Michael C. Tobes

Scintigraphic Localization of Pheochromocytoma

We used a new radiopharmaceutical agent, [131I]meta-iodobenzylguanidine ([131I]MIBG), to produce scintigraphic images of pheochromocytomas in eight patients. One day or more after injection, the only normal organ that displayed distinct concentrations of radioactivity was the urinary bladder. The [131I]MIBG was probably concentrated in adrenergic vesicles; in tissues where vesicles are numerous, such as pheochromocytomas, the radionuclide was retained for days. The spectrum of pheochromocytomas shown the scintigrams was broad: intra-adrenal and extraadrenal in location, benign and malignant in character, 0.2 to 65 g in weight, and with different hormone patterns in secretion. Tumors in four patients were not detected by computed tomography. In one patient, reoperation was undertaken only because the scintigram located the extra-adrenal tumors and thereby directed the surgeons exploration. The method offers hope of safe and reliable localization of pheochromocytomas in their many guises.

EFFECTS OF CARDIAC SYMPATHETIC INNERVATION ON CORONARY BLOOD FLOW

BACKGROUND The role of cardiac sympathetic nerves in regulating coronary blood flow is controversial. We sought to determine the degree to which cardiac efferent sympathetic signals modulate coronary blood flow. The heterogeneous sympathetic reinnervation in transplanted hearts provides a model for studying the vasomotor responses to adrenergic stimulation in reinnervated and denervated coronary territories of the same heart. METHODS We studied 14 cardiac-transplant recipients who had normal coronary arteries and no evidence of rejection and 8 normal subjects. We used positron-emission tomography with [(11)C]hydroxyephedrine, an analogue of norepinephrine, to delineate sympathetic innervation. Using [(13)N]ammonia, we measured myocardial blood flow at rest, during adenosine-induced hyperemia, and in response to sympathetic stimulation induced by cold pressor testing. RESULTS In the transplant recipients, the uptake of [(11)C]hydroxyephedrine was greater in the territory served by the left anterior descending artery (0.15+/-0.01) than in those served by the right coronary artery (0.07+/-0.01, P<0.001) or the circumflex artery (0.09+/-0.01, P<0.001). The basal flow was similar in all three regions, as was the percent increase in flow during hyperemia. However, the increase in flow in response to cold pressor testing was higher in the territory of the left anterior descending artery (46+/-10 percent) than in those of the right coronary artery (16+/-5 percent, P=0.01) or the circumflex artery (23+/-6 percent, P=0.06), although the changes in hemodynamics and levels of circulating catecholamines were similar. No such regional differences were observed in the normal subjects. CONCLUSIONS Increases in coronary blood flow in response to sympathetic stimulation correlated with the regional norepinephrine content in the cardiac sympathetic-nerve terminals. These findings suggest that cardiac adrenergic signals play an important part in regulating myocardial blood flow.

Platelet alpha2 adrenoreceptors in chronic congestive heart failure

Patients with chronic congestive heart failure (CHF) are known to have elevated plasma concentrations of norepinephrine. Although this elevation of catecholamines in plasma may facilitate myocardial contractility, it may also be toxic to the myocardium in the long term. The alpha2 adrenoreceptor located on noradrenergic nerve terminals regulates neuronal norepinephrine release by feedback inhibition. This receptor is also located on human blood platelets. This study determines, the status of platelet alpha;! adrenoreceptors in 16 patients with CHF (class I and II in 7 and class Ill and IV in 9) and in 26 normal volunteers. Specific high-affinity binding of the alpha* agonist 3H-clonidine and the alpha* antagonist 3H-yohimbine was used to determine the number (B,,,) of alpha* receptors and the dissociation constant (Ku) for the 2 ligands. In the control population, the B,,, (in fmol/mg protein) for 3Hclonidine was 33 f 2 and for 3H-yohimbine was 165 f 12. There was a 25 % difference in the maximum number of specific binding sites for 3H-clonidine in the class III/IV group (B,,, 24 f 2, p X0.05) and a 43 % difference in the maximum number of specific binding sites for 3H-yohimbine (B,,, 94 f 9; p <0.005). There was a smaller but nonsignificant difference in the number of receptors on platelets from patients in the class I and II group. The Ku’s were similar in all 3 groups. These differences correlated well with the increases in plasma norepinephrine levels between the normal group (273.6 f 44.1 pg/ml) and the class III/IV group (1333.5 f 244.9, p <0.0005). This study supports the hypothesis that increased levels of circulating norepinephrine in CHF lead to a decrease in platelet alpha* adrenoreceptors. Further studies should be performed to determine whether pharmacologic stimulation of these receptors might lead to a decrease in the neuronal release of that norepinephrine which might be toxic to the myocardium. Monitoring of platelet alpha* adrenoreceptor number may provide a guide to therapy of CHF.

L-kynurenine aminotransferase and L-α-aminoadipate aminotransferase. I. Evidence for identity

Summary The following observations strongly suggest that L-kynurenine aminotransferase (KAT) and L-α-aminoadipate aminotransferase (AAT) activities are associated with the same protein: 1. a similar distribution in the supernatant and mitochondrial fractions of kidney and liver of male rats, 2. a similar distribution in these fractions of kidney and liver of female rats, 3. a similar sex difference in the supernatant and mitochondrial fractions of kidney with no sex difference in liver fractions, 4. a similar pattern of inhibition with a homologous series of dicarboxylic acids with adipate being most effective, 5. competitive inhibition of KAT with d , l -α-aminoadipate (αAA), 6. substrate inhibition of KAT with α-ketoadipate (αKA), and 7. AAT activity in a highly purified preparation of KAT. A scheme for the interaction of the two activities in lysine and tryptophan metabolism is presented.

Analytical and chromatographic techniques in radiopharmaceutical chemistry

Thin-Layer Chromatography.- 1 Instrumental Evaluation of Thin-Layer Chromatograms.- Sample Application.- Mode Selection for Scanning Densitometry.- Instrumentation for Scanning Densitometry.- Instrument Parameters that Affect the Performance of Slit-Scanning Densitometers.- Protocol for Measuring the Sensitivity of a Slit-Scanning Densitometer.- Qualitative Sample Identification by HPTLC and Scanning Densitometry.- Quantitation of Separated Components by Scanning Densitometry.- Reagents Used to Enhance Fluorescence of Organic Compounds on Thin-Layer Plates.- Radiochromatography on Thin-Layer Plates.- Conclusions.- References.- 2 Radioanalytical Techniques: ITLC, TLC, Mini-Columns, and Electrophoresis.- ITLC.- Radio-TLC.- Mini-Columns.- Electrophoresis.- Conclusions.- Future Considerations.- References.- 3 Radio-Thin-Layer Chromatogram Imaging Systems-Performance and Design.- Instrumentation.- Resolution and Efficiency.- Performance and Operation of the TLC Imaging Systems.- Conclusion.- References.- 4 Detection of Radiochromatograms and Electropherograms with Position- Sensitive Wire Chambers.- The TLC Linear Analyzer.- Data Acquisition System.- Multiplate Detector.- Performance of the Linear Analyzer.- Quantitative Measurements.- Experience with the Linear Analyzer.- Determination of the Radiochemical Purity of Radiopharmaceuticals.- Summary.- References.- High Pressure Liquid Chromatography.- 5 Components for the Design of a Radio-HPLC System.- Columns and Packings.- Components of a Radio-HPLC System.- Conclusion.- References.- 6 Overall Radio-HPLC Design.- Components of the Basic System.- A More Complex Radio-HPLC System 130.- Additional Considerations.- A Specialized HPLC System.- Summary 147.- References.- 7 Quantitation of Radiolabeled Molecules Separated by High Pressure Liquid Chromatography.- Detection of HPLC-Separated Radioactive Compounds.- Detector Design.- Considerations in Use of Detector.- Applications.- Conclusion.- References.- 8 Flow Detector Designs: Build Your Own or Buy?.- Requirements.- Types of Flow Cells.- Data Acquisition and Analysis Considerations.- Conclusions.- References.- Applications.- 9 Radio-HPLC: Application to Organics and Metal Chelate Chemistry.- Stability Under HPLC Conditions.- TC-N2S2 Chelates.- Use of HPLC in Stereochemical Studies of Tc and Re Penicillamine Complexes.- HPLC in the Development and Analysis of HIDAs.- Phosphines.- Phosphonates.- Conclusions.- References.- 10 Concepts and Techniques Used in Metabolic Tracer Studies.- Requirements of Analytical Techniques for Metabolic Studies.- Applications in Tracer Kinetic Models.- Conclusions.- References.- 11. Development of No-Carrier-Added Radiopharmaceuticals with the Aid of Radio-HPLC.- Radio-HPLC.- Scaling Radiolabeling Reactions to nca Levels.- Identification of the nca Radiolabeled Compound.- Examples of nca Radiolabeling Experiments 238.- Conclusions.- References.- 12 From Cyclotron to Patient via HPLC.- HPLC Systems.- Columns.- Solvent Systems.- Dedicated vs General Instruments.- Detectors.- Pre-Columns and Filters.- Is HPLC Truly Necessary?.- Conclusions.- References.- 13 Potential Artifacts in the Chromatography of Radiopharmaceuticals.- Choice of Analytical Methods.- Radiochromatography.- Potential Artifacts/Inconsistencies in Radiochromatography.- Illustrative Examples.- Summary.- References.- 14 HPLC of Radiolabeled Antibodies.- 279.- Theory.- An HPLC System for the Analysis of Radiolabeled Antibodies.- Applications.- Discussion.- Conclusions.- References.

Adrenal cortical 11β-hydroxylase and side-chain cleavage enzymes. requirement for the A- or B-pyridyl ring in metyrapone for inhibition

The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical.

Kynurenine aminotransferase and α-aminoadipate aminotransferase: III. Evidence for identity with halogenated tyrosine aminotransferase

Abstract A nearly homogeneous preparation of α-aminoadipate (kynurenine) aminotransferase exhibited substantial activity with 3,5-diiodo-L-tyrosine, a major substrate for halogenated tyrosine aminotransferase. The new activity was found, according to heat inactivation and several inhibition studies, not to be attributable to contamination. Many of the properties previously reported for the two enzymes are identical or very similar. This paper lists these similarities and reports our observations of additional similarities of these activities in the supernatant and mitochondrial fractions of both rat kidney and liver. The properties of the purified enzyme and the noted similarities suggest that α-aminoadipate aminotransferase, kynurenine aminotransferase, and halogenated tyrosine aminotransferase activities are associated with the same protein. These activities are discussed in terms of a possible role in thyroid hormone metabolism.

OPPOSING ACTIONS OF Ca++ AND ATP PLUS Mg++ IN CONTROLLING THE KYNURENINE AMINOTRANSFERASE ACTIVITY OF ISOLATED RAT-KIDNEY MITOCHONDRIA

A new method of monitoring changes in the permeability of the mitochondrial inner membrane to substrates, based on mitochondrial kynurenine aminotransferase activity, is applied in this study of a requirement for Ca++ for optimal translocation. At pH 7.25, the Ca++ requirement was satisfied by endogenous (non-added) Ca++. The endogenous activity was decreased by 1.4 mM SrCl2, MnCl2, MgCl2, BaCl2, and LaCl3 and was unaffected by 1.4 mM NaCl, KCl, and NH4Cl. It was depressed to a minimum by 70 μM EGTA and rapidly restored by 70 μM CaCl2. The restored level was again depressed (rapidly) by added ATP + Mg++. The observations are discussed as a possible reflection of a known role of the Ca++Mg++ ratio in determining mitochondrial membrane permeability.