THE ACTION OF HISTAMINASE ON THE HISTAMINE CONTENT OF THE BLOOD OF THE RABBIT

RAP WILLIAMS, M.D., 1-I. L. ALEXANDER, 31.T)., AND T. KIRCHER, M.D.

ST. I,OTJIS, nr0.

s INCE t,he demonstration by Best and MeHenry (1930) of a his-

tamine-inactivating substance which they termed “histaminase,” considerable literature on the subject has appeared. Much of this is clinical, and there is little information concerning the action of his- taminase on histamine in the intact animal.

Atkinson and Ivy2 prepared histaminase according to the method of Best and MeHenry. When injected parenterally in doses more than sufficient to inactivate a given amount, of histamine in vitro, it had no effect on the gastric secretory response produced by the same amount of histamine. These findings are not in accord with those of Suzuki,3 who inhibited the action of histamine on gastric secretion in rabbits by giving histaminase intravenously one hour previous to histamine in- jection.

Guinea pigs injected with histaminase gave no symptoms of shock when histamine was administered soon thereafter, and, when sensitized, they were protected against anaphylact,ic shock following the parentera. injection of specific antigen.* These results have recently been disputed.j

The histaminase used in the experiments recorded here, as well as that reported in most of the recent literature, is a commercial product designated as T. 360.” Feli9 tested this material and found that it corresponded in every respect to the properties of histaminase reported by Best and MeHenry.

The quantitation of histaminase is based upon the amount of histamine inactivated by it. The procedures used to determine the quantity of histamine destroyed vary widely. The most prominent methods are the pharmacologic determination according to the technique of Barsoum and Gaddum’ or one of its modifications, and the measurement of the fail in blood pressure after injection into atropinized animals.

The above two methods were compared to that used in experiments here reported which consisted in measuring wheal formation after electrophoresis of the histamine-containing material into normal human skin. This was found to give satisfactorily comparable results.

EXPERIMENTAL DATA

Rabbits were chosen because of the comparatively high histamine con- tent of their circulating blood.

From the Department of Internal Medicine, Washington University, and Barnes Hospital.

*The supply of histaminase (called at Arst T. 360 and later Torantil) was fur- nished by the Department of Medical Research. Winthrop Chemical Co., Inc.

359

IlISTh3fIN.\Sl: (IJKIT) (nrc:.)

0.05 0.1 (1.2 0.:: 0.1 0.5 0.i

0 (I 0 L

tt I- i Itt

:. I + 0

Kesult.-1 unit of bistaminase (lot 7187) inactivated 0.2 nlg:. of histamine :Iftel eight hours and 0.1 xng. after twenty-four hours of incubation.;

Detcmainchm of ~~i.stcinhc or Ili.stcl)lli?lc-~~iiZ,e Szrhsftr?lce.--Tllis W ;LS done by electrophoresis into hmnan skin according to t,hc m&hod of L\brahamson and Gorin.” The apparal~ns consistcld of :I 45volt. “ I3 ” l)at- iery wired to a set of polrs through a milliammetcr ant1 a rheostat. Thea indifferent electrode was a metal eglindcr corrrctl with blotting papcl saturated with physiologic salt solution and held in the hantl. 1% t1 active electrode was a steel mesh grit1 1 (‘111. squaw applied to the skin 01 the outer asl)cct of tllc arm. To this was filstcnod blotting pal)cr moistened with the histamine solut iota IO 1~ tcstcd. Short,l;\- after the applicat,ion of a current of 2 nlilliaml)crcq i’or two minl~tes a wheal al)- Ijeared. Its size was est-imatcd fift.c>catl minlitcs aftw t tw current was interrupted.

This lnethod on most individuals will readily record dilutions of hist,amine base of 1 : l ,OOO,OOO and gives :L sharp differentiation between this di]ution and thoW of 1 : lOO,OOO ;llltl 1 :5.000,000. Tt W)lS fOlllld to

p(~mp:~l~e f:~~oral)ly with the pharmacologic met,hod of Barsoum and ( i ad- dnm and to be consistently reliable. ,\;o attempt was made to yuantitate ;Lcc~uratel>- the amount of histamine in each sample of 1)lood tested, and comparative values were recc~rdetl :IS .I. +, -t t, and 1 I I. TACSS than ly hist,amine per cubic centinleter of solution gave a + to 0 response.

*1 “unit” (n~anufacturer’s) of histalninilse xvi11 inacti\ratc I nrg. of histamine lwdrochloride.

+~l,~ discrepancy between the amount of histamine inactivated by 1 unit of bistaminase as declared bY the manufacturer and that indicated in Protocol I may be due to this method for the detection of histamine.

Determinution of the Effect of Uistnminrrse on Circulnting Nistunvhe. -Fasting rabbits were wcighed~ and the value of 60 C.C. of blood per kilogram of body weight used. The range of histamine in rabbit’s l~lood

has been estimated to average 1Oy.’ We assumed a maximum of 20~ pel cubic centimeter. The lattcl, Figaro \vas ernployd. The maximum amount of circulating histamine was thus estimated by multiplying the weight, in kilograms hy 60 1~s 20. ,\fter the quantity OF histamine inact,ivatcd in vitro IJ;V a sample of the hist~aminase had been dete~~mined 1)~ standard- ization, the amount of hist,aminase required to inactivwtc all of the cir- culating histamine was estimated (Protocols II and III).

nwTOCOL II

TIME OF BLEEDING

&fore injection 30 min. after injection 1 hour after injection 2 hours after injection 3 hours after injection 4 hours after injection 6 hours after injection R hours after injection

AFTER 8 ' AFTER %t IMXEDIATE IIOI’ES OF IIOTXS OF

ISPI-RATIOS Iscl-I~‘2’I’IoII

tt tt tt

2 0 0

tt tt

Controls

tt tt

t 0 0 0 0 t __-

.- -tt

t + 0 0 0 0 c

Before injection 2 C.C. blood +I/2 unit T. 360 tt 0 0 2 C.C. blood tq.05 mg. histamine ttt +t+ -t + + 2 C.C. blood to.05 mg. histamine ttt 0 0

t l/i unit T. 360 3 hours after injection

2 C.C. blood $0.05 mg. histamine -t t tt Rabbit A weighed 3.9 kg. Estimated maximum circulating histamine (20~ per CC.)

= 3.9 x 60 (CC.) x 20 = 4.28 mg. One unit T. 360 (lot 7187) was found to inactivate 0.2 mg’. of histamine in eight

hours (Protocol I). Therefore e or 23 + units would be required for this incuba- tion period.

Twenty-four units were injected intravenously. Blood s:kmples were tested for histamine immediately on withdrawal and after incubation for eight and twenty- four hours.

Blood was withdrawn before injcct,ion of histaminasc and at thirt,! minutes, one, two, three, four, six, and eight hours thereafter. This was done by cardiac puncture with a 22 gauge ncedlc under light, &he1 anesthesia. Each sample was tested immediately for histamine and again after eight and twenty-four hours of incubation.

Co?-relation Between Histamine Content rind Leucocytes of the Blood. -According to Code and Ing,” the histamine in the circulating blood of rabbits is carried in the 1~8~7 coat. probably in the polymorphonuclear cells, the granules of which arc eosinophilic. Total and differential white blood ‘cell counts were made on each blood sample withdrawn, and these were compared t,o the estimated amount of histamine present in

362 THE: JOIJRS.\I, OF’ i\LI,ER(;Y

that, sample. The differcnt,ial eorults r~orded t h(B ratio JWIWC(~II I tl(s granulocytes and mo~~ori~~~lea~ cells ( Protocol III ).

PRo’rclc~or. IJI

1 DEGREE OF SKIN WHEALING 1 J,EUCOCYTE COUNTS

AFTER 8 TIME OF BLEEDING

AFTER 24 TOTAL NO.1

IMMEDIATE HOIJRS OF HOURS OF INPIJ!3.1- INCUBA-

(PER CU.

TIOS ‘I’ION MM.)

__- _____ Before injection tt tt t t S,ROO 22 30 min. after injection t t t i +

GRANU- LOCYTF

-

1 hour after inj”ection tt 2 hours after injection +

0 ii

0 0

3 hours after injection 0 0 4 hours after injection 0 n 0 (i hours after injection rt + 0 S hours after injection t t tt +

11 hours after injection Controls had same values as in Protocol II.

2,950 10 90 1,400 12 88 6,250 65 2 5 7,600 77 23 9,800 84 16 9.500 72 / 28

MONONU- (!J.EAIIS

78

Rabbit C weighed 4.3 kg. l%timated circulating histamine = 4.3 X 60 X 20 = 5.16 mg.

One unit (lot 718i) inactivated 0.2 ng. in eight hours. Therefore $ 01’ 2.5.8

units would be required. Twenty-six units were injected as in Protocol II.’ Total and differential leucocyte counts were made on samples of blood withdrawn.

Granulocytes and mononuclear cells are recorded separately.

The protocols are re1)resentaGv-e. Of the twelve rabbits so t,reatcd, three showed but slight, reduction of histamine following injection of the calculated dose of histaminase. The remainder showed marked reduction. In all, however, the &ion of histaminasc was delayed for thirty minutes to t,wo hours as indicated in the protocols.

As further controls, histaminasc If-as heated to 80” P. .for thirty min- utes before injection in t,hree rabbits, and no reduction of histamine was noted at any time.

,Subcutaneous injections of hintaminasc were given to four rabbits, and the experiment was conducted thereafter as with the animals treated intravenously. In three there was qucstionablc, it’ any, reduc- tion of circulating histamine. Tn one rabbit, the calculated dose of his- taminase was given twice, and there was a derided diminution in cir- culating histamine after a delay of two hours following injection.

In almost every instance, inrluding ihosc in which inactivated hista- minase was given, there was a prompt initial fall in the total number of leucocytes lasting three to four hours. The percent,age of granulo- cytes, however, consistently rose to scvcral times the initial count. These findings are attributed t,o the injection of a foreign protein substance.

DISCWSION

From the experiments, it appears that histaminase given in large amounts intravenously has the power to inactivate temporarily most and

WILLIAMS ET AL. : HISTAMISE CONTENT OF BLOOD 363

frequently all of the circulating histamine Of interest is the fact that for a period of thirty minutes to two hours after inject,ion, a time when the histaminase concentration of 6he blood presumably should bc great- est, it had little or no effect whatever. This is shown when blood taken during this interval was incubated. Had histaminase been active then as in later samples, pronounced reduction of histamine should have occurred. Some three hours after injection t,he action of histaminasc was marked even wit,hout further incubation. In a few instances t.his effect was noted in one-hour samples. At no time was this result ob- tained in vitro, wherein four to six hours of incubation seemed essen- tial with the quantities of histaminase and histamine employed.

The reason for the initial delay in the action of histaminase is not known. Whether the enzyme leaves the circulating blood immediately or whether an antienzyme operates temporarily are problems that have not been studied. Ungar and Parrot reported that, whereas blood con- t,ains no demonstrable histaminase, a certain quantily appears imme- diately aft,er bleeding and diminishes markedly after one hour. This phenomenon would scarcely explain the results recorded here. More- over, these authors incubated blood wit,11 histamine for but two hours which, again, in our experience as well as that, of Felix and others, is far too short a time for inactivation to occur in vitro.

The action of histaminasc in every instance was but temporary and had essentially disappeared within eight hours.

The marked reduction of demonstrable histamine in the blood in the presence of an increased number of polymorphonnclear cells is of inter- est, since Code and Ing9 have demonstrated that this substance is con- tained in the white blood cells. There was no correlation between the histamine content and eit,her the total number of leurocytes or the rela- tive number of granulorytes, which morphologically did not appear abnormal. Zen, Ceder, and Crigler I” haye recently reported that the whit,e blood cells of the rabbit could be depressed markedly without any significant change in the blood histamine. They found indications that histamine is carried by the platelets. No platelet counts were made in the experiments here reported.

1. Histaminase injected intravenously into rabbits temporarily inac- tivates circulating histamine.

2. This action of histaminase is postponed for reasons unknown for thirty minutes to two hours after injection.

3. There is no correlation after the injection of histaminase between the histamine content of the blood and the total number of white blood cells or percentage of granulocytes present.

364 THE .JOTJRXAI, OF ALLERGY

REFERENCES

1. Best, C. H., and Mcllenry, E. IV.: The Inactiwtion of 11 istarninr, .J. Physiol. 70’ 349 . , 1930.

2. Atkinson, k. J., and Ivy, ,1. C. : T im Action of Histaminwc on the Gastric Secretory Response to Histamine and to a Meal, Am. ,I. Physiol. 107: I&S, 1934.

3. Suzuki, R.: Experimental Demonstration of the Distinctive Action of Hist:t- minase upon Histamine in the Living Organism, Taiwan Igalsi Zassi 36: 68, 1937.

4. Karadv. S.. and Browne. J. S. I,.: Effect of Bistaminnse Treatment on Bistamine and” ‘Anaphylactie Shock in Guinea Pigs; J. Immunol. 37: 463, 19x9.

5. Alexander, H. L., and Bottom, I).: The Failure of Histxminasc to Protect Guinea Pigs Against Histamine and Anxphylwtic Shock, J. Immunol. 39: 457, 1940.

6. Felix, J.: Experimental and Clinical Investigations of the Action of Hirta- minase Contained in Extracts of lhc Mucous Memhrnnc of the Intestines on Ilixtamine, Aeta med. Scandinar. 95: 1. 1938.

7. Barsoum, G.’ S., and Gaddum, J. H.: The Pharmacological Estimation of Adenosine and Histamine in Blood, J. PhTsiol. 85: 1, 19.15.

8. Abrahamson, H. A., and Gorin! M. II.: Relatton of Skin Permeability t,o Elwt~ro- phoresis of Biologically Active Materials into the Living Human Skin, J. Phys. Chem. 45: 335, 1939.

9. Codr. C. F.. and Ine. H. R.: The Isolation of Histamine From the \Vhitc C:ell Layer of Centyifuged Rabbit Blood, J. Physiol. 90: 501, 19:l’i.

10. Zon, Leo, Ceder, E. T., and Crigler, C. IV’.: The Presence of Histamine in the Platelets of the Rabbit, Pub. Ilealth Rep. 54: l!liS, 19X9.