Brit. J. Anaesth. (1970), 42, 698
A SOURCE OF ERROR IN THE DETERMINATION OF INHIBITOR CONSTANTS OF SERUM CHOLINESTERASE BY J. KING AND R. I. DIXON SUMMARY
The occurrence of low levels of serum cholinesterase (acylcholine acyl-hydrolase, E.C.3.1.1.8) in some individuals in the absence of disease or malnutrition and characterized by an unusual sensitivity to the muscle relaxant suxamethonium was first shown to be familial by Lehmann and Ryan (1956). Kalow and Genest (1957) then demonstrated qualitative differences in substrate affinities and in inhibition between what they termed the "usual" and "atypical" enzymes. They also showed that the genetic phcnotype could be determined by means of inhibition by the local anaesthetic dibucaine (cinchocaine, Nupercaine). The percentage inhibition, the dibucaine number (DN), of the "usual" phenotype was given as above 70, the "atypical" homozygote below 20, and the heterozygote between 40 and 70. Later work refined the value for normal individuals to 80 ± 3 , for heterozygotes to 62 ± 8 , and for atypical homozygotes to 22 ± 6 (King, 1965). Dibucaine numbers intermediate to these values had also been noted in patients sensitive to suxamcthonium and in their relatives, thereby suggesting further phenotypes (Lehmann et al., 1960). The use of fluoride inhibition gave a parameter to differentiate such genetic variants (Harris and Whittaker, 1961). These workers described cases heterozygous to the fluoride-resistant gene and both the usual and atypical genes, and Lehmann and associates (1963) reported the first instance of the fluoride-resistant homozygote. Recently, by means of sodium chloride inhibition (Harris and Whittaker, 1963; Whittaker,
1968a) a new cholinesterase phenotype sensitive to suxamethonium has been described (Whittaker, 1968b). The chloride numbers differ from both dibucaine and fluoride numbers in that suxame*thonium-sensitive individuals have the higher values and normal individuals the lowest numbers, that is 10 to 20. During the present survey a family has been discovered in which six out of the seven members studied have zero chloride numbers at 25 °C, which would suggest the existence of further variants. As part of an investigation into the kinetics of human serum cholinesterases the effect of temperature has been studied and certain anomalies affecting enzyme activities have been described (King and Dixon, 1969; King and Morgan, 1969). The present communication records the behaviour of the inhibitors usually employed in differentiating cholinesterase phenotypes when the reaction temperature is varied and points to a possible source of misinterpretation resulting therefrom. METHODS AND MATERIALS
No allowance was made for change in optimum benzoylcholine concentration or pH with temperature but following the scheme of King (1965) serum cholinesterase activity was estimated by the method of Kalow and Lindsay (1955) J. KING, asc., M.LBIOL., L J U . C , F.I.M.L.T., Department
of Biochemistry, Royal Infirmary, Glasgow; R. I. DIXON,
MJI.C.S.(ENG.)> L.R.CJ>.(LOND.),
Department of
Anaesthetics. North Lonsdak Hospital, Barrow-inFuraess, Lanes.
Downloaded from http://bja.oxfordjournals.org/ at University of Edinburgh on June 21, 2015
The effect of temperature on the inhibition of serum cholinesterase by dibucaine, fluoride and chloride has been studied. It is found that the dibucaine and chloride numbers together are capable of differentiating the inherited enzyme variants at 30° and 37°C. Fluoride numbers contribute little information at these temperatures; indeed fluoride inhibition is so sensitive to temperature change that unless rigid temperature control is maintained in current techniques, erroneous conclusions may be drawn. A family with unusual chloride numbers is revealed.
ERROR IN THE DETERMINATION OF INHIBITOR CONSTANTS
RESULTS
DISCUSSION
In the absence of a thermostatically controlled spectrophotometer it is the ambient temperature of the laboratory which dictates the reaction temperature of cholinesterase assays, and even in these laboratories the ambient temperature has been found to fluctuate between 18° and 32°C during the year. This is probably a general range for these islands but is probably wider elsewhere. It was an appreciation of such high ambient temperatures which doubtless prompted the International Union of Biochemistry (1965) not to accept the earlier recommendation of its Commission on Enzymes (I.U.B., 1961) that 25 °C be the standard temperature but to recommend instead that 30°C be used. In many parts of the world, however, even in these northern latitudes in summertime, to maintain a temperature of 30°C requires thermostatic cooling of the instrument. This difficulty, the fact that a valid index of some enzymes is only obtained at their physiological temperature (King and Dixon, 1969; King and Morgan, 1969), and the desirability of exploiting the increased activity at higher temperatures in the automation of enzyme assays, compeUingly suggest a universal adoption of 37 °C as the standard temperature for enzyme assays.
Typical patterns showing the temperature variation of dibucaine (DN), fluoride (FN) and chloride numbers (C1N) for the phenotypes E ^ E t D , E,» E,", E," E,», E," E t f , and Et« E,' are illustrated in figures 1, 2 and 3. It was found that sera from genotypes E ^ E,8 and Ej a E^ behaved in the same manner as from E ^ E ^ and E ^ E ^ respectively. Dibucaine inhibition (fig. 1) tended to show a maximum at about 20 °C, declining slowly thereafter and rapidly at temperatures in excess of 45 °C. This was a general trend except for sera from atypical homozygotes ( E / E^) which invariably exhibited a minimum value about 35 °C. However, it is not only with these three temBetween 20° and 30°C the percentage dibucaine peratures that assay of serum cholinesterase and inhibition, that is the dibucaine number, showed determination of inhibitor constants is involved. a minimal change of 2 for genotypes E, u Exf and Dibucaine numbers (Kalow and Genest, 1957) E, a E, f and changes of 4 to 6 for the other and presumably fluoride numbers (Harris and variants. Between 25° and 37 °C there is a change Whittaker, 1961) were determined at 26°C and in DN of about 11 in normal serum and 6 in the chloride numbers (Whittaker, 1968a) at 26.5 °C others. by including the respective inhibitor in the For chloride inhibition all variants showed a reaction mixture of the assay evolved by Kalow similar pattern (fig. 2), numbers falling to a mini- and Lindsay (1955). King (1965) reported that mum then increasing with temperature. The the fluoride numbers he recorded were only minima occurred between 30° and 40°C for the valid if the assay was performed in the neighfive phenotypes studied but in the family found bourhood of 25 °C and a decline in the dibucaine to have zero chloride numbers the minimum number, but more so in the fluoride number, with values appeared between 20° and 25 °C. Between increasing reaction temperature was illustrated by 20° and 30°C, chloride numbers changed by 4 to King (1967). 6 but less than this between 25° and 37°C. The present investigation confirms and extends The picture for fluoride inhibition (fig. 3) these latter findings and shows that dibucaine differs qualitatively, in that there are no maTima (fig. 1) and chloride numbers (fig. 2) are relatively or minima, and quantitatively, in that there were little affected by the reaction temperature much greater changes amounting to falls of 14 to between 20° and 40°C. At least their determina22 between 20° and 30°C and 17 to 25 between tion at 30° or at 37 °C would still provide a means of differentiating the cholinesterase pheno25° and 37°C.
Downloaded from http://bja.oxfordjournals.org/ at University of Edinburgh on June 21, 2015
simultaneously with inhibition by 10 dibucaine, 50 pM. sodium fluoride and 500 mM sodium chloride in a Unicam SP800 recording spectrophotometer with temperature control and scale expansion to a slave recorder. Sera from patients who had developed prolonged apnoea following suxamethonium administration, from their relatives and from laboratory staff were studied. The convention proposed by Motulsky (1964) is used throughout in describing the genetic variants of the first cholinesterase locus.
699
BRITISH JOURNAL OF ANAESTHESIA
700
EuEu
80
80 o D CO
70 «
70
e
a
a
u o ETEj
.
60
B
o
a
ber
jo
8
8 •
a
To JJ f
J50
9
-s J40
o
L •
Tl o t
u.
•
30
°
r#
fcTETI
20
.
•
•
.
.
°
o 0
1
30
»
•
8
'
•
:
•
§ •
10
•
• 20
30 40 Reaction Temperature (°C)
TO
50
30 40 Reaction Temperature ( ° O
FIG. 3 Variation of fluoride number with temperature.
FlG. 1
Variation of dibucaine number with temperature.
1 •
50
Key for all figs.: _ 40
1
E U E'/
'
• m
m>
•
*
E U E, f
•
E a E, f
Z 30
1
° f[°
)
1
W°
o o
10 -
'°
°
°
°
°
«
•
° *
:
.
•
* *
•
20
30 40 Reoctlon Temperature (°C)
50
FIG. 2 Variation of chloride number with temperature.
50
see text
Downloaded from http://bja.oxfordjournals.org/ at University of Edinburgh on June 21, 2015
J» | « . • I-
10
E7E7
9
60
20
I
ERROR IN THE DETERMINATION OF INHIBITOR CONSTANTS
ACKNOWLEDGEMENTS
The authors wish to thank Miss Judith Pottage, FXM.L.T., for technical assistance and Mrs P. Corkhill for keeping records and collecting specimens. This work was partly undertaken while one of us (R.I.D.) was in receipt of a grant from the Endowment Funds of Barrow and Furness Hospital Management Committee to investigate the incidence of serum cholinesterase variants in the Furness area.
Kalow, W., and Lindsay, H. A. (1955). A comparison of optical and manometric methods for the assay of human serum cholinesterase. Canad. J. Biochem., 33, 568. King, J. (1965). Practical Clinical Enzymology. London: Van Nostrand. (1967). Temperature standardization in clinical enzymology. Clin. Biochem., 1, 42. Dixon, R. I. D. (1969). A further factor contributing to inherited suxamethonium sensitivity. Brit. J. Anaesth., 41, 1023. Morgan, H. G. (1969). Temperature anomalies of serum cholinesterases. Enzym. biol. clin., 10, 346. Lehmann, H., Liddell, J., Blackwell, B., O'Connor, D. C , and Daws, A. V. (1963). Two further serum pseudocholinesterase phenotypes as causes of suxamethonium apnoM. Brit. med. J., 1, 1116. Ryan, E. (1956). The familial incidence of low pseudocholinesterase level. Lancet, 2, 124. Silk, E., Harris, H., and Whittaker, M. (1960). A new pseudocholinesterase phenotype? Acta gentt. {Basel), 10, 241 Morulsky, A. G. (1964). Pharmacogenetics; in Progress in Medical Genetics, Vol. I l l (eds. Steinberg, A. G. and Beam, A. G.). New York: Grune and Stratton. Whittaker, M. (1968a). The pscudocholinesterase variants: differentiation by means of sodium chloride. Acta genet. (Basel), 18, 556. (1968b). An additional pseudocholinesterase phenorype occurring in suxamethonium apnoea. Brit. J. Anaesth., 40, 579.
UNE SOURCE D*ERREUR DANS LA DETERMINATION DES CONSTANTES DTNHIBITION DE LA CHOLINESTERASE SERIQUE SOMMAIRE
L'effet de la temperature sur Finhibition de la cholinesterase serique par dibucaine, fluoride et chloride, a cti itudii. On a trouve^ que dibucaine et chloride ensemble sont capables de differencier les variantes enzymatiques inh&itees a 30°C et 37°C. Les donnees fluoride ne contribuent que peu d'informarion a ces temperatures; en effet, l'inhibition fluoride est si sensible aux modifications de la temperature, que dcs fausses conclusions sont tirees si la temperature n'est pas strictement contr61ee dans les techniques actuelles Les auteurs decrivent une famille avec des donnees chloride inhabituelles. EINE FEHLERQUELLE BEI DER BESTIMM U N G VON INHIBITORKONSTANTEN DER SERUM-CHOLINESTERASE
REFERENCES
Harris, H., and Whittaker, M. (1961). Differential inhibition of human serum cholinesterase with fluoride: recognition of two new phenotypes. Nature, 191, 496. (1963). Differential inhibition of "usual" and "atypical" serum cholinesterase by N a d and NaF. Ann. hum. Genet., 27, 53. International Union of Biochemistry (1961). Report of the Commission on Enzymes. Oxford: Pergamon. (1965). Enzyme Nomenclature. Amsterdam: Elsevier. Kalow, W., and Genest, K. (1957). A method for the detection of atypical forms of human serum cholinesterase: determination of dibucaine numbers. Canad. J. Biochem., 35, 339.
ZUSAMMENFASSUNG
Die Wirkung der Temperatur auf die Hemmung der Serum-Cholinesterase durch Dibucain, Fluorid und Chlorid wurde untersucht. Es wird festgestellt, dafi bei 30"C und 37°C die Dibucain- und Chlorid-Zahlen zusammen in der Lage sind, die ererbten EnzymVarianten zu verandern. Die Fluorid-Zahlen sind bei diesen Temperaturen nur wenig informativ; tatsachlich reagien die Fluorid-Hemmung so empfindlich auf eine Temperaturveranderung, dafl falsche Schlusse gezogen werden konnen, wenn nicht eine strikte Temperaturkontrolle bei den derzeit gebrauchh'chen Methoden aufrecht erhalten wird. Eine Familie mit einer ungewohnlichen Chlorid-Konzennation wird vorgestellL
Downloaded from http://bja.oxfordjournals.org/ at University of Edinburgh on June 21, 2015
types. On the other hand, the fluoride number (fig. 3) is shown to be extremely sensitive to temperature change and at 30° and 37°C does not provide a reliable means of distinguishing between the several cholinesterase variants. A more serious consequence affecting current practices, however, is the revelation that between 18°C and 32°C, the ambient temperature range in these laboratories, the fluoride number alters by 1.4 to 2.2 per degree in reaction temperature. From figure 3 it can readily be seen that an alteration in reaction temperature of 2° to 4°C suffices to change the fluoride number from one phenorype range to that of the next. The anomalous behaviour of atypical homozygotes in presenting a minimum dibucaine number about 35 °C was potentially interesting since this cholinesterase variant has been shown to have maximum activity about 32°C (King and Dixon, 1969; King and Morgan, 1969). However, the genotype E t a E / , which has an activity mayi'mum about 37°C, showed the same general relationship between dibucaine inhibition and reaction temperature as the remaining variants with activity maxima between 47°-51°C. Indeed no relationship was found between the effects of temperature on enzyme activities and on inhibitor constants. The dibucaine and fluoride numbers of the family in which six out of seven members had unusual low chloride numbers at 25 °C indicated that the individuals were apparently either normal E,11 E, n or normal fluoride-resistant heterozygotes, E, n E / . The anomaly occurred in both phenotypes and is obviously inherited.
701