Abstract
For assay of urine concentration of gonadotropic substances in normal or non-pregnant women it is necessary to prepare concentrated extracts which are not toxic to immature female rats. Complete recovery of active material is highly desirable. Tests on 6 published extraction methods have failed to yield satisfactory total recovery when applied to urine from pregnant women. The various steps in some of these processes have been studied, and sources of loss have been identified.
Decrease in potency from concentrating urine by low temperature evaporation is probably not so much a temperature effect as due to the adsorption on the urinary solids. Washing these solids with 33% acetone at pH 5 yielded significant amounts for each of 5 washings.
Acetone precipitations of gonadotropic material from urine are incomplete unless the acetone is 95% (20 volumes) at room temperature or 16 volumes at 38°C. The acetone precipitation alone does not remove all toxic materials.
Urine acidified to pH 5 with glacial acetic acid will allow of complete adsorption of gonadotropic substances by Lloyd's reagent or norit. Permutit was less active. Elution from norit was, however, incomplete with a number of reagents. From Lloyd's reagent partial recovery was possible with ammonia-free distilled water, 33% acetone, 50% acetone, or 0.1 N sodium hydroxide. Complete recovery was found with 50% aqueous pyridine, or alkalinized acetone (50%). Better yields were obtained with equal parts of C.P. acetone and 0.2 N sodium hydroxide than with two parts of alkali to one of acetone.
The following extraction method is proposed to secure non-toxic extracts from pregnancy urine without appreciable loss of gonadotropic potency.
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