Analogous biological methods have been (and in some cases are still) used for initially measuring quantities of many hormones. Thus, the potency of adrenocortical extracts were originally measured by their ability to promote survival of adrenalectomized mammals , alter
renal sodium excretion or promote the formation of liver glycogen . Thyroid extract activity
has been estimated by its ability to increase metamorphosis of tadpoles, and decrease the
suvival time of mice in closed jars (due to increased rate of oxygen usage). Changes in bloodglucose levels and glycogen content of rat diaphragm muscle can be used to gauge insulin
concentration, while corticotrophin can be measured by its ability to decrease the amount
of ascorbic acid in the adrenal cortex . Amounts of neurohypophysial hormones are still assessed by measurement of their ability to produce effects such as antidiuresis,increased blood
pressure, contraction of the uterus or promotion of 'milk let down' in mammals. Other assays of neurohypophysial hormones utilize biological responses in such diverse groups as
birds, reptiles, and amphibians.
Adequate quantitative biological.expression of a hormonal activity requires the adoption
of a 'standard', with which the strength of an 'unknown' extract can be compared. If the
precise chemical structure of the hormone is known, and it is available in a pure form, the
effect of a known amount (the 'standard') can be compared with the 'unknown,' and the
result expressed in terms of the weight or molecular concentration. If, on the other hand,
the exact chemical nature of the hormone is unknown, or if it is unavailable in a sufficiently
pure chemical form, an arbitrary' biological standard' can be made . This usually consists
of a suitably prepared and rreserved quantity of the endocrine tissue, which is assigned a
potency, usually in terms 0 units (D.) of a certain biological activity per unit weight. Thus,
the international standard for neurohypophysial hormones consists of dried and powdered
beef neurohypophyses, stored under refrigeration in the presence of a dessicant , Each milligram of this powder has been assigned 2 1.U. (international units) of vasopressor-antidiuretic activity, as measured by its ability to increase the blood pressure or produce an antidiuresis in rats, and 2 LD. of oxytocic activity, as shown by its ability to contract the rat
uterus . Pure neurohypophysial peptides have been made and one milligram of vasopressin
(ADH) is equivalent to 450 LD. activity, while one mgof oxytocin has 400 LU. A 'standard'
need not have an international status, though this helps comparison of results in different
laboratories, but can be made up for nresent and future reference by a particular investigator.
Identification of a hormone can be made by measuring its various activities and properties . Such 'profiles' or 'fingerprints', may utilize various biological actions, physico-chemical properties and imm unological behaviour. A pharmacologicalprofileof some of the many
activities of neurohypophysial hormones is shown in Table 2.1. Many biological actions may
be shared by several hormones, but the ratios of the potencies of the different hormones,
with respect to each of their activities, vary so that when several such criteria are used, almost
positive identification of a substance is possible. In addition to pharmacological criteria,
chemical and enzymological evidence can also be used to aid identification of the
neurohypophysial peptides; incubation with thioglycollate breaks the disulphide bridge in
the hormone and abolishes its actions, while tyros inase and trypsin also destroy the activities
of some such peptides. Chromatographic behaviour can also be used to aid identification
of hormones, though in some instances, as with oxytocin, mesotocin and isotocin, they can
behave similarly .
Despite the fact that the chemical structure of many polypeptide hormones is known,
their chemical determination is not feasible. This is partly due to their presence in low conentrations, especially compared to other protein materials that are also present. Separation
of the various components would be essential before chemical determination would be even
possible, while the tedious nature and inevitable losses make such procedures routinely inconceivable . Thus, apart from immunological procedures, biological methods have been retained as the most convenient, sensitive, specific and accurate ways for the determination
of the polypeptide hormones . In the instance of the neurohypophysial hormones, bioassay
procedures are still superior to immunoassay methods.
Identification of steroidhormones in tissues is often hampered by the small amounts of
material that may be present. In order to increase the concentrations, suspected steroidogenic tissues are often incubated in vitro prior to extraction and identification procedures . These methods can be refined further by addition of exogenous steroid substrates,
45
renal sodium excretion or promote the formation of liver glycogen . Thyroid extract activity
has been estimated by its ability to increase metamorphosis of tadpoles, and decrease the
suvival time of mice in closed jars (due to increased rate of oxygen usage). Changes in bloodglucose levels and glycogen content of rat diaphragm muscle can be used to gauge insulin
concentration, while corticotrophin can be measured by its ability to decrease the amount
of ascorbic acid in the adrenal cortex . Amounts of neurohypophysial hormones are still assessed by measurement of their ability to produce effects such as antidiuresis,increased blood
pressure, contraction of the uterus or promotion of 'milk let down' in mammals. Other assays of neurohypophysial hormones utilize biological responses in such diverse groups as
birds, reptiles, and amphibians.
Adequate quantitative biological.expression of a hormonal activity requires the adoption
of a 'standard', with which the strength of an 'unknown' extract can be compared. If the
precise chemical structure of the hormone is known, and it is available in a pure form, the
effect of a known amount (the 'standard') can be compared with the 'unknown,' and the
result expressed in terms of the weight or molecular concentration. If, on the other hand,
the exact chemical nature of the hormone is unknown, or if it is unavailable in a sufficiently
pure chemical form, an arbitrary' biological standard' can be made . This usually consists
of a suitably prepared and rreserved quantity of the endocrine tissue, which is assigned a
potency, usually in terms 0 units (D.) of a certain biological activity per unit weight. Thus,
the international standard for neurohypophysial hormones consists of dried and powdered
beef neurohypophyses, stored under refrigeration in the presence of a dessicant , Each milligram of this powder has been assigned 2 1.U. (international units) of vasopressor-antidiuretic activity, as measured by its ability to increase the blood pressure or produce an antidiuresis in rats, and 2 LD. of oxytocic activity, as shown by its ability to contract the rat
uterus . Pure neurohypophysial peptides have been made and one milligram of vasopressin
(ADH) is equivalent to 450 LD. activity, while one mgof oxytocin has 400 LU. A 'standard'
need not have an international status, though this helps comparison of results in different
laboratories, but can be made up for nresent and future reference by a particular investigator.
Identification of a hormone can be made by measuring its various activities and properties . Such 'profiles' or 'fingerprints', may utilize various biological actions, physico-chemical properties and imm unological behaviour. A pharmacologicalprofileof some of the many
activities of neurohypophysial hormones is shown in Table 2.1. Many biological actions may
be shared by several hormones, but the ratios of the potencies of the different hormones,
with respect to each of their activities, vary so that when several such criteria are used, almost
positive identification of a substance is possible. In addition to pharmacological criteria,
chemical and enzymological evidence can also be used to aid identification of the
neurohypophysial peptides; incubation with thioglycollate breaks the disulphide bridge in
the hormone and abolishes its actions, while tyros inase and trypsin also destroy the activities
of some such peptides. Chromatographic behaviour can also be used to aid identification
of hormones, though in some instances, as with oxytocin, mesotocin and isotocin, they can
behave similarly .
Despite the fact that the chemical structure of many polypeptide hormones is known,
their chemical determination is not feasible. This is partly due to their presence in low conentrations, especially compared to other protein materials that are also present. Separation
of the various components would be essential before chemical determination would be even
possible, while the tedious nature and inevitable losses make such procedures routinely inconceivable . Thus, apart from immunological procedures, biological methods have been retained as the most convenient, sensitive, specific and accurate ways for the determination
of the polypeptide hormones . In the instance of the neurohypophysial hormones, bioassay
procedures are still superior to immunoassay methods.
Identification of steroidhormones in tissues is often hampered by the small amounts of
material that may be present. In order to increase the concentrations, suspected steroidogenic tissues are often incubated in vitro prior to extraction and identification procedures . These methods can be refined further by addition of exogenous steroid substrates,
45