Burton V. Caldwell

Immunologic methods in steroid determination

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Preparation and Purification of Antibodies to Steroids

As discussed in previous papers at this symposium, steroids or other low-molecular-weight compounds are not inherently antigenic (Goodfriend and Sehon, 1970; and Gross, 1970). Landsteiner, however, demonstrated in the early 1900’s that antibodies directed against these low-molecular-weight compounds can be produced if they are chemically conjugated to a substance which is antigenic (see Landsteiner, 1946, for review). When conjugated to the antigen, these low-molecular-weight substances function as haptens.

Further studies on corticosteroidogenesis.v. 11β-hydroxylation of deoxycorticosterone by mitochondria incubated with malate, supernatant fraction and supernatant fraction + pyruvate + co2

Abstract Rat adrenal gland mitochondria (P2) utilize malate for generating intramitochondrial NADPH required for 11β-hydroxylation of deoxycorticosterone∗. High speed supernatant fractions (Sup.) of rat adrenal gland homogenates which are devoid of mitochondria and microsomes can replace malate when incubated with the rat adrenal gland mitochondria for the aforementioned steroid hydroxylation. The substance(s) in the Sup. which are responsible for intramitochondrial NADPH generation were found to be heat-labile and dialyzable so that small as well as large molecular weight substances appear to be implicated in the phenomenon of pyridine nucleotide reduction. P2 alone cannot utilize pyruvate + CO2 to form malate although the presence of the malate dehydrogenase (decarboxylating: NADP) (EC 1.1.1.40) in the P2 is strongly indicated. Thus, malate in P2 as a result of its oxidation via the intramitochondrial NADP-linked malate dehydrogenase (decarboxylating: NADP), generates NADPH and pyruvate+CO2. The Sup. on the other hand, in the presence of NADPH+CO2 converts pyruvate into malate. This malate in the Sup. + P2 combinations can be utilized by the P2 for intramitochondrial NADPH production necessary for corticosterone production from deoxycorticosterone. It is concluded, therefore, that the Sup. activity might be due to proteins (enzymes), small molecular weight substances like ions (Mg2+, Mn2+) or pyruvate (or those which can be metabolized in the Sup. to pyruvate) which function together to bring about the production of malate.

Solid-Phase Radioimmunoassay of Steroids

There have been several procedures reported which offer the new investigator interested in developing a radioimmunoassay for steroid hormones a choice of methods for separating the free from bound steroid. Midgley et al. (1969) have described a means for accomplishing separation, based on precipitation of bound antibody-steroid with a second antibody. This separation usually requires incubation for several days. Mikhail et al. (1970) separate free from bound estradiol by polymerizing the antibody, but this adds a high-speed centrifugation step. The recently described solid-phase system for estradiol (Abraham, 1969; Abraham and Odell, 1970) would appear to offer many advantages over the above procedures, particularly the unique feature of accomplishing the equilibration and the separation of free from bound steroid in a single step. It will be the principal aim of this paper to describe in detail the several essential criteria which must be examined for each antiserum, central to its use in the development of new methods for the radioimmunoassay of Steroids. Chemical, immunologic, and endocrinologic aspects of steroid-protein conjugates have been described by Lieberman et al. (1959). Pressman and Grossberg (1968, 1970) have discussed chemical and structural concepts which are involved in antibody-antigen interactions. However, the effects of certain physicochemical factors, such as ionic strength, hydrogen ion concentration, and temperature, on potential radioimmunoassay systems for progesterone and testosterone have not been previously examined in detail and will be described below as they apply to the solid-phase assay of these hormones.

Steroid hydroxylations by guinea-pig adrenal tissue fractions.

Abstract The effect of oxidizable substrates, α-ketoglutarate and isocitrate on 11β-hydroxylation in guinea-pig adrenal mitochondria has been studied. These substrates supported the conversion of Compound S (17α,21-dihydroxy-pregnene-3,20-dione) into cortisol as well as exogenously added NADPH in Ca 2+ -swollen mitochondria. Progesterone was also hydroxylated at the C-11β position but not at the C-21 position. Microsomes, on the other hand, when NADPH was added, converted pregnenolone, progesterone and 17α-hydroxyprogesterone into Compound S, but showed no steroid 11β-hydroxylating activity. Evidence obtained in incubations carried out with α-ketoglutarate and isocitrate in the presence of 2,4-dinitrophenol, oligomycin, amytal and antimycin A indicate that α-ketoglutarate utilization for steroid 11β-hydroxylation is dependent on activity of the classical electron chain. This activity can be related to high energy intermediates possibly needed for NADPH reduction arising from NADH oxidation via the energy-requiring transhydrogenase reaction. These reactions do not appear necessary for isocitrate utilization and isocitrate oxidation probably gives rise to intramitochondrial NADPH reduction as a result of a NADP + -linked isocitrate dehydrogenase. Data obtained in oxygen uptake studies with an antimycin A blocked system supplied with α-ketoglutarate, are in accordance with this conclusion. The high-speed supernatant fraction (103000 × g ) could partially replace α-ketoglutarate, isocitrate or Ca 2+ + NADPH, indicating that it contains a factor(s) (the physiological substrate?) which brings about intramitochondrial NADPH.

Physiologic Studies Using Antibodies to Steroids

Following the comprehensive presentation of Dr. Lieberman et al. in 1959 on the “Chemical, Immunological, and Endocrinological Properties of Steroid-Protein Conjugates,” several investigators have described studies utilizing the ability of steroids, when coupled to a carrier protein, to act as haptens and elicit the formation of specific antibodies in properly immunized animals. The previous papers in this symposium have dealt with the chemical and immunologic aspects of steroid-protein conjugates and the reader is referred to these publications for a detailed review of the pertinent literature (Goodfriend and Sehon, 1970; Gross, 1970; Thorneycroft et al., 1970; Pressman and Grossberg, 1970). The primary emphasis of this paper will be on the endocrinologic aspects and the possible use of active and passive immunization procedures in physiologic studies. In particular, evidence will be presented which will more clearly establish the role of estradiol in the control of luteinizing hormone (LH) secretion from the pituitary gland.

Solid Phase Radioimmunoassay of Estradiol-17β and Estrone

Publisher Summary This chapter discusses the solid phase radioimmunoassay of estradiol-17β and estrone. Sensitive and practical assays for various steroids have been developed using specific blood or target tissue proteins. The chapter describes the use of a specific immunoglobulin for the analysis, by radioimmunoassay, of picogram quantities of estradiol and estrone. Radioimmunoassay and competitive protein binding differ in the specific protein employed, and the methods used for separating the bound and free hormone. The chapter describes the use of a solid phase separation system, as modified for estradiol radioimmunoassay. A simple ether extraction of plasma should be sufficient to estimate total immunologically active estrogens because there is a negligible cross reaction with non-phenolic steroids. However, in immunoassay, there are difficulties with blanks when an ether extraction is performed.

Neutralization of Exogenous Estradiol Activity following Active Immunization Procedures

Publisher Summary Low molecular weight compounds such as steroids would not elicit the production of antibodies when present in the free form. However, when linked by covalent bonding to large antigenic molecules, such as bovine serum albumen (BSA) and injected as a conjugate into other species, antibodies of two types would form. Those directed against the naturally occurring antigenic determinant groups present on the BSA molecule and those directed against the artificial steroid haptens. Because of structural similarities between the free steroid and the steroid-protein haptenic groups, antibodies directed against these groups would bind the free steroid. This forms the basis of a sensitive radioimmunoassay for estradiol, which has been described by several investigators. Another application lies in the ability of the antibodies to neutralize the effects of endogenous or exogenous steroids in vivo, thus providing a tool for the study of the physiological effects of these hormones.