Geoffrey A. Lyles

The metabolism of aminoacetone to methylglyoxal by semicarbazide-sensitive amine oxidase in human umbilical artery

The aliphatic amine aminoacetone has been described previously as a product of mitochondrial metabolism of threonine and glycine. Here, aminoacetone is shown to be deaminated to methylglyoxal by supernatants obtained by low speed centrifugation (600 g/10 min) of human umbilical artery homogenates, and also by membrane fractions isolated by high speed centrifugation (105,000 g/60 min) of these supernatants. Metabolism of 100 microM aminoacetone was completely inhibited by 1 mM propargylamine and MDL 72145, drugs which are capable of inhibiting the membrane-bound semicarbazide-sensitive amine oxidase (SSAO) activity found in vascular smooth muscle cells, whereas 1 mM pargyline and deprenyl which are inhibitors of monoamine oxidase, were without inhibitory effect. Estimated kinetic constants (at pH 7.8) for aminoacetone metabolism were Km = 92 microM; Vmax = 270 nmol/hr/mg protein. In addition, aminoacetone was a competitive inhibitor (Ki = 83 microM and 128 microM in low speed supernatants and high speed membrane fractions, respectively) of [14C]benzylamine metabolism by SSAO in this tissue. Aminoacetone would appear to be an endogenously occurring amine with a Km for metabolism by SSAO far lower than other aliphatic and aromatic biogenic amines examined previously as potential physiological substrates for the human vascular enzyme and possible implications of this are discussed.

Deamination of methylamine by semicarbazidesensitive amine oxidase in human umbilical artery and rat aorta

The deamination of methylamine (MA) by amine oxidase enzymes has been studied and compared with that of benzylamine (BZ) in homogenates of rat aorta and human umbilical artery by means of a radiochemical assay to estimate the radiolabelled deaminated metabolites produced, and also a spectrophotometric assay to measure H2O2 formation during the metabolism of these substrates. The effects of various inhibitors used in these assays suggest that a semicarbazide-sensitive amine oxidase (SSAO) is predominantly if not wholly responsible for the deamination of both MA and BZ in these tissues. MA was found to have a relatively higher apparent Km (102 microM in aorta; 779 microM in umbilical artery) than BZ (6.8 microM in aorta; 207 microM in umbilical artery) for metabolism by SSAO in these tissues. However, these large differences between species in the apparent Km values for each amine indicate that the biochemical properties of SSAO in human and rat vasculature are not identical. SSAO in human umbilical artery was particularly active towards MA, with a Vmax which was approximately 70% greater than that for BZ as substrate, whereas in rat aorta the Vmax for MA was around 60% of that for BZ. MA is known to occur endogenously in man and other species, and the possibility that it may be a physiological substrate in vivo for SSAO is discussed.

Vascular smooth muscle cells: a major source of the semicarbazide-sensitive amine oxidase of the rat aorta

Several methods have been used to study the distribution of the semicarbazide‐sensitive amine oxidase (SSAO) within the wall of the rat aorta. After separation of the smooth muscle‐containing layers of the tunica media from the connective tissue of the tunica adventitia, much higher specific enzyme activity (measured with 1 μM benzylamine) was found in homogenates of the media than of adventitia. Similar results were obtained for MAO‐A (with 1 mM 5‐HT as substrate). SSAO activity was also considerably higher in homogenates of cells (predominantly smooth muscle) isolated from medial tissue by enzymatic dissociation with collagenase and elastase compared with homogenates of cells (mostly of connective tissue origin) from the adventitia. Histochemical staining resulting from SSAO activity (with benzylamine as substrate) occurred predominantly and intensely over the tunica media in rat aortic sections, although some occasional staining of adventitial sites was also observed. Staining was prevented by the SSAO inhibitors hydroxylamine (1 μM) and semicarbazide (1 mM), but not by the MAO inhibitor, clorgyline (1 mM). These results indicate that SSAO is associated predominantly, although not exclusively, with the smooth muscle cells in the rat aorta. Our findings that β‐aminopropionitrile (BAPN) is a reversible, competitive inhibitor (Ki around 2 times 10−4 M) of SSAO, in contrast to the irreversible inhibition of the connective tissue lysyl oxidase by BAPN reported by others, provides further evidence that these enzymes are not identical.

Methylamine metabolism to formaldehyde by vascular semicarbazide-sensitive amine oxidase.

The capacity of the vascular enzyme, semicarbazide-sensitive amine oxidase (SSAO), to metabolize methylamine to the potentially toxic product, formaldehyde, was tested using rat aortic homogenates and purified porcine aortic SSAO. Formaldehyde production in incubations of enzyme source with methylamine (1 mM) was detected by high performance liquid chromatography and product was confirmed by desorption chemical ionization mass spectrometry (DCI-MS). Inhibitor studies using the specific SSAO inhibitor semicarbazide and the monoamine oxidase inhibitor pargyline indicate that SSAO is responsible for metabolism of methylamine to formaldehyde. These results suggest the possibility that elevated methylamine found in several pathologic states (such as uremia and diabetes mellitus), or generated from exogenous sources, could result in overproduction of formaldehyde in tissues with high SSAO activity, especially blood vessels.

The Enhanced Daily Excretion of Urinary Methylamine in Rats Treated with Semicarbazide or Hydralazine May Be Related to the Inhibition of Semicarbazide-sensitive Amine Oxidase Activities

Abstract— The effects of amine oxidase inhibitors upon the daily urinary excretion of monomethylamine (MMA), dimethylamine (DMA), trimethylamine (TMA) and ammonia in the rat have been examined. Administration of hydralazine (5 mg kg−1) or semicarbazide (100 mg kg−1); drugs which irreversibly inhibit semicarbazide‐sensitive amine oxidases (SSAO) but not monoamine oxidase (MAO), enhanced MMA excretion by around three‐ to six‐fold above pretreatment levels, whereas no effect of pargyline (25 mg kg−1), a selective irreversible inhibitor of MAO was found. No apparent changes in DMA or TMA excretion in response to drug‐treatment were observed. Ammonia excretion also was generally unchanged except for an apparent marked increase (approximately four‐fold) over the 24 h following semicarbazide, a result which might be explained if ammonia is a degradation product of semicarbazide metabolism in the rat. With recent evidence that MMA is a substrate in‐vitro for SSAO activities, results here may indicate that SSAO or related enzymes are involved in endogenous MMA turnover.

Further Studies on the Metabolism of Methylamine by Semicarbazide‐sensitive Amine Oxidase Activities in Human Plasma, Umbilical Artery and Rat Aorta

Abstract— An ion exchange radiochemical assay has been developed to study the deamination of [l4C]methyIamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0.1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide‐sensitive amine oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 μM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol (mg prot.)−1 h−1, and in plasma of 48 nmol (mL serum)−1 h−1. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 μM (plasma), 222 μM (umbilical artery), and Vmax values of 28 nmol (mL serum)−1 h−1 (plasma) and 377 nmol (mg prot.)−1 h−1 (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 μM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 μM) and [14C]MA (800 μM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion. β‐Aminopropionitrile was found to be a reversible, competitive inhibitor (Ki of 165 μM) of [14C]MA metabolism in umbilical artery, inhibitory properties characteristic of those found previously for the effects of β‐aminopropionitrile upon BZ‐metabolizing SSAO activities in other tissues. The possibility that vascular and plasma SSAO activities may be involved in the endogenous turnover of the biogenic amine MA is discussed.

Properties of a Semicarbazide‐Sensitive Amine Oxidase in Human Umbilical Artery

Abstract— The metabolism of some aromatic amines by amine oxidase activities in human umbilical artery homogenates has been studied. The inhibitory effects of clorgyline showed that 5‐hydroxytryptamine (5‐HT) and tryptamine, 1 mM, were predominantly substrates for monoamine oxidase (MAO) type A, whereas MAO‐A and B were both involved in the metabolism of β‐phenylethylamine (PEA), 100 μM, and tyramine, 1 mM. About 20–30% of tyramine and PEA metabolism was resistant to 1 mM clorgyline, but sensitive to inhibition by semicarbazide, 1 mM, indicating the presence of a semicarbazide‐sensitive amine oxidase (SSAO). Benzylamine, 1 mM, appeared to be metabolized exclusively by SSAO with a Km (161 μM) at pH 7.8 similar to that found for SSAO in other human tissues. Tyramine and PEA were relatively poor substrates for SSAO, with very high apparent Km values of 17.6 and 13.3 mM, respectively, when determined in the presence of clorgyline, 10−3 M, added to inhibit any metabolism of those amines by MAO activities. However, kinetic studies with benzylamine indicated that clorgyline, 10−3 M, also appears to inhibit SSAO competitively such that the true Km values for tyramine and PEA may be about 60% of those apparent values given above. No evidence for the metabolism of 5‐HT or tryptamine by SSAO was obtained. The aliphatic amine methylamine was recently shown to be a specific substrate for SSAO in umbilical artery homogenates. We have used benzylamine and methylamine as SSAO substrates in histochemical studies to localize SSAO in tissue sections. Both amines promoted tissue staining which occurred predominantly over the medial layers of the vessel wall, and this staining was prevented by the presence of semicarbazide, 1 mM, but not by pargyline, 1 mM, in the reaction medium. The results support the notion that smooth muscle cells are an important site of SSAO activity in human blood vessels, and reinforce the possibility that methylamine, an endogenously‐occurring amine, may be a better candidate as a physiological substrate for SSAO in man, than several aromatic biogenic amines so far examined.

Inhibition of rat aorta semicarbazide-sensitive amine oxidase by 2-phenyl-3-haloallylamines and related compounds

The inhibition of semicarbazide-sensitive amine oxidase (SSAO) in rat aorta homogenates by some 2-phenyl-3-haloallylamines has been studied. Derivatives containing a fluorine atom were approximately three times more potent than the corresponding 3-chloroallylamines. These halogen-containing compounds were irreversible inhibitors of SSAO after preincubation with aorta homogenates; kinetic evidence for an initial competitive, reversible interaction (Ki around 0.4-0.6 microM) was found with two compounds (MDL 72145 and 72274). A similar Ki (approx. 0.7 microM) was obtained with 2-phenylallylamine (MDL 72200). However, this compound which lacks a halogen atom was a reversible inhibitor, even after preincubation. The use of a spectrophotometric assay to measure H2O2 production from amine metabolism demonstrated that MDL 72200 was a substrate (Km = 1.4 microM) for SSAO, with a Vmax approximately five times smaller than that of benzylamine (Km = 8.1 microM). Of particular interest in this study is the finding that (E)-2-phenyl-3-chloroallylamine (MDL 72274) is highly selective as an inhibitor of SSAO, compared with MAO-A or B activities, and may be a useful compound for investigating the importance of SSAO in animal tissues.

An allylamine derivative (MDL 72145) with potent irreversible inhibitory actions on rat aorta semicarbazide‐sensitive amine oxidase

(E)‐2‐(3, 4‐dimethoxyphenyl)‐3‐fluoroallylamine (MDL 72145) was found to be an extremely potent inhibitor of the semicarbazide‐sensitive amine oxidase (SSAO) in rat aorta homogenates. Considerable inhibition, which was not reversed by dialysis, could be produced under appropriate in‐vitro conditions at drug concentrations around 10 nM. The pseudo first order kinetics for time‐dependent inhibition by MDL 72145 (10–100 nM) were found to be consistent with a bimolecular reaction between enzyme and inhibitor with a rate constant for this step of 2 times 106 min−1 M−1. A similar rate of inhibition under an oxygen atmosphere to that obtained under nitrogen was produced upon incubation of enzyme with inhibitor, suggesting that oxidation of the inhibitor to an active metabolite was not required for its activity. Incubation of homogenates for very short periods (1 min) with inhibitor (0.05‐0.5 μM) and benzylamine (1–10 μM) as substrate indicated non‐competitive kinetics for the early interaction of enzyme with the drug. Benzylamine (50 μM), but not pyridoxal phosphate (100 μM), was able to protect SSAO from inhibition by 10 nM MDL 72145. However, pyridoxal phosphate alone appeared to produce some irreversible inhibition of the enzyme. Dialysis against buffer containing 50 μM or 1 mM benzylamine was unable to reactivate SSAO inhibited by 10 nM MDL 72145. It is concluded that MDL 72145 irreversibly inhibits SSAO by acting at, or near, the substrate binding site, but the exact nature of the complex formed remains to be identified.

Aminoacetone metabolism by semicarbazide-sensitive amine oxidase in rat aorta.

High speed (105,000 g/60 min) membrane fractions from rat aorta homogenates metabolized the aliphatic amine aminoacetone (AA) to methylglyoxal (MG) with a Km of 19 +/- 3 microM, and Vmax of 510 +/- 169 nmol MG/hr/mg protein. This deaminating activity appears to be due to a semicarbazide-sensitive amine oxidase (SSAO), which is associated with smooth muscle cells in blood vessels of the rat and other species. AA was a competitive inhibitor (Ki of 28 +/- 6 microM) of the metabolism of benzylamine, a synthetic amine often used as an assay substrate for SSAO. AA is produced endogenously from mitochondrial metabolism of threonine and glycine, and thus could be a physiological substrate for SSAO, whereas the production of MG by SSAO could have cytotoxic implications for cellular function.