The principles of the double-isotope derivative technique was first shown by Keston et al. (125) for the identification and assay of specific
6. CHEMICAL ASSAY OF ADRENOCORTICOSTEROIDS 333 amino acids. The method makes use of two isotopes of different energies so that one may be measured in the presence of the other. One isotope added with an internal standard is used to correct for losses throughout the procedure while the second isotope serves to measure the amount of unknown substance. After purification to constant isotope ratio, calcula
tions based only on the two isotope measurements yield the amount of unknown.
The double-isotope method for corticoids was necessary because of the extremely small amount to be found in blood. Avivi et al. (126) introduced the use of carbon-14 and tritium labeled acetic anhydride to make the labeled aldosterone derivative. The simultaneous measurement of beta emitting isotopes now possible by liquid scintillation counting has stimulated research in their use. Aldosterone, which occurs in blood in very low concentration, was analyzed by Kliman and Peterson in 1960 (24). The method was soon used in clinical and basic research studies and adapted for other steroids (121). The double-isotope method can be performed on about 5 ml of peripheral plasma or 20 ml of urine if it contains at least 0.01 μg of the corticosteroid that is to be measured. An example of the method for aldosterone in urine is as follows:
1. Addition of internal standard: A small known amount of pure carbon-14 labeled aldosterone is added to a 20-ml urine sample. The amount of this aldosterone recovered at the end of the experiment gives a measure of the efficiency of the whole assay.
2. Derivative formation: The urine sample is hydrolyzed, extracted, dried, dissolved in benzene-pyridine, and acetylated with tritium-labeled acetic anhydride. Thus, a fixed amount of tritium labeled-acetate per microgram is added to all corticoid present.
3. Purification: The published methods separate the double labeled aldosterone from contaminants by multiple paper chromatographic separation. In the case of aldosterone it can be oxidized and chroma-tographed again for more certain identification. In order to easily identify the aldosterone, unlabeled aldosterone is added to give an amount which will be visible on a paper chromatogram using ultraviolet light.
4. Measurement: The purified aldosterone containing both tritium and carbon-14 labels is dissolved in a toluene-phosphor solution and placed in an automatic, dual channel, liquid scintillation spectrometer.
Since the energies of the two isotopes differ, they can be measured in separate windows of the energy spectra. The instrument provides a printed record from which to make calculations, or the instrument can be connected directly to a card punch. The punched cards can then be used in a computer programmed to do all the calculations.
5. Computation: The amount of aldosterone-1 4C recovered, divided
334 GRANT G. SLATER
by the amount added X 100, gives the percentage recovery. The tritium is corrected for losses through the procedure, then the tritium count is converted to a corresponding value in micrograms of aldosterone, the conversion factor being determined by the specific activity of the tritium-labeled acetic anhydride reagent. Actually more than one corticosteroid could be determined on the same sample, if different chromatographic fractions were used. The yield of each corticosteroid is found in the differ-ent fractions isolated during chromatography, and concdiffer-entrations are computed from the final carbon-14 and tritium content of each corti-costeroid. For each corticosteroid the tritium content measures total steroid, while the carbon-14 content measures the yield of internal standard. Even the small amount of tracer aldosterone added is subtracted to get the exact amount of original aldosterone.
V I . OTHER PHYSICAL METHODS
A number of other physical methods have been applied to the separa-tion and identificasepara-tion of adrenocortical steroids. Ultraviolet and infrared absorption spectra are still used for identification when sufficient corti-costeroid is obtainable for the method. Countercurrent distribution has been used for separation, purification, and even for identification, but this method has not found widespread use. Recently, Kornel (127) has used high voltage paper electrophoresis to effect a separation of a number of naturally occurring steroid conjugates and applied this method to the separation of cortisol-4-l 4C conjugates in human plasma (128).
A major contribution to all methods is the use of reagents of high purity. Recently some of the larger chemical companies have placed on the market solvents which contain very low levels of contamination, so low that further purification should be unnecessary in most of the known methods. The use of highly purified reagents should, when properly applied, help to increase the sensitivity of the methods and permit in the future the measurement of the minor steroids where previously it was not possible.
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