EUROPEAN JOURNAL OF PHARMACOLOGY 19 (1972) 180-190. NORTH-HOLLAND PUBLISHING COMPANY
AN IMPROVED METHOD F O R THE DETERMINATION OF HISTAMINE RELEASE IN MAN: ITS APPLICATION IN STUDIES WITH PROPANID1D A N D THIOPENTONE*
W. L O R E N Z * * , A. D O E N 1 C K E , R. M E Y E R , H.J. R E I M A N N , J. K U S C H E , H. B A R T H , H. G E E S I N G , M. H U T Z E L a n d B. W E I S S E N B A C H E R
Institute of Clinical Chemistry and Clinical Biochemistry, and Division of Anesthesiology, Surgery Outpatient Clinic, University of Munich and Division of Experimental Surgery' and Pathological Biochemistry,
Surgery Clinic of Universi(v of Marburg/Lahn, W.-Germany
Received 22 September 1971 Accepted 28 March 1972
W. LORENZ, A. DOENICKE, R. MEYER, H.J. REIMANN, J. KUSCHE, tt. BARTH, H. GEESING, M. ttUTZEL and B. WEISSENBACtlER, An improved method for the determination of histamine release in man: its application in studies with propanidid and thiopentone, European J. Pharmacol. 19 (1972) 1 8 0 - 1 9 0 .
tlistamine release by propanidid and thiopentone was demonstrated with the use of a highly sensitive and specific method for determining histamine in human plasma. 5 min after i.v. administration of propanidid and 3 rain after thiopentone, the plasma histamine level was increased by 350 and 4205~ respectively. In whole blood, hista- mine release could be demonstrated only when the basophil content remained constant. This was the case in about 50% of test persons after injection of propanidid, but not after administration of thiopentone where the basophil content decreased in all probands by about 35%. The increase of the histamine concentration in plasma was 3 ng/ml and in whole blood, 24 ng/ml, t t a l f m a x i m u m gastric acid secretion was elicited by injection of propanidid and thiopentone. It was increased by very rapid application of propanidid and diminished by premedication with atropine by about 30%. The changes in plasma histamine and gastric secretion were parallel over 30 rain, whereas tachycardia and peripheral arterial hypotension lasted for only 3 4 min.
During i.v. infusion of histamine a correlation could be shown between the doses infused, ( 1 5 - 9 0 ng/kg)/min, and the elevation of the plasma histamine concentration. 3 ng/ml plasma were measured at a dose of 145 ng/kg)/min, which induced a half-maximmn gaslric secretion, but was without effect on blood pressure and heart rate. It is concluded that only the increase of plasma histamine concentration, not that of whole blood, represented the release of free, pharmacologically active histamine. In normal test persons, histamine release by propanidid and thiopentone is without significant clinical consequences, but it is important in anaphylactoid incidents occurring during anesthesia with these substances.
Plasma histamine Histamine release Propanidid Thiopentone
1. I N T R O D U C T I O N
H i s t a m i n e release b y drugs u s e d b e f o r e a n d d u r i n g a n e s t h e t i c a d m i n i s t r a t i o n in m a n a n d a n i m a l s h a s
* Members of SFB 37 'Restitution und Substitution innerer Organ& of Deutsche Forschungsgemeinschaft.
** Requests for reprints should be send to Professor Dr. W. Lorenz, Division of Experimental Surgery and Pathological Biochemistry, Surgery Clinic of University Marburg/Lahn, Robert-Koch-Strasze 8.
b e e n p r o v e n b y n u m e r o u s a u t h o r s , since its first ob- s e r v a t i o n b y A l a m et al. ( 1 9 3 9 ) in t h e s k e l e t a l m u s c l e o f dogs ( f o r reviews see P a t o n , 1 9 5 6 , 1 9 5 7 , 1 9 5 9 ; M u r p h y , 1 9 6 2 ; R o t s c h i l d , 1 9 6 6 ; L o r e n z and Werle, 1 9 7 1 ) . S o m e t i m e s d i r e c t m e t h o d s h a v e b e e n used, w h e n h i s t a m i n e i t s e l f was m e a s u r e d in b o d y fluids or in tissues. M o r e o f t e n , h o w e v e r , i n d i r e c t m e t h o d s h a v e b e e n e m p l o y e d w h e n clinical s y m p t o m s a n d c h a n g e s o f p h a r m a c o l o g i c a l l y m e a s u r a b l e p a r a m e t e r s , s u c h as gastric s e c r e t i o n , d e c r e a s e in p e r i p h e r a l arte-
W. Lorenz et aL, Determination o f histamine release 181 rial blood pressure, tachycardia and increase in bron-
chial resistance, were concluded to be a result of his- tamine release. These symptoms and measurable reac- tions are, however, specific for histamine only in a limited sense.
Direct proof of histamine release by drugs adminis- tered before and during anesthesia is extremely diffi- cult to obtain in humans, despite availability of sim- ple specific assay methods for determination of hista- mine in whole blood, because an increase of free, pharmacologically active plasma histamine is masked by the nearly 100-fold higher concentrations of hista- mine stored in the blood within the basophil granu- locytes (Code, 1952; Lorenz and Werle, 1971).
The histamine content of human plasma is so low, that its determination has in the past been especially difficult (Adam et al., 1957; Graham et al.,1968).
However, with an improved, highly sensitive and spe- cific method for plasma histamine determination (Lorenz et al., 1970a; Lorenz et al., 1972) it has been possible, to show histamine release in man by 2 anes- thetic drugs often used in European clinics: propa- nidid and thiopentone. Recent information of ana- phylactoid incidents during anesthesia with both sub- stances led us to this investigation. To demonstrate that the increased concentration of plasma histamine was actually free, pharmacologically active histamine, gastric acid secretion, heart or pulse rate and periph- eral arterial blood pressure were also measured. To determine whether histamine release in man can be demonstrated by histamine assay of whole blood, the histamine concentration and the density of basophilic granulocytes in whole blood, in which histamine in humans may be localized almost exclusively, were also measured.
2. MATERIALS AND METHODS
2.1. Materials
2.1.1. Drugs
Propanidid (Bayer, Leverkusen): a colloidal solu- tion consisting of propanidid (3-methoxy-4-(N,N-di- ethylcarbamoylmethoxy)-phenylacetic acid-n-propyl ester), Micellophor (20%), NaC1 and phosphate salts, as well as water, prepared according to instructions of
the company (Wirth and Hofmeister, 1965; Scholtan and Lie, 1966).
Micellophor ® (Bayer Leverkusen): a colloidal solu- tion consisting of 20% Micellophor = ORPE (oleum ricini polyoxyethylate Elberfeld), NaCI and phos- phate salts, as well as water, as stated by the manufac- turer. Micellophor contains the hydrophobic conrpo- nents of Cremophor EL ® (BASF, Ludwigshafen), which was used in diluted form until 1967 as a sol- vent for propanidid (Doenicke et al., 1968), and con- sists of ricinoleic acid, polyglycol esters and glycerol, polyglycol ethers.
Thiopentone (Penthotal ®, Abbott, lngelheim).
Atropinum sulfuricum (from University's phar- macy).
Antazoline (Antistin @, Ciba, Basel).
2.1.2. Reagents
Histamine dihydrochloride puriss. (Fluka, Buchs), o-phtaldialdehyde puriss, p.a. (Fluka, Buchs) (recrys- tallized from petrol ether p.a., b.p. 40 60°), metha- nol, n-heptane, inorganic acids and bases as Uvasol @ (Merck, Darmstadt), n-butanol (for chromatography, Riedel de Haen, Seelze near Hannover), Dowex 50W-X8, H*, mesh 200 400 (Serva, Heidelberg).
Unless otherwise stated, all substances were dissolved in twice distilled water.
Heparin for biochemical research (180 l.U./mg)*
( H o f f m a n n - L a Roche, Grenzach)), 0.1 N NaOH for titration of the gastric juice (Titrisol @, Merck, Darm- stadt), TiJrk's solution for leucocyte counts (acetic acid-gentian violet), May-Grunwald's solution (eosin-methylene blue) and Giemsa stain ( a z u r - eosin methylene blue) (Merck, Darmstadt).
2.2. T e s t persons
The investigations were carried out on 143 volun- teers. The test persons had healthy case histories and in particular were devoid of allergic reactions, gastric diseases or complaints, and had normal pulse rate and blood pressure in sitting and prone positions, normal leucocyte counts and/or normal differential blood pic- ture. The test persons consisted of 140male and 2 female students and anesthetists between the ages of 22 and 31 years, weighing between 54 and 100 kg;
* We thank Professor Dr. H. Gastpar, Munich, for supplying this substance.
one test person (A. Doenicke) was himself one of the Investigators in this paper.
3.3. Seqrrevr~e arid urrungemettr 0,f ittvrs~igaliott The tests were conducted f‘tu11 7 a111 to 9 a111 in a quiet well-lit room. The anaesthetic was administered during spontaneous respiration. The test persons. on whom all investigative parameters were measured simultaneously. were handled in the following se- quence and arrangement (fig. 1 ).
Blood Pressure
Venous Blood.
e Blood Histamw
Fig. I. Scheme of the arrangement of investigations in the test persons. I.or explanations we test under Matemls and hlethods.
To begin with. a nasogastral tube with X-ray con- trast strips (Levin No. I?- 14) was introduced into the stomach. the position of which was radiologically controlled and. if necessary. corrected. After the tube was fixed on his nose, the test person laid down on a sofa with the upper body slightly raised. Leads for the ECG were attached to the extremities and a sphygmomanometer (Riva- Rocci) to measure blood pressure was fastened to the right upper arm. Then. in the right and left under-arms, polyethylene cannulae (Braunule@. Braun. Melsungen) were inserted into superficial veins, the right one being used for the in- jection of the anesthetic. solvent and other drugs, the left one being used for withdrawing blood for hista- mine assay in plasma and whole blood, as well as for leucocyte counts and blood smears. Capillary blood from the finger tips of the left hand was also used to determine leucocyte counts and the differential blood pictures.
If 1 or 2 of the parameters were not simultaneous- ly measured, then the sequence of those still to be investigated remained. as stated above. To carry out this program, a very well-practiced team of at least
5 persons, including 7 anesthetists. was absolutely necessary.
When in any one of the investigations. ;I solvent and agent, or an agent at various conditions, were employed on one and the s;lnle test person, then the sequence of application of both solutions was alter- nated. Thus. when in one person Micellophor’“, WiIS
used first and then Propanidid. in the next test person Propanidid was used first and then the solvent. The time lapse between the first and second investigation was always 2- 4 weeks.
The order as described above was also adhered to in studies with histamine infusion. Gastric acid secre- tion was not determined since the dosc~- effect rela- tionship between histamine infusion and gastric acid secretion in humans is known from the literature (Hanson and Ivy. 1948: Adam et al., 1954; Lawrie et al., 1964; Lorenz and Pflcger, 1968).
The histamine solution was prepared using a steril- iLed stock solution from the University’s pharmacy.
diluting it with physiological saline. and then drawing it up into a SO-ml polyethylene syringe. The hista- mine concentration of the fluid within the syringe
was determined after the investigation by tluoromct- ric assay. There was no loss of histamine caused b>, an adsorption onto the syringe surface.
I.v. infusions were given into the right arm using the infusion apparatus Unita II (Braun, Melsungen).
while blood was withdrawn from a cuhital vein of the left arm, as in the other investigations. At the begin- ning, 0.9% NaCl solution was infused, followed by the application of histamine from a separate syringe after which physiological saline was again infused.
To avoid any side effects from infusion of the highest dose (headache, flush, etc.). the antihistaminic drug antazoline, 2 mg/kg, was injected i.v. immedi- ately prior to infusion.
2.4. Deterntimbon of’the various parmerers
2.4.1. Histamine assay in whole blood and plasma From the left underarm, 5.0 ml of blood were withdrawn with a polyethylene syringe 15 min before and immediately before, injection of the anesthetic or solvent. as well as at intervals of 3, 6. 10. 30 and 30 min after the i.v. application. The blood samples were put immediately into centrifuge tubes contain-
ing 5.0 ml 1 N HC104 and centrifuged for 5 min at
W. Lorenz et al., Determination of histambw release 183 1800g. 4.0 ml of the supernatant were stored for
several days in a freezer at - 2 0 ° C and used later for histamine assay according to Lorenz et al, (1970a,
197 la) (Dowex method).
lnnnediately after the withdrawal of blood sanr- pies for whole blood histamine determination, 19.5 ml of blood were withdrawn with a polyethyl- ene syringe containing 2 mg heparin dissolved in 0.5 ml of 0.9% NaC1 solution and centrifuged imme- diately for 30min at 1000g and l - 2 ° C . 6.0ml of plasma were mixed with 2 ml of 2 N HC104 and centrifuged, for 10 min at 1800g. The whole super- natant was filtered through a filter paper and was either used immediately for histamine assay or stored in the freezer at -20°C, and histamine assayed several days later. Histamine was determined on a Zeiss spec- trofluorometer ZFM 4 at a temperature of 21-+0.5°C according to Lorenz et al. (1970a, 1972) (combined method).
Two types of blanks were used, in one 3 N HCI was added to the mixture before the o-phtaldialde- hyde solution (Shore et al., 1959, reversed blanks), in the other the plasma was substituted by fresh twice distilled water (histamine-free) and carried along throughout the procedure (reagent blanks). The high sensitivity of our method was partly due to strict control of the reagent blanks which should not be more than l(F/o higher than the reversed blanks (on the contrary see Van Redlich and Glick, 1965). On the other hand, the low concentration of o-phtaldial- dehyde used in our method, 0.05%, w/v, diminished both blanks considerably and thus increased the sen- sitivity of our assay.
2.4.2. Determination of gastric acid secretion (Lorenz et al., 1969; Lorenz, 1971).
After complete emptying of the stomach with a manually operated syringe, the nasogastral tube posi- tion was again tested by instillation of 20 ml of 0.9%
NaCI solution (37°C) and reaspiration. Then basal secretion was collected by suction at intervals of 2 rain over a period of 30 min. When atropine had to be used, a dose of 0.01 mg/kg was administered i.v.
15 rain after the beginning of the collection. At the end of the 30 min, the anesthetic was injected and the gastric juice withdrawn by suction every rain and collected in portions corresponding to the blood withdrawal, i.e., in 2 3-rain portions, 1 4-min portion
and 2 10-rain portions. The volume of gastric juice was measured and the acid output determined for each portion by titration with 0.1 N NaOH upto pH 7.0 using a glass electrode. The acid output was given in nwal HCl/min (Lorenz et at., 1969).
2.4.3. Determination of heart rate and blood pressure The heart rate (beats/rain) was determined from the average period length in lead I1 of the standard ECG (Heinecker, 1970). The arterial blood pressure (in mm Hg) was determined by indirect, sphygmano- metric measurement (Riva-Rocci) according to the method of Burton (1965). Because the test persons were all young people (very little or no arteriosclero- sis!), the blood pressure values obtained by the in- direct method would have been similar to those ob- tained by a direct method. Indeed, the blood pressure values measured by indirect methods during anesthe- sia with propanidid agreed with those obtained by direct methods (Henschel and Buhr, 1965; Avrucki and Zinovjev, 1969; Soga and Beer, 1971).
2.4.4. Determination of basophil granulocyte content in peripheral venous and capillary blood
At the same time as the blood was withdrawn for histamine assay, 2 blood smears and I leucocyte count were made with venous and capillary blood.
The leucocyte count was performed according to Von Mtiller et al. (1962), while the blood differential was determined according to Pappenheim, whereby 1000 cells from each sample were counted. The con- tent of basophils in venous and capillary blood was determined according to Hamerston et al. (1956) (number of leucocytes/ram 3 and per cent of baso- phils in the blood smear). As the results of Moore and James (153) and Hamerston et al. (1956) have shown, the values from such an indirect method agree with those front a direct method. The content of basophils in the blood is given as the number of cells/ram 3.
3. RESULTS
3.1. Plasma histamine levels during inJusion o f hista- mine and its correlation to heart rate attd blood pres- sure
With the highly sensitive histamine assay used in this study, an increase of plasma histanrine concentra- tion was measured after infusion of relatively low doses
184 W. Lorenz et aL, DetermiHation of histamine release
Blood
~o]P r e s s u r e , o o t
[mmHg] :(~.J
A n t a z o h n e I i v 2 m g / k g )
0
l___!_ r _!_ t . . .
0 • 0 0 0 0 0 0 0 0 0 0 0 0Heart 8o] }~,}
Rate 7o ~__~$~t__~}__(j~ ~
[ b e a t s / r a i n I 6 0
P asma ° I iill
Histamine
Concert- 4o
tration 2oi t
[ n g / m l
] i i
/ l
H's'='oe'°{
,n,os,on 4
[
ng/" kl~l rain
Il ~ / / / / / / / / / ~
0 3 0 6 0 9 0 120 135
Time i ...
Fig.2. Plasma histamine concentration, blood pressure and heart rate after intravenous infusion of increasing doses of histamine. Mean value + S.E.M., 3 probands were tested. For further conditions see Materials and Methods.
o f h i s t a m i n e , ( 1 8 - 9 0 n g / k g ) / m i n . F u r t h e r m o r e , t h e r e was a r e l a t i o n s h i p b e t w e e n the d o s e s o f hista- m i n e i n f u s e d and the e l e v a t i o n o f p l a s m a h i s t a m i n e level (fig.2).
D o s e s o f h i s t a m i n e up t o (45 ng/kg)/min did n o t p r o d u c e c o m p l a i n t s or clinical s y m p t o m s in any o f the 3 test p e r s o n s . No e f f e c t s on b l o o d pressure or h e a r t rate w e r e o b s e r v e d , b o t h relatively sensitive in- d i c a t o r s o f h i s t a m i n e release. Clinical r e a c t i o n s (a slight t a c h y c a r d i a and in o n e case o n l y , also a slight h e a d a c h e ) were o b s e r v e d a f t e r i n f u s i o n o f ( 9 0 n g / k g l / m i n , d e s p i t e a n t i h i s t a m i n e m e d i c a t i o n .
3.2. Histamine release b.v propanidid and thiopentone 3.2.1. C o n c e n t r a t i o n s o f plasma h i s t a m i n e a f t e r i.v.
in j e t tion
A f t e r i.v. a d m i n i s t r a t i o n o f p r o p a n i d i d , in d o s e s c o m m o n l y used for a n e s t h e s i a in m a n , the plasma h i s t a m i n e levels i n c r e a s e d s i g n i f i c a n t l y to a b o u t 350%
o f the n o r m ( t a b l e 1): t h e h i g h e s t value was fl)und 5 rain a f t e r i n j e c t i o n . T h e h i s t a m i n e c o n c e n t r a t i o n re- m a i n e d at an i n c r e a s e d level even t h o u g h a n e s t h e s i a b y p r o p a n i d i d h a d l o n g since b e e n e n d e d . At least 30 rain a f t e r t h e e n d o f i n j e c t i o n was r e q u i r e d for h i s t a m i n e c o n c e n t r a t i o n to a p p r o x i m a t e to t h e initial levels ( t a b l e 1). The i.v. a d m i n i s t r a t i o n o f a t r o p i n e did n o t significantly a f f e c t the c o n c e n t r a t i o n o f plas- m a h i s t a m i n e , a l t h o u g h this agent has b e e n p r o v e n to
Table l
Plasma histamine concentrations in man before and after the i.v. injection of propanidid, Micellophor ® and thiopentone. Mean values ± S.D. With propanidid, 7 mg/kg, i.v., injection time 20 sec, and Micellophor ® 0.15 ml/kg, i.v., injection time 20 sec, 12 male test persons were tested, with thiopentone, 5 mg/kg, i.v., injection time 20 sec, 8 male probands were studied. Statistical sig- nificance was calculated between the values at zero time and those at several times after the injection. For further conditions see
Materials and Methods.
Time after Propanidid Micellophor ® Thiopentone
injection
(min) Histamine Signifi- liistamine Signifi- Histamine Signifi-
content (ng/ml) cance content (ng/ml) cance content (ng/ml) cance
0 0.85 + 0.33 - 0.86 -+ 0.36 - 0.80 +_ 0.32
1 2.10 + 1.30 p < 0.005 1.03 + 0.63 p < 0.4 -
3 - 0.88 + 0.50 4.2 _+ 3.1 p < 0.01
5 3.80 + 2.10 p < 0.005 0.92 +- 0.58 - -.
6 - - 2.9 _+ 1.3 p < 0.001
10 2.40 + 1.70 p < 0.01 0.94 ± 0.76 - 2.0 + 0.9 p < 0.005
20 1.50 _+ 0.55 p < 0.005 0.88 + 0.69 1.6 -+ 0.64 p < 0.001
30 1.10 +- 0.52 p < 0.2 0.89 +_ 0.50 1.0 + 0.48 p < 0.4
W. L o r e n z et aL, Determination o f histamine release 185 be a histamine liberator in humans, dogs and cats
(Schachter, 1952; Rotschild, 1966).
For a long time, it has been assumed that the sol- vent of propanidid, Cremophor ®, or presently Micel- lophor ® released histamine ira humans since they liberate histamine in dogs and cats (Lorenz et al., 1971b). In this study, the test persons who were treated with propanidid also received Cremophor ® and Micetlophor ® alone. The increase of plasma hista- mine concentration, about 20%, was not statistically significant (table 1). Thus, it can be concluded that propanidid (anesthetic drug plus solvent), and not Cremophor ® or Micellophor ®, releases histamine in h u l ] l a n s .
Thiopentone, ira anaesthetic doses, also elevated plasma histamine concentration (table 1). The degree o f elevation a p p r o x i m a t e d that obtained after ad- ministration o f propanidid; also the concentration of plasma histamine fell at a p p r o x i m a t e l y the same rate as after propanidid. The solvent used for thiopentone, 0.9% NaC1 solution did not lead to any increase of plasma histamine levels even when injected rapidly (see also fig.2).
3.2.2. Histamine concentrations and basophil con- tents in whole b l o o d after injection o f propanidid, Micellophor ® and t h i o p e n t o n e
After i.v. injection o f propanidid, the histamine concentrations and basophil contents o f whole blood were not uniformly changed (fig.3).
(a) In those cases where the basophil content re- mained constant, the histamine concentration in- creased significantly. However, this increase, approxi- mately 30%, did not agree with the increase o f plasma histamine concentrations previously obtained, which was smaller than the 24 ng/ml observed in whole blood. It must therefore be assumed that histamine was briefly increased in c o m p a r t m e n t s o f the blood other than plasma (see discussion); the level o f hista- mine in whole blood was already decreased after 30 min.
(b) In those test persons in which whole-blood histamine concentration was not significantly changed, the number o f basophils decreased signifi- cantly (fig.3). This could have been due to sequestra- tion or partly also to degranulation o f basophils.
Ideally histamine assay and basophil count should be made on the same blood sample, because there was a
~
r o p a n i d i d ,,, 7~,k~) BasophilsG r u u p ] . . . . G r o u p ~ - -
°oooeo- 2
tration
6 g g lb 20 3b ~
Fig.3. Histamine c o n c e n t r a t i o n and basophil c o n t e n t in h u m a n whole blood: two kinds o f reaction to propanidid.
Mean values _+ S.E.M. 29 male probands were tested with propanidid, 7 mg/kg, i.v., injection time 20 sec. They were divided into 2 groups which reacted to the anesthetic in a different manner: (1) 13 probands, who showed no more than ± 10% difference in their basophil content in blood at any time investigated after the injection of propanidid ( o - - - o ) . (2) 16 probands, who showed more than -+ 10%
change of their basophil content in blood (o---o). The values at zero time were obtained from blood samples with- drawn immediately before the injection of propanidid. Statis- tical significance according to table 1. Statistical significance:
( o - - - o ) no significance; o - - o , 0/3 p<O.02, 0/6 p<0.05, 0/10 p<0.1; (o- e) 0/3 p<0.05, 0/6 p<O.1, 0/10p<0.05, 0/20 p<0.02, 0/30 p<0.1; (o o) no significance. For further conditions see Materials and Methods.
large difference in the basophil content o f venous and capillary blood; and no correlation between these values could be found (r = 0.37, p < 0 . 4 , n = 10).
The histamine concentration and the basophil con- tent in whole blood remained unchanged after ad- ministering Cremophor ® and Micellophor ®. After in- jection of thiopentone, there was a decrease o f the basophil content in practically all cases (fig.4). The histamine concentration of whole blood remained un- changed, in accord with one o f the test groups receiv- ing propanidid (see fig.3).
3.2.3. Gastric acid secretion after i.v. administration of propanidid, Micellophor ® and thiopentone
Rapid injection o f propanidid resulted in approxi- mately halfmaximal gastric acid secretion (table 2).
The increased acid o u t p u t parallel the increased plas- ma histamine concentration (fig.5); basal secretion was reached 30 min after injection. Micellophor @ did
186 W. L o r e n z et al., Determination o / h i s t a m i n e release
Thiopentone ~,~ 5mglkgl
l
Basophils
L~ ellS/ram3 i 80
~o }
Blood 7o]
Histamine 6o Concen- 50
tration
3 d 6 20 3o"
Time t ~,,, l:ig.4. Itistamine concentration and basophil content in human whole blood after injection of thiopentone. Mean values +- S.EM. 22 male probands were tested with thiopen- tone, 5 mg/kg, i.v., injection time 20 sec. Atropine, 0.01 mg/kg, i.v., was given as premedication 10 rain before thiopentone. Zero time is defined as in fig.3. Statistical sig- nificance according to table 1 (o----o), 0/3 p<0. l, 0/6 p<0.02, 0/10 p<0.05, 0/20 p<0.2; (o e), no significance.
[:or turther conditions see Materials and Methods.
not significantly increase gastric secretion (table 2).
After an extremely rapid injection of propanidid (as used often in the past and referred to as 'one-shot injection'), gastric acid secretion was much more profuse than after a slower injection (table 2). Proba- bly, slow injection o f the drug resulted in less hista- mine being released, thus characterizing propanidid as a histamine liberator (Paton, 1956). This is somewhat surprising since propanidid is a neutral substance, whereas all other histamine liberators are basic com- pounds. It is furthermore remarkable that with the very rapid injection the highest rate of gastric acid secretion was reached not less than 10 rain after injec- tion of the a n e s t h e t i c ( t a b l e 2).
The effect o f histamine on gastric acid secretion is usually reduced, but not completely blocked, by atro- pine (Ivy and Bachrach, 1966). When propanidid was administered 10 min after injection of atropine, gas- tric secretion was reduced approximately 31Y} (ta- ble 2). Therefore the increase of histamine in human plasma after injection o f propanidid represents an in- crease o f free, pharmacologically active histamine.
Injection of t h i o p e n t o n e also stimulated gastric acid secretion in a similar manner to propanidid (ta- ble 3); for safety purposes, t h i o p e n t o n e was not ad-
Blood ,2~
Pressure
i m m H ~ ) ~oo
80
Blood 7o
Histamine Bo [ nglml }
50 e o
B a s o p ~ l s [ c e l l s l ~ 3 1 60
40
Acid o~
O u t p u t ms [ m v a l l m m ]01
0O5 O0
P l a s m a a 1
Histamine 2Ol [ , - ~ t l , o I ~ /
, I - -
06 6
, Tr'rTi lTrti
. . . . / , }
\
/
5 *0 ~5 20 ~
Time t ~,0 ]
Fig.5. Histamine concentration in whole blood and plasma, basophils, gastric acid secretion, blood pressure and heart rate after i.v. injection of propanidid. Mean values + S.E.M., 5 probands were tested after injection of propanidid, 7 mg/kg, i.v., injection time 20 sec. In each test person all parameters were studied simultaneously, l leart rate e--- o,
blood pressure . - - e . Blood pressure is given as systolic- diastolic pressure. [:or fu['thcr conditions sec Materials and Methods.
ministered withoLtt atropine premeditation, l.v. injec- tion of 10 ml physiological saline, the solvent of thiopentone, did not stimulate gastric acid secretion.
3.2.4. Heart rate and blood pressure after injection of propanidid, Micellophor ® and thiopentone
The heart rate was increased during the first 5 min after propanidid injection (fig.5). Systolic and diastol- ic blood pressure were significantly decreased during the first 2 min only that is, before the maximum in- crease in plasma histamine concentration (fig.5).
Therefore, it is improbable that the initial tachycardia and the acute peripheral arterial h y p o t e n s i o n are re- lated to the release of histamine into the plasma, In contrast, stimulation o f gastric acid secretion and de- crease of basophils in the blood paralleled the in- crease of plasma histamine concentration (fig.5). The
W. Lorenz et al., Determination o f hisramhte release 187
Table 2
Gastric acid secretion in man after injection of Micellophor ® and propanidid at various conditions. Mean values ± S.D.
Propanidid, 7 mg/kg, i.v., injection time 20 sec, if n o t otherwise stated. Micellophor ®, 0.15 ml/kg, i.v., injection time 20 sec.
Atropine, 0.01 mg/kg, i.v., l 0 rain before propanidid. Propanidid, slow means, an injection time of 40--50 sec: propanidid, rapid means, an injection time of 5 - 7 sec. n = nmnber of male probands tested, in each series o f experiments the sanle test persons were investigated. The basal secretion before the injection of atropine (last column) was 0.016 _+ 0.019 mval/min. Statistical signifi-
cance was calculated as in table 1, the p-values are added in parentheses. For further conditions see Materials and Methods.
Time after Gastric acid o u t p u t 0rival/rain) injection
(min) Propanidid Micellophor ® Propanidid Propanidid Propanidid Propanidid
(slow) (rapid) + atropine
n = 9 n = 9 n = 11 n = 11 n = 9 n = 9
0 0.03 ± 0.04 0.04 _+ 0.05 0.02 a 0.02 0.02 ± 0.02 0.02 ± 0.01 0.01 ± 0.01
3 0.28 -+ 0.28 0.09 ± 0.12 0.13 ± 0.12 0.14 ± 0.12 0.32 -+ 0.29 0.15 ± 0.16
(p < 0.02) (p < 0.3) (p < 0.005) Ip < 0.005) (p < 0.011 (p < 0.025/
6 0.25 +_ 0.21 0.09 ± 0.10 0.09 ± 0.11 0.18 ± 0.23 0.26 ± 0.21 0.20 _+ 0.23
(p < 0.011 (p < 0.3) (p < 0.05) (p < 0.05) (p < 0.005) (p < 0.025)
10 0.20 + 0.17 0.08 +_ 0.08 0.10 ± 0.13 0.25 ± 0.24 0.14 ± 0.10 0.11 + 0.13
(p < 0.02) (p < 0.3) (p < 0.11 (p < 0.0051 (p < 0.005) (p < 0.05)
20 0.10 + 0.09 0.07 ± 0.08 0.07 ~+ 0.11 0.12 ± 0.11 0.09 _+ 0.10 0.10 +_ 0.11
(p < 0.11 ( - ) (p < 0.1t qr~ < 0.02) (p < 0.05) {.p < 0.05)
30 0.05 _+ 0.07 0.03 ± 0.04 0.03 ± 0.03 0.07 _+ 0.09 0.03 ± 0.(i14 0.05 _+ 0.06
( - i ( ) t ) ~ - ) ( ) ( p < 0 . 0 5 1
Table 3
Gastric acid secretion in man after injection of thiopentone.
Mean values -+ S.D. Thiopentone, 5 mg/kg, i.v., injection 20 sec. Atropine, 0.01 mg/kg, i.v., 10 rain before thio- pentone. Basal secretion before atropine 0.021 + 0.015 (reval/
rain). 13 male probands were tested. Statistical significance was calculated as in table 1. For further conditions see Mate-
rials and Methods.
Time after Gastric acid
injec tion ou tpu t
(min) (mval/min)
Significance
0 0.01 ± 0.01
3 0.11 +- 0.01 p < 0.001
6 0.19 -+ 0.13 p < 0.001
10 0.13 ± 0.12 p < 0.005
20 0.05 -+ 0.09 p < 0.1
30 0.04 + 0.09 p < 0.1
60 0.02 + 0.02 p < 0.3
h i g h e s t p l a s m a h i s t a m i n e c o n c e n t r a t i o n r e a c h e d , 3.1 n g / m l , w o u l d n o t h a v e b e e n e x p e c t e d f r o m t h e r e s u l t s o f h i s t a m i n e i n f u s i o n s t u d i e s ( f i g . 2 ) , 1o p r o - d u c e t a c h y c a r d i a o r h y p o t e n s i o n .
A f t e r i n j e c t i o n o f M i c e l l o p h o r ®, t h e r e w a s n o c h a n g e in h e a r t r a t e o r b l o o d p r e s s u r e .
F o l l o w i n g t h i o p e n t o n e i n j e c t i o n , t h e r e w a s a slight a n d s h o r t l a s t i n g i n c r e a s e in h e a r t rate a n d a s l i g h t , b u t s i g n i f i c a n t , h y p o t e n s i o n ( f i g . 6 ) ; as w i t h p r o p a n i d i d , t h e s e c h a n g e s o c c u r r e d b e f o r e t h e m a x i m u m i n c r e a s e in p l a s m a h i s t a m i n e c o n c e n t r a t i o n . S t i m u l a t i o n o f g a s t r i c a c i d s e c r e t i o n a n d t h e d e c r e a s e o f b a s o p h i l s in t h e b l o o d parallel t h e i n c r e a s e o f p l a s m a h i s t a m i n e ( f i g . 6 ) . T h e r e f o r e , a n d as w i t h p r o p a n i d i d , t h e r e w a s n o c o r r e l a t i o n b e t w e e n t h e c h a n g e s o f h e a r t rate a n d b l o o d p r e s s u r e a n d t h e r e l e a s e o f h i s t a m i n e i n t o t h e p l a s m a ; c i r c u l a t o r y c h a n g e s d u e t o r e l e a s e d h i s t a m i n e w e r e n o t e x p e c t e d f r o m a p l a s m a h i s t a m i n e c o n c e n - t r a t i o n o f 3 . 2 n g / m l .
4. D I S C U S S I O N
D e t e r m i n a t i o n o f h i s t a m i n e in b l o o d p l a s m a is a p r e r e q u i s i t e f o r t h e d e m o n s t r a t i o n o f h i s t a m i n e re- l e a s e in m a n b y a n e s t h e t i c s . T o d a t e n o s u c c e s s f u l m e t h o d s e x i s t e d f o r m e a s u r i n g s u c h l o w i n c r e a s e s o f p l a s m a h i s t a m i n e c o n c e n t r a t i o n . F u r t h e r m o r e , t h e e a r l i e r m e t h o d s w e r e n o t s p e c i f i c f o r h i s t a m i n e ( L o r e n z e t al., 1 9 7 2 ) .
188 IV. Lorenz el aL. Determination o f histamine release
B l o o d ~2o
Pressure
so eo
B l o o d zo
H i s t a m i n e eo
[ no/n ]
r,o
!,2o Heart
~ i Rate [ blatlg~]
%++_-
B a s o p h i l s [ c . d s t ~ 3 ] 4O
OlS A c ~ t O u t p u t o~
[ mval/r~nl OO5
O0 " ~
Histamme /
20 /
~o
o o / P
o s Io 15 20
T i m e ¢ ~,o I
Fig.6. Histamine concentration in whole blood and plasma, basophils, gastric acid secretion, blood pressure and heart rate after i.v. injection of thiopentone. Mean values _+ S.E.M., 5 probands were tested after the injection of thiopentone, 5 mg/kg, i.v., injection time 20 sec. In each person all param- eters were studied simultaneously. Heart rate, • •, blood pressure •----•. For further conditions see Materials and Meth- ods.
Only the methods of Adam et al. (1957) and Graham et al. (1968) produced results in agreement with our values (Lorenz et al., 1970a, b, c; Doenicke and Lorenz, 1970).The 'histamine values' found by the other authors, over a period o f more than 30 years (for a survey see Lorenz et al., 1972), were either up to 100 times too high or were below the limit of sensitivity of the m e t h o d (e.g. below 3 ng/ml). The high 'histamine values' were due to substances fluo- rescing after condensation with o-phthaldialdehyde which were not, or not sufficiently, separated from histamine during the purification procedure (Lorenz et al., 1970a). The less than satisfactory methods used in previous investigations makes necessary revi- sion of all results on histamine release in humans which are based upon plasma histamine assay.
Determination of histamine in human blood as a measure of histamine release is afflicted with a num-
ber of problems. As Paton (1957) has already re- marked, the determination must include a determina- tion of the content of tire corpuscular elements in the blood to be assured that their contents have not al- tered. Since histamine in human blood is almost ex- clusively located in the basophil granutocytes (Graham et al., 1955; Lorenz el al., 1972), histamine concentration in whole blood is dependent on the absolute density of these cells in the blood. The den- sity of these cells was altered in approximately 50c,~
of cases after propanidid and in ahnost all cases after thiopentone.
In those test persones receiving propanidid and with a constant blood basophil content, the histamine concentration in whole blood increased to approxi- mately 24 ng/ml, whereas the plasma histamine level only increased to approximately 3 ng/ml. Thus, the increase of histamine in whole blood did not corre- spond to the increase in plasma histamine, and above all, the histamine was not pharmacologically active, as shown by the results of the infusion experiments (fig.2). A facultative, short-term uptake in eosinophil granulocytes and e r y t h r o c y t e s should be considered (Code et al., 1964; Lindell and Viske: 1961: Lorenz and Werle, 1971). The eosinophil content increased in propanidid and thiopentone narcosis (Lorenz and Doenicke, unpublished). However, it is not known if the eosinophils store so much histamine under our ex- perimental conditions.
The big difference between plasma and whole blood histamine levels in humans (relationship ap- prox. 1 : 100) is the reason why only large increases o f plasma histamine, as seen in severe incidents (Lorenz et al., 1972a), can dependably be measured by histamine determination in whole blood, and then only when the basophil content remains constant.
However, since the histamine increase in whole blood is not entirely free histamine, the determination of this amine in human whole blood as a m e t h o d for determining histamine release by anesthetics and mus- cle relaxants (Westgate and van Bergen, 1962: Lorenz et al., 1969; Doenicke and Lorenz, 1970), is of lim- ited value and should be combined with indirect measures such as gastric secretion, heart or pulse rate and blood pressure.
Indirect methods for the determination of hista- mine release are not specific. Therefore, several o f the following measurements should be included to en-
W. L o r e n z et al., Determination o f histamine release 189 hance specificity o f the assay: d e t e r m i n a t i o n o f b l o o d
plessure decrease in peripheral circulation, the so- called ' d e l a y e d depressor response' ( M c l n t o s h and Paton, 1949); t a c h y c a r d i a ; h e m o c o n c e n t r a t i o n : gas- tric secretion; increase o f bronchial resistance u p t o b r o n c h o s p a s m ; erythenra, e d e m a (skin tests), etc. The p r e v e n t i o n o f clinical s y m p t o m s by antihistaminics is also an indirect m e t h o d o f proving h i s t a m i n e release a l t h o u g h specificity o f antihistaminics is m u c h less than is n o r m a l l y assumed (Werle and Lorenz, 1970).
In the future, all indirect m e t h o d s s h o u l d be com- b i n e d w i t h a d e t e r m i n a t i o n o f plasma histamine. The value o f such a c o m b i n e d p r o c e d u r e lies above all in d e m o n s t r a t i n g the p h a r m a c o l o g i c a l and p a t h o p h y s i o - logical effectiveness o f h i s t a m i n e released i n t o the plasma. The results p e r m i t the f o l l o w i n g c o n c l u s i o n s relating to h i s t a m i n e release by p r o p a n i d i d and thio- p e n t o n e : (1) in n o r m a l persons, i.v. injections o f b o t h anesthetics release h i s t a m i n e i n t o the plasma, in quan- tities sufficient to s t i m u l a t e gastric acid secretion, but n o t to p r o d u c e reactions in the circulatory, system;
increase o f heart rate is one o f the m o s t sensitive reactions o f the c i r c u l a t o r y system to h i s t a m i n e ( L e c o m t e , 1956); (2) in n o r m a l persons, the changes in the c i r c u l a t o r y system, e.g. t a c h y c a r d i a and h y p o - tension, observed in the first m i n u t e s after i n j e c t i o n o f b o t h anesthetics, c a n n o t be a t t r i b u t e d to a release o f h i s t a m i n e i n t o the plasma ( L o r e n z e t a l . , 1972a).
H o w e v e r , release o f histamilm f r o m the heart c a n n o t be e x c l u d e d as a cause o f these reactions and this aspect m u s t be separately s t u d i e d ; (3) in persons w i t h a b n o r m a l i t i e s relating to h i s t a m i n e storage or catabo- lism, as well as in those with an 'allergic diathesis', release o f large a m o u n t s o f h i s t a m i n e by p r o p a n i d i d or t h i o p e n t o n e , sufficient to e v o k e strong reactions from the c i r c u l a t o r y system c a n n o t be e x c l u d e d . Such incidents with p r o p a n i d i d are described in an- o t h e r paper ( L o r e n z et al., 1972a).
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