Observations on biochemical abnormalities in hereditary angioneurotic edema

Observations on biochemical abnormalities in hereditary angioneurotic edema Kenneth L. Burden, Ph.D., Jose T. Quetq’, M.D.,* Orville U.D., and Jolhn P. McGone,rn, 3l.D., Houston, Texas

C. Thomas,

Serum specimens collected from patients wSth hereditary angioneurotio edema showed low titers for inhibitors of serum kallikrein., and, when obtained shortly after an episode of edema, were deficient in available substrate for kallikrein activi’ty. Patients were more sensitive to intradermul injection of serwm kallikrein than normal controls. Serum also showed an abnormally low content of inhibi,tor for a’ctivated C’l-esterase, and the capacity of Cl-esteruse to cause severe permeability-increasing reactions in human skin, ancl with special intensity in patients’ skin, was demonstrated. Other skin tests suggested the possible participation in the edematous process of PP/Dil. The exact role of these permeability factors and the precise identity of the specific inhi,bitor(s) which are lacking or deficient remain uncertain.

H

ereditary angioneurotic edema is a relatively rare familial, allergy-like disease1-4 characterized by recurrent episodes of acute, circumscribed noninflammatory edema, involving the skin or the mucous membranes, especially those lining the pharynx, larynx, and gastrointestinal tract. Since the pioneering studies of Landerman, Webster, Becker, and Ratcliffe,“> B this disease has been recognized as an inherited disorder of vascular permeability. The two principal permeability globulins which are readily activated in serum by dilution or other simple means, PE’/Di17 and serum kallik.rein,8~ o are in some way involved.1° Probablv Y also playing a part is the Hageman facto+ I2 which may be regarded as a third permeability globulin. Its apparently participates in the From the Departments of Microbiology and Pediatrics, Baylor University College of Medicine. This investigation mas supported in part by Research Grant AI-01563 and in part by Training Grant TI-AI-47, National institute of Allergy and Infectious Diseases, United States Public Health Service. Presented in part at the 64th Annual Meeting of the American Society for Microbiology, Washington, 1). C., May 6, 1964, and at the Fifth International Congress of Allergology, Madrid, Spain, Oct. 12, 1964. Beceived for publication Feb. 8, 1965. *Fellow in Pediatric Allergy, Postdoctoral Trainee, Training Grant TI-AI-47, National Institute of Allergy and Infectious Diseases, United States Public Health Service. 546

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activation of both PF/Dil and kallikrein.“‘, 13.I4 Kagen, Leddy, and Becker” have recently shown that serum kallikrein and PF/Dil are not one substance, as was formerly believed, but two distinct factors. Both exist in inactive form in serum but are activated by a number of the same procedures, e.g., by dilution and incubation in glass vessels. They split. TANe (p,-toluene sulfonpl L-arginine methyl ester) and are inhibited by soybean trypsin inhihitor (SBTI) . Injected into guinea pig skin they cause increased capillary permeabi1ity.l” Landerman and associatesz. 6 administ,ered Evans blue intravenously to their patients and to normal individuals, thrn 10 minutes later injected diluted autologous and heterologous serum intracntancousl>-. Their index patient gave larger and more intense bluing reactions when injected intradermally with her own diluted serum, than when inoculated with similarly diluted serum from normal individuals. Her serum and skin evidently lacked the inhibitor required to prevent the permeability-increasing effect. Also, the patient shoal-ed an abnormally strong response to the intradcrmal injection of partially purified serum kallikrein. Further, direct, I1lCasU1’CrtlCl1t,sin vitro show4 that the level of inhibitor for serum kallikrein in the patient’s serum was low in comparison with that in normal scra. These findings suggested that, the gcnctically dctcrmined biochemical defect in hereditary angionc>nrotic c~dema might be the lack of inhibitor for the prrmeabilty globulins. ln 1963, a new dimension to the problem was added when Donaldson and Evans”’ reported the absence of the normal inhibitor for the activated first component of complcmc~nt, (“I-thsterasc, in the I~lood of several patients with the typical hereditary edema. In the same year, Ratnoff and I,epow’~ demonstrated t,hat purified human C’l-estcrase acts as a permeability-increasing agent in guinea pig skin. These results indicated that inhibitors for two different natural enzyme systems affecting permeability may be lacking in hereditary angiontlurotic edema. Kagcn and I:ccker,l” however, found that the inhibitor of C’I-esterase also inactivated both purified PF/Dil and strum kallikrein. The working hypothesis these authors currently favor. thcrcforc, is to re:qard the disease as one characterized by the genetically determined lack of a single inhibitor, capable of inactivating both the pcrmcability globulins and C’l-csterase.lo The case for a significant role of VI.-esterase in the etiology of the heredit.ary edema has been strengthened by the recent work of Austrn and associates.‘“, 33 These investigators placsent intlircct evidence of C’I -cst,erase activity during the illness. Affected pt~ons could be detccte(I hy titrating the sw~m1 content of the sccontl componc>nt of complrmcnt ((22). Members of families with the disease wcrc found to be scverc>ly dcplctcd of (1’2, and the titers were ditninishcd in relation to the occnrrencc of active symptoms. The authors suggest that determination of the concentration of (“2 may scrv(b as a simple method of differentiating hereditary anpioneurotic edema from ot,her types of angioedcma. Vurthcr cvidcncc for the role of (:‘I-c&erase is pro\riclt?(l by the presence in the circulating blood of some patients during attacks of a “C’l-esterasc-like” cstcrase, active against n-acet?-1-L-tvl.osine ethyl ester (ATE?), and inhibited by C’l-cstcrasc inhibitor.” This esterase \vas not usually

demonstrable in periods beOween episodes of edema. The inheritance ot’ the C’l-esterase inhibitor is being studied closely.2” In the present report are described some limited studies on blood specimens from two patients with active disease and specimens from other members of the family. Between March 18, 1963, and March 20, 1964, eight blood samples were collected from the index patient A. T,. ; two from his mother (II. TJ.) aged 50; two from each of A. L.‘s children, aged 7, 11, and 14 years; and two from A. W., first cousin of A. Ii., aged 22 years. A. L’. was a 33-year-old whit,c man who had experienced repeated episodes of edema for 15 years or more, necessitating hospitalization on several occasions. His mother (M. L.) had only minor symptoms during the period of observation, but in ea,rlier years had suffered more severe attacks at frequent intervals. Patient 8. W. had first shown classical symptoms of hereditary angioneurotic edema a few months before she was seen by us. A. L.‘s son, aged 14, occasionally experienced acute abdominal distress of a nature characteristic of the gastrointestinal manifestations of the disease. The other two children, both females, aged 7 and 11, were asymptomatic. The family history was typical fo’r this condition. Twelve cases, including one death. arc known within 5 generations. METHODS Blood

AND

MATERIALS

specimens

Contact of the blood with glass was avoided in collecting and handling the original samples in order to prevent activation of serum kallikrein ; this permitted activation to be carried out subsequently, under controlled conditions, as desired. The blood wa,s drawn with siliconized syringes and needles, placed in polyethylene test tubes, and allowed to stand at room temperature until firml;v clotted, then centrifuged at 10,000 r.p.m. for 30 minutes at 5’ C. Portions of the serum not tested at once were frozen and stored in plastic tubes at -20” C. Esterase

tests

The csterolytic activity of C’l-esterase and various serum samples were tcstcd with the use of two synthetic substrat,es, p-toluene-sulfonyl-L-arginine methyl ester (TAMe) and n-acetyl-L-tyrosine ethyl ester (ATEe). The change in titratable acidity after incubation of the enzyme or serum with the substrate at 37’ C. for periods of 15 minutes to 1 hour was determined. Hydrolysis of the substrate was measured by a microformal titration2”, 24 by using 0.05N sodium hydroxidr, a manostat microtitrator, and a Beckman Zeromatic pH meter to determine the micromoles of acid liberated in a given time. One esterase unit was taken to be that volume of enzyme (or serum) which hydrolyzed an amount of substrate equivalent to a net titra,tion of 0.01 ml. of 0.05N NaOH. One unit of esterase inhibitor was contained in that volume of serum or other material which inhibited 10 esterase units. Tests

for

kallikrein

inhibitor

The reduced kallikrein-inhibiting activity of serum from A. L. as compared with tha,t from normal persons, when added to acetone-activated human serum,

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was observed directly in the Schultz-Dale apparatus. For quantitative results, t.ests for kallikrein inhibitor were carried out with TAMe as the substrate in a manner similar to that described by Landerman and co-workers.” One normal serum or more was always included in each series of tests with pat,ients’ sera. The esterase activity against TAMe of a mixture of equal parts of the serum under test and a preparation of partially purified human plasma kallikrein” were determined before and after incubation of the mixture for 30 minutes at 37’ C. The difference in esterase activity in the presence and in the absence of the serum gave a measure of its inhibitory effect. A control on the e&erase activity of the serum itself, in which buffer was subst.ituted for kallikrein, was also run. The results were expresspa in terms of esterase inhibitor units pet milliliter of serum. Because of lack of material for further assays of this kind other tests were performed by use of an acetone-activated pooled normal human serum, which had stood for 90 minutes at room temperature, as the source of kallikrein. Such serum always had high activity in Schultz-Dale tests with the rat uterus, as w-cl1 as in TAMe esterase tests, and both of these activities were completely suppressed by SBTI. The acetone-treated serum could be frozen and stored for several weeks without change of csterase activity at. the first melting. Three mixtures were made: (1) 0.5 ml. (1:2) acetone-activated serum plus 0.5 ml. phosphate buffer, pH 7.4; (2) 0.5 ml. (1.2) acetone-activated serum plus 0.5 ml. serum under test (1.2) ; (3) 0.5 ml. buffer plus 0.5 ml. serum under test (1.2). Following incubation at 37O C. for 20 minutes, 0.6 ml. from each tube was added to 1.4 ml. 0.057154 TAMe solution and incubation at 37O C. continued for an additional 60 minutes.5 The final concentration of TAMe was 0.04M. At 0 time, and again at 60 minutes of t,his incubation period, duplicate 0.3 ml. aliquots were removed from each tube, added to an equal amount of 37 per cent neutral formalin, and titrated for micromoles of 0.05N NaOH required to bring the sample to a convenient pH. Results were multiplied by 0.33 t.o give micromoles of XaOH required per 1.0 ml. aliquot of t,he mixbure. Inhibit,ion units were calculated on the basis of 1.0 ml. of the serum under test. Tests for C’l -esterare

inhibitor

Lyophilized purified human C’l-esterase ( C’l-E) was reconstituted with wa.ter, divided into 0.5 ml. aliquots, and frozen. When needed, the thawed 0.5 ml. volume was diluted with 0.05M phosphate buffer, pH 7.4, ionic strength 0.15, to give the desired standard. It was found that a higher esterase titer was obtained if the diluted C’l-E solution was allowed to activate by standing in a beaker of crushed ice for 2 hours, rather than at 37’ C. for 15 minutes as recommended.2” Under these circumstances, the 0.5 ml. came to possess 10 unit,s of esterase activity on incubation for 15 minutes wit.h a 1.611 solution of ATEe in methyl cellosolve. A supply of C’l-esterase inhibitor (El), also sent to us from Dr. Lepow’s laboratory, was described as having approximately 50 to 60 per cent purity. To test for C’l-E inhibitor in serum the following three mixtures were set up: (1) 1.88 ml. buffer plus 0.5 ml. C’l-E (10 units) ; (2) 1.88 ml. buffer plus

0.5 ml. serum (1:5) ; (3) 1.38 ml. buffer plus 0.5 ml. serum ( 1:5) plus 0.5 1~11. C’l-E. After incubation, 10 minutes at 37” C., 0.125 ml. ATEe was nddcd to each tube. This made the fina,l concentration of ATEe 0.08BI. Duplicate 0.5 ml. aliquots were taken at 0 minutes and at 15 minutes, added to an equal amount of 37 per cent neutral formalin, and titrated for micromoles of N&H required to bring the pH to 7.2. Calculat,ions were based on the combined results of the two 0.5 ml. aliquots, i.c., micrornoles of alkali required per 1.0 ml. aliquots. Inhibition units were calculat.ed on the basis of 1.0 ml. of the serum under test. Schultz-Dale system

tests in

normal

and

assays

and

in

of

the

patients’

intrinsic

kinin-forming

serum

A sensitive Schultz-Dale apparatus, by which smooth muscle contractions were recorded elect,ronically wibh a Physiograph,” was used to follow the appearance and disappearance of active serum kallikrein and of kallidin in serum from patients and from normal persons. Actiration of the serum was carried out by dilution and glass contact, by treatment with acetone, or by acidifica.t.ion.2”, 26 To measure smooth muscle-stimulating activit,y, the uterus from a rat (injected intramuscularly 24 hours previously with 100 mg. est,radiol) was used. Permeability

tests

in

guinea

pig

and

human

skin

The method of Miles and Wilhelrn2i and Elder and Wilhclmzs was used in tests of skin permeability in guinea pigs and was adapted to trials in human beings as done by Stewart and Bliss.“” RESULTS Inhibition

of

kallikrein

Ten norrnal sera assayed with Webster’s purified human plasma kallikrein preparation, and 10 with kallikrein-containing, acet,one-activated serum, showed averages of 2.52 and 2.9-l units of kallikrein inhibitor per milliliter of serum, respectively. The titers of the 20 norrnal sera ranged from 1.3 to 5.3 inhibitor units, with an average of 2.73 (i 0.97) units per milliliter. The kallikrein-inhibiting capacity of 5 samples of A. TJ.‘s serum was 0.0, 0.0, 1.3, 1.3, and 2.3 units, averaging 0.98 inhibitor unit per milliliter. His mother’s serum and that of the younger daughter had titers of 1.7 inhibitor units per milliliter, and the other children had higher than normal levels (A.0 and 5.3 units). These limited data indicate that t,hc level of kallikrein inhibitor ma.p btr negligible but may vary from time to time in the same pa.tient and on occasion overlap the values found in normal persons. The average of 0.98 unit in ,4. L’s serum was 36 per cent of the normal mean. Inhibition

of

C’l -esterase

Our tests did not show the complete: lack of inhibitor for C’l-esterase (El) which has been reported.‘” The titers, however, were low, and never approached normal levels. Five separate serum samples from A. L. showed 0.6, 1.8, 2.0, 2.3 *E

& M Instrument

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and 2.3 inhibitor units, respectively, averaging 1.8 El units per milliliter. At the time (Feb. 4, 1964) when the titer was lowest (0.6 unit), the patient had marked swellings of both hands and had residual edema of the throat from an attack during the preceding 48 hours. The same serum specimen showed another abnorma.lity to the greatest degree-it completely lacked the capacity to exhibit normal kallikrein activity, as described below. Aside from this example, the individual titers do not permit any meaningful correlation with the clinical state of the patient. Ten normal sera, tested under strictly comparable conditions, gave from 5.0 to 9.6 C’l-esterase inhibitor units per milliliter, with an average of 6.9 (t 1.4) units. This is slightly higher than the range for normal sera (5.8 f 1.8 inhibitor units) reported by Donaldson and Evans.lG The patient’s mother and his children ha.d relatively high titers-8.3, 9.6, 8.9, and 7.9 El units per milliliter, respectively. Variation serum

in was

kallikrein activated

activity in

when

patient’s

vitro

Normal human serum will regularly develop capacity to contract the estrous rat uterus after activation of the serum by glass contact and dilution, or by acetone, wit,h incubation at room temperature. The active principle is inhibited by SBTI and, otherwise, has the properties of serum kallikrein; no bradykinin or other plasma kinin is formed.2” Acid treatment of normal serum is regularly followed by t,he appcara.nce of strong and persistent smooth muscle contracting activity, which at first has the characterist.ics of serum kallikrein (inhibited by SBTI) , then later those of bradykinin (not inhibited by SBTI) .25. 26 In contrast, serum specimens from our patients collected at different times, and activated bp these procedures, often showed a sharply limited capacity t,o exhibit kallikrcin activity. Samples from A. Id. on the only two occasions (Dec. 27, 1963, and March 20, 1964) when he was entirely free of symptoms responded normally, whereas the one specimen which totally lacked kallikrein activity was obtained shortly after an acute edematous episode, as noted above. Partial loss of normal kallikrein release was observed in the 4 other specimens from A. L. and in each case the patient had had some edema or abdominal cramps in the preceding 24 to 48 hours. These findings suggest that there was a degree of correlation between the failure of the different blood samples to show normal kallikrein activity and the occurrence of active discasc in the patient during the preceding day or t,wo. It was found that lack of the usual concentration of substrate for kallikrein appeared to be the explanation for t.hc deficiency in kallikrein activity (Fig. 1). The following materials were added in equal volume to separate portions of heparinized plasma (obtained from A. L. on Oct. 25, 1963) which failed to contract, the rat uterus after acid treatment : (n) partially purified human serum kallikrein (Webster) ; (C) the eluate made with 7 per cent NaCl solution from glass beads which had been incubated with fresh normal human serum for 15 minutes, then washed repeatedly with 0.9 per cent. KaCl solution, and thus contained mostly Hageman factor (plus some other plasma constituents) ; (11) normal serum heated at 61’ C. for 30 minutes, which contained substrate only;

552

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.T. Allergy 196’5

6

(minutes

I

0

30

60 TIME

OF

90

120

CHALLENGE

Effects of adding various components of normal serum to a specimen plasma which showed no smooth muscle stimulating activity on the treatment. (a) patient’s plasma alone; (B) after adding partially kallikrein; (C) after adding Hageman factor preparation (eluate from with normal serum) ; (D) after adding normal serum heated at 61” C., only) ; (E’) after adding normal serum heated at 56” C. for 3 hours man factor).

of patient A. L’s rat uterus after acid purified human serum glass beads incubated 30 minutes (substrate (substrate, plus Hage-

and (E) normal serum heated at 56” C. for 3 hours, which contained principally substrate and some Ha.geman factor.‘” (A) is patient’s plasma alone. Neither the addition of preformed kallikrrin nor of Hagema,n factor alone supplied the needed materials, whereas normal activity was restored by wpplying additional substrate, and continued when Hageman factor was also added (Fig. 3, E). Hematological tests showed tha.t the patient’s blood contained the normal amount of Hageman factor; hence, it was concluded that the material needed for normal kallikrcin act,ivity must be additional subst,ratc. It is conceivable that substrate for kallikrcin was depleted during attacks and remained in low concentrat,ion for a day or two the.reafter. Permeability

tests

in guinea

pig

skin

Diluted normal and patient’s serum which had been incubated in glass tubes and shown to contain active kallikrein by simultaneous Schultz-Dale tests gave the cxpect.ed skin responses. In 1 25 dilution, after 60 minutes’ incubation at room temperature, active kallikrein was no longer present, and normal serum ceased to cause skin reactions. IJnder t,he same conditions, however, A. L.‘s

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serum continued to produce lesions. The causative permeability-increasing factor was inhibited by 0.6 mg. per milliliter SBTI, and was probabmly PF/IM,” which is most active in human serum at 1 :‘L5 dilution.‘” The capacity of active C’l-esterase alone to produce sizable bluing lesions in the guinea pig skin, as described by Ratnoff and Lepow,” was confirmed. Also the more intense reactions produced by mixtures of Cl-e&erase and serum,“” especia.lly A. L.‘s serum, were okserved. Permeability

tests in the skin of patients

A. 1. and

A. W. and in normal

controls

Both the unactivated, unincubated serum of patient A. L. ( 1: lo), presumably containing no kallikrein, and also the serum diluted 1 :25 and activated in glass for 20 minutes so that it did contain active kallikrein, had greater permeabilityincreasing effects in A. L.‘s own skin than in the nornml controls. The serum of patient A. IV., diluted 1:25 and incubated in glass for 60 minutes so that it no longer contained a,ctive kallikrein at that t,ime, also produced a more intense response in her skin than in the control (diameters 10.5 mm. vs. 7.5 mm., respectively), and there was a lesser reaction to another normal serum similarly prepared (8.5 mm. diameter). These responses are in agrecmcnt with the findings of Landerman and associates,G who called attention to the sensitivit! of patients’ skin to their own diluted serum. Acetone-activated serum kallikrein (showing peak smooth muscle stimulating activity on Schultz-Dale tests) produced a greater response and more intense bluing in the skin of A. L. and A. I\‘. than in normal skin. At a dilution of

Fig.

2

Dermal response of the normal skin (N) and the skin of patient activated wrum kallikrein at peak smooth muscle stimulating activity. (A$) 1:612 dilutions. Note the absence of reaction with the higher skin.

A.

IV. (d,l) dilution

(2’) to acetone1:32 dilutions; in the normal

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J. Allergy 1965

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Response of patient (A,1) 0.1 U. C’l-E; 0.025 U. C’l-E and

A. W.‘s skin to C’l-esterase and mixtures of (B,1) 0.025 U. C’l-E; (A$) 0.025 U. C’l-E 1 U. EI. Note that there is no wheal (erythema

C’l-esterase and inhibitors. and 0.1 mg. SBTI; (B,b) only) at site B,d.

1:512, the kallikrein preparation gave a negative response in normal skin but still produced a 9 mm. diameter wheal and bluing in A. W.‘s skin (Fig. 2). A slight surrounding erythema was noticed in the patients’ skin but none in the normal skin. Both patients and controls noticed a burning sensation to’ injected kallikrein. Strong reactions (Fig. 3) followed the inject,ion of 0.025 to 2.0 units of active C’l-esterase, and of mixtures of S’ILesterase and the homologous and heterologous sera. These responses were characterized by a large wheal, deep bluing, and an intense surrounding erythema, and were definitely more severe in the patients’ skin. The reaction to 0.025 unit was not inhibited by antihistamine. Burning sensation was not felt after injections of C’I-esterase. In general, the response of the patients’ skin to the various prrmeabilityincreasing substances and mixtures tested was definitely greater than that, shown by the normal controls. Also, the lesions persisted for more than 3 hours, whereas those in the normal skins had largelg faded by that time. (Fig. 4). DISCUSSION

In agreement with reports of previous investigators, the diluted serum from our patients was found to contain a permeability factor distinctly causing

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4

Appearance of skin permeabilit? rractions 3 hours after intracutaneous inJections.

in normal

control

(X)

and

in patient

-L. IV.

(P)

greater responses in the patients’ own skin than in the skin of normal persons. The patients wcrc also more sensitive to intradermal injections of scruln kallikrein. In general, the reactions to the various pernlcabilit?--irlcreasiny substances and mixtures tested were definitely more intense and more persistent in patients’ skin than in normal controls. A probable role of active serum kallikrein in the causation of the edema is suggested by the finding (also in agreement with other studies) of low levels of kallikrein inhibitor in the blood, and by the further observat,ion that several serum specimens collected within ‘21 to 48 hours after attacks of edema. showed a total or a partial lack of substrate for serum kallikrein. It is tempting to assume that excessire kallikrein activity in viva during the preceding active l)hase of the clinical disease may have depleted the available supply of this substrate. In the first reported trials in human beings, our tests demonstrated the powerful permeability-increasing capacity of activated purified C’l-cstcrase in the human skin. The finding of measurable amounts of inhibitor for C’l-csterase in the swum of patient A. L. is a result contrary to the reported complete absence of this inhibitor.16 The variability in the amounts of inhibitor found in different spccimcns from this same patient, collected at, different times, would

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suggest that we are dealing hew with an enzyme system in a dynamic state. In any case, the highest level of C’I-estcrase inhibitor found was no more than 33 per cent of the normal average. d possible explanation for our results is the use by us of a microtitrat,or and pH meter in the esterase tests for the miwoform01 titration, rather than t,he somewhat less sensitive calorimetric method employed by Donaldson and Evansl’; It is also possible that the slight modifications we introduced into the inhibitor tests ma.y have reduced their specificity. The precise rausc of the characteristic noninflammatory localized hereditary angioncurotic edema remains uncertain. Syst,ematic studies on patients during and between attacks, with many blood specimens collected at frequent intervals, will be needed, toget,her with furthcr experiments with purified reagents.‘” Among the pertinent, questions to be answrrcd are the rclativc role of the permeabi1it.y globulins on the one hand, and the complement enzyme system on the other hand, and the true identity of the inhibitor (or inhibitors) whose lack permits the lesion to’ develop. From our limited oh~servations, it, appears that in affcctcd persons an>- in viva mechanism that could actiyatv C’l-esterase, and/or serum kallikrcin or PF/DiI, coupled with pressure or trauma at a particular site, could result in localized vascular damage and t:dema. The marked responsiveness of the patients’ tissues (as illustrated in the skin) to these permeability-increasin g agents may ~11 br an important factor. The authors acknowledge the technical assistance of Miss Gloria Schultz, 3Iiss Dallas Pung, and Mrs. Stewanna, Lewis, and extend thanks to the volunteers who served as controls on the skin tests. C’l -cstt~rase and its inhibitor wew provided by Drs. Irwin TT. Lepow and Jack Pransky, Western Rcwrrw University Institute of Pathology, Cleveland, Ohio : and partially purified human plasma kallikrein was supplied by Dr. Marion E. Webster, Laboratory of Cardiac Physiology, Sational lnstitutrs of H(lxlth, Bethesda, Maryland. Thcx inter& of Dr. James R. Old and Dr.
REFERENCES TV.: Hereditary Angio-neurotic Oedcma, Am. .J. M. Sc. 95: 36!, 1888. 2. Spaulding, W. B. : Methyl Testosterone Therapy for Hereditary Episodx Edema (Hcreditary An@oneurotic Edema), Ann. Int. Med. 53: 739, 1960. 3. Trigg, J. W.: Hereditary Angioneurotic Edema. Report of a Case With Gastrointestinal Manifestations, New England J. Med. 264: 761, 1961. 4. Landerman, N. S.: Hereditary Angioneurotic Edema. I. Case Reports and Revicrv of the Literature, J. ALLERGP 33: 316, 1962. 5. Landerman, N. S., Webster, M. E., Becker, F. L., and Ratcliffe, H. E.: Hereditary Angionrurotic. Edema. II. Deficiency of Inhibitor for Serum Globulin Factor and/or Plasma Kallikrein, J. AI,LERGY 33: 330, 1962. 6. Landerman, N. S., Becker, E. L., and Kateliffe, H. E.: Increased Cutaneous Response to Diluted Autologous Serum in Hereditary Angio-edema, Lancet 1: 1053, 1960. 7. Miles, A. A.: Local and Systemic Fa.rtors in Shock, Fed. Proc., Suppl. No. 9, 20: 141, 1961. 8. Merle, E., and Trautschold, I.: Kallikrcin, Kallidin, Kallikrein Inhibit,ors, Ann. Sew York Acad. SC. 104: 117, 1963. 9. Webster, M. E., a.nd Pierce, J. B.: Studies on Plasma Kallikrein and Its Relationship to Plasmin, J. Pharmacol. & Exper. Therap. 130: 484, 1960. 10. Becker, E. L., and Kagen, L.: The Permeability Globulins of Human Serum and the Biochemical Mechanism of Hereditary Angioneurotic Edema, Ann. New York Acad. Sc. 116: 866, 1964. 1. Osler,

ilbnorn~alities 11. Ratnoff, Deficiency 12. Ratnoff, Human 13. Margolis, SC. 37:

in hereditnry

angioneurotic

edema

557

0. D., and Colopy, J. E.:

A Familial Hemorrhagic Trait Associated With a a Clot-promoting Fraction of Plasma, J. Clin. Invest. 34: 602, 1955. 0. D., and Miles, A. A.: The Induction of Permeability-increasing Activity in Plasma by Activated Hageman Factor, Brit. J. Exper. Path. 45: 328, 1964. J. : Hageman Factor and Capillary Permeability, Australian J. Exper. Biol. of

239,

1959.

iVebster, M. E., and Ratnoff, 0. D.: Role of Hageman Factor in the Activation of Yasodilator Activity in Human Plasma, Xature 192: 180, 1961. The Prescncc of Two Permeability 15. Kagen, L. J., Leddy, J. S., and Reeker, E. L.: Globulins in Human Serum, J. Clin. Invest. 42: 1353, 1963. 16. Donaldson, V. H., and Evans, R. R.: A Biochemical Abnormality in Hrreditary hngioneurotic Edema. Absence of Rrrum Inhibitor of C’l-e&erase, Am. J. Med. 35: 37, 1963. 17. Ratnoff, 0. D., and Lepow, I. H.: (‘omplement as a Mediator of Inflammation. Enhancement, of Yascwlar Pcrmeal)ility by Purified IIuman C’l-estwaw, J. Exper. Xcd. 118: 681, 14.

1963.

18. 19. 20. 21. 22. 23. 24. 25. 26.

Tnhibition of P(wneabilitp Globulins by C’l-&wasc Kagen, L. J., and Becker, E. L.: Inhibitor, Fed. Pro?. 20: 613, 1963. The Measurement of Second Componrnt of Human ComXustcn, K. F., and Beer, F.: b’l’lpo Cells, J. Immunol. 92: 946, 1964. plement, ((72”“) by Its Interaction With EAC’I, Austen, K. F., Sheffer, A. L., and Seymour, J. L.: The Detection of Hereditary hngioneurotic Edema and Certain Immunologic Reactions of Man by Specific Titration of the Second Component of Human Complement, J. Clin. Tnvcst. 43: 1234, 1964. Abst. Donaldson, V. H., Charache, P., and Rosen, F. R. : Activation of C’l-&erase iu Hereditary Angioneurotic Edema, J. Lab. & Clin. Med. 62: 873, 1963. AlIst. P., R,osen, F. S., and Janeway, (“. A.: (‘harache, P., Donaldson, V., Pensky, J., Fireman, Genetic Abnormalities in Hrrwlitary Angioneurotic~ Edrma, J. Clin. Invest. 43: 11’50, 1964. Abs;t. RatnoR, 0. D., and Lepow, I. H.: Some Proptors in Human Plasnla: The Effect of Acid. Acetone, Chloroform. Heat and Euelobulin Henaration. J. Phvsiol. 166: ,, 406.

1963.

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Xil&, A. A., and Wilhelm, D. L.: Enzyme-like Glolmlinn From Swum Reproducing thra Vascular Phenomena of Inflammation. 1. An Activable Permeabilitv Factor and Its Itlhihitor in Guinea Pir Serum. Brit. J. Exner. Path. 36: 71. 1955. L 28. Elder, J. M., and \&elm, h. L.: Enzynqe-like Globulins &on, Serum Reproducing tht, Vascular Phenomena of Inflammation. V. Activable Permeability Factor in Human Strum. Brit. J. Exper. Path. 39: 335, 1958. 29. Stewart, P. B., and Bliss, J. Q.: Permeability-increasing Factor in Diluted Human Plasna, Brit. J. Exper. Path. 38: 462, 1957. R. I)., Abernethy, R. XV., Ratnoff, 0. D., and Lepow, T. H. : Enhanwmmt of Jras30. Hmink, cwlar Prrmcability by Mixtures of C’l-&erase and Normal Strum, Proc. Sot. Exp,:r. Biol. R; Med. 116: 280, 1964. 27.