noted a very gradual spontaneous exposure of annexin V binding sites during storage of purified erythrocytes, reaching a level of approximately 20,000 sites per cell after 30 days. Measurements could also be made directly on diluted whole blood specimens. In samples freshly drawn from 35 normal donors, a mean number of 276 sites per cell were present; this increased to 858 sites per cell after storage of specimens at 4 degrees C for 24 hours. Measurement of annexin V binding to samples from patients with sickle-cell anemia revealed a marked increase in binding (mean of 12,430 sites per cell for all samples); serial measurements in a patient hospitalized with sickle-cell crisis showed a progressive decline in annexin V binding over a period of 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)

1) To examine a fluorescence polarization (FP) assay with an independent set of data that contained more cases of respiratory distress syndrome (RDS) than a previous study, 2) to determine whether the same reference ranges are applicable to infants born to diabetic women, and 3) to evaluate whether adding the lecithin-sphingomyelin ratio (L/S) would substantially improve the prediction of RDS among women with an intermediate FP value (between 0.26-0.289).We identified 389 women who had FP analysis performed at the University of Washington Medical Center from February 1986 to October 1988 and who delivered within 3 days of amniocentesis. We reviewed the medical records of these women and their infants to extract information for our study.For FP values of 0.26 or greater, the sensitivity and specificity for prediction of RDS were 90.2 and 84.6%, respectively, compared with 100 and 82.0% in the previous study. For FP values of 0.29 or greater, the sensitivity and specificity were 62.8 and 94.2%, respectively (80.8 and 96.2% in the previous study). Among diabetics, an FP result below 0.26 was associated with the same low risk of RDS as among non-diabetics. Among the patients with FP between 0.26-0.289, the addition of L/S did not provide a clinically useful improvement in the prediction of fetal lung maturity.The NBD-PC FP assay can be used as the sole test of fetal lung maturity in most clinical circumstances.

We evaluated potential screening protocols for alpha-thalassemia in a group of 80 patients whose genotypes were determined by Southern blot analysis with alpha- and zeta-globin DNA probes. Erythrocyte inclusion bodies were measured by a modified brilliant cresyl blue test. Erythrocyte indices and iron status were also measured. The brilliant cresyl blue test reliably detects couples at risk for hemoglobin Bart's hydrops fetalis. Measurement of the number of inclusion bodies differentiates the alpha-thalassemia genotypes in the absence of a coincident beta-chain synthesis deficiency, such as hemoglobin E or beta-thalassemia. The test appears to identify patients, such as those with the Thai and Filipino deletion variants, whose alpha-thalassemia cannot be definitively characterized by DNA testing when only alpha- and zeta-globin probes are used in the analysis. We also found evidence of elevated serum ferritin levels in many patients with deletion of two or three alpha-globin genes. This study shows that most routine screening for alpha-thalassemia can be performed with three simple tests: (1) the brilliant cresyl blue inclusion study, (2) erythrocyte indices, and (3) iron studies. Analysis with DNA probes is needed in only some circumstances.

We have shown recently that the calcium-dependent phospholipid-binding protein annexin V (placental anticoagulant protein I) can be used to study the exposure of anionic phospholipid after platelet activation. In this study we have further examined the mechanism of this process. Collagen-induced exposure of annexin V binding sites correlated directly with increased ability to support activity of the reconstituted prothrombinase complex. The potency of annexin V as an inhibitor of platelet prothrombinase was the same as its Kd for platelets. Prior incubation of platelets with 5'-p-fluorosulfonylbenzoyladenosine or p-chloromercuribenzenesulfonate had no significant effect on annexin V binding. Similarly, inhibition of platelet cyclic endoperoxide synthesis by acetylsalicylic acid or indomethacin did not inhibit annexin V binding. Staurosporine inhibited collagen-induced, but not A23187-induced, annexin V binding. Agents that increase intraplatelet cyclic nucleotides partially inhibited collagen-induced annexin V binding. Thus, collagen-induced exposure of anionic phospholipid appears to depend primarily on increases in intraplatelet free calcium and may be independent of ADP- or endoperoxide-mediated pathways. Binding sites for annexin V on microparticles derived from collagen-stimulated platelets were demonstrated by flow cytometry and gel filtration. In addition, prior incubation of platelets with 100 nM annexin V inhibited factor Va binding to both platelets and platelet-derived microparticles. These results support the concept that the procoagulant effect of platelets and platelet-derived microparticles is mediated by calcium-induced exposure of anionic phospholipids.

Although DNA analysis based on the polymerase chain reaction (PCR) offers potential advantages for screening newborns for sickle cell disease, few data are available concerning the reliability of PCR-based tests for such screening. We describe a protocol for detecting the A, S, and C alleles of the beta-globin gene in dried blood from phenylketonuria screening cards. This method is based on PCR and detection with allele-specific oligonucleotide probes. Results of a blind comparison of PCR analysis of the dried blood with hemoglobin electrophoresis of whole-blood samples agreed for 80 of 81 samples. The single discrepancy is probably not attributable to a failure of the PCR method, but rather to limitations of the electrophoresis method. The PCR method should be a highly accurate means of detecting beta-globin alleles in routine genetic screening with dried blood already collected for (e.g.) phenylketonuria screening.

The recent discovery of the cystic fibrosis gene enables DNA-based testing for the direct identification of the deletion of three basepairs coding for phenyalanine at codon 508, the major mutation responsible for the disease. This mutation can be detected by analysis of amplified DNA with allele-specific oligonucleotide probes. We have simplified the procedure of Kerem et al. (Science 1989;245:1073-80) so that the assay can be routinely completed in one working day, starting with an extracted DNA sample. Addition of salmon-sperm DNA to the product of the polymerase chain reaction greatly improved the quality of the hybridization signal. The precision of the method was evaluated by blind analysis and interpretation of results for 100 specimens from 25 patients. The same result was obtained for each patient analyzed separately four times, and four independent observers agreed on the interpretation of results for all 100 specimens. No specimen required repeat analysis to produce interpretable results. We conclude that this method is reliable and convenient for routine clinical laboratory use.

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.

Placental anticoagulant protein-I (PAP-I), a member of the lipocortin protein family, is a potent in vitro anticoagulant whose in vivo function is unknown. Very low levels of PAP-I were present in plasma of normal volunteers (0 to 5 ng/ml) and in randomly chosen plasma specimens from hospitalized patients (0 to 28 ng/ml). Review of selected hospital records did not reveal any single clinical entity that correlated with plasma levels. PAP-I was also found in amniotic fluid (12 to 107 ng/ml) and in conditioned medium of cultured endothelial cells (49 +/- 20 ng/ml). Gel filtration experiments showed that PAP-I was intact and uncomplexed in plasma and amniotic fluid. The protein was fairly abundant intracellularly: 4080 +/- 2560 ng/mg total protein in cultured umbilical vein endothelial cells; 178 +/- 109 ng/mg in platelets; 564 +/- 384 ng/mg in leukocytes; and 8.4 +/- 4.3 ng/mg in erythrocytes. The levels of PAP-I increased in platelet-rich plasma after stimulation of platelets with arachidonic acid but not after stimulation with ADP, epinephrine, thrombin, ristocetin, or collagen. These data suggest that PAP-I probably does not function as a circulating natural anticoagulant in normal persons.

We recently identified residues 185-224 of the light chain of human high molecular weight kininogen (HMWK) as the binding site for plasma prekallikrein (Tait, J.F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). In the present study, we have further defined the primary structure requirements for binding of HMWK to factor XI and prekallikrein. In a competitive fluorescence polarization binding assay, a 31-residue synthetic peptide (residues 194-224 of the HMWK light chain) bound to prekallikrein with a Kd of 20 +/- 6 nM, indistinguishable from the previously determined value of 18 +/- 5 nM for the light chain. We also prepared three shorter synthetic peptides corresponding to different portions of the 31-residue peptide (residues 205-224, 212-224, and 194-211), but these peptides bound to prekallikrein more than 100-fold more weakly. Factor XI also bound to the same region of the HMWK light chain, but at least 58 residues (185-242) were required for optimal binding (Kd = 69 +/- 4 nM for the light chain; Kd = 130 +/- 50 nM for residues 185-242). The four synthetic peptides inhibited kaolin-activated clotting of blood plasma with potencies paralleling their affinities for prekallikrein and factor XI. Peptide 194-224 can also be used for rapid affinity purification of prekallikrein and factor XI from plasma.

We clinically evaluated, retrospectively, our improved fluorescence polarization assay for fetal lung maturity. The procedure requires 0.5 mL of amniotic fluid and a standard clinical laboratory fluorescence polarimeter (TDx Analyzer, Abbott Laboratories). We measured the L/S ratios for 93 freshly collected amniotic fluids, uncontaminated with blood or meconium, collected within three days of delivery. The fluids were stored frozen for eight to 32 months, then thawed and assayed for net fluorescence polarization. Fourteen of the infants developed respiratory distress syndrome; five, transient tachypnea of the newborn; and 74, no respiratory distress. The polarization assay and lecithin/sphingomyelin ratio had equivalent receiver operating characteristic curves, indicating no difference in their clinical performance. Although a prospective study with fresh amniotic fluid specimens will be necessary to establish a definitive reference range, the present study shows that this assay can be used to rapidly predict fetal lung maturity.

We describe a fluorescence polarization assay for use in predicting fetal lung maturity, which is suitable for the TDx Analyzer (an automated fluorescence polarimeter). The assay requires 0.5 mL of amniotic fluid and approximately 30 min, and involves the fluorophore 1-palmitoyl-2(6-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl)amino]caproyl) phosphatidylcholine. Reproducible polarization measurements depend on proper regulation of incubation time and temperature, but variations in the concentrations of fluorescent probe and amniotic fluid have little effect on measured polarization and therefore little effect on assay precision. Working solutions of the fluorescent probe are stable for at least nine months when stored at -20 degrees C and pH 5. Interferences include erythrocytes, serum, bilirubin, meconium, and lidocaine.

We studied the ability of fragments of the light chain of human high molecular weight kininogen to bind to plasma prekallikrein. In a competitive fluorescence polarization assay, kallikrein-cleaved light chain (light chain-2; residues 49-255), a cyanogen bromide fragment (residues 185-242), and a tryptic peptide (T-7; residues 185-224) had binding affinities of approximately 20 nM, equivalent to the value for the intact light chain (residues 1-255) of high-molecular-weight kininogen. In contrast, fragments consisting of residues 49-184 and 243-255 showed no binding activity (Kd much greater than 1,000 nM). Direct titrations of fluorescein-labeled derivatives of light chain-2 and peptide T-7 with prekallikrein confirmed that T-7 retained full binding activity for prekallikrein (Kd = 12 +/- 2 nM for labeled light chain-2; Kd = 7 +/- 1 nM for labeled T-7). These results localize the binding site of high molecular weight kininogen for prekallikrein within a region of 40 amino acids (residues 185-224) that resides in the near carboxyl terminus of the light chain of kininogen.

We assessed the analytical performance of an improved fluorescence polarization assay for use in evaluating fetal lung maturity and compared results with the lecithin/sphingomyelin ratio. During a three-month period 150 patients' samples were assayed by clinical laboratory personnel with both techniques. Values for the lecithin/sphingomyelin ratio correlate closely with net fluorescence polarization values (r = -0.85), less closely with net fluorescence intensity (r = 0.65). Background fluorescence intensity and polarization varied widely, indicating a need to correct measurements for endogenous fluorescence. Net fluorescence polarization values have a CV of 0.32% within-run, 1.07% between-day. A comparison of two amniotic fluid centrifugation procedures showed no significant difference in such values. For both methods, however, such values are slightly but significantly higher than those obtained for amniotic fluids without prior centrifugation. Short-term storage (less than 30 days) of uncentrifuged amniotic fluid samples at -20 degrees C does not significantly affect results.

Abbau von Aldosteron (C21H28O5) mit NaJO4 oder CrO3 gab neben Formaldehyd ein Lacton (III) (Nr. 874), das nach Hydrierung und Erhitzen mit Hydrazin und Na-Athylat ein Gemisch von neutralen und sauren Stoffen lieferte. Aus den sauren Anteilen liessen sich 3 β, 11 β-Androstan-18-saure-17-carbonsaure-lacton-(1811) (X) sowie Derivate der 3 β, 11 β-Dioxy-5 α-atiansaure- (XV) in reiner Form isolieren. Das Lacton X wurde durch seinen krist. Methylester Nr. 876 (XI), sein Acetylderivat Nr. 882 (XII) und den aus Nr. 876 (XI) erhaltlichen 3-Keto-methylester Nr. 877 (XIX) charakterisiert. XIX konnte auch aus 21-Monoacetyl-aldosteron nach Dehydrierung mit CrO3, milder Verseifung, Abbau mit NaJO4, Methylierung, Hydrierung mit Pt und Dehydrierung mit CrO3 gewonnen werden. Die IR.-Spektren der genannten Stoffe werden wiedergegeben. Fur Aldosteron ergab sich die Formel eines 18-Oxo-corticosterons Ib; in Losung durfte es vorwiegend in der Cyclohalbacetalform Ia vorliegen.

The peripheral conversion of dehydroisoandrosterone (D) to plasma androstenedione (A) and testosterone (T) was studied by constant infusion of labeled D and isolation of both precursor and product steroid from plasma. The metabolic clearance rate (MCRd) of D in 8 normal subjects was 1640 ± 93 (se) liters/day. The transfer constant , the proportion of D converted to A as measured in plasma, was 6.2 %. The transfer constant , the proportion of D converted to T in plasma, was only 0.7%. The product of the MCRd and available values for plasma D concentration indicates a plasma production rate of 7 mg/day. From the production rate and transfer constants it can be calculated that D is a minimal peripheral precursor of both plasma A and T in the normal adult female. In abnormal states of virilization with normal or moderate increases in D production, the increased plasma A and T in these states are not due to peripheral conversion of D in blood. The experimentally determined value of 0.7 % is in reasona...

Adrenal cells have been prepared, using crude collagenase, from the separated capsular and decapsulated portions of adrenals from intact rats. Microscopic examination and consideration of steroid outputs suggest that preparations from decapsulated glands consist of zona fasciculata-reticularis cells, while those from capsular glands consist mainly of zona glomerulosa cells with some zona fasciculata contamination. The yield of cells from the 2 preparations was of the order of 2 ×105 cells/adrenal for decapsulated glands and 1 ×lO5/adrenal for capsular glands. The endogenous output of 18-OH B, aldosterone, 18-OH DOC and corticosterone from the incubations of capsular and decapsulated gland cells has been examined. Three of the 4 steroids are produced by both types of cells; aldosterone production is virtually confined to capsular gland cells. The effect of ACTH, angiotensins, potassium and serotonin added in vitro on the output of corticosterone by decapsulated gland cells and that of corticosterone and al...

Die Isolierung von «Subst. Z» aus Nebennierenextrakten wird beschrieben. Der Stoff erwies sich als identisch mit 4,ω-Dihydroxy-3-methoxy-acetophenon, das zum Vergleich durch Synthese in reiner krist. Form bereitet wurde. Auch die Synthese des stellungsisomeren 3, ω-Dihydroxy-4-methoxy-acetophenons («Subst. Iso-Z») wird beschrieben.

Since the end of 1947 a series of 18 cases of thyroid carcinoma have been investigated at the hospital using tracer doses of radio-iodine I131, and of these one was found to concentrate I131 in metastatic deposits that were known to be present. The percentage uptake was such that an attempt to carry out treatment using larger amounts of the isotope was made. This paper is a clinical report upon this case, and whilst a cure is not claimed, it illustrates some of the problems which may arise in this new method of treatment, together with some conclusions which may be drawn from a study of the physical data. After our preliminary tracer studies it was decided that thyroidectomy would be desirable before giving doses of I131 sufficient to treat the metastases. Our reasons for this were threefold. Firstly, it was feared that a high percentage concentration of a large dose of the isotope in the thyroid might lead to a thyrotoxic crisis, as has been reported by Trunell et al (1947). Secondly, irradiation effects...

Aldosterone metabolism has been shown to be altered in pregnancy. The increased conversion of intravenously administered aldosterone-(3)H to the acid-labile conjugate in the urine (aldosterone 18-glucuronide) has again been observed. The urinary yield of intravenously administered aldosterone-(3)H as aldosterone 18-glucuronide in 16 pregnant subjects of 13.4+/-0.9 (SE)% was significantly higher (P 0.001) than in 11 nonpregnant subjects (seven males and four females) of 7.3+/-0.5 (SE)%. After combining oral ((14)C) and intravenous ((3)H) administration of aldosterone, the (14)C/(3)H ratios of urinary metabolites (free aldosterone, tetrahydroaldosterone glucuronide, and aldosterone 18-glucuronide) were measured and were expressed as a per cent of administered dose. From these data the splanchnic extraction of aldosterone was calculated. The splanchnic extraction was significantly lower in pregnant as compared to nonpregnant subjects, although previous work indicated no change in protein binding of aldosterone in pregnancy.However, the data on the (14)C/(3)H ratio of other metabolites suggested that a large part of the increased aldosterone 18-glucuronide metabolite in pregnancy is formed in the splanchnic circulation; also, there appeared to be increased tetrahydroaldosterone glucuronide formation extrasplanchnically.

Die Bestimmung von Aldosteron, seine Isolierung in Kristallen und seine Eigenschaften werden beschrieben.

A new crystalline compound has been isolated from beef adrenal extracts. In three different assay methods using epinephrectomised rats or dogs it was found to be from 30 to 100 times as potent as cortexone (11-deoxy-corticosterone) or its acetate.

A simple demountable counter is described suitable for the assay of tracers emitting low energy β-rays. Satisfactory characteristics have been obtained with a helium-ether mixture flowing through the counter at atmospheric pressure. Using this mixture, the counter has a plateau length of 400 V., a plateau slope of 0.01%/V. and a threshold potential of approximately 3250 V. A comparison is made with other methods which have been used for the assay of C14 and a table is included showing their relative sensitivity.

1. A method involving centrifugation through phthalate ester oils is described for the rapid separation of isolated cells from suspending medium. Intracellular potassium concentrations and intracellular water space values for isolated adrenal zona glomerulosa and zona fasciculata cells are given. 2. The effects of angiotensin II and a high external potassium concentration (8·4 mmol/l) on intracellular potassium contents, intracellular water spaces and hence intracellular potassium concentrations of purified glomerulosa cells have been investigated. Corticosterone secretion was also measured. Previously reported results showing a significant increase in potassium content with high external potassium were confirmed. However, angiotensin II slightly, but significantly, decreased potassium content. No significant change in intracellular water space or intracellular potassium concentration was observed with either stimulus. 3. Both stimuli significantly increased corticosterone production. There was no correlation between the percentage increase in potassium content or concentration and the percentage increase in steroid output after stimulation with high external potassium. 4. It is concluded that angiotensin II does not stimulate steroidogenesis in adrenal glomerulosa cells by raising the intracellular potassium concentration. The small, but statistically insignificant, increase caused by high-potassium medium is unlikely to be causally linked to steroidogenesis.

We describe a fluorescence polarization assay for use in predicting fetal lung maturity, which is suitable for the TDx Analyzer (an automated fluorescence polarimeter). The assay requires 0.5 mL of amniotic fluid and approximately 30 min, and involves the fluorophore 1-palmitoyl-2(6-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl)amino]caproyl) phosphatidylcholine. Reproducible polarization measurements depend on proper regulation of incubation time and temperature, but variations in the concentrations of fluorescent probe and amniotic fluid have little effect on measured polarization and therefore little effect on assay precision. Working solutions of the fluorescent probe are stable for at least nine months when stored at -20 degrees C and pH 5. Interferences include erythrocytes, serum, bilirubin, meconium, and lidocaine.