such as progesterone, that are usually isotopically labelled to facilitate identification of any
products that may be formed . Such procedures, utilizing added substrates , can be used to
demonstrate the pr esence of certain steroidogenic enzymes, but do not indicate whether
or not the products are normally formed or released.
Although in the past steroid hormones were principally assayed and identified by biological means, these are becoming less frequent as sophisticated chemical and radioisotopic
methods become available. Steroid hormones can be separated from other biological substances, and from each other by extraction and partition in suitable solvents such as chloroform, methylene chloride or ethyl acetate, followed by such procedures as gel filtration and
chromatography. They can be measured by colourimetric methods such as that utilizing
the yellow colour developed with phenylhydrazine (PORTER-SILBER reaction for cortisol
and corticosterone), or by using their ability to fluoresce after treatment with ethanolicsulphuric acid, sodium hydroxide or potassium terbutoxide. Steroid hormones can also be
measured by gas chromatography and rad ioisotope methods, such as the double - isotope
dilution method, which is particularly sensitive for hormones, like aldosterone, that are
present at low concentrations. Isotopes of the steroids can be util ized in man y quantitative
procedures, and especiall y to assess losses of material that inevitably occur during their
purification and preparation for assay. Identification ofsteroid hormones is generally based
on their ph ysico-chemical beh aviour, this includes such things as :
1. Chromatographic mobilities in different solvent systems as compared with parallel
standards which, in order to assist their location, can be isotopically labelled. Changes In
chromatographic behav iour following acetylation of the steroids can be used as an additional
criterium for identification.
2. Characteristic colour reactions such as tak e place with PORTER-SILBER reagent and
tetrazolium blue.
3. Fluorescence, either natural or after treatment with such agents as ethanolic sulphuric
acid or sodium hydroxide.
4. Measurement of ultra-violet absorption spectra in sulphuric acid , ethanol or methanol
and infra-red spectra in chloroform.
A consensual definitive statement regarding th e problems and desired criteria for iden -
tification and measurement of steroid hormones has recently been made (BROOKS et al.,
1970).
f) Immunoassay
Immunoassay procedures, especially those using isotopically labelled hormones, have in recent years allowed great strides to be made in the measurement of various protein hormones.
Specific antibodies can be made to most peptide hormones, though in some instances when
the molecule is small , like the neurohypophysial hormones, it is first conjugated to a larger
molecule like serum albumin. Peptide normones are labelled with 131 1 or
125
1, and these react
with the specific antibody to form a complex. When unlabelled hormone, either as a'standard' or 'unknown', is add ed, it competes with the labelled hormonefor the ant ibody so that
the rat io of ' bo und' to 'free' labelled hormone decreases. The 'bound' and 'free' components
are separated by absorption onto materials like charcoal, cellulose or a second antibody,
and the radioactivity of each counted. The methods are not free of problems and the presence
of interfering substances can necessitate special separation procedures. The use of immunological assays has been largel y confined to mammalian hormones, though the y have been
used to indicate the degrees of structural similarity of peptide hormones from different vertebrates. Their potential use, as a tool for investigating the different peptide hormones
throughout the vert ebrate series, is considerable.
2. The Pituitary Gland
The pituitary gland plays a central role in the maintenance and regulation of the
secretions of three other endocrine glands, produces several additional hormones
47
products that may be formed . Such procedures, utilizing added substrates , can be used to
demonstrate the pr esence of certain steroidogenic enzymes, but do not indicate whether
or not the products are normally formed or released.
Although in the past steroid hormones were principally assayed and identified by biological means, these are becoming less frequent as sophisticated chemical and radioisotopic
methods become available. Steroid hormones can be separated from other biological substances, and from each other by extraction and partition in suitable solvents such as chloroform, methylene chloride or ethyl acetate, followed by such procedures as gel filtration and
chromatography. They can be measured by colourimetric methods such as that utilizing
the yellow colour developed with phenylhydrazine (PORTER-SILBER reaction for cortisol
and corticosterone), or by using their ability to fluoresce after treatment with ethanolicsulphuric acid, sodium hydroxide or potassium terbutoxide. Steroid hormones can also be
measured by gas chromatography and rad ioisotope methods, such as the double - isotope
dilution method, which is particularly sensitive for hormones, like aldosterone, that are
present at low concentrations. Isotopes of the steroids can be util ized in man y quantitative
procedures, and especiall y to assess losses of material that inevitably occur during their
purification and preparation for assay. Identification ofsteroid hormones is generally based
on their ph ysico-chemical beh aviour, this includes such things as :
1. Chromatographic mobilities in different solvent systems as compared with parallel
standards which, in order to assist their location, can be isotopically labelled. Changes In
chromatographic behav iour following acetylation of the steroids can be used as an additional
criterium for identification.
2. Characteristic colour reactions such as tak e place with PORTER-SILBER reagent and
tetrazolium blue.
3. Fluorescence, either natural or after treatment with such agents as ethanolic sulphuric
acid or sodium hydroxide.
4. Measurement of ultra-violet absorption spectra in sulphuric acid , ethanol or methanol
and infra-red spectra in chloroform.
A consensual definitive statement regarding th e problems and desired criteria for iden -
tification and measurement of steroid hormones has recently been made (BROOKS et al.,
1970).
f) Immunoassay
Immunoassay procedures, especially those using isotopically labelled hormones, have in recent years allowed great strides to be made in the measurement of various protein hormones.
Specific antibodies can be made to most peptide hormones, though in some instances when
the molecule is small , like the neurohypophysial hormones, it is first conjugated to a larger
molecule like serum albumin. Peptide normones are labelled with 131 1 or
125
1, and these react
with the specific antibody to form a complex. When unlabelled hormone, either as a'standard' or 'unknown', is add ed, it competes with the labelled hormonefor the ant ibody so that
the rat io of ' bo und' to 'free' labelled hormone decreases. The 'bound' and 'free' components
are separated by absorption onto materials like charcoal, cellulose or a second antibody,
and the radioactivity of each counted. The methods are not free of problems and the presence
of interfering substances can necessitate special separation procedures. The use of immunological assays has been largel y confined to mammalian hormones, though the y have been
used to indicate the degrees of structural similarity of peptide hormones from different vertebrates. Their potential use, as a tool for investigating the different peptide hormones
throughout the vert ebrate series, is considerable.
2. The Pituitary Gland
The pituitary gland plays a central role in the maintenance and regulation of the
secretions of three other endocrine glands, produces several additional hormones
47