Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35).

The metabolism of high-density lipoprotein-associated cholesteryl esters (HDL-CE) in liver cells is not well understood. We studied the possible role of lysosomal and extralysosomal pathways on such metabolism by measuring the uptake and hydrolysis of HDL-CE in H-35 rat hepatoma cells. Incubation of cells with [3H]cholesteryl ester-labeled HDL led to the intracellular accumulation of both 3H-free cholesterol and [3H]cholesteryl ester. The ratio of 3H-free cholesterol/[3H]cholesteryl ester increased with an increase in incubation time even in the presence of chloroquine. Because chloroquine did not inhibit the conversion of cholesteryl ester to free cholesterol, the hydrolysis of HDL-CE may have been catalyzed by an extralysosomal enzyme, perhaps by neutral cholesteryl ester hydrolase (NCEH). When we incubated cells with increasing concentrations of HDL, NCEH activity increased. This increase in enzyme activity was not inhibited by the addition of chloroquine. A complex of dimyristoylphosphatidylcholine (DMPC)/apo HDL/cholesteryl ester enhanced the activity as well as native HDL. Neither the DMPC/apo HDL nor the DMPC/cholesteryl ester complex affected the activity, suggesting that apo HDL may be required for the uptake of HDL-CE. The present study demonstrated that the extralysosomal hydrolysis by NCEH is operating in the metabolism of HDL-CE in hepatoma cells.

Corticosteroids in human blood. VIII. Cortisol metabolites in plasma of normotensive subjects and patients with essential hypertension.

Results of our previous studies revealed a derangement in the peripheral metabolism of adrenal steroids in patients with essential hypertension. To investigate further this finding, all indIVidual free and conjugated metabolites of cortisol were isolated, identified and quantitated in plasma of 14 normotensive subjects and 13 patients with benign, uncomplicated essential hypertension, following iv administration of a tracer dose of [4-14-C] cortisol. In addition, plasma levels of endogenous cortisol were determined at 8 AM and 4 PM in all the subjects examined. The results obtained revealed the following statistically significant differences between normotensives and hypertensives: 1) Mean plasma concentrations of cortisol metabolites reduced in ring-A with nonreduced 20-ketone, tetrahydrocortisol, tetrahydrocortisone, and their 5alpha-epimers, were 30% lower in the hypertensives; since these steroids constitute the bulk of the major group of cortisol metabolites--the glucuronide conjugates, plasma levels of this group of conjugates measured in toto were also found to be significantly lower in the hypertensives. 2) Concentrations of cortisol metabolites with non-reduced ring-A (delta-4-3-keto configuration preserved) but with reduced 20-ketone and/or hydroxylated at C-6, 20alpha- and 20beta- dihydrocortisol, 6alpha- and 6beta-hydroxycortisol, and 6-hydroxy-20-dihydrocortisol (all 4 isomers), were 73%, 48% and 68% respectively, higher in the hypertensives; since these steroids constitute the bulk of the sulfate-conjugated and nucleoside-complexed metabolites of cortisol, plasma levels of these groups of metabolites, measured in toto, were also found to be higher in the hypertensives. No significant difference was found between normotensives and hypertensives in the AM and PM plasma levels of cortisol. These findings, in conjunction with the results of our studies on urinary corticosteroid metabolites, which yielded identical findings, provide evidence for a decreased activity of hepatic cortisol-delta-4-hydrogenase enzyme system and increased activities (presumably compensatorily) of cortisol-20-reductase and 6-hydroxylase enzyme systems in patients with essential hypertension. The interrelation of these findings with those of other investigators studying steroid metabolites in hypertension, points to the corticosteroid metabolizing enzymes may be an etiological factor in essential hypertension.

Prolactin and growth hormone responses to psychological stress in normal and neurotic subjects.

In order to study the response of plasma prolactin (PRL) to acute psychological stress and to compare it with that of growth hormone (GH), the mirror drawing test (MDT) was performed in 20 normal controls (11 male, 9 female) and 22 neurotic patients (12 male, 10 female). Plasma PRL and GH were measured serially before, during and after the test. In controls, the test caused no significant change in plasma levels of either hormone. In neurotic males, the response of PRL to the test was not consistent, whereas, in neurotic females, plasma PRL level rose significantly following the test. Increase of GH, on the other hand, was apparent in the neurotics of both sexes. The correlation between the responses of the two hormones in the neurotics was low and non-significant. The results indicate that although the psychoendocrine coping mechanism in the neurotics works less effectively for both PRL and GH, the two hormones may have different psychological correlates.

Growth hormone and cortisol responses to psychological stress: comparison of normal and neurotic subjects.

The mirror drawing test (MDT) was performed to induce acute psychological stress in 9 normal controls and 10 neurotic subjects. Plasma growth hormone (GH) and cortisol were determined serially before, during, and after the test. In controls the MDT caused no significant change in plasma GH level, while in neurotics plasma GH increased progressively following the test. The increase of cortisol also tended to be greater in neurotics as a group, but there was considerable overlap in individual responses. The maximum increments of GH in neurotics correlated inversely with those of cortisol. The results indicate: 1) effective psychological coping mechanisms operate in normal man to keep the hormonal response minimum. 2) GH response is a more adequate indicator than cortisol response to psychological stress in neurotics. 3) GH and cortisol may have different psychological correlates in neurotics.

Role of nitric oxide and superoxide anion in leukotoxin-, 9,10-epoxy-12-octadecenoate-induced mitochondrial dysfunction.

The present study was carried out to explore the involvement of nitric oxide (NO) and superoxide anion (O2.-) in Leukotoxin (Lx)-induced suppression of mitochondrial respiration. Glutamate- and succinate-dependent oxygen consumption and cytochrome c oxidase activity were assayed. Lx-induced mitochondrial damage was significantly attenuated by the pretreatment of lung with 4 x 10(-4) M NG-monomethyl-L-arginine (L-NMMA) or 500 units/ml superoxide dismutase (SOD) in ex vivo. However, L-NMMA plus SOD pretreatment showed no additive effect on the recovery of mitochondrial functions. The same assay was performed after the exposure of intact mitochondria to NO containing solution (1.25 x 10(-5) M) or 0.1 mM KO2/18-Crown-6 solution, which generated O2.-(6.4 x 10(-5) M). NO, but not O2.-, significantly inhibited the respiration of isolated mitochondria in vitro. Thus, there were great discrepancies in the involvement of NO and O2.- between ex vivo and in vitro system. Together with the previous reports, these facts suggested that the mechanisms by which NO and O2.- probably from vascular constituent cells inhibit mitochondrial respiration function of isolated perfused rat lung may not be simply due to their direct reactions with mitochondrial electron transport chain components, but may rely on the formation of peroxynitrite, and/or peroxynitrite-derived oxidants.

Relation of angiographically defined coronary artery disease and plasma concentrations of insulin, lipid, and apolipoprotein in normolipidemic subjects with varying degrees of glucose tolerance.

We investigated the association between hyperinsulinemia and changes in lipid, lipoprotein, and apolipoprotein that would increase the risk of coronary artery disease (CAD) independent of glucose tolerance. A coronary angiogram was recorded in 127 male subjects, including 41 with normal glucose tolerance, 41 with impaired glucose tolerance, and 45 with non-insulin-dependent diabetes mellitus (NIDDM). Subjects were divided into 2 groups according to results: the group with CAD (n = 94) and the group with normal coronary arteries (n = 33). All subjects were normolipidemic (total cholesterol < 230 mg/dl and triglycerides < 150 mg/dl). The CAD group had a significantly lower plasma level of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apo A-I) and a higher level of apolipoprotein B (apo B) than the normal group with normal glucose tolerance. In considering subjects with impaired glucose tolerance or NIDDM, the CAD group had a significantly lower plasma level of HDL cholesterol and apo A-I and a significantly higher plasma level of total cholesterol, triglycerides, and apo B than the normal group. In each of the subjects with normal and impaired glucose tolerance, and NIDDM, the elevation of plasma insulin concentration during both the complete test period and the early phase of an oral glucose challenge was significantly higher in the CAD than in the normal group. In all subjects, graded reductions in HDL cholesterol and apo A-I and graded increases in plasma total cholesterol, triglycerides, and apo B were observed with increasing tertiles of the postglucose challenge measurements of insulinemia.(ABSTRACT TRUNCATED AT 250 WORDS)

OP-1206, a prostaglandin E1 derivative. Effects of oral administration to patients with chronic lung disease.

In order to assess the clinical effectiveness of an oral prostaglandin E1 derivative, OP-1206, five patients with chronic obstructive pulmonary disease and two with pulmonary fibrosis were studied from the standpoint of hemodynamics and nine others with chronic lung disease from the standpoint of respiratory function. Oral intake of OP-1206 resulted in a significant decrease in the pulmonary arterial pressure (p less than 0.01), total pulmonary vascular resistance (p less than 0.01), pulmonary arteriole resistance (p less than 0.01), and total systemic vascular resistance (p less than 0.05), and an increase in the cardiac index (p less than 0.05) and oxygen delivery (p less than 0.01) with insignificant changes of PaCO2, PaO2 and pH. There was no clinical improvement of lung function after OP-1206 intake. OP-1206 is a potent vasodilator, improving cardiac performance in patients with chronic lung disease and possibly preventing the progress of cor pulmonale in this kind of patient.

Bronchial hyperresponsiveness in patients with chronic congestive heart failure.

To investigate the relationship between pulmonary congestion and bronchial responsiveness, we measured bronchial responsiveness to acetylcholine in 51 patients with left heart disorders. The measurement of bronchial responsiveness was performed by inhaling doses of acetylcholine chloride (0.08 to 20 mg/ml) and calculating the PC20-FEV1. The median value for PC20-FEV1 was above 20 mg/ml in the subjects without history of congestive heart failure (n = 18), was 5.29 mg/ml in the subjects with clinical evidence of congestive heart failure in the past days (n = 18; p less than 0.01), and was 5.74 mg/ml in the subjects with clinical evidence of congestive heart failure at the time of study (n = 15; p less than 0.01). The hemodynamic variables by cardiac catheterization and the clinical symptoms were not correlated with the grade of bronchial responsiveness. These results suggest that the bronchial responsiveness was increased in most of the patients with chronic congestive heart failure. We concluded that continuous pulmonary congestion may contribute to the pathogenesis of bronchial hyperresponsiveness.

Bronchial hyperresponsiveness to acetylcholine in patients with vasospastic angina pectoris.

STUDY OBJECTIVE: To investigate the similarity between coronary vasospasm and bronchial spasm. DESIGN: Nonrandomized, case-control study. SETTING: Referral-based clinics for cardiac and pulmonary disease at one secondary care center. PATIENTS: Seventeen patients with vasospastic angina pectoris (VSAP) and 14 patients with chest pain syndrome (CPS). INTERVENTIONS: Medications prohibited: those with known effects on bronchial responsiveness. MEASUREMENT: Induction of coronary vasospasm: ergonovine maleate (10, 20, 40 micrograms) injection into coronary arteries during coronary angiography. Bronchial responsiveness to acetylcholine (ACh): acetylcholine chloride (0.08 to 20 mg/ml) inhalation and calculation of the provocative concentration of ACh (PC20-ACh) that revealed 20 percent fall in FEV1. RESULTS: The median value for PC20-ACh in patients with VSAP, 7.80 mg/ml, was significantly lower than that in patients with CPS, > 20.0 mg/ml (p < 0.01 by Mann-Whitney U test). The PC20-ACh in patients with VSAP, however, was correlated neither with the responsive threshold of ergonovine maleate, which induced coronary vasospasm, nor with the duration from the latest angina attack. CONCLUSION: These results suggest that bronchial responsiveness was increased in most patients with VSAP, but not with CPS. We therefore speculate that patients with VSAP may also have hypercontractibility to ACh of noncoronary systemic smooth muscles.

Negative Feedback by Corticosterone is the Major Mechanism Responsible for Corticotropin-Releasing Factor-Induced Desensitization.

Abstract Subcutaneous infusion of ovine corticotropin-releasing factor (oCRF) to male rats at a rate of 0.1 mug/h for 4 days did not alter the rise of plasma adrenocorticotropin (ACTH) and corticosterone induced by 3-min ether exposure. In contrast, 1.0 mug/h oCRF for 4 days virtually abolished the ACTH response to ether, whereas a substantial corticosterone response was preserved. Intravenous administration of phenoxybenzamine (5 mg/kg) prior to ether stress completely inhibited the corticosterone response. Plasma ACTH and corticosterone responses in chronic CRF-treated rats to an intravenous bolus injection of 2.0 mug oCRF were also markedly blunted by pretreatment with subcutaneous oCRF 1.0 mug/h for 4 days. Adrenalectomized rats given corticosterone in the drinking fluid at a concentration of 80 mug/ml showed a plasma corticosterone pattern mimicking the normal diurnal rhythm. Basal plasma ACTH and thymus weight were within normal limits. In these rats, the magnitude of ACTH rise to ether stress did not differ between the chronic CRF-treated rats and the vehicle-treated rats. In cultured pituitary cells prepared from animals infused with oCRF 1.0 mug/h for 4 days, the basal and CRF-stimulated ACTH release was reduced by 46%. We conclude that among the possible mechanisms proposed for 'desensitization' during long-term infusion of CRF, negative feedback by elevated corticosterone at both brain and pituitary levels is the primary factor. The results also suggest the existence of non-ACTH-mediated catecholaminergic systems in the stress-induced adrenocortical activation.

Endothelin-1 potentiates leukotoxin-induced edematous lung injury.

We tested the hypothesis that leukotoxin (Lx), a cytochrome P-450-dependent linoleate product of leukocytes, can stimulate the release of endothelin-1 (ET-1) from the lung and further that exogenous ET-1 synergizes with Lx to produce edematous lung injury. In isolated rat lungs perfused with Earle's balanced salt solution, Lx (10 mumol) alone caused lung edema and increased the perfusate and lung tissue ET-1 levels. The combination of ET-1 (5 nM) and Lx (5 mumol), at concentrations that by themselves did not increase wet lung weight, significantly increased wet lung weight, wet-to-dry lung weight ratio, as well as the lung effluent lactate dehydrogenase activity. Pretreatment with BQ-123 (5 x 10(-6) M), an endothelin A receptor antagonist that significantly attenuated the ET-1 (5 nM)-induced increase in pulmonary arterial pressure (Ppa) and pulmonary capillary pressure (Ppc), suppressed the edematous lung injury generated by the combination of ET-1 and Lx, suggesting that the edema-enhancing effect of ET-1 in Lx-treated lungs occurred through endothelin A receptor-dependent elevation of Ppa and Ppc. Elevation of the pulmonary venous pressure in Lx-treated lungs (13.5 cmH2O) mimicked the effect of ET-1 on Ppa and Ppc and produced a degree of lung edema that was comparable to that after combined ET-1 + Lx treatment but without increase in the perfusate lactate dehydrogenase. These data support the idea that ET-1 and Lx promote lung edema in a synergistic fashion.

Stimulation of the hypothalamic-pituitary-adrenal axis by epidermal growth factor.

The effects of mouse epidermal growth factor (mEGF) on the hypothalamic-pituitary-adrenocortical axis were studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Both intravenous (i.v.) and intracerebroventricular (i.c.v.) injections of mEGF (5-20 ng: 8.3-33.3 pmol) produced significant, dose-related increases in plasma ACTH and corticosterone concentrations. The potency of mEGF is 1/20-1/50 of that of rat corticotropin-releasing factor (rCRF), and pretreatment with 150 micrograms alpha-helical CRF (9-41) completely abolished the effects of the two peptides. mEGF in concentrations ranging from 10 pM to 10 nM did not significantly affect ACTH release from dispersed anterior pituitary cells. It also failed to alter ACTH secretion in response to rCRF. These results indicate that mEGF stimulates the pituitary-adrenocortical axis through a CRF-dependent mechanism.

Effects of neonatal treatment with monosodium glutamate on circadian locomotor rhythm in the rat.

In order to study the effects of treatment with monosodium glutamate (MSG) during the neonatal period on the intrinsic circadian timekeeping system in rats, the locomotor activity of blinded MSG-treated and control (saline-treated) rats was analyzed with power spectral analysis and cross-correlation. In contrast to a robust free-running circadian rhythm in the control rats, a significant shortening of the circadian period and rapid decomposition into ultradian components were noted in the MSG-treated rats. Computer-assisted stereometry of the hypothalamic nuclei revealed that, in addition to the well-known severe damage in the arcuate nuclei (ARC), the volumes of the suprachiasmatic nuclei (SCN) and ventromedial hypothalamic nuclei (VMH) were also reduced significantly in the MSG-treated rats. Although no gross histological damage was apparent in either the SCN and VMH, neonatal MSG treatment appears to impair the function of SCN to integrate many minor oscillations in the brain into a single, definite and precise circadian period.

The effect of Cu2+ on rat pulmonary arterial rings.

In the current study, Cu2+ was tested for its ability to relax vessels and to accumulate cyclic GMP (cGMP) in rat pulmonary artery employing rat extrapulmonary arterial rings. Cu(2+)-induced relaxation was endothelium and concentration (in the range from 10(-7) to 10(-4) M) dependent. The content of cGMP in the rings was increased 1.7-fold with 10(-4) M Cu2+. NG-Monomethyl-L-arginine abolished both the copper-induced relaxation and the increase in cGMP of rings. Cu2+ and zaprinast, which inhibits phosphodiesterase activity, caused a synergistic increase in cGMP level in the rings, suggesting that Cu2+ enhanced cGMP level through a mechanism different from that of zaprinast, probably as a consequence of elevated accumulation of nitric oxide (NO). The magnitude of vasorelaxation observed due to simultaneous addition of Cu2+ and acetylcholine was additive, not synergistic. Cu2+ did not augment relaxation induced by exogenously added NO donor. These results suggest that Cu2+ elevates NO level in the rings not by prolonging the half-life of NO, but by activation of endothelial nitric oxide synthase and subsequently potentiating the action of NO on vascular tone.

Plasma thrombomodulin concentration in diabetes mellitus.

Thrombomodulin is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. Recently, it was found that soluble forms of thrombomodulin exist in plasma. Although the physiological significance of circulating thrombomodulin is presently obscure, it may reflect injury of the endothelial cell. In the present study, we examined plasma thrombomodulin concentrations in 106 Type 2 (non-insulin-dependent) diabetic patients. Plasma thrombomodulin was determined by a sandwich ELISA employing monoclonal anti-thrombomodulin antibodies. The patients with proteinuria had higher plasma thrombomodulin concentrations (61.0 +/- 36.0 ng/ml) compared to the patients without proteinuria (33.6 +/- 9.5 ng/ml, P less than 0.001) and control subjects (32.8 +/- 6.5 ng/ml, P less than 0.001). Plasma thrombomodulin concentrations were positively correlated with the level of serum creatine, blood urea nitrogen, urinary albumin and urinary beta 2-microglobulin (P less than 0.001 for each), but not with fasting plasma glucose, hemoglobin A1c or fructosamine. Elevated plasma thrombomodulin was also observed in the patients with pre-proliferative (63.4 +/- 28.9 ng/ml) or proliferative retinopathy (57.4 +/- 34.7 ng/ml), but not in the patients with non-proliferative retinopathy (33.5 +/- 12.9 ng/ml) or those without retinopathy (32.4 +/- 8.9 ng/ml). Even in the 81 diabetic subjects without proteinuria as determined by a dip and read method, and whose serum creatinine was lower than 1.0 mg/dl, the plasma thrombomodulin concentration was significantly higher in the patients with pre-proliferative (41.5 +/- 4.4 ng/ml) and proliferative retinopathy (41.0 +/- 12.8 ng/ml) compared to the patients without retinopathy (32.2 +/- 8.8 ng/ml) and those with non-proliferative retinopathy (31.9 +/- 7.8 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of lipoprotein receptors of the isolated liver parenchymal cells prepared from the streptozotocin-induced diabetic rats.

Characteristics of lipoprotein receptors of the isolated liver parenchymal cells prepared from the streptozotocin-induced diabetic rats were investigated. Streptozotocin-induced diabetic rats fed 1.0% cholesterol showed the exaggerated hypercholesterolemia as compared to control rats fed 1.0% cholesterol. The present study was designed to elucidate the role of lipoprotein receptor mechanisms of liver parenchymal cells in the diabetic dyslipoproteinemia. 125I-labeled lipoproteins (rat beta-VLDL, human LDL2 or rat HDL3) were incubated with liver parenchymal cells isolated by liver perfusion using collagenase. According to the Scatchard analysis, the apparent dissociation constant (kd) and maximum beta-VLDL binding (Bmax) for the higher affinity binding site in the diabetic rats (n = 6) were (11.9 +/- 5.1) X 10(2) ng/ml and 307.5 +/- 145.2 ng/10(6) cells, respectively. These binding characteristics of the diabetic rats were not significantly different from the control rats. Furthermore, there were no significant differences in the binding characteristics of human LDL2 and rat HDL3 between the diabetic rats and the control rats. The data presented suggest that significant role of alteration of lipoprotein receptor characteristics in liver parenchymal cells is not played in the diabetic dyslipoproteinemia.

Decreased ketonaemia in the monosodium glutamate-induced obese rats.

Plasma concentrations of total ketone bodies, acetoacetate (AcAc) and 3-hydroxybutyrate (3-OHBA) in monosodium glutamate (MSG)-induced obese rats were measured. MSG-treated rats showed higher Lee's indices, shorter naso-anal and tail length, and a more marked intraperitoneal fat deposition than control rats. Plasma concentrations of glucose, free fatty acid, triglyceride and phospholipids were significantly increased in the MSG-treated rats as compared to the control rats (24 weeks-old). Plasma levels of total ketone bodies, AcAc and 3-OHBA were all decreased in the MSG-treated rats as compared to control rats. The ratio, 3-OHBA/AcAc in the MSG-treated rats were not different from those in the control rats.

Endothelin-3 stimulates the hypothalamic-pituitary-adrenal axis.

Endothelin-3 (ET-3) is a member of the novel vasoconstrictive peptide family, identified in porcine central nervous system. Intravenous bolus injection of 1000 pmol/kg of ET-3 in freely moving rats caused significant increases in plasma ACTH and corticosterone levels, almost equivalent to those of 100 pmol/kg of rat corticotropin-releasing hormone (rCRH). The action of ET-3 was virtually abolished by pretreatment of CRH-antagonist, alpha-helical CRH. When ET-3 was added to cultured anterior pituitary cells, neither direct stimulation of ACTH release nor potentiation of rCRH action was noted. The results indicate that ET-3 may function as a neuropeptide and stimulation of the CRH-neurons, direct or inderect, is mainly responsible for activation of ACTH and corticosterone release.

Studies on steroid conjugates: IX. Urinary excretion of sulfate conjugated metabolites of cortisol in man.

Following i.v. administration of [4-14C]cortisol, various sulfate conjugated metabolites of cortisol in urine were identified and their respective excretion rates measured. The results obtained demonstrated the following: 1) sulfate conjugates as a group are excreted considerably slower than glucuronide conjugates; 2) sulfate conjugates of steroids with non-reduced ring-A (C-21 sulfates) are excreted (and presumably formed) much faster than steroid-3-sulfates, which require reduction of the ring-A prior to the conjugation; 3) the excretion of C-3 sulfates of ring-A reduced steroids with glycerol side-chain (cortols and cortolones) is significantly faster than those of the corresponding steroids with dihydroxyacetone side-chain (THF, THE and their 5alpha-isomers); 4) the relative concentrations of C-21 sulfates of steroids with ring-A intact (FK, EK, ER, epiER and 6beta-hydroxycortisol) are much higher than the concentrations of C-21 glucuronides of these steroids.

Characterization of antisera to 2-hydroxyestradiol and 4-hydroxyestradiol using 6-(O-carboxymethyl)oxime- and 17-hemisuccinate-bovine serum albumin conjugates and radioimmunoassay.

For radioimmunoassay of the catechol estrogens, four hapten-bovine serum albumin (BSA) conjugates were prepared from 6-oxo-2-hydroxyestradiol 6-(O-carboxymethyl)oxime, 2-hydroxyestradiol 17-hemisuccinate, 6-oxo-4-hydroxyestradiol 6-(O-carboxymethyl)oxime and 4-hydroxyestradiol 17-hemisuccinate by coupling with BSA, employing the mixed anhydride method. The antisera elicited in rabbits by immunization with these antigens showed high affinity and specificity for 2-hydroxyestradiol or 4-hydroxyestradiol with cross-reactivities to a few structurally related estrogens. The specificity of antisera obtained is discussed in relation to the site of attachment of the hapten to BSA.