Enzymatic Inhibition of Diphosphonate: A Proposed Mechanism of Tissue Uptake, Lecture notes of Nuclear medicine

Download Enzymatic Inhibition of Diphosphonate: A Proposed Mechanism of Tissue Uptake and more Lecture notes Nuclear medicine in PDF only on Docsity! Enzymes have been proposed as tissue recep tors that bind O9mTc.stannous diphosphonate and its analogs. Incubation of diphosphonate with several enzymes demonstrated inhibition of acid and alkaline phosphatase activity but showed no effect on glutamic oxalacetic transaminase and lactate dehydrogenase activity. Complete re versal of the diphosphonate-induced inhibition of alkaline phosphatase activity occurred when calcium ion was added to the reaction. The spe. cificity of calcium to induce reversal was dis pelled when magnesium ion gave identical results. Diphosphovuite-induced inhibition of acid phosphatase, however, was not reversed by calcium or magnesium. The chemical adsorption of diphosphonate to hy droxyapatite crystals of bone was proposed by Fran cis and associates in I 969 ( 1 ,2 ) . The skeletal uptake of oDmTc@stannousdiphosphonateand its analogs, 99@'Tc-stannous polyphosphate and DftmTc@stannous pyrophosphate, is attributed to the same reaction. The process. however, fails to elucidate the mecha nism of extraosseous uptake of these radiopharma ceuticals when neither calcification nor active bone formation are demonstrable. These agents have been observed to concentrate in recent infarcted heart muscle (3) , infarcts of the cerebral cortex (4) , and in breast carcinomas (5). Our efforts to resolve this dilemma have led us to hypothesize the likely presence of tissue receptors that selectively bind the labeled phosphate radio pharmaceuticals. Support for this hypothesis and the possible character of the receptor was provided when histochemical stains of biopsied tissue showing in creased DomTcstannous diphosphonate uptake dem onstrated qualitative increased amounts of acid and alkaline phosphatase (6) . This report is an exten sion of these observations and assesses the in vitro effect of stannous diphosphonate on enzyme ac tivity. MATERIALSAND METHODS Enzyme kinetics. Five enzymes—acid phosphatase from wheat germ, alkaline phosphatase from Escheri chia coli, alkaline phosphatase from chicken intes tine, glutamic oxalacetic transaminase from porcine heart, and lactate dehydrogenase from rabbit mus cle—were obtained commercially from the Worth ington Biochemical Corp., Freehold. N.J. Enzymatic activity was determined by adding enzyme to the appropriate substrate and measuring the end prod uct spectrophotometrically with a Beckman Acta Ill spectrophotometer. The substrates, reactions, end product, and spectrophotometer wavelengths (nm) for the enzymes are shown in Table I. A cuvette reaction vessel was used to study the enzyme kinetics. The total reaction volume was 1.5 ml and the activity of each enzyme was measured prior to adding the diphosphonate. Stannous diphos phonate, obtained from Diagnostic Isotopes, Upper Saddle River, N.J., was added in three different con centrations ( I , 2, and 4 @mole)to the reaction vessels of each enzyme and their activity was moni tored for preselected time periods to determine in hibition. Two observations were made at different times for each of the reactions. No significant van ations in pH were recorded in the reaction vessel throughout the studies. Calcium and magnesium ion effects. Calcium chloride (CaCl2) , in concentrations ranging from 0.025 to 2.3 @mole,was added to acid phosphatase and alkaline phosphatase systems containing stan nous diphosphonate and its effect on the enzyme activity was determined. This was repeated later using the enzymes without stannous diphosphonate. The systems were monitored for either 6 or 10 mm Received Aug. 29, 1974; revision accepted Dec. 4, 1974. For reprints contact: R. A. Holmes. Nuclear Medicine Div., Milwaukee County Medical Complex, 8700 W. Wis consin Ave., Milwaukee, Wis. 53226. 352 JOURNAL OF NUCLEAR MEDICINE ENZYMATIC INHIBITION OF DIPHOSPHONATE: A PROPOSED MECHANISM OF TISSUE UPTAKE A. Mjchael Zjmmer,All T. Isitman,and RichardA. Holmes Milwaukee County Medical Complex, Medical College of Wisconsin, Milwaukee, Wisconsin EndproductEnzyme Substrate Reaction measured Absorbance (nm) Salicylic acid 300 TABLE 1. TECHNIQUE TO DETERMINE ENZYME ACTIVITY Acid phosphatase (wheat germ) o-Carboxyphenylphos phate o-Carboxyphenylphos phate —@salicylic acid + phosphate Alkali ne phosphatase p-Nitrophenylphos (E. coli + chicken intestine) phate p-Nitrophenylphosphate -+ p-Nitrophenol + phos p-Nitrophenol 405 phate Glutomic oxalacetic trans aminase (porcine heart muscle) a-Ketoglutarate a-Ketoglutarote oxoloce tate + NADH -@ malate NADH disappearance 340 + NAD Lactate dehydrogenase (rob- Lactate bit muscle) Lactate + NAD pyruvate + NADH NADH 340 to determine enzyme activity. The procedures were repeated using magnesium chloride (MgCl@6H@O) in a concentration of I .2 jzmole in both acid and alka line phosphatase systems. The results of each reac tion were plotted on linear graph paper with ab sorbance in nanometers on the ordinate and time in seconds on the abscissa. RESULTS The effect of diphosphonate on the enzyme sys tems is shown in Figs. I and 2. Figure 1A describes the linearity of the acid phosphatase activity over a 10-mm period and the effective decrease (inhibi tion ) of the enzymatic activity during the same time interval with the addition of increasing amounts of stannous diphosphonate. One micromole of stannous diphosphonate caused 26% , 2 @mole28% , and 4 @rnole29% inhibition of acid phosphatase activity 10 mm after initiating the reaction. The results are only qualitative and show no stoichiometric relation ship to the diphosphonate concentration. Adding stannous chloride to the acid phosphatase system did not change the linearity of the enzyme activity. Figure 1B demonstrates the effect of diphospho nate on alkaline phosphatase (chicken intestine) activity. The enzyme shows significant and propor tional inhibition with increasing concentrations of diphosphonate. Seventy-seven percent, 83% , and 89% inhibition were recorded for 1, 2, and 4 @mole of diphosphonate, respectively, 6 mm after initiating the reaction. The results were identical for alkaline phosphatase from E. coli. Figure 2 graphs the effect of diphosphonate on the activity of glutamic oxalacetic transaminase and lactate dehydrogenase, respectively. Both enzymes showed no inhibition of their activity with increasing diphosphonate concentrations. Adding calcium ion to the enzyme system was predicated on the assumption that its presence was important, if not essential, to skeletal uptake of 9tsmTcstannous diphosphonate and its analogs. Cal cium ion alone did not alter the linear activity of either acid or alkaline phosphatase (Fig. 3 ) . How ever, when calcium was added to the diphosphonate inhibited alkaline phosphatase systems, the inhibi tion was completely reversed (Fig. 3A) . The 2 B FIG. 1. Effectof diphosphonate(DiP)on acidphosphatase (AcP) activity (A) and alkaline phosphatase(AlP) activity (B). 015 . #cP £AcP+l@mDIP •AcP42MmDIP 0 AcP+4Mm DIP I0 A . AlP C AlP . l@m DIP CAIPs 2um DIP OAIP+4um DIP 0.10 ThVt(MIN)--' Volume 16, Number 5 353