`
`Complement I: 147—159 (1984)
`
`01984 S. Karger AG, Basel
`0253—5075/34/0013-0147sz.7s/o
`
`Quantification of C1 -Inhibitor Functional Activities by
`Immunodiffusion Assay in Plasma of Patients with Hereditary
`Angioedema @ Evidence of a Functionally Critical Level of
`Cl-Inhibitor Concentration
`
`P.J. Spc'ith 3. B. Wi‘ithrich 1’, R. Bt‘t’tlera
`
`4‘ Blood Transfusion Service SRC, Central Laboratory, Berne;
`bDermatological Clinic, Allergy Unit, University Hospital, Zurich, Switzerland
`
`Key Words. Attenuated androgens - Hereditary angioedema - Cl-inhibitor « C4 -
`Immunodiifusion assay
`
`Abstract. The relationShip of Cl-inhibitor (Cl-INH) concentration and apparent func-
`tional activity was investigated in 11] plasma samples from 21 patients with the common
`form of hereditary angioedema (HAE). Functional Cl-lNH was analyzed by means of a modi-
`fied version ofimmunodilfusion assay. Down to a Cl-lNl—I level of approximately 0.075 g/l
`(38% of normal) apparent Cl-lNl—l functions were found within the normal range, while
`below this level functional adequacy of Cl-lNH could no longer be ascertained. When C4
`concentrations, considered to reflect approximately functional Cl-lNH, were related to Cl-
`INH antigen levels of individual samples, a relationship emerged which was identical to that
`between Cl-INH concentration and apparent function. No attacks of edema could be associ-
`ated with Cl-lNH concentrations above 0.075 2/], while it was possible to associate attacks
`with concentrations below this level. In experiments where patient plasma and normal plasma
`were mixed in various ratios or where HAE plasma was replaced by purified Cl-lNH, an
`increase in Cl-lNH antigen to concentrations of 0.06-0.08 g/l was followed by a sharp rise in
`apparent functions to normal values. The rise of functional C l-lNl-l became moderate when
`Cl-IN H antigen further increased. The results supported the idea of a functionally critical
`level ofCl-INl'l in the common form ofHAE.
`
`Introduction
`
`All these inhibitors become modified by the
`serine proteases which they inactivate [I].
`
`The serine proteases inhibited by Cl-lNH
`is a regulatory
`Cl-inhibitor (Cl-INH)
`are the active forms of the two enzymatic
`plasma protein. This uz-glycoprotcin belongs
`subunits of the first component of comple-
`to a class of inhibitors such as al-pi'oteinase
`inhibitor, antithrombin III and antiplasmin. ment, Clr and C15, and also plasmin, kalli-
`
`This materislwss copied
`stzhe NLM and may be
`Subjnt USCcpyright law:
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`148
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`Spéith/Wiithrich/Biitler
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`krein and coagulation factors Xla, XIIa and
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`XIIf[2—8]. In addition, Cl-INH controls the
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`autoactivation of zymogen Clr [9].
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`A variety of clinical symptoms may be
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`associated with reduced functional activity of
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`Cl-INH. In hereditary angioedema (HAE)
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`the reduced Cl-INH functional activity in-
`duces recurrent attacks of edema of the ex-
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`tremities, the gastrointestinal tract, the face
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`or the larynx, and death may result from air-
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`way obstruction [10]. Affected persons are
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`heterozygous for a defect of the Cl -INH pro-
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`tein gene [1 1]. In the common form of the
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`disease, the reduction in functional activity is
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`due to Cl-INH concentrations being only 5—
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`30% of normal. In the variant form of the
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`disease, immunochemically detectable con-
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`centrations of Cl-INH are normal or ele-
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`vated, but a dysfunctional mutant protein is
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`synthesized [12]. A reduction of functional
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`Cl-INH may also be acquired; such condi-
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`tions may or may not be associated with
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`reduced antigen levels [13, 14]. Furthermore,
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`functional
`inadequacy of Cl-INH can be
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`transient without any decrease in the level of
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`Cl-INH [15]. Thus the unequivocal diagno-
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`sis of all Cl-INH deficiency stages is based
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`on the demonstration of a functionally inade-
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`quate Cl-INH.
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`Recently, Ziccara’i and Cooper [16] de-
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`scribed a simple and easily performable assay
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`system for assessment of Cl-INH functional
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`activity. Unlike other methods, no special-
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`ized techniques are needed and all reagents
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`are commercially available. Furthermore,
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`this assay system is the only one where one of
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`the natural substrates of Cl-INH, Clr, in its
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`physiologic concentration serves directly as
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`an indicator of functional activity of the in-
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`hibitor. So far a drawback of this assay sys-
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`tem was the lack of a valid method to quan-
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`tify functional Cl-INH.
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`A modification of the immunodiffusion
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`assay is proposed in this study which allows
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`quantification of functional Cl-INH. It was
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`used to investigate routinely the plasma of a
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`large number of patients for functional C1-
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`INH and to select those patients suffering
`from the common form of HAE. A further
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`aim of this study was to reinvestigate the
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`effect of attenuated androgens in the com—
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`mon form of HAE. Indeed,
`the literature
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`reports that therapy with impeded androgens
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`prevents attacks of angioedema and normal-
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`izes the concentration of the fourth compo—
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`nent of complement. However, there are con—
`troversies about the effect on the level of Cl-
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`INH antigen [17—23]. The results presented
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`in this study suggest that there is a function—
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`ally critical level of Cl-INH concentration of
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`approximately 0.075 g/l (38% of normal),
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`above which apparently normal function of
`Cl-INH and thus normal concentrations of
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`C4 seem to be assured in the plasma ofpatients
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`affected by the common form of HAE.
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`Patients and Methods
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`Subjects
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`21 patients were studied, 15 women and 6 men, all
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`suffering from the common form of HAE. In each
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`case, diagnosis was made on the basis of clinical and
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`family history as well as on serological findings. Of the
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`111 plasma samples analyzed, 22 were from 13 pa-
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`tients receiving no therapy. 2 of these 13 patients were
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`eventually treated with attenuated androgens and 1
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`with tranexamic acid. 5 samples were collected from 3
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`patients receiving therapy with tranexamic acid.
`I of
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`these patients was later treated with danazol. 7 pa-
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`tients were managed by danazol or stanozolol (Win-
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`throp, Basel, Switzerland). 84 samples originated from
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`this group of patients.
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`Laboratory Methods
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`Heat-aggregated human lgG (aIgG) was prepared
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`by heating a solution of 16 mg/ml for 20 min to 63 °C.
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`After cooling and centrifugation for 15 min at 1,500 g
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`This mate rial was copied
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`aims NLM and may be
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`SL5 Eject U5 Earp-yright Laws
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`Functionally Critical Level of C l-INH Concentration
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`149
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`INH. The method proposed estimates func-
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`tional Cl-INH by calculating the slope of the
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`regression line formed when the added aIgG
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`concentrations are plotted against ratios of
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`immunologically detectable Clr in the 5 ali-
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`quots of the recalcified plasma sample to be
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`analyzed. To obtain a significant coefficient
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`of regression (r2), it proved useful first to cal-
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`culate the percentages of immunologically
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`detectable Clr in the 5 aliquots. The Clr con-
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`tent of plasma pooled from 50 healthy blood
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`donors served as reference (=100%). Accu-
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`racy of Clr quantification from day to day
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`was i 5 % (n = 45). To avoid a dependency of
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`apparent functional activity of Cl-INH on
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`levels of Clr, calculation of the ratios of
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`immunologically detectable Clr in the ali-
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`quots A0_4 was a prerequisite. Calculation in
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`parallel of the regression lines in individual
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`serum and recalcified citrate plasma samples
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`from 50 healthy blood donors generally re-
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`sulted in a higher r2 value for the recalcified
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`plasma samples. Quantification of functional
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`Cl-INH in EDTA plasma proved not to be
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`possible, even though Ca2+ and Mg“ were
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`added in a 10-fold excess.
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`In each run of analyses the slope of the
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`regression line of recalcified plasma pooled
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`from 50 healthy blood donors was also esti-
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`mated and was set at 100% (in fig. 1, b =
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`—0.577). The percentage of Cl-INH func-
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`tional activity in plasma from a HAE pa-
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`tient is reflected by the value of the slope of
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`the regression line (in fig. 1, b = -—0.196) and
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`can be calculated as given in this example
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`(fig. 1):
`(—0.196/—0.577) X 100% = 34%.
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`The accuracy of the determination within 1
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`day was found to be i 10% (n = 15) and
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`from day to day to be i 13% (n = 45). The
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`normal range of functional Cl-INH levels
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`varied between 70 and 135% (95% confi-
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`dence interval).
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`the protein concentration was adjusted to exactly
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`15 mg/ml.
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`Concentrations of C4 and Cl-INH were deter-
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`mined nephelometrically (Hyland) and by single radial
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`immunodiffusion (RID), respectively. Standards from
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`Atlantic Antibodies (Merz + Dade, Diidingen, Swit-
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`zerland) and from Hyland (Travenol, Switzerland)
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`served as references. Purified, functionally active C1-
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`INH was kindly donated by II.P. Kocher, University
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`of Geneva, Switzerland.
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`Quantification of Functional CI—INH by the
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`Immunodiffusion Method
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`9 volumes of blood were drawn into siliconized
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`glass tubes containing 1 volume of0.11 mol/l trisodium
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`citrate. After adequate mixing and centrifugation,
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`plasma was stored in plastic tubes at —70 °C. To 495 pl
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`plasma 5 pl of 2 mol/l CaClz solution was added. The
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`formed clot was removed and the supernatant was
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`divided into 5 aliquots of 80 ul (A04); aIgG solution
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`was added to 4 of the aliquots to obtain final concen-
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`tration of 0.25 (A1), 0.5 (A2), 1.0 (A3) and 1.5 mg
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`aIgG/ml (A4). The volumes of the aliquots were ad-
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`justed to 100 pl with isotonic saline. To the 5th of the
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`aliquots (A0) 20 ul isotonic saline was added. The mix-
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`tures were incubated for 60 min at 37 °C. Incubation
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`was stopped by the addition of6 ul of0.2 mol/l EDTA,
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`pH 7.4. In each run, pooled plasma from 50 healthy
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`blood donors was treated the same way. Clr levels in
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`A04 were assessed by RID in 1 % (w/v) agarose, pH 8.0,
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`containing 3.25% (v/v) ofan IgG fraction ofgoat anti-
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`human Clr (Atlantic Antibodies). The Clr contents of
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`various dilutions of the aliquot of the pooled plasma
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`that did not receive aIgG served as a calibration curve.
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`Ratios ofClr were formed by dividing the percentage of
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`immunologically detectable Clr in A04 by the C 1 r level
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`in A0. In order to quantifiy Cl—INH functional activity,
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`the logarithms of the added aIgG concentrations were
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`plotted against ratios ofClr in the aliquots and the slope
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`of the regression line was calculated.
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`Results
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`Quantification of Functional CI-INH by
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`Immunodiffitsion Assay
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`The immunodiffusion assay of Ziccardi
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`and Cooper [16] was modified in such a way
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`as to allow quantification of functional C1-
`
`
`
`Th is mate rial was copied
`
`
`
`
`
`
`aims NLM and may DE
`
`
`
`
`EL: Eject U5 Copyright Laws
`
`
`
`
`
`
`Page 3 of 13
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`Page 3 of 13
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`

`

`ISO
`Spath/Wiithrich/Bfitler
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`Functional Adequacy of C1 -INII in the
`Common Form of HAE
`Figure 2 gives an overview of the relation-
`ship between C 1 -INH concentrations and ap-
`parent functions in clinical conditions. These
`include the common and the variant form of
`
`HAE (sample of the variant form kindly pro-
`vided by C. Alper. Boston. USA), acquired
`Cl-INH deficiency with complete remission
`and normalization of various complement
`parameters for 6 months after surgical re-
`moval ofa giant myoma of the fundus uteri,
`deficiencies and dysfunctions of components
`ofcomplement other than Cl-lNH, hypo- or
`agammaglobulinemia, immune complex dis-
`eases, acute and chronic idiopathic thrombo-
`cytopenic purpura, malignancies, bacterial
`and viral infections and urticaria. The results
`
`indicate a correct quantification of functional
`Cl-lNH in cases of the acquired and variant
`forms of angioedema. However, in l
`l l recal-
`cifted plasma samples from 21 patients with
`the common form of HAE, unexpectedly
`high functional activities associated with
`subnormal levels of (‘l-INH were observed.
`
`A closer view of (‘l-lNH antigen levels and
`related functions in these samples is given in
`figure 3a. The association of apparently nor—
`mal function and abnormally low Cl-INH
`level was found almost exclusively in recal—
`cificd plasma from patients treated with at-
`tenuated androgens. Among the 23 samples
`with Cl-lNH levels above 0.075 g/l (38% of
`normal) only 1 sample (4%) displayed an
`apparently inadequate Cl-INH function. In
`contrast, among the 88 samples with Cl-INH
`concentrations below 0.075 g/l 65 (74%)
`showed functionally inadequate Cl-lNH.
`In the range of 0035—0075 g/l Cl-lNH
`antigen the functional activities could vary
`from 10 to 135% ofnormal, and in 1 sample
`an apparent Cl-lNH function as high as
`
` V7r“fr )Iv..'J
`
`airy, vl'l’lWJv mghul
`
`2‘:w'_>
`._')
`'3
`3-}(I
`
`Fig. l. Quantification ofCl-INH functional activ-
`ity in HAE using the immunodilTusion method. The
`regression lines between the concentrations of added
`algG and the ratios of immunologically detectable C l r
`in aliquots (A04) of recalcified plasma pooled from
`healthy blood donors (0) and in aliquots of recalcified
`plasma from a patient (0) are shown. The coefficients
`of correlation (r3) in the examples given were 0.999 (D)
`and 0.99] (0), respectively.
`
`156% emerged (open circle in fig. 3a). This
`and all other samples with extremely diver~
`gent Cl-INH concentrations and apparent
`functions were collected from patients with
`manifest infections or from patients where a
`subclinical infection was strongly suspected.
`
`Thus, the most extemc Cl-lNH concentra—
`tion and related function (0.02 g/l and 85 %,
`respectively; one of the open triangles in
`fig. 3a) were seen in the plasma from a pa-
`tient with an abscess on his neck. All other
`markedly divergent concentrations and func-
`tions were observed only in recaleified
`plasma from members ofa particular family.
`All these patients were heterozygous for the
`Cl-INH and the factor I
`(C3b/C4b-INA)
`protein gene, and some of them even showed
`
`this material wascopied
`It the NLM and MW be
`Subject US Copyright Laws
`
`Page 4 of 13
`
`Page 4 of 13
`
`

`

`l5l
`Functionally Critical Level ol‘(‘l-lNH Concentration
`
`150
`
`10!} .
`
`so -
`
`
`
`Fig. 2. (.‘l-lNH concentrations and
`apparent functions in 364 rccalcificd
`plasma samples analyzed routinely. The
`symbols indicate samples from patients
`with the common form of HAE under
`various therapeutic regimens (0), ac-
`quired angioedema before and after sur-
`gical intervention (D). the variant form
`of HAE (A). the common form ol'HAE
`after infusion of (‘l-lNH concentrate
`(I), HAE under discussion (0) and
`plasma samples from members of HAE
`families (A), or from subjects with var-
`ious other diseases (see text) ( + ). Nor-
`mal 95% range ofimmunologically de-
`tectable Cl -lNH is given by the vertical
`and of functional (‘l-lNH by the hori-
`zontal. partially dotted. lines.
`
`a
`
`w—w-I
`Fewxici'tdl
`
`‘11
`O
`Cielrlll Ll!lll'.]"ll‘ tjll
`
`0.7
`
`0.3
`
`(1/.
`
`depressed plasma concentrations of C4-bind-
`
`ing protein. Levels of C3 were usually found
`below the lower limit ofthe normal range and
`the patients had prolonged phases of infec-
`tions.
`
`Figure 3a does not clearly indicate the re-
`lationship of (‘l-INH concentration and
`
`function in the samples analyzed. However,
`when Cl-lNH antigen was ranged in classes
`of 0.02 g/l, as indicated by bars in figure 3b.
`the means of CI-INH concentrations and
`
`apparent functions of the individual classes
`
`clearly indicated a nonlinear relationship.
`Two approaches calculating the relationship
`between concentration and function were
`
`made. In a first attempt, the regression lines
`of concentrations and functions in samples
`with Cl-INH levels in the ranges 0.012—0.08
`and 0.08—0.13 g/l, respectively. were calcu-
`lated. For this purpose, individual Cl-lNH
`concentrations and functions of plasma sam-
`
`plcs as given in figure 3a were considered.
`The two regression lines are shown in figure
`3b. In a second attempt the regression line
`was calculated after logit transformation of
`apparent (‘l -lNH function. Such transforma-
`tion requires values below 100%. Thus,
`it
`was reasonable to do a calculation using the
`mean values of functional Cl-INH as given
`
`in figure 3b. The use of the mean values
`ensured that the corresponding functional ac-
`tivities of an equivalent of Cl-lNH antigen
`up to a concentration of 0.09 g/l were taken
`into consideration. The retransformed values
`
`of the calculated regression line are given in
`figure 3b. Both calculations indicated that a
`concentration of Cl-lNH antigen of 0.07—
`
`0.08 g/l represents a threshold level for Cl-
`lNH function.
`
`The concept of a functionally critical level
`of Cl-INH concentration was further sup-
`
`ported by relating CI-lNH concentration to
`
`This material was copied
`at the ML“ and may be
`Subjut US Copyright Law:
`
`Page 5 of 13
`
`

`

`I52
`Spa'lh/Wiithrich/Bfillcr
`
`
`
`
`150
`
`0
`
`a: 10
`I
`Z
`,I_
`U 50
`a
`5
`U
`«
`1.5
`
`o
`
`c
`
`°
`A
`o .
`o
`.C . .
`O.
`.
`.
`a .
`a0...-
`.“
`o ..
`o
`.0
`.‘ O
`o 55.
`‘0’ °
`v
`t
`D
`0 all.
`“g -- °
`
`f—‘I—I
`A
`
`0
`._L
`
`04
`
`°
`
`a
`
`M
`
`d
`
`1
`
`l
`
`’1
`
`T‘b
`
`._
`
`FT
`
`,
`
`I
`
`ll
`
`_
`
`a
`“" I
`M
`e
`
`‘
`
`c
`
`.
`
`_
`
`°
`
`0
`
`e
`
`'
`
`I
`
`’I
`
`/I
`
`0.3
`
`02
`
`01
`
`0
`
`a
`OI
`C~
`"’
`t‘
`.5."
`E1
`a
`
`0
`
`AA A. o
`A
`0A A!
`A
`'1. "
`$.A...’ ‘
`.
`o
`‘ 91‘ °
`W
`a...
`«J
`v
`g D
`
`.V DE:
`’0
`le ' ‘1—fi' TT r
`" mgrflT'Ti—T
`0.05
`0.1
`0.15
`U 05
`O 'l
`Ci-lNH antigen, gll
`
`A
`
`‘
`
`.
`
`,’
`
`’
`
`___
`
`._
`
`,
`
`o
`
`0.15
`
`Fig. 3. Quantification of Cl-lNH and C4 antigen
`and functional (‘l-lNH in lll individual plasma sam-
`ples from 2| patients with the common form ofHAE.
`The vertical lines indicate the lower limit of the nor-
`mal range (—2 SD) of Cl-lNH concentration. The
`horizontal lines represent the lower and the upper lim-
`its ofthe normal range (1 2 SD) ofapparcnt ('l-lNH
`function, respectively.
`:1 Plot of individual Cl—INH
`antigen levels against apparent functional activities.
`The therapeutic management of the patients at the
`time when the samples were collected is indicated: no
`therapy (C1), therapy with tranexamic acid (v). therapy
`with danazol (e) or slanozolol (A). A or o = Samples
`from patients with present or recent bacterial or viral
`infections and therapy with danazol or stanozolol; a =
`Cl-lNH concentrations and functions in samples
`from an asymptomatic woman, a member of the
`family with concomitant heterozygoty for the C l -INH
`and the factor I protein gene, herself heterozygous only
`for Cl-lNH. b Means ( :1 SD) ofCl-lNH concentra-
`tions and functions as can be calculated when ranges
`
`of Cl-lNH antigen levels are formed. Limits of the
`ranges were 6—15, 16-25.
`56—67 % of normal con-
`centration (100% = 0.]96 g/l). 0 = Calculated curve
`of mean (‘l-lNH concentrations versus logit trans-
`formed and retmnsformed mean functional activi-
`ties; ——— = regression lines between individual (‘1-
`lNH concentrations in the ranges 6-40 and 41-67%
`of normal and associated functions.
`respectively.
`c C l-INH antigen levels and manifestation of attacks
`of edema. o = Cl-lNH concentrations in plasma col-
`lected after an attack; 0 = mean concentrations or
`two different quantifications. During the period of
`quantification an attack was observed. but the time
`between the plasma collections was too long to per-
`mit direct correlation of (‘l-lNH concentration and
`attack of edema. d Individual Cl-lNH levels, plotted
`against C4 concentrations. The symbols used are the
`same as in a. e Means (i 1 SD) ofCl-lNH and (‘4
`concentrations as can be calculated when ranges of
`C l-INH antigen levels are formed. Symbols and ex-
`planations as in b.
`
`This material was copied
`at the ML" and may be
`Subject US Cowman: Laws
`
`Page 6 of 13
`
`

`

`153
`Functionally Critical Level of Cl-lNH Concentration
`
`
`
`
`
`
`
`
`
`
`
`
`manifestations of angioedema. Cl—INH con-
`
`
`
`
`
`
`
`
`centrations above 0075 g/l (38% of normal)
`
`
`
`
`
`
`
`
`
`could not be related to an attack of edema,
`
`
`
`
`
`
`
`
`while in all samples collected after an attack
`
`
`
`
`
`
`
`Cl-INH concentrations were below 0.075 g/l
`
`
`
`
`
`
`
`(fig. 3c). Attacks were most frequent when
`
`
`
`
`
`
`
`
`Cl-INH levels were below 0.035 g/l (18% of
`
`normal).
`
`
`
`
`
`
`
`
`Relationship between (“I-[NH and ('4
`
`
`
`
`
`Concentrations in the Common Form of
`HAE
`
`
`
`
`
`
`In HAE a close relationship between func-
`
`
`
`
`
`
`
`
`tional Cl-INH and levels of C4 antigen is
`
`
`
`
`
`
`
`assumed. The assumption of such a close
`
`
`
`
`
`
`relationship is widely accepted and serves
`
`
`
`
`worldwide for routine laboratory approxima—
`tion of functional Cl-INH in HAE. Thus.
`
`
`
`
`
`
`
`
`
`
`
`quantification ofC4 levels by routine labora—
`
`
`
`
`
`
`tory technique in parallel to Cl-lNH func—
`
`
`
`
`
`
`
`tion offered a chance to verify the correctness
`
`
`
`
`
`
`
`ofthe method proposed for quantification of
`
`
`
`
`
`
`
`Cl-INH function. Figure 3d depicts the rela-
`tionship between Cl-INH and C4 concentra-
`
`
`
`
`
`
`
`
`
`
`
`tion ofindividual samples. Analogous to the
`
`
`
`
`
`
`
`results as given in figure 3a , Cl-INH concen—
`
`
`
`
`
`
`
`trations above 0.075 g/l were associated with
`
`
`
`
`
`
`C4 levels within the normal range. Further-
`
`
`
`
`
`
`
`more, Cl-INH antigen within a range of
`
`
`
`
`
`
`
`0035—0075 g/l could be associated with C4
`
`
`
`
`
`concentrations of 0.03—0.25 g/l. Finally, be—
`
`
`
`
`
`
`
`low a Cl-INH concentration of 0.035 g/l C4
`
`
`
`
`
`
`
`levels were greatly reduced. When the same
`
`
`
`
`
`
`
`procedure was applied to results presented in
`
`
`
`
`
`
`
`
`figure 3d as was applied to parameters in fig-
`
`
`
`
`
`
`
`
`
`
`ure 3a to obtain the results in figure 3b, a
`
`
`
`
`
`nonlinear relationship between Cl-INH and
`
`
`
`
`
`
`C4 concentrations became apparent (fig. 3e).
`
`
`
`
`
`Again a Cl-INH threshold level of0.07—0.08
`
`
`
`
`
`
`
`g/l was found, above which normal C4 con-
`
`
`
`
`
`
`centrations seemed to be assured in patients
`
`
`
`
`
`
`
`suffering from the common form of HAE.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CI-INII Functions in illixtures ofllAE
`and Normal Plasma and in IIAE Plasma
`
`
`
`
`
`
`
`
`
`
`Replaced by Purified C1 -INH
`
`
`
`
`
`
`
`The results in figure 3a reflect the fluctua-
`tion in Cl-INH concentration and apparent
`
`
`
`
`
`
`
`
`
`
`
`
`function in the plasma of various HAE indi-
`
`
`
`
`viduals receiving various therapeutic regi-
`
`
`
`
`
`
`
`mens. The results imply, but do not conclu-
`
`
`
`
`
`sively prove, a nonlinear relationship be-
`tween Cl-INH concentration and apparent
`
`
`
`
`function. To obtain more evidence of the
`
`
`
`
`
`
`
`
`
`
`
`
`existence ofa functionally critical level ofCl-
`
`
`
`
`
`INH concentration, recalcified plasma from
`2 individuals with the common form of HAE
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`was used for further in vitro studies. At the
`
`
`
`
`
`
`
`time of plasma collection both patients were
`
`
`
`
`
`
`
`receiving tranexamic acid and thus had low
`Cl-INH concentrations and functions. The
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`concentration of factor B in the plasma from
`
`
`
`
`
`
`
`1 of these patients was elevated, indicating
`
`
`
`
`
`
`
`
`that
`there was, or recently had been. an
`
`
`
`
`inflammatory
`process.
`The
`recalcifled
`
`
`
`
`
`
`
`
`
`plasma ofthe patient with a normal factor B
`concentration was mixed in various ratios
`
`
`
`
`
`
`
`
`
`
`
`
`with recaleified plasma from a healthy per—
`
`
`
`
`son. Cl-INH concentration and apparent
`
`
`
`
`
`
`function of the mixtures were quantified and
`
`
`
`
`
`
`
`
`the results are shown in figure 4. To demon-
`
`
`
`
`
`strate the relationship between Cl-INH con-
`
`
`
`
`
`centration and apparent function, the regres-
`
`
`
`
`
`
`
`sion lines ofthese parameters within Cl-INH
`
`
`
`
`
`concentration ranges of 0.02—0.06 and 0.06—
`
`
`
`
`
`
`0.18 g/l, respectively, were calculated. Again
`
`
`
`
`
`
`a disproportionately rapid rise of apparent
`
`
`
`
`function over
`immunologically detectable
`
`
`
`
`
`Cl-INH emerged in the Cl-INH concentra-
`
`
`
`
`
`
`tion range of 0.02—0.06 g/l. This dispropor-
`tion was reversed as soon as Cl-lNH concen-
`
`
`
`
`
`
`
`
`tration exceeded a value of 0.06 g/l.
`
`
`
`
`
`
`
`
`
`
`
`
`The study described above was repeated
`
`
`
`
`with plasma containing elevated concentra-
`tions of factor B. A rise in Cl-INH concen-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thia material was applied
`
`
`
`at the NLM and may be
`
`
`
`
`
`
`Subject US Copyright Laws
`
`
`
`
`
`Page 7 of 13
`
`Page 7 of 13
`
`

`

`154
`Spa‘th/Wiithrich/Biillcr
`
`tration from 0.035 to 0.065 g/l was associ-
`ated with an overshoot
`in apparent func-
`tional activity reaching values of 180%. A
`further rise in Cl-lNH antigen as a result of
`the addition of normal recalcificd plasma re-
`sulted in a constant decrease of apparent
`functional activity to the values of the recal-
`cified normal plasma (fig. 5). Replacement of
`the HAE plasma containing elevated concen-
`trations of factor B by increasing amounts of
`purified Cl -1NH led again to an initially dis-
`proportionate rise in apparent functions; at a
`concentration of0.075 g/l the apparent func-
`tion of Cl-INH was approximately 100%
`and subnormal concentrations of Cl-lNH
`
`around 0.1 g/l were associated with apparent
`
`functions above the upper limit of normal. A
`rise in Cl-lNH concentration from 0.1 to
`
`0.35 g/l showed little effect on functional Cl-
`lNH (fig. 5).
`
`1‘/1
`
`VJ)
`
`
`
`
`
`'1-l\.‘l-_‘1.t.
`
`“‘é.
`v-;-lc,
`
`r——fi'
`C 1
`”()5
`(J
`",1 11111 'lll'lq‘fll' g/l
`
`r
`
`l 7 t
`010
`
`
` ,FA.
`
`I
`
`-e
`
`I
`111‘;
`
`Fig. 4. Concentrations and functions ofCl-lNH in
`rccalcified mixtures with varying ratios of HAE
`plasma and normal plasma. Regression lines of Cl-
`lNH functions and concentrations in the ranges 0.02—
`0.06 and 006-0. 18 g/l. respectively. were calculated.
`Vertical and horizontal lines indicate normal ranges of
`(‘l-INH concentration and apparent function.
`
`Discussion
`
`The results of this study suggest that there
`is a functionally critical level of Cl-lNH in
`recalcifted plasma. This evidence was ob-
`tained by analysis of l
`l
`l recalcified plasma
`samples from 2| patients with the common
`form of HAE under various types of therapy,
`and also from recalcified HAE plasma sup-
`plemented by recalcified normal plasma or
`replaced by purified Cl-lNH. The existence
`
`ofa functionally critical level ofCl-INH was
`
`found to be rooted in a nonlinear relationship
`between Cl -1NH concentration and apparent
`function. The value of the functionally criti-
`cal
`level was found to be approximately
`0.075 g/l (38% of normal); indeed. the func-
`tional adequacy of (Tl-INH, the concentra-
`tions of C4 within the normal range and the
`absence of attacks of edema were guaranteed
`
`as long as the antigen concentration did
`not
`fall below this threshold level. Thus.
`
`this is the first study to explain the obser-
`vation of this and other reports [18—23] of
`normal
`(‘4 concentrations
`in association
`with subnormal levels of (‘l-lNH in HAE
`
`(fig. 3).
`At the time when the preliminary results
`of this 4-year study were presented [24]. no
`other reports existed which could support the
`idea ofa nonlinear relationship between (‘1 -
`INH concentration and function; indeed all
`
`reports analyzing concentrations and func-
`tions of Cl-lNH by various methods fa-
`voured a linear relationship [25—31]. How-
`ever, in the meantime four publications pre-
`sented data which are compatible with a
`functionally critical level ofCl-INH concen-
`tration. Very recently, Ghebrahiwc! et a1. [32]
`studied the spontaneous breakdown of he-
`
`l'his material was copied
`it the NLM and may be
`Subject US Copyright Law:
`
`Page 8 of 13
`
`

`

`155
`Functionally (‘ritical Level of (‘l-lNH (‘oncentration
`
`Fig. 5. Concentrations and func-
`tions of (.‘l-lNH in a recalcilied
`mixture of HAE plasma with ele-
`vated factor B level and normal
`plasma (0) or purified ('i-INH (A).
`The indicated relationships of Cl-
`lNH concentrations and functions
`as they are given by the drawn
`curves are approximations and were
`not calculated. The normal
`range
`(RlD) is indicated by vertical lines,
`the apparent normal range of func-
`tional (II—[NH by horizontal lines.
`
`L l—INH antigen, git
`
`OO‘j
`
`01
`
`molytic complement in diluted sera obtained
`
`ccntration and function was reported by
`
`from patients with HAE. They found that the
`concentration ofCl -INH in NHS is in excess
`
`ofwhat is required to prevent the breakdown
`ofhemolytic complement in the diluted sam—
`
`ples. ln 1 HAE serum sample with no detect-
`able functional (‘l-lNH but substantial he-
`
`the
`molytic complement prior to dilution.
`replacement of highly purified and function-
`ally active C‘l—lNH resulted in a dose-depen-
`dent prevention of spontaneous complement
`reduction which reached its maximum effect
`at concentrations around 50% of normal. In
`
`[33]
`another publication. Yolvingmn et al.
`showed Cl-lNH concentrations of approxi-
`mately 0.08 g/l in sera of patients with HAE
`in association with normal and abnormal
`
`results of the immunodiffusion assay. Below
`Cl-INH concentrations of approximately
`0.075 g/l
`the immunodilTusion assay indi-
`cated a functionally diminished (Tl-[NH in
`all samples, whilst above a concentration of
`
`Doe/«'3 ct al. [34]. They analyzed in vitro the
`inhibition of the C4-converting activity after
`activation of C1 by aIgG of definite size in
`the presence of various amounts ofCl-INH.
`The maximum C4 consumption that could
`be reached at high concentrations of algG
`could be significantly reduced at a Cl-lNI-l:
`C4 ratio of only 0.6 although in vivo the
`value of normal Cl-INH:C4 ratio is 5-10.
`
`The fourth publication which supports the
`findings of this study is by Ziccardi [9]. He
`analyzed the concentration dependence of in-
`hibition by Cl-lNH of spontaneous Cl acti-
`vation. Although a different method was
`used to quantify functional Cl-INH, the re-
`sults obtained were similar to those of this
`
`study. A rise in Cl-lNH concentration from
`25 to 35% of normal was associated with a
`
`rise in apparent inactivating capacity of C [-
`lNH from 20 to 70% of normal. Variations
`in Cl-INH concentration above 35% of nor-
`
`approximately 0.095 g/l functional (‘l-INH
`was apparently normal. Further evidence oi'a
`
`nonlinear relationship between (7l-INH con-
`
`mal resulted only in minor changes of appar-
`ent functional activity of Cl-INH. Although
`Ziccara'i'r [9] results and those of this study
`
`This material wsseopied
`It the NLM and "my be
`Sobjcct USCopyright Law:
`
`Page 9 of 13
`
`

`

`156
`Spéith/Wiithrich/Butler
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`accord, it must be borne in mind that two
`different functions of Cl-INH were ana-
`
`
`
`
`
`
`lyzed.
`The existence of a threshold level of anti—
`
`
`
`
`
`
`
`
`
`
`
`
`
`gen for adequacy of functional Cl-INH is
`
`
`
`
`
`
`
`also plausible if the disappearance of Cl-
`
`
`
`
`
`
`
`INH and the decrease in C4 hemolytic titers
`
`
`
`
`
`is studied following the intravenous adminis-
`
`
`
`
`
`tration of partially puri