Journal of Clinical InvestigationVol. 44, No. 3, 1965

Complete Pseudocholinesterase Deficiency: Genetic andImmunologic Characterization *

W. E. HODGKIN,t E. R. GIBLETT, H. LEVINE, W. BAUER, ANDA. G. MOTULSKYt(From the Departments of Medicine and Genetics, University of Washington, and King County

Central Blood Bank, Seattle, Wash., and Middlesex Memorial Hospital,Middletown, Conn.)

The plasma enzyme pseudocholinesterase (acyl-choline acyl hydrolase) is composed of a seriesof related glycoproteins that comprise no morethan 0.017o of the total plasma proteins (1). Itsbiological function in normal metabolism is notfully understood (2). Pseudocholinesterase isessential for the rapid breakdown of the musclerelaxant suxamethonium (succinylcholine), whichis frequently used in surgery and electroshocktherapy. Thus, if pseudocholinesterase activityis defective or markedly decreased, prolongedmuscle relaxation and apnea may follow the ad-ministration of the drug (3, 4).

Pseudocholinesterase activity is markedly in-hibited by a number of compounds. There are,however, two variant forms of the enzyme, whichare characterized by decreased susceptibility toinhibition by the local anesthetic dibucaine (5, 6),on the one hand, and by fluoride (7) on theother. The formation of these enzymes is pre-sumably controlled by allelic mutants of the nor-mal gene, E1u, which determines the usual esterase(8, 9). The dibucaine-resistant enzyme is pro-duced by the gene called Ela, for which 3 to 4%oof the population are heterozygous (10). Theother gene E1l, associated with fluoride-resistantesterase production, has a much lower frequency.Individuals who are either homozygous for thesegenes (i.e., E1a/Ela and E1l/El') or who possess

* Submitted for publication September 15, 1964; ac-cepted November 30, 1964.

Aided by grants HE 03091 (A.G.M.) and HE 05780(E.R.G.) from the National Institutes of Health and bya grant from the Middletown United Fund.

t Fellow of the National Institutes of Health. Presentaddress: Department of Pediatrics, University of Ver-mont College of Medicine, Burlington, Vt.

4 Address requests for reprints to: Dr. A. G. Motulsky,University of Washington School of Medicine, Seattle,Wash. 98105.

both genes (E1a/Elf) are susceptible to prolongedapnea after suxamethonium administration.

A fourth gene, probably situated at the pseudo-cholinesterase locus, is associated with completeabsence of enzyme activity in the homozygote andhas been referred to as the "silent" gene (E1l).Only two homozygotes have been described (11,12). These otherwise normal subjects had pro-longed apnea when given suxamethonium. Thisreport describes a kindred containing two addi-tional individuals who are homozygous for the"silent" gene. Study of these patients and theirrelatives provided further data of the genetics ofthe silent gene. New data are presented to sug-gest that in such rare homozygotes no significantamount of antigenically related cross-reactingmaterial is produced.

MethodsCase reports

Case 1. Patient G. Cl. (11-1; Figure 1) a 53-year-old white male of Irish origin was hospitalized in 1958for elective repair of a ventral hernia. His parents werenot related. Past history revealed hypertension of 3years' duration, auricular fibrillation, and digitalis ther-apy for 2 years. Physical examination revealed an obesewhite male with a blood pressure of 180/110, moderatecardiomegaly, and a grade one apical systolic murmur.Aside from a ventral hernia, no other abnormality wasnoted.

Laboratory studies showed a hemoglobin of 16.4 gper 100 ml, hematocrit 50%, and leukocytes 6,600 withnormal differential count. Urinalysis was normal. Non-protein nitrogen was 25 mg per 100 ml, and cholesterolwas 320 mg per 100 ml. The electrocardiogram showedauricular fibrillation and suggested left ventricular hyper-trophy.

Anesthesia preparatory to the hernia repair was in-duced with intravenous sodium pentothal. Cyclopropanewas followed by nitrous oxide, ether, and oxygen. Atotal of 235 mg of succinylcholine was given intra-venously. Muscle relaxation and apnea were prolonged toapproximately 3 hours, during which artificial ventilation

486

I

EU

IE

COMPLETEPSEUDOCHOLINESTERASEDEFICIENCY

PEDIGREE OF KINDRED WITH COMPLETEPSEUDOCHOLINESTERASEDEFICIENCY

I 2

gL2

2 3

EU 0 435 0'\ III 116

I1 g_;2 3 4 15 6

189 179 255 10 218 181 147 64

2 F7t1S134 THomozygotes for complete pseudo-

Cd $! S!)9 < cholinesterase deficiency Els ElsEU 290 239 219 190 255 314 233 Heterozygotes for complete pseudo-

cholinesterose deficiency El E1sEU Esterase unitMean of 143 controls 226 E U 53 (or)

FIG. 1. PEDIGREE OF KINDRED STUDIED. Dibucaine and fluoride numberswere normal in all individuals.

was required. Intermittent hypotension occurred for24 hours postoperatively and was treated with intra-venous metaraminol and with 500 ml of whole blood.The remaining hospital course was uneventful, and sev-

eral liver function tests before discharge were normal.The patient subsequently expired as the result of a cere-

bral vascular accident.Case 2. Patient G. Co. (II-2: Figure 1), a 52-year-

old white female and sister of patient II-1, entered thehospital on May 16, 1961, for treatment of an acutetraumatic injury to her right hand. Physical examina-tion showed no abnormalities other than injury. Anes-thesia was induced with intravenous sodium pentothal,then maintained with cyclopropane. A total of 300 mg

of succinylcholine was given intravenously. Muscle re-

laxation and apnea persisted for approximately 4 hours.During the period of apnea, pulmonary congestion ap-

peared and was treated with 8 mg of lanatoside D intra-venously. Her condition gradually improved, all signsof pulmonary congestion disappearing within 24 hours.She was maintained on digoxin for several weeks andhas continued in excellent health since.

Serum specimens from these two patients and theirrelatives were used in the experiments described in thispaper.

Pseudocholinesterase activity

Enzymatic activity of serum cholinesterase was meas-

ured by the method of Kalow and Lindsay (13) usingbenzoylcholine as the substrate.

Dibucaine number

Inhibition of enzyme activity was determined by themethod of Kalow and Genest (5) where the "dibucainenumber" represents the per cent inhibition of enzyme

activity.

Fluoride number

Inhibition of enzyme activity was determined by themethod of Harris and Whittaker (7) where the "fluoridenumber" represents the per cent inhibition.

Starch gel electrophoresisVertical starch gel electrophoresis was performed for

6 to 18 hours at 4 v per cm at 40 C by the method ofSmithies (14) using the discontinuous buffer system ofPoulik (15). One-half of the gel was stained for pro-

tein with amido black, and the other half stained foresterase activity by the method of Harris, Hopkinson,and Robson (16) using as substrate a-naphthyl or j3-

naphthyl acetate and, as the diazo salt, fast red TR or

diazo blue B.

Preparation of antipseudocholinesterase

Five mg of a human pseudocholinesterase preparationestimated to contain 90% impurities was mixed withFreund's adj uvant and inj ected in 0.5-ml amounts intothe foot pads of two rabbits. One week later a subcu-taneous injection of 1 mg of the enzyme preparation inFreund's adjuvant was administered, followed in 2 weeksby a similar injection intraperitoneally and, 1 day later,intravenously.

Immunodiffusion

The technique of double diffusion in agar (17) was

employed using human serum as the antigen in theperipheral wells and rabbit antipseudocholinesterase inthe central well. The observed precipitin bands were

shown to be due to antibodies against human globulinshaving no esterase activity. Because of its very smallcontent of cholinesterase, it was possible to use human

1 Obtained from Sigma Chemical Company, St. Louis,Mo.

487

HODGKIN, GIBLETT, LEVINE, BAUER, AND MOTULSKY

serum to absorb out the unwanted antiglobulin antibodiesfrom the rabbit serum, which thus retained most of itsantipseudocholinesterase activity. The reaction of thisantibody with its corresponding antigen was not visibleas a precipitin band until the agar plate was stained witha-naphthyl acetate and fast red TR (see above).

IiiinunnoelectrophoresisWith a method similar to that described by Grabar

(18) and staining techniques described by Uriel (19),immunoelectrophoresis of normal serum was carried outon 31- X 4-inch photographic glass plates, employing 1%Ionagar dissolved in 0.1 M barbital buffer, pH 8.6.After electrophoresis, the linear troughs were first filledwith 100 IAI of normal serum in dilutions of 1: 4 to1: 64. When this diluted serum had diffused into theagar, the troughs were charged with 100 IAl of anti-human pseudocholinesterase rabbit serum diluted 1: 8,and the plates were incubated at 40 C for 72 hours.Washing in two changes of phosphate buffer pH 7.1during a 48-hour period was followed by staining foresterase as described above. When the antipseudo-cholinesterase antibodies had been neutralized by thepreviously added human serum, no enzyme activity was

30-

25-

,, 20-

Iz 15-

10

I_

5-

LAI

visible. On the other hand, if the diluted human serumcontained little or no antigenic material to neutralize theantibody, a brilliantly stained esterase band becameapparent.

ResultsPseudocholinesterase levels

The mean serum esterase activity of 142 ap-parently normal random blood bank donors was226 U with a SD of ± 53, as shown in Figure 2.Esterase levels determined on the family membersare indicated in Figure 1. All members of thekindred had normal dibucaine and fluoride num-bers. The index cases II-1 and II-2 had nodetectable pseudocholinesterase activity and weretherefore considered homozygotes for the "silent"gene. Their five children, III-1 through III-5,obligate heterozygotes for the "silent" gene, hada mean esterase activity of 153 + 49.2, a valuesignificantly different from the control mean atthe 1%o level. However, only III-3 had a level

Mean 2260' + 53

100J0 200 T36o0-2 0J -1 0 mean +I cT +20C

400

Esterase UnitsFIG. 2. ESTERASE LEVELS OF 143 BLOOD BANK DONORSWITH NORMALDIBUCAINE AND

FLUORIDE NUMBERS. Heterozygotes for silent gene (E10E1") include the five obligatoryheterozygotes (III-1 through III-5) plus 1I-3, II-4, and III-6, who had esterase levelsbelow 2 SD of the mean.

Esterase units of 143 normal controls Eu EjuEsterase units of hetero. for complete pseudocholinesterosedeficiency Eju E1s

* Esterase units of homo. for complete pseudocholinesterosedeficiency Els ES

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COMPLETEPSEUDOCHOLINESTERASEDEFICIENCY

Esterose Activity -Mixtures of NormalSerum and Serum with 0 CholinesteraseActivity

75 50 25%Normal Serum

FIG. 3. ESTERASEACTIVITY AS PER CENT OF TOTAL AC-TIVITY OBSERVEDWHENDILUTED WITH SERUMOF INDEXCASES. Note lack of inhibition of normal esterase ac-tivity.

deviating by more than 2 SD from the normalmean. Other family members, II-2, 11-4, andIII-6, had esterase activities below 2 SD of themean of normals and undoubtedly were alsoheterozygotes for the "silent" gene.

Effect of mixing serum specimens

In order to determine whether the sera lackingany enzyme activity might contain an inhibitorsubstance, mixtures of enzyme-deficient serumwith normal serum were prepared in the propor-tions indicated in Figure 3. Absence of an in-hibitor is demonstrated by the linear decrease inesterase activity that followed a curve expectedfrom dilution alone. Fluoride and dibucainenumbers were similarly unaffected.

Starch gel electrophoretic pattern

As previously described by Harris and hisco-workers (16), DFP (diisopropyl fluorophos-phate) inhibitable cholinesterase activity of nor-mal serum appeared in one major and three minorzones on starch gel (Figure 4). Essentiallyidentical patterns were obtained with the com-mercial pseudocholinesterase preparation as wellas with the sera of individuals homozygous andheterozygous for the dibucaine-resistant type ofenzyme variant. None of these bands wasdemonstrable in the sera of the two subjectshomozygous for the "silent" gene. Three addi-tional zones of esterase activity, previously de-scribed by Uriel (19) and Harris and his co-workers (16) in the zones corresponding to a-

and 8-lipoprotein and albumin were detected inall serum specimens from normal individuals,homozygotes for the atypical allele, El", andhomozygotes for the silent gene. The bands as-sociated with lipoprotein were more clearly visual-ized when 83-naphthyl acetate was utilized as theenzyme substrate.

Immunologic studies

Immunodiffusion. Serum specimens from ho-mozygotes for the silent gene (S) were alternatedwith normal serum (U) in the peripheral wellsof agar immunodiffusion plates, and the centralwell was filled with rabbit antihuman pseudo-cholinesterase (AB). Figure 5 shows that theesterase-stained precipitation bands formed withthe normal serum (U) as antigen were not de-flected from entry into the wells containing en-zyme-deficient serum (S). Since the esterase

Albumin

Cl1C2

C3

C4

Origin

a b c dFIG. 4. STARCHGEL ELECTROPHORESISOF SERUM. Slot

c is serum from a normal individual (EluElu). C4 rep-resents the principal isozyme of pseudocholinesterase.This band and three minor pseudocholinesterase bands(C-C,) are normally observed. Slot a: note identicalelectrophoretic mobility of C1-C4 in serum from homo-zygotes (EaEla) for the atypical, presumably structurallymutant, enzyme. Slots b and d are from patients II-2and II-1, respectively; these homozygotes for the silentallele (E,1E,') lack all pseudocholinesterase bands. Notethat albumin has esterase activity under these experi-mental conditions. As expected, homozygotes for thesilent allele preserve the esterase activity of albumin.

489

'tZ6

(aIR

f -3Klb

tz

7"7'

HODGKIN, GIBLETT, LEVINE, BAUER, AND MOTULSKY

JFIG. 5. IMMUNODIFFUSION. (Drawn after photo-graph.) Normal serum (U) is alternated in the roundwells with serum of Patient II-2 (S) who has completepseudocholinesterase deficiency. Antipseudocholinesteraserabbit serum (AB) is p!aced in the center well. Thearc-like bands represent the enzyme-stained precipitinbands indicating a reaction of normal serum (U) withthe anticholinesterase rabbit serum (AB). Lack ofdeflection of the bands from the wells with patient'sserum (S) is consistent with absence of cross-reactingmaterial (CRM) in this serum. If any CRM were

present, the precipitin bands would not tend to terminatein the wells containing the sera without pseudocholin-esterase activity.

activity localized the protein, this pattern is con-

sistent with the absence of cross-reacting proteinwithout enzymatic activity from the abnormalserum.

No precipitating lines were observed betweenthe wells containing normal and acholinestera-semic serum consistent with the absence of serum

antibody against pseudocholinesterase in homozy-gotes for the silent gene.

"Blocking" experiments. The possible presence

of a gene product with neither enzymatic nor

antigenically cross-reactive activity, which mightblock the reaction between cholinesterase andanticholinesterase, was tested in the followingmanner (20). Antihuman cholinesterase serum

was placed in the central well. Human serum

with normal pseudocholinesterase activity, towhich had been added an equal quantity of seri-ally diluted serum from the patients with no

pseudocholinesterase activity, was placed in theperipheral wells. The serum from the patientsfailed to affect the equal intensity of the resultingprecipitin lines. This finding is consistent withthe absence of an immunologically related sub-stance blocking the antigen-antibody reaction.

Immunoelectrophoresis

Immuoelectrophoretic experiments were de-signed to demonstrate the presence in humanserum of antibody-neutralizing activity. Normalhuman serum in dilutions up to 1: 8 containedsufficient cholinesterase antigen to neutralize therabbit antihuman cholinesterase. Similar resultswere obtained when dilutions of serum fromhomozygous individuals (E1aEja) with the mostcommon type of mutant enzyme were used toprecharge the troughs. Thus it appears thatalthough the enzyme in such individuals is struc-turally altered, it reacts with the antinormal cho-linesterase and is present in normal quantities.Other kinds of studies on the nature of the mu-

tant (21, 22) have provided similar evidence. Inan identical experiment, serum (from II-2) with-

TABLE I

Pseudocholinesterase El mutants

Esteraselevel Dibucaine Fluoride

Genotype Phenotype Relative % number number Frequency*

Normal response to suxamethoniumEiuEiu U 100 71-83 57-88 96%-97%EluE 1a I 78 52-69 42-55 3%o- 4%EiuEis U 65 71-83 57-68 -1/150EluElf UF 80 71-78 50-55 ?

Prolonged response to suxamethoniumE1aE ia A 25 15-25 20-25 -1/3,000E18E18 S 0 1/40,000-1/160,000EifE1f F 50 64-67 34-35 Very rareE aEi, A 20 15-25 20-25 -1/8,000ELE1f IF 60 47-53 41-39 ?EJfE18 F Not described yet

* These frequencies are based on actual observation for the E1LE1u genotype and have been calculated for the othersas discussed by Simpson and Kalow (8) and Motulsky (9).

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COMPLETEPSEUDOCHOLINESTERASEDEFICIENCY

out pseudocholinesterase activity was substitutedfor normal serum. Even when this specimen wasused undiluted, there was no evidence of antibodyneutralization, again indicating the absence ofany demonstrable cross-reacting material.

Discussion

The complete absence of pseudocholinesteraseactivity from the sera of these two patients andof the other two reported cases (11, 12) wasnot associated with any detectable impairment ofhealth. Liver tissue obtained by biopsy of onepatient contained no detectable enzyme (12).Since that organ is the site of pseudocholines-terase production, the total lack of enzyme in in-dividuals homozygous for the silent allele is con-firmed. The enzyme is therefore completely dis-pensable, and its normal physiological role, ifany, can be compensated by other systems.

Two variants of pseudocholinesterase (the di-bucaine and the fluoride-resistant enzyme) havebeen previously well characterized (4) and theirheredity studied. Family data fit the hypothesisthat both variants are determined by mutant genes(Ela and El') allelic to the gene determining thenormal enzyme (Elu) (4, 6, 23, 24). Table Ipresents all of the known phenotypes and geno-types of the pseudocholinesterase system alongwith their associated laboratory findings andpopulation frequencies. Individuals sensitive tosuxamethonium are either homozygous or mixedheterozygous for the mutant genes E1R and Elfor the "silent" gene. All presently available pedi-gree data and considerations based on populationgenetics suggest that the "silent" gene is an alleleof the normal esterase gene and its two allelicmutants (8, 9, 11, 23). The kindred presentedhere provides further evidence against the al-ternative hypothesis (8) that the gene determin-ing complete absence of pseudocholinesterase isnonallelic but specifically suppresses the E1a generather than the Elu gene. In that case the geno-type of silent gene homozygotes would be E1l/Ela, ss and its frequency rare. Silent gene hetero-zygotes would be of genotype Ela/Elu, Ss. Onequarter of the children of matings from suchheterozygotes with normals (E1a/E,'uSs x E1u/EluSS) would be expected to be of genotype Ela/E111,SS and have the characteristics of the atypicalheterozygote with intermediate dibucaine resist-

ance. Among 14 offspring of such matings [onereported by Harris, Whittaker, Lehmann, andSilk (23), one by Liddell, Lehmann, and Silk(11), five by Simpson and Kalow (8), andseven (i.e., IV-1-IV-7) in the present paper],no individual of this type was found. The prob-ability of not finding such individuals under thenonallelic hypothesis is (3/4)14 = 2%. Althoughmore offspring from the critical matings are re-quired to disprove this more remote hypothesis,allelism of the silent gene with the E1u and El,gene is the most likely possibility, both statisti-cally and biologically. The silent gene is there-fore designated in Table I as El".

Liddell and associates (4, 11) demonstrated in-hibition of normal pseudocholinesterase activityby serum from their patient with complete pseudo-cholinesterase deficiency. These findings are un-like those shown in Figure 3 where no inhibitionof normal pseudocholinesterase could be shown.This discrepancy raises the problem of a differenttype of mutation or possibly of antibody produc-tion against pseudocholinesterase by transfusionin Liddell and associates' patient (4, 11). Suchantibodies might inhibit normal pseudocholines-terase activity. No antipseudocholinesterase anti-bodies could be demonstrated in our two sibs withcomplete pseudocholinesterase deficiency.

A number of genetic systems with enzyme de-ficiency have been studied with immunologicalmethods. In Neurospora as well as in Escherichiacoli, there are many mutants with no tryptophanesynthetase enzymatic activity. These have beenclarified on the basis of serological cross-reactivitywith the wild type enzyme as cross-reacting ma-terial (CRM) positive or CRMnegative (25, 26).

In studies on human subjects, no CRMwas de-tected in muscle phosphorylase deficiency (27) andin the Japanese type of acatalasemia (28, 29).The results of a very recent investigation sug-gested that in the Swiss type of acatalasemia,trace amounts of catalase with the same physico-chemical properties as normal catalase could bedemonstrated (30). In the case of glucose-6-phosphate dehydrogenase deficiency, Marks andTsutsui (31) have shown that the enzyme isolatedfrom the red cells of subjects with the two majorvariants have immunological identity with thenormal enzyme. The immunological studies de-scribed in this paper indicate that individuals

491

HODGKIN, GIBLETT, LEVINE, BAUER, AND MOTULSKY

with an inherited complete absence of serumpseudocholinesterase do not produce an antigeni-cally similar protein detectable by the methodsused.

A variety of genetic mechanisms could lead tothe present findings. Considering that the silentgene mutation is allelic, an operator negative mu-tation (32-34) at the pseudocholinesterase locusis an attractive hypothesis. However, at the pres-ent time, proof for this type of mutation does notexist yet in mammalian systems.

Structural gene mutation affecting directly orindirectly both antigenic and enzymatic "active"sites would also lead to complete absence of en-zyme activity. The problem is similar to thatpresented by other human mutations causing ab-sence of gene product. A variety of genetic mecha-nisms including structural mutations can lead tothe absence of a protein or enzyme. At our pres-ent state of knowledge, a definite conclusion re-garding the specific nature of the mutation is notyet possible in such cases.

The mean pseudocholinesterase activity of fiveobligatory heterozygotes was 67%o of normal inour study and 717o of normal in six obligatoryheterozygotes of Simpson and Kalow (8). Simi-lar observations were made by Harris and his co-workers (35). It appears that the control ofpseudocholinesterase activity, as that of other en-zymes (36), is exerted on the genic level and notby feedback repression (37), so that individualswith only one mutant gene for pseudocholinester-ase have less enzyme activity than normals. How-ever, these individuals, unlike most heterozygotesin other systems, produce two-thirds, rather thanone-half, of the enzyme activity of the normalhomozygote.

SummaryTwo sibs with complete absence of pseudocho-

linesterase activity were discovered in an Ameri-can-Irish family. In both instances, prolongedapnea following succinylcholine administration wasobserved, but no other pathologic effect of theenzyme deficiency was apparent.

The family data were compatible with the hy-pothesis that "a pseudocholinesterasemia" repre-sents the homozygous state for a gene that de-termines complete absence of pseudocholinesteraseactivity. Genetic considerations from this and

previously reported families indicate that this geneis allelic with the two more frequently observedstructural gene mutations affecting pseudocho-linesterase.

Starch gel electrophoresis of serum from theindex cases showed absence of the four isozymebands associated with normal pseudocholinesteraseactivity. The electrophoretic pattern of these iso-zymes in subjects with the common structural("dibucaine resistant") mutation of this enzymewas indistinguishable from normal.

Heterozygotes for the rare "silent" allele hadsignificantly less but more than half of normalcholinesterase activity.

Serum with complete absence of pseudocholines-terase activity failed to inhibit normal pseudocho-linesterase activity.

Potential gene products in the sera with no en-zyme activity were tested by immunologic meth-ods. No cross-reacting or blocking material couldbe demonstrated by immunodiffusion and immuno-electrophoretic techniques. Although these find-ings would be expected with an operator type ofmutation, certain structural mutations could leadto similar results.

Acknowledgments

The expert technical assistance of Mrs. Nancy Morrowand Miss Nancy Scott is gratefully acknowledged.

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