George D. Prell

Aspects of histamine metabolism

ConclusionsProgress in learning the role of histamine in physiology and pathology has been impeded by difficulties in accurately measuring histamine and by the deficiencies of methods to measure its metabolites. The availability of specific, sensitive and rapid methods to measure histamine has helped in understanding the role of histamine in disease. Measuring histamine alone may provide an incomplete indication of the role of histamine in disease or in any other process. For histamine is metabolized by multiple pathways, and the kinetics of these enzymatic activities (as well as the rate of synthesis of histamine) determine the steady-state levels of histamine in tissue and in body fluids. Measurements of both histamine and its metabolites would contribute, and may be essential, to the understanding of the role of histamine in disease, just as measurements of the metabolites of other biogenic amines have been critical to understanding of their roles in diseases. Yet another reason that compels measurements of metabolites is evidence that some of the metabolites of histamine are pharmacologically, perhaps physiologically, active.

Interaction of clozapine with the histamine H3 receptor in rat brain.

We examined possible interactions between neuroleptics and the histamine H3 receptor and found an interaction of clozapine with this receptor. In competition binding experiments, using the H3 antagonist, [125I]‐iodophenpropit, we observed a Ki of 236 ± 87 nM. Functionally, clozapine was studied on the H3‐mediated inhibition of [3H]‐5‐hydroxytryptamine ([3H]‐5‐HT) release from rat brain cortex slices. Clozapine acts as an antagonist with an apparent KB value of 79.5 nM.

[3H]‐thioperamide as a radioligand for the histamine H3 receptor in rat cerebral cortex

1 The purpose of the present study was to characterize the binding of the histamine H3 receptor antagonist, [3H]‐thioperamide, to rat cerebral cortical membranes. 2 The binding of [3H]‐thioperamide, to rat cerebral cortical membranes reached equilibrium after incubation with [3H]‐thioperamide after 8–10 h at 4°C. Equilibrium was maintained for up to 18 h of incubation. Addition of 1 μm (R)‐α‐methylhistamine rapidly dissociated [3H]‐thioperamide from its binding sites. From these kinetic experiments a dissociation constant of 0.3 nM was obtained for [3H]‐thioperamide. 3 Saturation experiments with [3H]‐thioperamide using 1 μm (R)‐α‐methylhistamine to define nonspecific binding were best analysed according to a single site model. A dissociation constant (KD) of 0.80 ± 0.06 nM (n = 3) and a maximal number of binding sites (Bmax) of 73 ± 20 fmol mg−1 protein (n = 3) were obtained for the binding of [3H]‐thioperamide to rat cerebral cortical membranes. 4 Saturation experiments with [3H]‐thioperamide using 0.3 μm iodophenpropit to define nonspecific binding were best analysed according to a two site model. For the high affinity [3H]‐thioperamide site a KD value of 1.1 ± 0.3 nM (n = 3) and Bmax value of 162 ± 108 fmol mg−1 protein (n = 3) were obtained whereas KD and Bmaxvalues for the low affinity site were 96 ± 19 nM and 4346 ± 3092 fmol mg−1 protein (n = 3), respectively. 5 Using 5 nM [3H]‐thioperamide, the binding was hardly displaced by H3 agonists within concentration‐ranges expected to bind to the histamine H3 receptor. Under these conditions, [3H]‐thioperamide binding was fully displaced by various H3‐antagonists, yet most H3 antagonists showed Ki values different from those expected for the histamine H3 receptor. 6 Using 0.3 nM [3H]‐thioperamide, 50–60% of the total binding was potently displaced by the H3 agonists histamine, (R)‐α‐methylhistamine, (S)‐α‐methylhistamine, imetit and immepip. Displacement of the binding of 0.3 nM [3H]‐thioperamide binding exhibited clear stereoselectivity for the R and S isomers of α‐methylhistamine. 7 Binding of 0.3 nM [3H]‐thioperamide was completely displaced by several H3 antagonists (thioperamide, iodophenpropit, iodoproxyfan, and burimamide) and biphasic displacement curves were obtained; the Ki values for the high affinity site corresponded well with the expected values for the H3 receptor. Antagonists fully displaced the binding of 5 nM [3H]‐thioperamide with affinities comparable to the low affinity site found with 0.3 nM [3H]‐thioperamide. 8 Ondansetron and haloperidol did not displace binding of 5 nM [3H]‐thioperamide at concentrations at which the former are known to bind to 5‐HT3 or σ receptors, respectively. On the other hand, nonselective cytochrome P450 inhibitors displaced the binding of 5 nM [3H]‐thioperamide from both rat cerebral cortical membranes and rat liver microsomes. 9 It is concluded that the histamine H3 antagonist, [3H]‐thioperamide, can be used as a radioligand to study the histamine H3 receptor in rat brain, provided that subnanomolar concentrations are used in displacement studies. Moreover, the specific binding should be defined with an H3 agonist, since most H3 antagonists share with [3H]‐thioperamide a low affinity, high density, non‐H3 receptor binding site(s) in rat brain. The latter is probably due to binding to cytochrome P450 isoenzymes.

Lack of a Precursor‐Product Relationship Between Histamine and Its Metabolites in Brain After Histidine Loading

Abstract: Levels of histamine and its major metabolites in brain, tele‐methylhistamine (t‐MH) and tele‐methylimidazoleacetic acid (t‐MIAA), were measured in rat brains up to 12 h after intraperitoneal administration of l‐histidine (His), the precursor of histamine. Compared with saline‐treated controls, mean levels of histamine were elevated at 1 h (+ 102%) after a 500 mg/kg dose; levels of t‐MH did not increase. Following a 1,000 mg/kg dose; levels mean histamine levels were increased for up to 7 h, peaked at 3 h, and returned to control levels within 12 h. In contrast, levels of t‐MH showed a small increase only after 3 h; levels of t‐MIAA remained unchanged after either dose. Failure of most newly formed histamine to undergo methylation, its major route of metabolism in brain, suggested that histamine was metabolized by another mechanism possibly following nonspecific decarboxylation. To test this hypothesis, other rats were injected with α‐fluoromethylhistidine (α‐FMHis; 75 mg/kg, i.p.), an irreversible inhibitor of specific histidine decarboxylase. Six hours after rats received α‐FMHis, the mean brain histamine level was reduced 30% compared with saline‐treated controls. Rats given His (1,000 mg/kg) 3 h after α‐FMHis (75 mg/kg) and examined 3 h later had a higher (+112%) mean level of histamine than rats given α‐FMHis, followed by saline. Levels of t‐MH and t‐MIAA did not increase. These results imply that high doses of His distort the simple precursor‐product relationship between histamine and its methylated metabolites in brain. The possibility that some His may undergo nonspecific decarboxylation in brain after His loading is discussed. These findings, and other actions of His independent of histamine, raise questions about the validity of using His loading as a specific probe of brain histaminergic function.

Measurement of histamine metabolites in brain and cerebrospinal fluid provides insights into histaminergic activity

Measurements of the concentrations of histamines metabolites,tele-methylhistamine (t-MH) andtele-methylimidazoleacetic acid (t-MIAA), in brain have been used to evaluate histamine turnover in brains of animals, and the same measurements in CSF have been used to infer histaminergic activity in brains of man. In regions of rat brain, half-lives of histamine are shorter than those of dopamine, 5-hydroxytryptamine and norepinephrine. Studies of human CSF suggest that brain histaminergic activity increases with age and is higher in females than in males.

Diurnal fluctuation in levels of histamine metabolites in cerebrospinal fluid of rhesus monkey.

In samples of ventricular cerebrospinal fluid (CSF) that were collected from a conscious, restrained rhesus monkey at intervals of 30–90 min, levels of the histamine metabolites,tele-methylhistamine (t-MH) andtele-methylimidazoleacetic acid (t-MIAA), were determined by gas chromatography-mass spectrometry. Levels of t-MH and t-MIAA each showed time-related fluctuations. Peak and trough concentrations of t-MIAA, the product of t-MH, paralleled, but lagged about 2 h behind, the levels of t-MH. Within the first 3 h of illumination, metabolite levels increased more than 3-fold; they fell sharply within the first 3 h of darkness. Mean levels of t-MH and t-MIAA were significantly higher during periods of illumination than of darkness. Fluctuations in the levels ofpros-methylimidazoleacetic acid (p-MIAA), an endogenous isomer of t-MIAA that is not a histamine metabolite, were markedly different from those of t-MH or t-MIAA; p-MIAA levels peaked only at the middle of the dark period. The time-related fluctuations in levels of t-MH and t-MIAA, but not p-MIAA, are similar to the daily rhythmic changes observed in monkey CSF for the levels of other central neurotransmitters and peptide neurohormones.

Influence of age and gender on the levels of histamine metabolites and pros-methylimidazoleacetic acid in human cerebrospinal fluid

The metabolites of histamine, tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA), were measured in cerebrospinal fluid (CSF) from 47 subjects with neurological disorders and healthy controls. In lumbar CSF, concentrations of these metabolites were significantly correlated. Levels of t-MH, t-MIAA and their sum (which represents virtually all histamine metabolized in brain) were significantly higher in CSF from older subjects and were positively correlated with age. Females had higher levels of histamine metabolites than males. Males had higher levels of pros-methylimidazoleacetic acid (p-MIAA), an isomer of t-MIAA that is not a metabolite of histamine. Levels of p-MIAA increased with age among men. These results are in contrast to those of age-related effects on levels of other aminergic transmitter metabolites in CSF and suggest that metabolic activity of histamine in brain may increase with age.

Histamine Metabolites in Cerebrospinal Fluid of the Rhesus Monkey (Macaca mulatto): Cisternal‐Lumbar Concentration Gradients

Abstract: Similar to metabolites of other aminergic transmitters, histamine metabolites of brain, tele‐methylhistamine (t‐MH) and tele‐methylimidazoleacetic acid (t‐MIAA), could have a concentration gradient between rostral and caudal sites of CSF. To test this hypothesis, cisternal and lumbar CSF samples were collected in pairs from eight monkeys (Macaca mulatta), and levels of t‐MH and t‐MIAA were measured by gas chromatography‐mass spectrometry. pros‐Methylimidazoleacetic acid (p‐MIAA), an endogenous isomer of t‐MIAA that is not a histamine metabolite, was also measured. Cisternal levels (in picomoles per milliliter, mean ± SEM) of t‐MH (9.9 ± 1.4) and t‐MIAA (40.8 ± 7.6), but not of p‐MIAA (9.7 ± 1.2), exceeded those in lumbar CSF (t‐MH, 1.8 ± 0.3; t‐MIAA, 6.8 ± 0.9; p‐MIAA, 8.6 ± 0.6) in every monkey. The magnitudes of the mean cisternal‐lumbar concentration gradients fort‐MH(6.6 ± 1.1)and t‐MIAA (6.5 ± 1.3) were indistinguishable. These gradients exceed those of metabolites of most other transmitters. There was no gradient for the levels of p‐MIAA. The cisternal, but not lumbar, levels of t‐MH and t‐MIAA were correlated. There was no significant difference between the means of the metabolite concentration ratios (t‐MIAAJ.t‐MH) in cisternal (4.0 ± 0.4) and lumbar (4.4 ± 0.9) CSF. The steepness of these gradients suggests that levels of t‐MH and t‐MIAA in lumbar CSF might be useful probes of histaminergic metabolism in brain.

Imidazoleacetic Acid, a γ‐Aminobutyric Acid Receptor Agonist, Can Be Formed in Rat Brain by Oxidation of Histamine

Abstract: It is generally accepted that in mammalian brain histamine is metabolized solely by histamine methyltransferase (HMT), to form tele‐methylhistamine, then oxidized to tele‐methylimidazoleacetic acid. However, histamines oxidative metabolite in the periphery, imidazoleacetic acid (IAA), is also present in brain and CSF, and its levels in brain increase after inhibition of HMT. To reinvestigate if brain has the capacity to oxidize histamine and form IAA, conscious rats were injected with [3H]histamine (10 ng), either into the lateral ventricles or cisterna magna, and decapitated 30 min later. In brains of saline‐treated rats, most radioactivity recovered was due to tele‐methylhistamine and tele‐methylimidazoleacetic acid. However, significant amounts of tritiated IAA and its metabolites, IAA‐ribotide and IAA‐riboside, were consistently recovered. In rats pretreated with metoprine, an inhibitor of HMT, labeled IAA and its metabolites usually comprised the majority of histamines tritiated metabolites. [3H]Histamine given intracisternally produced only trace amounts of oxidative metabolites. Formation of IAA, a potent GABA‐A agonist with numerous neurochemical and behavioral effects, from minute quantities of histamine in brain indicates a need for reevaluation of histamines metabolic pathway or pathways in brain and suggests a novel mechanism for interactions between histamine and the GABAergic system.

Presence and Measurement of Imidazoleacetic Acid, a γ‐Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid

Abstract Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic‐mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N,15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n‐butyl(N‐ethoxycarbonyl)imidazole acetate and n‐butyl(N‐methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean ± SEM) of free IAA were 140 ± 14 pmol/g and 2.7 ± 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at γ‐aminobutyrate receptors, it merits examination as a regulator in brain.