Histamine as a marker for hydroxyl radicals

During inflammation an influx of neutrophils and release of mediators from mast cells (such as histamine) take place. The stimulated neutrophils can produce reactive oxygen species (ROS). One of these ROS is the highly reactive hydroxyl radical (OH.). It would be interesting to be able to quantify the extent of ROS formation. We investigated if histamine which is present at the inflammation site can serve as an endogenous marker for the formation of OH.. We found that histamine after incubation with OH. gave two distinct products in our HPLC system. One of the products gave the same characteristics as the synthesized 2-imidazolone derivative of histamine. This suggests that this derivative will be formed when histamine is incubated with OH..


Introduction
Neutrophils are the most prominent migratory cdlular elements in most forms of acute inflammation, particularly during the initial .stages. These cells will kill and digest bacteria at the inflammation site (phagocytosis). Activation of neutrophils includes a stimulation of the membrane bound enzyme, NADPH oxidase. This enzyme is responsible for the one-electron reduction of molecular oxygen to a superoxide anion radical. Subsequent reactions lead to the formation of other reactive oxygen species (ROS) such as hydrogen peroxide (H202) hypochlorous acid (HOCl) and hydroxyl radicals (OH'). [1][2][3] These ROS can cause injury to cells and tissues. In several pathological processes such as myocardial injury and lung diseases ROS 4 7 are thought to be involved.
Basophil leukocytes and tissue mast cells are inflammatory cells that are found in virtually all human tissues. These cells can release a variety of chemical mediators, including histamine, upon appropriate stimulation. 8 Studies have shown that ROS released extracellularly from phagocytosing neutrophils at an inflammation site can cause degranulation and histamine release from mast cells.9-11 In vivo acetylsalicylic acid had been suggested as a marker for OH" formation. 2 This study was undertaken to investigate whether histamine could serve as an OH" marker. The advantage of histamine is that it is already present at the site of inflammation and no exogenous compounds have to be administered.
All other chemicals were of analytical grade and used without additional purification. Incubations with OH': The OH" were generated via three different incubation conditions. The first system contained 100 M FeC13, 100 I.tM EDTA, 1001.tM ascorbic acid and I mM H202 inphosphate buffer (20mM KHiPO4, pH 7.0). The second system contained 5 mM ascorbic acid and 0.05 mM CuSO4 in the phosphate buffer 14 and the third system contained 50 mM H202 and 0.5 mM CuSO4 in the phosphate buffer. 5 In all of these OH" generating systems histamine (2.7 mM)was added for a period of time at 37C. The reaction mixtures were injected directly on the column. OPA-derivatization: Histamine, the synthesized 2-imidazolone derivative of histamine and the collected samples from the above mentioned HPLC system were post-column derivatized with OPA by a continuous flow reaction system according to Yamotodani 16 with some modifications. The samples were injected on a reversed phase C8 column and were eluted with 0.16 M KI-I2PO4 containing 0.5 mM SOS at a flow rate of 0.6ml/min. The eluate from the column was mixed first with 0.1% OPA solution at a flow rate of 0.12ml/min, subsequently 2.5M NaOH was added (0.12ml/min). The solution was mixed in a reaction coil made of polyetheretherketone tubing (8m x 0.5mm I.D.), obtained from Bester (Amstelveen, The Netherlands) and thermostated at 50C, and then 1 M HNO3 was added (0.2ml/min). The pH of the final reaction mixture was 2.5. The fluorescence intensity was measured at 450 nm with excitation at 350nm by a Hewlett Packard 1046 Programmable Fluorescence Detector.  8 This compound gave a peak at 6.0 min in our HPLC system (data not shown). In this study we were interested in the product formation of histamine in the presence of OH'. Histamine, dissolved in phosphate buffer, eluted at 10.6 min in our HPLC system (data not shown). When histamine was incubated with a OH" generating system like FeCI, EDTA, ascorbate and H202 for 3h at 37C, peaks at the retention times of 6.0 and 7.6 min appeared next Synthesis of the 2-imidazolone derivative of histamine: The 2-imidazolone derivative of histamine was synthesized according to the method of kerfield and Dahlen v (Fig. 1). Histamine was hydrolysed to 1,4 diamino-buta-2-one. Condensation of the diamine ketone with potassium cyanate gave the 2-imidazolone derivative which was isolated as its hydrochloride salt. The 2-imidazolone derivative of histamine was characterized by H-NMR.

Results
The synthesized 2-imidazolone derivative (see Materials  EDTA, 100tM ascorbic acid and mM H202. This sample was incubated for 3 h at 37C and revealed products at 6.0 (peak 1) and 7.6 min (peak 2) next to histamine which eluted at 10.2 min (peak 3). The paxis represents the absorbance intensity (arbitrary units), the x-axis represents the retention time (min). to the histamine peak at 10.2 min (Fig. 2). In the literature other OH" generating systems have been reported such as ascorbate/Cu 2+ according to Uchida 4 and a system containing H202/ Cu2+. 5 When histamine was incubated with ascorbate/Cu 2+ for 5h at 37C again two fairly separated peaks appeared at 6.2 and 7.6 min and histamine was found at 10.9 min (Fig. 3). When histamine was incubated in the H202//Cu 2+ system for 5 h at 37C, the reaction products in the chromatogram appeared at 6.0 and 7.6 min (Fig. 4). Surprisingly, histamine was not seen in the chromatogram in this system. A possible explanation for this could be that all the hista-mine might be complexed by the metal ion Cu 2 +. However, a simple complexation of Cu 2+ and histamine could not explain this as the absorbance of histamine (at 210nm) remained unchanged after an incubation of histamine and Cu2+. Another small absorbance maximum appeared at 256nm in the presence of Cu + (F g. 5).
After OPA derivatization of the synthesized 2imidazolone derivative of histamine the product eluted at 4.4 min. The collected product (at 6.0 min) gave after OPA derivatization products at 4.4 and 7.2 min (Fig. 6). The product which appeared at 7.2 min originates from histamine. CuSO4. This sample was incubated for 5 h at 37C and revealed products at 6.0 (peak 1) and 7.6 min (peak 2). No histamine peak was observed. The y=axis represents the absorbance intensity (arbitrary units), the x-axis represents the retention time (min). Discussion mechanism had been proposed for the ascorbate/Cu 2+ system. Here it was suggested that the Histamine is a low-molecular weight biogenic histidine residue will complex with Cu2+. Then amine and is involved in numerous physiological an electron will transfer from ascorbate to the and pathophysiological processes including aller-Cu 2 +-His complex, giving a Cu+-His complex. gic reactions, vasodilatation and vasoconstrictions, This latter complex reacts subsequently with gastric acid secretion, and neurotransmission. 19 molecular oxygen to give an 02 adduct. It is Histamine is formed by decarboxylation of the probable that the His-Cu+-O2 complex is folamino acid histidine, this reaction is catalysed by lowed by generation of the His-Cu 2 +-O the enzyme t-histidine decarboxylase. The major-complex. The authors now assume that the ity of histamine in humans is stored in the gran-metal-peroxo complex like Cu+-O2 or Cu 2+ules of circulating basophils and tissue mast ceils.
O can react directly with the ligand (histidine It is metabolized via two major pathways. In residue) itself, finally leading to the imidazolone humans, histamine is primarily methylated to 1-product. [25][26][27][28] However, in our study we were methylhistamine by the enzyme histamine methyl-unable to find similar differences when histamine transferase. This product is converted to 1-methylwas incubated with the ascorbate/Cu 2+ or the imidazole-4-acetic acid by the enzyme mono-HiO2/Cu 2+ system. Both situations revealed at amine oxidase. In the other pathway, histamine is least two products with same retention times. oxidized by diamine oxidase (histaminase) to These retention times of the products were idenimidazole-4-acetic acid (via imidazole-acet-tical with those found in the Fe+/HiOi/EDTA/ aldehyde), much of which is conjugated with ascorbate incubation, i.e. 6.0 and 7.6 min. ribose and is excreted as the riboside. 2 '21 Results have been reported about the oxida-Earlier studies have been conducted with the tion of histamine in the presence of ascorbate compound imidazole and OH'. The radicals were and Cu 2 +, in which the substrate histamine is generated via pulse radiolysis of aqueous solu-completely broken down into aspartic acid. tions of imidazole and it was found that OH" add Hydantoin-5-acetic acid had been identified as an 29 injecat the C2 and C5 positions of the imidazole. 22'2 intermediate in this conversion. When we Samuni et al. even suggested an H abstraction ted aspartic acid or hydantoin-5-acetic acid on from the NH of the imidazold, but this process our HPLC system retention times of 1.0 and 0.95 occurred only under basic conditions (pH 10min were found respectively (data not shown). 12). The mechanism involved addition of the Although the incubations revealed a broader OH" (at all pH values) and then the OH adduct band around 1 min (Figs 3 and 4), these comcould undergo a base-catalysed water elimination pounds cannot explain the peaks found at 6.0 involving the OH and the H from NH. 2 Uchida and 7.6 min. When we injected the synthesized 2et al. 24 have tested a larger molecule which con-imidazolone derivative of histamine, a peak at 6.0 mined an imidazole, i.e. N-benzoylhistidine. The min was found (data not shown). This could OH" were generated in an ascorbate/Cu 2+ indicate that the product of 6.0 min might be the system. Oxidation of the imidazole group was 2-imidazolone derivative of histamine. Similar assumed to be initiated at the C2 position of the results were found for N-acetylhistamine incuimidazole group to yield an imidazolone deriva-bated in a ascorbate/Cu 2+ system. The ascorfive, N-benzoyl-J3-(2-oxo-imidazolonyl)alanine, as bate-mediated reaction was shown to occur the main product. Other minor products were mainly at the imidazole group in histamine yieldvarious ring-ruptured products and products ing a product that should have the structure of 4which were tentatively formed by the hydrogen [2-(acetylamino)ethyl]-2,3-dihydro imidazole-2abstraction of the z and 13 position of the subone. Another indication that suggests that the strate. However, when another OH" generating product eluted at 6.0 min might be the 2-imidasystem such as H202/Cu 2+ was used, the same zolone derivative of histamine, is that both the ring-ruptured products were found. Surprisingly synthesized imidazolone derivative of histamine the imidazolone derivative could not be detected and the isolated product at 6.0 min behave in this system. 5 The authors pointed to a serious similar in the OPA derivatization system (Fig. 6).
difference between the H202/Cu 2+ and the We are aware that a definite characterization ascorbate/Cu 2+ system, as the data from the should be obtained with mass spectrometry, HiO2/Cu 2+ system were basically distinct from although the presence of SDS in the eluent was a that of the ascorbate/Cu 2+ system, major disturbing factor to obtaining a proper In the H202/Cu 2+ system an addition of the mass spectrum of the products. Histamine and OH" was suggested to generate an imidazole probably also its derived products in OH" genradical, followed by reaction with oxygen to give erating systems are relative small molecules and ring-ruptured products. 5 Another reaction quite difficult to identify with mass spectrometry as a relative high concentration of SDS is present. The reason for this is that SDS will be fully fragrnented and in this way will disturb the low molecular area of the mass spectrum. Even after a purification by extraction of SDS the sample was still not pure enough for mass spectrometry. We have tried to use a cation-exchange column in the HPLC system, but then instead of SDS the rather high potassium phosphate concentration disturbed the mass spectrum. Functional experiments on the fight atria of guinea-pigs revealed that the synthesized 2-imidazolone derivative of histamine showed no agonistic or antagonistic activity on the H2 histamine receptor (data not shown).
In summary, we found that histamine incubated together with a OH" forming system gave several products, one of which is probably the 2imidazolone derivative of histamine. This product has a distinct retention time compared to the rest of the products formed. This could mean that biological compounds can be analysed for this 2imidazolone derivative in order to determine if ROS such as OH" have been involved. The compound histamine could then be an endogenous marker for OH'. It has been reported that the concentration of histamine, which will be released at the site of an inflammation can be up tO 10 -3 M. 2 The charm of using histamine as an endogenous marker for OH', above other biomarkers discussed by Hageman et a/..3 is that both OH" and histamine are released at the site of the inflammation. The release of histamine can even be stimulated by ROS. The involvement of OH" in this pathological process might then be indicated by the presence of the 2-imidazolone derivative of histamine.