ANALYTICAL BIOCHEMISTRY 109, 142- 146 (1980)

A Sensitive and Specific Radiometric Method for the Measurement of Plasma Histamine in Normal Individuals

M. J. BROWN, P. W. IND, P. J. BARNES, D. A. JENNER, AND C. T. DOLLERY

Received March 19. 1980

A radioenzymatic method for assaying histamine using histamine-N-methyltransferase from guinea pig brain was modified to increase its sensitivity, precision, and specificity. This was achieved by incorporation of a thin-layer chromatography step and use of N,- methyl histamine as an internal standard in each sample. No prior extraction of the plasma samples is required, permitting the assay of 30 samples in 1 working day. Hista- mine was detected in the plasma of 17 normal volunteers, at a level of 3.4 r 0.69 nmoV liter (range 0.82 to 4.7 nmoliliter). In 19 asthmatics, a low-peak expiratory flow rate was found to be associated with a plasma histamine concentration above this “normal” range.

Histamine has long been known to be a potent bronchoconstrictor, and its release from mast cells is a probable factor in the induction of some forms of asthma (1,2). The generally disappointing response to antihistamines may suggest that histamine is not the most important mediator released by mast cells (3); nevertheless, a measure of its release might be expected to provide an index of mast cell discharge, since his- tamine is undoubtedly a major constituent of the mast cell granules (4). It has there- fore been desirable to develop a satisfactory method for its determination in plasma. Existing assays-both fluorometric and radiometric-do not meet the standards of sensitivity, reproducibility, or specificity required (5-9). Furthermore, relatively little attention has been paid to the problem of providing a true measure of the free plasma concentration in a blood sample whose total histamine content is several orders of magnitude higher; and where, on the other hand, the presence of the enzyme diamine oxidase may degrade the histamine as the sample is collected (10).

The radioenzymatic assays are based on the conversion of histamine to [3H]- or [‘4C]methylhistamine in the presence of histamine-N-methyltransferase and “H- or S-[*4C]adenosyl-L-methionine (7-9,11,12). In the modification described here, experience with the similar catechol-O-methyltrans- ferase-based assay used for the measurement of catecholamines has been exploited. Specificity and sensitivity have been in- creased by incorporation of a thin-layer chromatography stage in the assay, and precision by use of an internal standard in each sample.

MATERIALS AND METHODS

Histamine - N - methyltransferase was prepared from guinea pig brains ( 13). S- [3H]- adenosyl-L-methionine, specific activity 60-85 Ci/mmol, was obtained from the Radiochemical Centre, Amersham. Solvents were obtained from BDH, sodium tetra- phenylboron from Alldrich, and N,-methyl histamine from Calbiochem. Other chemicals were purchased from Sigma.

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142

RADIOMETRIC MEASUREMENT OF PLASMA HISTAMINE 143

For assay, three loo-p.1 aliquots of un- extracted plasma were used. To one of these was added 10 ~1 of 0.01 M HCl containing 1 ng histamine and 0.1 ng N,-methyl histamine; the remaining pair received 10 ~1 of 0.01 M

HCl containing only N,-methyl histamine (1 ng). Blanks consisted of 100 ~1 of pooled plasma (separated from blood collected into lithium heparin) previously incubated at 25°C for 2 h with 1 mgiml diamine oxidase. All samples received 100 ~1 of an incubation mixture consisting of 50 ~1 enzyme (hista- mine-N-methyltransferase), 100 mM Tris, pH 7.9, 10 mM EDTA, 5 $Zi S-[3H]adenosyl- L-methionine and 1 mM reduced glutathione. Incubation was at 25°C for 2 hr. The reac- tion was stopped by placing the samples on ice and adding 50 ~1 of a mixture containing 1 M borate, pH 8.0; tetraphenylboron sodium, 5 mgiml; and 25 PM unlabeled methylated histamine and N,-methylhistamine (cold carriers). Diethyl ether (2 ml) was then dispensed into all tubes. These were then vortexed for I min. and the phases sepa- rated by centrifugation. The aqueous layer was frozen in a mixture of acetone and solid carbon dioxide, allowing the ether to be decanted into a fresh set of tubes con- taining 50 ~1 of 0.01 M HCl and 4 mM cold carriers. All tubes were vortexed and cen- trifuged as before. On this occasion the ether was discarded and the aqueous layer was spotted on Whatman tic’ plates with a pre-adsorbent origin. After drying, the plates were developed in chloroform, methanol, and 70% ethylamine 32:6:4. After I h the plates were removed and dried. The methylated histamine bands were visualized using an iodopalatinate spray. The spots were scraped into tubes containing 0.8 ml 0.05 M

NH,OH. After vortexing for 1 min. 0.2 ml Tris. pH 7.0, and 10 ml toluene containing I% diethylhexyl phosphate were added. The tubes were stoppered and again vortexed for 1 min. After centrifugation and freezing

’ Abbreviations used: tic, thin-layer chromatography: PEFR, peak expiratory flow rate.

of the aqueous layer, the toluene was de- canted into scintillation vials containing 0.4 ml Permafluor (Packard).

The histamine content of a plasma sample (ngiml) was calculated as follows:

cm - cpm, X

cm, - cpm, x 10. cpm - cpm, cph - cpm,

where cpm, = mean counts per minute of the l-methyl histamine spots from the dupli- cate tubes to which only the N,-methyl histamine standard was added initially.

cpm, = mean counts per minute for hista- mine blanks (mean of 6 tubes).

cpm,, = mean counts per minute for N,,- methyl histamine blanks.

cpm, = mean counts per minute for the I-methyl-N,,-methyl histamine spots from the duplicate tubes to which only the N,- methyl histamine standard was added ini- tially.

cpm, = counts per minute for the l-methyl- N,,-methyl histamine spot from the tube to which both standards were initially added.

cpm, = counts per minute for the l-methyl histamine spot from the tube to which both standards were initially added.

Mcusurenwnt of plasma histamine c’on- centrution in normuls rend asthmatics. A resting, nonfasting plasma histamine level was measured in 17 healthy males, aged 24-36. Blood was collected from an in- dwelling venous cannula (no. 19 gauge butterfly, Abbott) primed with heparinized saline (1 U/ml). The blood (5 ml) was trans- ferred to chilled plastic tubes containing 50 ~1 0.5 M EDTA buffered to pH 7.4. These were immediately spun at 4°C for 15 min at 1OOOg. The plasma was stored at -80°C till assayed. Nineteen asthmatics (13 male, 4 female, aged 15 to 62) were studied on an occasion when their peak expiratory flow rate (PEFR) was less than 70% of their minimum predicted. A repeat study was possible in 11 of these subjects at a time when they attained a PEFR within their predicted range. No subject was re-

144 BROWN ET AL.

ceiving chronic therapy at the time of their exacerbation, though some had recently received inhaled beta-agonists.

RESULTS

The assay was found to be linear from 0 to 50 ng when a plasma blank was spiked with increasing amounts of histamine (Fig. 1). The sensitivity (defined as the lowest hist- amine concentration giving twice the back- ground counts) was 5 pg added to a IOO-~1 sample: that is, 50 pgiml. The assay was specific for histamine; this was tested by taking 50 ng of the following compounds through the assay: histidine, noradrenaline, adrenaline, dopamine, phenylethanolamine, 1 -methyl histamine, 4-methyl histamine, N,- methyl histamine, imidazole acetic acid. tyr- amine, and serotonin. None was found to give counts higher than the blank. N,-methyl histamine was, however. a substrate for the enzyme and could thus be used as an internal standard. However, a given amount of N,-methyl histamine gave generally fewer cpm in the assay then the same amount of histamine. To correct for this in the cal- culation of results, each triplet of samples included one tube to which equal amounts (1 ng) of the two amines were added in order to provide a correction factor in the calculation. The use of an internal standard yielded a high precision: a pooled sample with a histamine content of 0.90 nmol/ liter (100 pg/ml) gave intra- and interassay coefficients of variation of, respectively, 4.9 and 7.8%. In order to obtain this degree of reproducibility, it was necessary to use plastic syringes and tubes, to collect samples into EDTA (not heparin) as described, and to separate the plasma immediately after collection. Providing that samples were stored at the temperature indicated. his- tamine levels remained stable over a 6-month period: the interassay coefficient of varia- tion was no higher than 7.8% when aliquots of pooled plasma were assayed at 6-month intervals.

FIG. I. Standard curve obtained when 100 ~1

plasma blanks were assayed with increasing amounts

of histamine (0 to 50.000 pg). Each point represents

the mean ? SD of three estimations.

The plasma concentration in the normal volunteers was 3.4 + 0.69 nmol/liter (range 0.82 to 4.7 nmol/liter). In the asthmatics, the plasma histamine level when their PEFR was low was 7.6 + 1.9 nmoliliter (range 5.4 to 34.3 + 6.7 nmouliter). In the 11 asthmatics who were studied when their PEFR was within the predicted range, the histamine level was 5.1 ? 0.98 nmoVliter (range 2.7 to 9.6 nmoliliter). This represents a significant fall in plasma histamine con- centration in these individuals (p < 0.01, Student’s paired t test). There was no over- lap between the plasma histamine con- centration in the normals and the asthmatics during exacerbation; however, these groups are not directly comparable since they were not matched for age, sex, or race.

DISCUSSION

The present assay represents an improve- ment in the sensitivity and precision over previously described methods for measuring histamine, and provides for the first time a reliable means for detecting changes in plasma histamine concentration in normal individuals as well as asthmatics (8,9). The improved sensitivity results mainly from the use of a tic stage to reduce the background to very low levels (Fig. 1): and this is true

RADIOMETRIC MEASUREMENT OF PLASMA HISTAMINE 145

despite the use of a much higher specific activity methyl donor than used by other workers. This provides a higher sample to blank “signal” than is otherwise possible: this fact was exploited in most recent modi- fications of the catechol-O-methyltransferase assay for catecholamines to obtain a similar increase in sensitivity over previous methods (14,15). The use of a plasma blank also yields slightly lower counts than a water blank; this was felt to be more accurate since methyltransferase activity is lower in plasma than in buffer alone, and this should be allowed for in choice of blank (1.5).

The addition of an internal standard to each sample enables the precision of the double-isotope assays to be retained with- out the loss of sensitivity entailed in these by the use of S-[‘-‘C]adenosyl-L-methionine. The lack of precision of previous single iso- tope methods seriously preclude the assay from being used to obtain a possible index of mast cell discharge, since small physiological changes in plasma histamine concentration could not be accurately detected.

The tic stage almost certainly improves also the specificity of the assay. First, another substraste for the histamine-N-methyltrans- ferase enzyme was found-namely, N,- methyl histamine-whose methylation prod- uct would not be separated from that of histamine without tic. Although N,-methyl histamine is not known to be an endogenous amine, it has not in fact been previously possible to detect it. Second, the prepara- tion of histamine-N-methyltransferase used in the assay is only a crude purification: an ammonium sulfate fractionation only is performed on the supernatant fraction of the tissue homogenate (13). Several tissues, including brain, have nonspecific methyl- transferase activity which catalyzes the methylation of a large number of amines (16,17). This nonspecific methyltransferase is present in the supernatant fraction of the cells, and is unlikely to be adequately re- moved during the preparation of the hista- mine-N-methyltransferase. It therefore

seems desirable to include a chromatographic step in the assay in order to render the assay specific for histamine. Use of the tic plates described allows the assay to be completed within 1 working day, during which one person can handle 25-30 samples.

The accuracy of previous measurements may also have been compromised by the sample collection. Most workers have used lithium heparin as anticoagulant. This fails to take account of the diamine oxidase in plasma (10). Reproducibility of results was found to be increased by inhibition of this enzyme. This can be achieved by EDTA, which chelates the copper contained in the enzyme.

The plasma levels of histamine reported here illustrate the success of the method in measuring circulating levels in both normal individuals and asthmatics. The differing results obtained from asthmatics under varying circumstances are compatible with previous findings (8,9,18). They suggest the possible use of plasma histamine meas- urements as an index of mast cell release, since discharge of mast cell mediators is likely to be involved in the causation of some forms of asthma (19). More formal measurements have now been made of changes in plasma histamine in normals and asthmatics induced by various physiological and pharmacological stimuli; these results will be reported elsewhere.

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146 BROWN ET AL.

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