experiences with a test for the determination of guanosine-3,5-monophosphate

2
122 6.3 Z. Anal. Chem., Band 279, Heft 2 (1976) Experiences with a Test for the Determination of Guanosine-3,5-monophosphate P. Wunderwald, R. Heinze, and U. Deneke Boehringer Mannheim GmbH, Forschungszentrum Tutzing Erfahrungen mit einem Test zur Bestimmung yon Guanosin-3,5-monophosphat Best. von Guanosin-3,5-monophosphat; Proteinbindung Recently for the determination of cGMP a radio- receptor assay with a binding protein from silk moth (bombyx mori) was published by Fallon and Wyatt [1 ]. With this test the following results were obtained: 1. The cGMP-silk moth protein complex is stable over a long time, so the test is suitabIe for determina- tions in series. 2. The detection limit of 0.2 pmole cGMP, the measured precision and accuracy prove that the test is suitable for many examinations. 3. The test is not disturbed by guanosine derivatives. As it is disturbed by salts and by an excess of cAMP samples from biological material must be purified in many cases. 4. For the test purifications of samples described in the literature [5] are valid. Often the simultaneous change of cAMP and cGMP in biological material is under consideration. So it seemed to be desirable to measure both cyclophos- phates simultaneously. In order to prove the validity of the combination of the new test [1] for measuring cGMP and the Gilman test [2] for measuring cAMP the following samples were analyzed: a) a synthetic mixture of cAMP and cGMP, b) human urine, c) rat lung. Methods cGMP was measured with the silk moth protein binding test [1] using the test combination No. 15266 from Boehringer Mannheim. cAMP was determined according to Gilman [2] using the test combination No. 15 290 from Boehringer Mannheim. cAMP and cGMP in Krebs Ringer Phosphate Buffer. To the synthetic mixture ethanol/Zn acetate was added and the Zn ions precipitated as ZnCO3 [6]. After removing the cations with Dowex 50 [5] cAMP and cGMP were separated on a polyethylene (PEI) cellulose column [5] using 5 mM acetic acid for washing, 50 mM acetic acid for elution of cAMP and 50 mM ammonium formiate for elution of cGMP. cAMP and cGMP in Human Urine. In human urine cAMP and cGMP were measured after 1:100 dilution without purification or after PEI cellulose chromatography [5] as described for the synthetic mixture. cAMP and cGMP in Rat Lung. The tissue was prepared as described by Schultz et al. [6] up to removing the cations by a Dowex 50 column. Then the separation of cAMP and cGMP was performed on a PEI cellulose column [5]. For determination of the recovery of the purification methods 14C-cAMP and 3H-cGMP were added as tracer before purification. The recovery of radioactivity (the quench was determined by internal standardization) and the amount of added tracer were considered for evaluation. Results For the first evaluation of a suitable purification method for measuring cGMP with the new method and cAMP according to Gilman [2] simultaneously a mixture of cAMP and cGMP in Krebs Ringer phosphate buffer was analyzed. The purification method according to Schultz et al. [5, 6] proved to be valid as can be seen from the results given in Table 1. The recovery of cGMP (107 ~) was very good, the recovery of cAMP rather high (132~) but possibly still acceptable, if it is considered that the errors of determination of initial quantity, recovery of radio- activity and determination of cAMP after purification may be additive. For urine the question had to be answered whether purification for measuring cAMP and cGMP is necessary. Table 1 shows, that the cAMP and cGMP values are only slightly elevated compared to the values from purified samples. Normal values in human urine differ widely in literature. According to Steiner et al. [7] cGMP concent/'ation may vary between 0.6 and 2.6 gM, for cAMP concentrations between 0.6 gM and 19 gM [3,4] are published. The found values for cGMP and cAMP given in Table 1 are within this range, the cAMP/cGMP ratio being about 5:1. In a control experiment cAMP and cGMP was eliminated quantitatively from urine. These results may be a hint, that for many purposes purification of samples from human urine may not be necessary for the measurement of cAMP and cGMP as described. cGMP and cAMP were also measured in rat lung, were the levels of cGMP are reported to be rather high. Gilman [2] reported a cGMP content of 2.7pmoles/mg protein and a cAMP content of 7.0 pmoles/mg protein. If it is assumed, that 1 mg wet weight will give approximately 0.1 mg protein, a content of 0.3 pmoles cGMP and of 0.7 pmotes

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Page 1: Experiences with a test for the determination of guanosine-3,5-monophosphate

122

6.3

Z. Anal. Chem., Band 279, Heft 2 (1976)

Experiences with a Test for the Determination of Guanosine-3,5-monophosphate

P. Wunderwald, R. Heinze, and U. Deneke

Boehringer Mannheim GmbH, Forschungszentrum Tutzing

Erfahrungen mit einem Test zur Bestimmung yon Guanosin-3,5-monophosphat

Best. von Guanosin-3,5-monophosphat; Proteinbindung

Recently for the determination of cGMP a radio- receptor assay with a binding protein from silk moth (bombyx mori) was published by Fallon and Wyatt [1 ]. With this test the following results were obtained:

1. The cGMP-silk moth protein complex is stable over a long time, so the test is suitabIe for determina- tions in series.

2. The detection limit of 0.2 pmole cGMP, the measured precision and accuracy prove that the test is suitable for many examinations.

3. The test is not disturbed by guanosine derivatives. As it is disturbed by salts and by an excess of cAMP samples from biological material must be purified in many cases.

4. For the test purifications of samples described in the literature [5] are valid.

Often the simultaneous change of cAMP and cGMP in biological material is under consideration. So it seemed to be desirable to measure both cyclophos- phates simultaneously. In order to prove the validity of the combination of the new test [1] for measuring cGMP and the Gilman test [2] for measuring cAMP the following samples were analyzed:

a) a synthetic mixture of cAMP and cGMP, b) human urine, c) rat lung.

Methods

cGMP was measured with the silk moth protein binding test [1] using the test combination No. 15266 from Boehringer Mannheim.

cAMP was determined according to Gilman [2] using the test combination No. 15 290 from Boehringer Mannheim.

cAMP and cGMP in Krebs Ringer Phosphate Buffer. To the synthetic mixture ethanol/Zn acetate was added and the Zn ions precipitated as ZnCO3 [6]. After removing the cations with Dowex 50 [5] cAMP and cGMP were separated on a polyethylene (PEI) cellulose column [5] using 5 mM acetic acid for washing, 50 mM acetic acid for elution of cAMP and 50 mM ammonium formiate for elution of cGMP.

cAMP and cGMP in Human Urine. In human urine cAMP and cGMP were measured after 1:100 dilution without

purification or after PEI cellulose chromatography [5] as described for the synthetic mixture.

cAMP and cGMP in Rat Lung. The tissue was prepared as described by Schultz et al. [6] up to removing the cations by a Dowex 50 column. Then the separation of cAMP and cGMP was performed on a PEI cellulose column [5].

For determination of the recovery of the purification methods 14C-cAMP and 3H-cGMP were added as tracer before purification. The recovery of radioactivity (the quench was determined by internal standardization) and the amount of added tracer were considered for evaluation.

Resul ts

For the first evaluation of a suitable purification method for measuring cG MP with the new method and cAMP according to Gilman [2] simultaneously a mixture of cAMP and cGMP in Krebs Ringer phosphate buffer was analyzed. The purification method according to Schultz et al. [5, 6] proved to be valid as can be seen from the results given in Table 1. The recovery of cGMP (107 ~ ) was very good, the recovery of cAMP rather high (132~) but possibly still acceptable, if it is considered that the errors of determination of initial quantity, recovery of radio- activity and determination of cAMP after purification may be additive.

For urine the question had to be answered whether purification for measuring cAMP and cGMP is necessary. Table 1 shows, that the cAMP and cGMP values are only slightly elevated compared to the values from purified samples. Normal values in human urine differ widely in literature. According to Steiner et al. [7] cGMP concent/'ation may vary between 0.6 and 2.6 gM, for cAMP concentrations between 0.6 gM and 19 gM [3,4] are published. The found values for cGMP and cAMP given in Table 1 are within this range, the cAMP/cGMP ratio being about 5:1. In a control experiment cAMP and cGMP was eliminated quantitatively from urine. These results may be a hint, that for many purposes purification of samples from human urine may not be necessary for the measurement of cAMP and cG MP as described.

cGMP and cAMP were also measured in rat lung, were the levels of cGMP are reported to be rather high. Gilman [2] reported a cGMP content of 2.7pmoles/mg protein and a cAMP content of 7.0 pmoles/mg protein. If it is assumed, that 1 mg wet weight will give approximately 0.1 mg protein, a content of 0.3 pmoles cG MP and of 0.7 pmotes

Page 2: Experiences with a test for the determination of guanosine-3,5-monophosphate

6. Radioimmunoassay. II

Table I. Determination of cAMP and cGMP in different samples

123

Sample cAMP found cGMP found

Mixture with 234 pmoles cAMP/ml and 27 pmoles cGMP/ml

Human urine (0.6-19 IxM cAMP and 0.6-2.6 ~tM cGMP)

not purified purified

0.7 pmoles cAMP/mg wet weight [2] Rat lung 0.3 pmoles cGMP/mg wet weight [2]

309 pmoles/ml (132 ~o) 29 pmoles/ml (107 ~)

9.7 gM 1.8 gM 7.2 gM 1.4 gM

pmoles , pmoles 5.1 mg wet weight 0.2 mg wet weight

cAMP is calculated per mg wet weight. While the found cGMP content of rat lung given in Table 1 is within the reported range, we found higher values for cAMP.

References

1. Fallon, A. M., Wyatt, G. R.: Anal. Biochem. 63, 614 (1975)

2. Gilman, A. G. : In: Advances in cyclic nucleotide research, Vol. 2, p. 9. P. Greengard and G. A. Robison, eds. New York: Raven Press 1972

3. Kuo, J. F., Lee, T.-P., Reyes, P. L., Walton, K. G., Do- nelly, T. E., Greengard, P. J. : Biol. Chem. 247, 16 (1972)

4. Michal, G., Wunderwald, P.: In: Methoden der enzyma- tischen Analyse 2, Hrsg. H.U. Bergmeyer, 3. Aufl., S. 2186. Weinheim: Verlag Chemie

5. Schultz, G., Boehme, E., Hardman, J. G.: In: Methods in enzymology, Vo�91 38, S. P. Colowick and N. O. Kaplan, eds., p. 9. New York: Acad. Press 1974

6. Schultz, G., Hardman, J. G., Schultz, K., Davis, J. W., Sutherland, E. W. : Proc. Nat. Acad. Sci. U.S.A. 70, 1721 (1973)

7. Steiner, A. L., Wehmann, R. E., Parker, C.W., Kipnis, D. M. : In: Advances in cyclic nucleotide research, Vol. 2, P. Greengard and G. A. Robison, eds., p. 51. New York: Raven Press 1972

6.4

Vergleichende radioimmunologische Untersuchungen zur Bestimrnung von Proteohormonen mit verschiedenen Testkombinationen

Lieselotte Nocke-Finck, H. H. von Burgsdorff, H. Breuer und W. Geiger

Inst. f. Klin. Biochemie der Universitfit Bonn und Universitgts-Frauenklinik K61n

Comparative Studies on the Radioimmunological Determination of Proteohormones using Different Test Combinations

Best. von Proteohormonen; Radioimmunoassay; verschiedene Testkombinationen

Dutch die Einffihrung des Radioimmunoassay (RIA) in die klinisch-chemische Diagnostik ist eine neue Dimension der Empfindlichkeit (10-12 g) und damit die M6glichkeit er6ffnet worden, Hormone auch. in kleinen Mengen yon K6rperflfissigkeiten, insbesondere Blut, quantitativ zu bestimmen. Auf den ersten Blick erscheint die Durchffihrung radioimmuno- logischer Analysen einfach und problemlos, zumal

inzwischen zahlreiche Testkombinationen kommerziell erh/iltlich sind. Es gibt jedoch zahlreiche Fehler- m6glichkeiten, auf die der groge Kreis yon Unter- suchern, der mit den Schwierigkeiten des RIA nicht so sehr vertraut ist, aufmerksam gemacht werden soll- te. An Hand von Vergleichsuntersuchungen mit ver- schiedenen im Handel befindlichen Testkombina- tionen sowie mit Verfahren, die in langj/ihriger Arbeit selbst entwickelt wurden, wird im folgenden fiber einige Probleme des RIA berichtet. Dabei wurden folgende Hormone in die Studie einbezogen: Thyreotropes Hormon (TSH), luteinisierendes Hor- mon (LH), menschliches Wachstumshormon (HGH) und menschliches Prolaktin (HPRL).

Die Bestimmung von TSH erfolgte mit Hilfe kommerzieHer Testkombinationen verschiedenen Ursprungs (Serono, Frei- burg; CEA-IRE-SORIN, c/o Isotopendienst-West, Sprend- lingen ; Byk-Mallinckrodt, Dietzenbach-Steinberg) sowie nach der Methode von Geiger [1]. Alle vergleichenden Bestimmun-