White matter protection with insulin-like growth factor 1 and hypothermia is not additive after severe reversible cerebral ischemia in term fetal sheep.

Moderate cerebral hypothermia significantly improves survival without disability from perinatal hypoxia-ischemia. However, protection is partial. Insulin-like growth factor 1 (IGF-1) plays a key role in oligodendrocyte survival and myelination. The purpose of this study was to test the hypothesis that the combination of IGF-1 plus hypothermia could reduce postischemic white matter damage compared with hypothermia alone. Unanesthetized near-term fetal sheep received 30 min of cerebral ischemia, followed by either an infusion of 3 µg of IGF-1 intracerebroventricularly from 4.5 to 5.5 h plus cooling from 5.5 to 72 h (IGF-1 + hypothermia; n = 8), vehicle infusion plus cooling from 5.5 to 72 h (vehicle + hypothermia; n = 12), sham cooling plus sham infusion (ischemia control; n = 12) or sham ischemia (n = 5). The fetal extradural temperature was reduced from 39.4 ± 0.1°C to between 30 and 33°C. White matter was assessed after 5 days. Ischemia was associated with severe loss of CNPase-positive oligodendrocytes in white matter compared with sham ischemia (380 ± 138 vs. 1,180 ± 152 cells/field; mean ± SD; p < 0.001). Delayed hypothermia reduced cell loss (847 ± 297 cells/field, p < 0.01, vs. ischemia control), but there was no significant difference between vehicle + hypothermia and IGF-1 + hypothermia (1,015 ± 211 cells/field; NS). Ischemia was associated with increased caspase 3 expression in white matter (216 ± 41 vs. 19 ± 18 cells/field; p < 0.001). Hypothermia reduced numbers of activated caspase 3-positive cells (116 ± 81 cells/field; p < 0.05), with no significant difference between vehicle + hypothermia and IGF-1 + hypothermia (91 ± 27 cells/field; NS). In conclusion, delayed cotreatment with IGF-1 plus hypothermia after ischemia was associated with an improvement in white matter damage similar to that achieved by hypothermia alone.

Phonons and oxygen diffusion in Bi2O3 and (Bi0.7Y0.3)2O3.

We report investigation of phonons and oxygen diffusion in Bi2O3 and (Bi0.7Y0.3)2O3. The phonon spectra have been measured in Bi2O3 at high temperatures up to 1083 K using inelastic neutron scattering. Ab initio calculations have been used to compute the individual contributions of the constituent atoms in Bi2O3 and (Bi0.7Y0.3)2O3 to the total phonon density of states. Our computed results indicate that as temperature is increased, there is a complete loss of sharp peak structure in the vibrational density of states. Ab initio molecular dynamics simulations show that even at 1000 K in δ-phase Bi2O3, Bi-Bi correlations remain ordered in the crystalline lattice while the correlations between O-O show liquid like disordered behavior. In the case of (Bi0.7Y0.3)2O3, the O-O correlations broadened at around 500 K indicating that oxygen conductivity is possible at such low temperatures in (Bi0.7Y0.3)2O3 although the conductivity is much less than that observed in the undoped high temperature δ-phase of Bi2O3. This result is consistent with the calculated diffusion coefficients of oxygen and observation by quasielastic neutron scattering experiments. Our ab initio molecular dynamics calculations predict that macroscopic diffusion is attainable in (Bi0.7Y0.3)2O3 at much lower temperatures, which is more suited for technological applications. Our studies elucidate the easy directions of diffusion in δ-Bi2O3 and (Bi0.7Y0.3)2O3.

Formulating the alginate-polyornithine biocapsule for prolonged stability: evaluation of composition and manufacturing technique.

Alginate encapsulation is one of the most widely used techniques for introducing cell-based therapeutics into the body. Numerous encapsulation methodologies exist, utilizing a variety of alginates, purification technologies, and unique polycationic membrane components. The stability of a conventional alginate formulation encapsulated using a commercially available technique and apparatus has been characterized extensively. The current study employs an encapsulation protocol and ultra-pure alginate pioneered at the University of Perugia. The enhanced microcapsules were produced, characterized, and implanted into the brain, peritoneal cavity, and subcutaneous space of Long-Evans rats. After 14, 28, 60, 90, 120, and 180 or 215 days, capsules were explanted and the surface was analyzed using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Image analysis was carried out to measure changes in diameter and wall thickness. FTIR peak analysis and surface morphology from SEM indicated that the enhanced encapsulation technique and formulation produced a stable biocapsule capable of survival in all sites, including the harsh peritoneal environment, for at least 215 days. Preimplant analysis showed a marked increase in the structural integrity of the enhanced formulation with improved elasticity and burst strength compared with the baseline formulation, which remained stable for less than 60 days. The enhanced microcapsule composition showed advantages in physical strength and longevity, indicating that small changes in encapsulation methodologies and materials selection can dramatically impact the stability and longevity of alginate microcapsules and their contents.

Morphology and development of the reticuloperidial ascomata of Auxarthron conjugatum.

Light and electron microscopy showed that the reticuloperidium of thick-walled hyphae, characteristic of the mature ascoma of Auxarthron conjugaturn, originated from branches that grew from the broad, gyre-like hyphal loops making up the ascomatal initials. Within the developing peridium, short, acropetally proliferating chains of prototunicate asci each arose from a single crozier and matured from base to tip. The walls of young asci were two-layered but evanesced as they matured with the outer layer dissolving before the inner one. Distal asci in some chains retained the inner wall, detached from adjacent asci by septum schizolysis and when transferred to fresh media produced germ tubes and mycelium. Ultraviolet epifluorescent staining with a DNA intercalator (Hoechst) indicated that these spore-like asci probably contained diploid nuclei. In normal asci, ascospores had an inner, electron lucent primary wall and a three-layered secondary wall. The deposition pattern of the middle layer of the secondary wall created the distinctive array of pits and ridges characteristic of the ascospores in this taxon. The production of ascospores, spore-like asci and arthroconidia, along with the tendency of ascospores to adhere in a mass, is interpreted as contributing to the reproductive flexibility and inoculum potential of A. conjugatum. In all respects the ascomata of A. conjugatum differed substantially from the morphologically similar taxon, Myxotrichum arcticum. These findings underscore the benefit of using DNA-based phylogenies in concert with cytological and ultrastructural observations for exploring selective pressures behind homoplasious characters and revealing novel structural features.

Development of fetal thymocytes in organ culture: effect of corticosteroids.

Corticosteroids affect the development of fetal foregut-derived organs in which epithelial-mesenchymal interactions are associated with the developmental process. The thymus is one such organ and is profoundly sensitive to corticosteroids when mature. In this study corticosterone (CS) effects on fetal thymocyte development were investigated using a fetal thymus organ culture system which allows the growth, differentiation, and function of developing thymocytes to be monitored in vitro. CS inhibited, but did not block growth of fetal thymocytes, although the appearance of mature thymocytes was inhibited, similar to previously reported effects of interleukin 2 (IL2). CS enhanced the proportion of Mac1+, Ia+ and FcR+ cells and maintained high levels of IL2 receptor (IL2R) positive immature cells. Functional cytotoxic cells were detected in CS-treated organ cultures which expressed a Thy 1-, CD8- phenotype, atypical for thymus derived killer cells. While this cytotoxicity may be stimulated by CS, it could simply be due to a relative depletion of the main pool of thymocytes. These cytotoxic cells may have a role in directing apoptotic mechanisms occurring during thymocyte development.

The effects of insulin-like growth factor (IGF)-1, IGF-2, and des-IGF-1 on neuronal loss after hypoxic-ischemic brain injury in adult rats: evidence for a role for IGF binding proteins.

Insulin-like growth factor (IGF)-1, IGF binding protein (IGFBP)-2, and IGFBP-3 are expressed in the rat brain in regions of neuronal loss by 3 days after hypoxic- ischemic (HI) brain injury and IGF-2 somewhat later. Central administration of rh-IGF-1 after HI injury reduces neuronal loss in vivo. To clarify the mode of action of IGF-1 and the potential role of IGFBPs, the effects of IGF-1, IGF-2, des(1-3)-N-IGF-1 (des-IGF-1), an analogue of IGF-1 with low affinity for IGFBPs, and IGF-1 combined with IGF-2 were compared 2 h after administration into the lateral cerebral ventricle after an HI injury. Unilateral HI was induced in adult rats by right carotid artery ligation followed by 10- min exposure to 6%O2. The extent of neuronal loss was determined in the cortex, striatum, hippocampus, dentate gyrus, and thalamus 5 days later. Central administration of 20 micrograms IGF-1 (n = 17) reduced neuronal loss in all regions (P < 0.01). Neither 20 micrograms IGF-2 (n = 17), 2 micrograms des-IGF-1 (n = 10), nor 20 micrograms des-IGF-1 (n = 17) reduced neuronal loss. There was a trend towards a reduction in neuronal loss after 150 micrograms des-IGF-1 (n = 20). IGF-2 alone increased neuronal loss in the hippocampus and dentate gyrus compared with the same regions in vehicle-treated animals (P < 0.05). Coadministration of 30 micrograms IGF-2 blocked the neuroprotective effects of 20 micrograms IGF-1 (n = 18, P < 0.05) and reduced the accumulation of [3H]IGF-1 in the injured hemisphere (n = 4) (P < 0.05). These observations suggest a role for IGFBPs in targeting the neuroprotective actions of IGF-1. IGF-2 may antagonize the protective effect of IGF-1 by displacing it from IGFBPs.

Leukemia inhibitory factor: a novel bone-active cytokine.

A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.

Spontaneous remission of Cushing's syndrome in a patient with an adrenal adenoma.

A 23-yr-old male student presented with clinical and biochemical evidence of Cushing's syndrome. One month later, his elevated plasma and urinary adrenal steroids had returned to normal. At surgery, an adrenal adenoma was removed from his right side. We postulate that he either underwent a temporary spontaneous remission of his disease without treatment, prior to surgery, or that his adenoma secreted glucocorticoids in a cyclical fashion.

Developmental changes in the expression patterns of IGFs, type 1 IGF receptor and IGF-binding proteins-2 and -4 in perinatal rat lung.

The insulin-like growth factors (IGF-I and IGF-II), their receptors and binding proteins (IGFBPs) are endogenously expressed in a number of tissues including the lung during fetal and neonatal development. This endogenous autocrine/paracrine IGF 'system', together with endocrine sources, contributes to the regulation of lung cell proliferation. We investigated the expression of the mRNAs encoding IGF-I, IGF-II, the type 1 IGF receptor (IGF-T1R) and two IGF-binding proteins (IGFBP-2 and IGFBP-4) in rat lung during the perinatum. These were compared in lung with surfactant apoprotein A (Sp-A) mRNA levels. mRNA in extracts of fetal tissues collected between day 17 of gestation (17f) and day 9 after birth (9d) was estimated by Northern blot or RNase protection analysis. At day 20 of gestation IGF-I, IGF-T1R and IGFBP-4 mRNA levels were higher in lung than liver (all P < 0.01), whereas IGF-II and IGFBP-2 mRNA levels were higher in liver than lung (each P < 0.02). The expression of IGF-1, IGFBP-2 and IGFBP-4 in lung was high before birth (days 17-20f) but decreased to low levels at days 21f, 22f or at birth (1d) but increased in the neonatal lung. IGF-II expression in lung was high at 17f but decreased before birth and remained low after birth. The IGF-T1R was expressed at moderate levels before birth, decrease before birth but peaked at days 2-5 after birth. The decrease in expression of these growth regulators before birth expression of these growth regulators before birth was matched by an increased in Sp-A expression which was clearly seen at day 20f, peaked at 1d and then was clearly seen at day 20f, peaked at 1d and then was maintained at high levels after birth. Primary cell cultures of 18f lung epithelia express IGFBP-2 while fibroblasts from the same animals express only IGFBP-4. Cells grown from 22f lung tissue express IGFBP-2 and IGFBP-4 at lower levels, behaving in vitro as they do in vivo. The contrasting levels of expression of different components of the IGF system in the fetal lung and liver indicate organ-specific regulation. IGFBP-2 and IGFBP-4 expression in different cell types within lung but with similar temporal changes suggests cell-specific regulation, perhaps by a common agent. The patterns by a common agent. The patterns of expression of IGF-I, IGF-T1R, IGFBP-2 and IGFBP-4, but not IGF-II, in developing lung correspond to previously described phasic changes in lung cell proliferation rates. The nadir in expression of these four major components of the lung IGF system occurs in the saccular phase when the lung begins to differentiate, probably under the influence of certain endocrine agents.

Fetal rat lung epithelium has a functional growth hormone receptor coupled to tyrosine kinase activity and insulin-like growth factor binding protein-2 production.

Although growth hormone (GH) receptor (GHR) mRNA and protein are present in fetal tissues such as the lung, there is little evidence that GH mediates growth in the fetus. We have identified functional responses to GH in fetal rat lung epithelia and suggest a possible role for GHR in the developing lung. GHR mRNA in lung extracts was high before birth at day 16 of gestation (16f), decreased to low levels at day 22f but increased again after birth. At day 20f GHR mRNA levels were higher in lung than in liver, whereas growth hormone binding protein mRNA levels were approximately equal in lung and liver. Stimulation of primary cell cultures of day 19f lung epithelia with GH caused increased tyrosine phosphorylation in specific proteins, demonstrating functional GHR. Lung fibroblasts isolated at the same time did not respond to GH. Ligand and Northern blot analysis of the epithelial cultures revealed that GH stimulation increased insulin-like growth factor binding protein-2 (IGFBP-2) activity and mRNA. These experiments demonstrate the functional activity of GHR, specifically in fetal lung epithelium. We suggest that one role for GH in vivo may be indirectly to modify insulin-like growth factor activity in the developing fetal lung by increasing IGFBP-2.

Synthesis of dehydroepiandrosterone and dehydroepiandrosterone sulphate by the human adrenal.

The role of pregnenolone sulphate in adrenal steroid biosynthesis and the ability of the human adrenal gland to synthesize and secrete dehydroepiandrosterone (DNA) and dehydroepiandrosterone sulphate (DHA sulphate) was investigated. The presence of pregnenolone sulphate and DHA sulphate was demonstrated by measuring their concentrations in human adrenal tissue. Pregnenolone sulphate was metabolized in vitro mainly to free steroids, including DHA and cortisol, as well as directly to DHA sulphate in some cases. Similar results were obtained upon perfusion of the adrenal gland in situ with [14C]pregnenolone and [13H]prenenolone sulphate as the substrates and isolating the metabolites from the adrenal venous blood. Dehydroepiandrosterone sulphate was derived mainly from the sulphation of free DHA. The hydrolysis of DHA sulphate did not appear to make a significant contribution to the amounts of DHA synthesized under these conditions. The adrenal secretion of DHA and DHA sulphate by eight patients undergoing adrenal-ectomy was determined by measuring the concentrations of these compounds in samples of adrenal and peripheral venous blood taken simultaneously. In one patient secretion of DHA and DHA sulphate was equivalent whilst in the remainder there was much greater secretion of DHA.

Fetal and placental glucose and amino acid uptake in the spontaneously hypertensive rat.

Until late in gestation, fetal spontaneously hypertensive rat (SHR) is growth retarded. Fetal growth rate increases after placental hypertrophy occurs between fetal days 18 and 20. The increase in placental mass may result in improved transfer of macro nutrients to the fetus and thus stimulate growth. In this study, fetal and placental uptake of the glucose analog 3-O-methyl glucose (3MG) and the amino acid analog amino-isobutyric acid (AIB) from the maternal circulation were compared in the SHR and Wistar-Kyoto (WKY) on days 16, 20 and 22 of gestation. Placental 3MG uptake (d/min/g tissue wet weight) was decreased in the SHR on days 20 and 22 but no differences were observed in fetal 3MG uptake. Increased placental mass in the SHR meant that total placental 3MG uptake was greater in the SHR. Placental uptake of AIB (d/min/g tissue wet weight) was much lower in the SHR (on days 16, 20 and 22) and the decrease was not compensated for by increased placental mass. Fetal uptake of AIB was decreased on days 20 and 22 (P<0.05). AIB uptake (d/min/g tissue wet weight) by the carcass and the internal organs (brain, heart, kidney, liver and lung) was also lower in the SHR. These findings indicate that although fetal growth in the SHR increases rapidly in late gestation following placental hypertrophy, it does so despite a pronounced deficit in amino acid uptake.

CNS grafts of rat choroid plexus protect against cerebral ischemia in adult rats.

The present study examined the neuroprotective effects of choroid plexus isolated from adult rats and encapsulated within alginate microcapsules. In vitro, conditioned media from cultured choroid plexus produced a marked, dose-dependent protection of embryonic cortical neurons against serum deprivation-induced cell death. In vivo studies demonstrated that a one-hour middle cerebral artery occlusion in adult Wistar rats produced profound motor and neurological impairments 1-3 days after stroke. In contrast, stroke animals transplanted with encapsulated choroid plexus cells displayed a significant reduction in both motor and neurological abnormalities. Histological analysis 3 days post-transplantation revealed that choroid plexus transplants significantly decreased the volume of striatal infarction. This is the first report demonstrating the therapeutic potential of transplanted choroid plexus for stroke.

The movement of IGF-I into the brain parenchyma after hypoxic-ischaemic injury.

The movement of peptides from CSF into the parenchyma is thought to be slow and diffusion limited. However IGF-I can reduce neuronal loss at distal sites when given centrally 2 h after hypoxic-ischaemic (HI) injury. The present study determined the distribution of [3H]IGF-I given into the lateral ventricle after unilateral HI injury in adult rats. Radioactivity in the injured cortex peaked immediately after administration then rapidly declined. Autoradiography demonstrated radioactivity in the perivascular spaces and in the corpus callosum and external capsule of the injured hemisphere. HPLC and radioimmunoassay confirmed a rise in intracerebral IGF-I levels (from 159 +/- 9 to 401 +/- 88 ng g-1). These data suggest that injury can enhance the movement of IGF-I into the cerebrum via the white matter tracts and perivascular spaces.

Neuroprotective effects of Gly-Pro-Glu, the N-terminal tripeptide of IGF-1, in the hippocampus in vitro.

Insulin-like growth factor 1 (IGF-1) plays a critical role in CNS development. IGF-1 can block neuronal apoptosis in vitro and in vivo. IGF-1 is thought to be cleaved into des-N-(1-3)-IGF-1 and an amino terminal glycine-proline-glutamate (GPE tripeptide). Here we report a neuroprotective role for GPE tripeptide, with enhanced survival of the CA1-2 hippocampal neurons following an excitotoxic insult in vitro. Binding and displacement studies suggest uniquely distributed sites of action within the rat including the hippocampal CA1-2, pyriform cortex, amygdala, choroid plexus, blood vessels and to a lesser extent in the cortical regions. A similar pattern of binding was seen in the human. This finding could lead to new strategies to reduce neuronal death after injury and in disease.

Collagen content of human amniotic membranes: effect of gestation length and premature rupture.

The collagen content of amniotic membranes was measured in samples obtained at delivery from patients with and without premature rupture of the membranes (PROM). In samples from patients with PROM the collagen content (343 microgram/mg) was significantly lower than in samples from patients without PROM (373 microgram/mg) (P less than .005). The collagen content decreased between 32 and 40 weeks' gestation from 446 to 362 microgram/mg (r = .588; P less than .001) in patients without PROM and from 393 to 332 microgram/mg (r = -.362; P less than .05) in patients with PROM. The latent period between membrane rupture and delivery was not associated with a decrease in collagen content. The changes in amnion collagen during gestation and the differences observed with PROM suggest that weakening of the amnion in preparation for rupture may be determined partly by factors controlling the synthesis and degradation of collagen.

Intracerebral transportation and cellular localisation of insulin-like growth factor-1 following central administration to rats with hypoxic-ischemic brain injury.

Insulin-like growth factor-1 (IGF-1) has been shown to be neuroprotective when administered centrally following hypoxic-ischemic (HI) brain injury. However, the cerebral distribution and site of action of IGF-1 after intracerebroventricular (i.c.v.) administration are not known. A unilateral HI brain injury was induced in adult rats by a modified Levine method. Either 3H-IGF-1 alone, or in combination with unlabelled IGF-1, was administered into the lateral ventricle 2 h after injury. The activity of 3H-IGF-1 signal in the potentially injured cortex was compared between two treatment groups using image analysis. The regional distribution and cellular localisation of 3H-IGF-1 were examined autoradiographically in potentially injured hemispheres at 0.5 and 6 h after administration. Tritiated IGF-1 was detected predominantly in the pia mater, perivascular spaces and subcortical white matter tracts 0.5 h after administration and decreased by 6 h (p<0.05). The signals associated with the perivascular spaces and pia mater were not blocked by unlabelled IGF-1, suggesting non-saturable binding in these brain areas. IGF-1 signal was co-localised with IGF binding protein (IGFBP)-2 immunostaining in the white matter tracts where the signal was blocked by unlabelled IGF-1, suggesting competitive association. IGF-1 signal associated with neurons and glia was maximal in the cerebral cortex and less in the CA1-2 subregion of the hippocampus which were blocked by unlabelled IGF-1 (p<0.05). The signals from cortical neurons did not decrease 6 h after administration, suggesting specific and persistent binding to these cells. Our results indicate that centrally administered IGF-1 can be translocated to neurons and glia via the perivascular circulation and the ependymal cell-white matter tract pathways.

Effects of glucocorticoids and beta-adrenoceptor agonists on the proliferation of airway smooth muscle.

An increase in airway smooth muscle is a characteristic feature of asthma. Because beta-adrenoceptor agonists and corticosteroids are commonly used in the treatment of asthma we have studied the effects of these medicines on the growth of airway smooth muscle. These agents were incubated with bovine airway smooth muscle cells for 40 h for measurement of thymidine incorporation and 64 h for measurement of cell counts. Salbutamol inhibited thymidine incorporation (IC50 = 60 nM) and led to a reduction in cell number (IC50 = 10 nM). At 10 microM there was a 14.6 +/- 2.6% reduction in cell number. Salmeterol also inhibited the growth of the airway smooth muscle cells but the effect did not plateau at 10 microM. At this concentration there was an 89.5 +/- 3.6% reduction in thymidine incorporation and a 44.1 +/- 5.2% reduction in cell number. Cortisol and beclomethasone dipropionate were more potent than salbutamol in inhibiting thymidine incorporation with IC50 values of 5 nM and 0.2 nM respectively. Cortisol 100 nM led to a 16.6 +/- 6.5% reduction and beclomethasone dipropionate 3 nM led to a 17.8 +/- 5.8% reduction in cell number. If similar effects occur in man and in vivo, these medicines could act directly on airway smooth muscle to inhibit the development of hyperplasia.

Key enzymes of prostaglandin biosynthesis and metabolism. Coordinate regulation of expression by cytokines in gestational tissues: a review.

Preterm labor is frequently associated with ascending intrauterine infection, accompanied by leukocytes infiltration and enhanced local production of cytokines and other inflammatory mediators. The resulting amplification of the inflammatory response, and of prostanoid production in particular, is postulated to be a principal mechanism of infection-driven preterm labor. In this review the effects of pro- and anti-inflammatory cytokines are discussed with respect to the expression of enzymes involved in three key steps of prostanoid biosynthesis and metabolism: liberation of arachidonic acid (AA), conversion of AA to bioactive prostanoids, and prostanoid catabolism. We suggest that by exerting coordinate actions on all three key steps, through multiple molecular mechanisms, inflammatory cytokines acutely up-regulate prostanoid production in intrauterine tissues.

A radioimmunoassay for 7 alpha-hydroxy dehydroepiandrosterone in human plasma.

A radioimmunoassay for plasma 3 beta, 7 alpha-dihydroxy-5-androsten-17-one (7 alpha-hydroxy DHA) has been developed using anti-sera raised against 3 beta, 7 alpha-dihydroxy-5-androstene-17 beta-carboxyl-bovine serum albumin conjugate and [1, 2 (n) - 3H] 7 alpha-hydroxy DHA as the radioligand. Significant cross reactivity was found with 3 beta, 7 alpha-dihydroxy-5-pregnen-20-one (44%), 3 beta, 7 beta-dihydroxy-5-androsten-17-one (6%), 3 beta, 6 beta-dihydroxy-4-androsten-17-one (2.5%), 3 beta-hydroxy-5-androsten-17-one (DHA, 2%), 3 beta, 7 beta-dihydroxy-5-pregnen-20-one (2%) and 7 alpha-hydroxy-4-androstene-3, 20-dione (1%). 7 alpha-Hydroxy DHA was extracted from plasma and separated from cross-reacting factors using alumina micro-columns. The separation of bound and free steroid was achieved using dextran-coated charcoal. The concentration of 7 alpha-hydroxy DHA in the plasma of breast cancer patients was significantly lower than the concentrations in the plasma of normal women, hospitalized women suffering from non-endocrine diseases and patients with benign breast disease. The decrease in the concentration of 7 alpha-hydroxy DHA in the plasma of pregnant women was not significant.