Imaging MAO-A Levels by PET Using the Deuterium Isotope Effect

MONOAMINE OXIDASE (MAO)

MAO is one of a class of metabolic enzymes involved in the disposition of biogenic and xenobiotic amines by oxidative metabolism. In common with other amine oxidases, such as copper amine oxidase and benzylamine oxidase, MAO catalyzes the oxidation of the α-carbon of aliphatic amines, resulting in scission of the C—N bond and producing the corresponding aldehyde or ketone and amine (or ammonia) (Fig. 1).

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FIGURE 1.

Diagrammatic mechanism of MAO-catalyzed amine oxidation.

The enzyme was discovered and its biochemical scope was determined in the middle of the 20th Century (5). In 1988, Johnston (6) described 2 subtypes of the enzyme based on their selective inhibition. Subtype A (MAO-A) is inhibited by clorgyline and preferentially catalyzes oxidation of serotonin, whereas MAO-B is inhibited by l-deprenyl and preferentially acts on phenethylamine and benzylamine. Subsequent work has shown that the isozymes are distinct proteins coded by separate genes, although they share considerable homology in their genetic sequence (7). The proposed mechanism of MAO-catalyzed oxidation outlined in Figure 1 involves formation of a substrate–enzyme complex and oxidation via a flavin adenine dinucleotide (FAD) cofactor to an imine (Schiff’s base), which hydrolyzes to the corresponding aldehyde or ketone and residual amine, or ammonia if the original amine was primary (8). MAO inhibitors (MAOI) have applications as therapeutic drugs—for instance, in the treatment of depression (9) and Parkinson’s disease (10). Patients receiving MAOI therapy are admonished to avoid fermented food such as cheese to avoid hypertensive episodes (so-called tyramine crises or the “cheese effect”) (11,12) that result from inhibition of metabolism of the vasoactive constituent tyramine. Dietary tyramine is normally metabolized by MAO, largely by MAO-A in the intestinal wall (13).

DEUTERIUM ISOTOPE EFFECT

It is a basic tenet of isotopic tracer theory that isotopes of an element, having the same number of protons but a different number of neutrons, have the same chemical properties. Chemical properties are controlled by the configuration of orbital electrons, the number of which is determined by the number of protons in the nucleus. Nevertheless, the atomic mass (determined by the number of protons plus neutrons) does have an effect on certain chemical properties that depend on bond deformation, such as infrared absorption bands and rates of bond-making or -breaking reactions. Thus, whereas compounds with different isotopic composition undergo the same chemical reaction pathways, the rates of these reactions are not precisely the same, an example of the ponderal effect—that is, physicochemical properties influenced by differences in mass. The isotope effect is greater in the lighter elements, where the relative change in atomic mass is greater, and greatest in the lightest element, hydrogen, in which adding neutrons to the nucleus doubles or triples the atomic mass for deuterium (²H, D) and tritium (³H, T), respectively. The kinetic isotope effect has become a useful tool in the determination of the mechanism of chemical and biochemical reactions. In general, a large isotope effect indicates that the bond containing the isotopic label is involved in the rate-determining step—that is, the slowest step in a sequence of intermediate reactions. The difference in rate of reaction of isotopomers (compounds that differ only in the isotopic composition of one or more atoms) has even been used as a device to direct synthetic sequences via a deuterated intermediate that does not appear in the final product (14). Amine oxidases show significant isotope effects. Substitution of the α-hydrogen atoms of biogenic amines does not affect binding of the substrate to the enzyme but reduces the rate of oxidation by factors up to 5.5 (8). Studies of stereospecifically labeled amines have shown that MAO selectively catalyzes loss of the pro-R α-hydrogen atom (Fig. 1), in contrast to other amine oxidases, which affect the pro-S atom or are not selective (15).

DUAL-TRACER MAO IMAGING

Fowler and coworkers at Brookhaven National Laboratory (4) have used these properties to design radiotracer suites to improve in vivo quantitation and help define specific uptake, generating radiotracers for MAO-A (¹¹C-clorgyline and its deutero analog ¹¹C-clorgyline-D₂) (16) and for MAO-B (¹¹C-deprenyl and ¹¹C-deprenyl-D₂) (17). In an elegant study design, native enzyme levels are estimated by PET and arterial plasma analysis after administration of ¹¹C-labeled tracer, followed by a second study with the specifically deuterium-substituted isotopomer. By comparing the model-derived measure of MAO activity for the deuterio- versus protio-tracer, additional information about the specificity of radiotracer uptake is obtained. Those areas with greatest difference between deuterium and protium correspond to areas where the radiotracer interacts most directly with the enzyme. A modified 2-tissue, 3-compartment model (18) is used to derive the measure of enzyme activity. In such a model, transfer between arterial plasma and organ is represented by K₁ and k₂, respectively; the ratio K₁/k₂, often assigned the designation λ, represents a measure of permeability or tissue uptake. Transfer between nonspecific tracer (combining free and nonspecifically bound) and enzyme-bound tracer is given by rate constants k₃ and k₄, respectively. Setting the k₄ term (rate of dissociation from specifically bound compartment) to zero accounts for the irreversible trapping of radiotracer during metabolic oxidation (19). The k₃ term is related not only to specific binding to the protein but also to the catalytic bond-breaking process. For most tissues, the most robust model parameter was found to be the product λk₃ except for the lung, where k₃ alone was more reliable. This phenomenon has been investigated further and found to be related to the larger permeability term (K₁/k₂) in smokers (20).

Thanks to the short half-life of the ¹¹C label (20.2 min), both the protio- and deuterio-forms of the radiotracer can be studied in the same individual within a few hours, thus avoiding the confound of changing physiologic states on different days and the risk of losing a study because the subject fails to return for the follow-up scan. To perform such a program effectively requires substantial development and validation of methods, to provide the capability to synthesize multiple tracers for radiopharmaceutical administration in a short time, perform rapid and accurate plasma analysis for unmetabolized tracer, and perform quantitative PET acquisition and analysis. The Brookhaven group has demonstrated this paradigm in a series of basic and applied papers (16,17,19,21–23) in the study of MAO-A and MAO-B in brain and peripheral organs.

The results of applying this paradigm to MAO-A activity in lungs, as reported in this issue of the Journal, has revealed insights into the effects of smoking that go beyond the obvious and direct effects of nicotine and other major components of tobacco smoke. As the authors point out, among the main effects of smoking, smoking exerts synergistic effects so as to increase the plasma levels of vasoactive amines, both by stimulating their release and by inhibiting their metabolism and clearance through inhibition of MAO activity. Overall, this study report, which may be viewed as the culmination of a large body of work to lay the foundation and develop the data, has revealed intriguing facets of the effect of smoking on the lungs. As in many scientific investigations, the questions it has raised may turn out to be more informative than the questions it answers. In any event, these techniques provide tools that promise to lead to a broader understanding of the biochemistry of amine metabolism in vivo.