SERUM-PSEUDOCHOLINESTERASE IN DEPRESSION WITH NOTABLE ANXIETY

563

SERUM-PSEUDOCHOLINESTERASE IN DEPRESSION WITH NOTABLE ANXIETY LOUIS ROSE M.R.C.S. CHIEF

DEPARTMENT OF PSYCHOLOGICAL

ASSISTANT, MEDICINE, ST. BARTHOLOMEW’S HOSPITAL, LONDON, E.C.1

DAPHNE A. DAVIES Ph.D. Lond. BIOCHEMIST

M.D.

HERMANN LEHMANN Basle, Sc.D. Cantab., F.R.C.P., F.R.I.C., F.C.Path. DIRECTOR

MEDICAL RESEARCH COUNCIL ABNORMAL HÆMOGLOBIN RESEARCH

UNIT, UNIVERSITY DEPARTMENT

OF

BIOCHEMISTRY, CAMBRIDGE

INTEREST in plasma-pseudocholinesterase has been centred on low levels which could indicate a decrease in liver function, or cause a prolonged response to the musclerelaxant suxamethonium which normally is destroyed by pseudocholinesterase (Bovet-Nitti 1949). Low serumpseudocholinesterase is found when the response to suxamethonium is very prolonged (Bourne et al. 1952, Evans, Gray, Lehmann, and Silk 1952) and the length of action may usually be related directly to the individual serum-enzyme activity (Evans, Gray, Lehmann, and Silk

1953). High levels of enzyme activity have been reported, in anxiety (Richter and Lee 1942), in psoriasis (Helmeczy and Nagy 1954), and in nodular goitre (Koster and Kisch 1943). We have observed high levels in alcoholism. Pseudocholinesterase, now defined as acylcholine acylhydrolase (A.C.A.H., Enzyme Commission no. 3.1.1.8) exists as several genetically determined variants. The most frequent form is the normal (usual or typical) A.c.A.H. The unusual or atypical A.c.A.H. which can be expected in the homozygous state in one in three thousand to four thousand people in Britain (Kattamis, ZannosMariolea, Franco, Liddell, Lehmann, and Davies 1962) produces a notably prolonged apnoea, as a reaction to the usual dose of the muscle-relaxant suxamethonium. The incidence of heterozygotes is about 3%. The level of the normal enzyme is high enough in these heterozygotes to

protect them against a prolonged suxamethonium apnoea, but since the activity is lower than normal the heterozygotes never show a particularly brief response. Two other variants of A.C.A.H. can be recognised. One is called " fluorideresistant " A.C.A.H. because it is less inhibited by fluoride than the normal A.C.A.H., its ability to hydrolyse suxamethonium lies somewhere between that of the normal (typical) and that of the atypical variants. Another gene determines almost complete absence of A.C.A.H. activity and is called the " silent gene ". Homozygotes respond to suxamethonium with prolonged apnoea, but heterozygotes resemble in their reaction the homozygotes for the typical and the atypical genes respectively. The investigation arose from an observation by one of us (L. R.) at St. Bartholomew’s Hospital, that suxamethonium seemed to act for a shorter time when it was given to psychiatric patients before electroconvulsive therapy (E.C.T.) than when it was administered before surgery. An obvious possibility was that the psychiatric patients had a higher A.C.A.H. level. Since in normal homozygotes the activity is higher than in all other genotypes, these other phenotypes, possibly, occurred less often in psychiatric patients; but since their incidence is not greater than a few per cent in the normal population, it was unlikely that the difference in response could be

related

to

genetical

variation. Nevertheless it

was neces-

sary to investigate both the enzyme activity and the enzyme type of psychiatric patients. A higher A.C.A.H. level in psychiatric patients than in normal controls has been reported by Richter and Lee (1942), who referred to still earlier work by Tod and Jones (1937). These workers found that in 27 patients with anxiety the A.C.A.H. level varied from 2.5 to 7,9 units, whereas in other groups-10 epileptics, 24 schizophrenics, and 43 patients with melancholia-the ranges were, respectively, 1 -9-5-2; 2’1-5,1; and 2-4-5-3. Similarly Richter and Lee (1942) examined 205 subjects (12 normals, 12 surgical control patients, 12 patients with anxiety neurosis, and 163 newly admitted psychiatric patients taken at random). Levels of more than 5-8 units were found in 10 of those 205, and 9 of them had been diagnosed as anxious The 10th was an anxious and or depressed or both.

These investigations were tentative, but linked with the finding of low activities in catatonic stupor. The connection between lower enzymeactivities in some retarded states and malnutrition was not explained and, possibly, this was why the work was not given the attention it deserved. Plum (1960) and Antebi and King (1962) have reported that a wide variety of abnormal mental states show raised A.C.A.H. activity. Plum (1960) did not consider this to be caused by any of the many drugs used in a mental hospital over four years. Antebi and King (1962) found that 5 of 26 schizophrenics had raised A.C.A.H. levels.

unstable

they

man.

were

Methods and Clinical Material Methods Laboratory A.C.A.H. activity of serum or plasma (heparinised) was measured by incubation in a micromanometer. Acetylcholine (as substrate) was dissolved in Krebs-Ringer bicarbonate-buffer (Ammon 1933, McArdle 1940). For each mole of acetylcholine hydrolysed to choline and acetic acid, one mole of carbon dioxide is released. One unit of enzyme activity is defined as that which hydrolyses one micromole of acetylcholine per minute per ml. serum at 37°C. When heparinised plasma was used an additional 5 mg. of dry heparin was added to the micromanometric vessel to prevent clotting of the plasma on dilution with the Krebs-Ringer bicarbonate-solution.

Enzyme Type The unusual (atypical) form is less easily inhibited by dibucaine (cinchocaine, nupercaine) than the normal form (Kalow and Genest 1957) and it is also less inhibited by fluoride (Harris and Whittaker 1961). The fluoride-resistant form resembles the atypical enzyme in its resistance to fluoride, but resembles the typical variant in being more easily inhibited by dibucaine. The percentage inhibition at pH 7,4 and 26.5OC of a 5 x 10-5 M solution of benzoylcholine by serum or plasma diluted 1/100 is measured in the presence of 10-5 M dibucaine and 5 >; 10’° M fluoride, and is then expressed as the dibucaine number (D.N.) or the fluoride number (F.N.). Table I lists the D.N. and F.N. of the different genotypes arising from the three TABLE

IŃSUXAMETHONIUM

SENSITIVITY

A.C.A.H.

WITH DIFFERENT

TYPES

OF

564 genes for the normal, atypical, and fluoride-resistant forms of A.c.A.H. The homozygotes for the silent gene are recognised by the complete absence of enzyme activity, and the heterozygotes have the D.N. and F.N. of whatever other enzyme type is present.

TABLE IV-SERUM-PROTEIN AND A.C.A.H. LEVELS IN PSYCHIATRIC f

PATIENTS

All the six

phenotypes were used as controls during the investigation. Serum-protein Estimation Serum-protein was measured by the biuret method (King and Wooton 1956). Patients and Controls The normal level was known from previous work (Tod and Jones 1937, McArdle 1940, Richter and Lee 1942, Lehmann, Cook, and Ryan 1957), but we examined the serum or plasma of 40 patients with skin diseases such as eczema, acne, and boils who were being investigated for their glucose tolerance in connection with another research project, and also 15 healthy members of staff. 100 psychiatric patients selected for E.c.T. came from St. Bartholomew’s Hospital, and 15 serum samples came from patients with depression with anxiety features seen in Cambridge. The 115 patients were grouped as follows: depression with notable anxiety (42); retarded depressive state (26); agitated depressive state (25); and depressive state and schizophrenia TABLE II-NORMAL VALUES OF A.C.A.H.

significantly higher mean A.C.A.H. In patients with psoriasis the mean was high (significant only at 2% level). Kunkel and Ward (1947) observed high serum-A.C.A.H. in nephrosis and suggested that in this disease the liver a

increased amount of serum-albumin to comfor the loss by the kidneys, and that the production pensate of albumin and A.C.A.H. by the liver might be controlled jointly. We examined the possibility that A.C.A.H. was high in the psychiatric patients in association with a rise in serum-albumin, perhaps secondary to salt depletion or under the influence of the drugs such patients receive. There was no difference in albumin level between 16 patients with A.C.A.H. levels of more than 4-5 units and 10 patients with enzyme levels varying from 2.8-3-8 units. There were no abnormal or even low normal serumelectrolytes, which might have caused a removal of water from the plasma and thereby a rise in protein values makes

an

(table iv). A.C.A.H. levels fell with the of the clinical condition, but there was improvement no change in the serum-protein nor in the serum-

In

a

few

patients the

electrolytes. *

1 unit

hydrolyses lM acetycholine

per minute per ml.

serum at

37°C.

(22). 34 patients with psoriasis and 16 alcoholics, in whom advanced cirrhosis had been excluded, were also examined. Results

Enzyme Type The 115 psychiatric patients, the 34 patients with psoriasis, and the 16 alcoholics were all phenotypically normal homozygotes and so genetical considerations were not

important.

Normal Levels

The enzyme activity in the control sera and plasma corresponded well with the values reported elsewhere (see table 11). Levels in

Psychiatric Cases

The values for the psychiatric patients are given in table 111. The diagnoses are grouped into depression with anxiety, retarded depressive state, agitated depressive state, and depressive state, and schizophrenia. Each group had a significantly higher value of serum-A.c.A.H. than the control group. The small sample of alcoholics also showed

Other States Associated with

High A.C.A.H.-levels

The observations on psychiatric patients are significant and support the earlier reports of Tod and Jones (1937) and Richter and Lee (1942). We have not excluded the influence of drugs on enzyme levels in the psychiatric patients, even though we have accumulated, over the past fifteen years, information which excluded the effect of numerous tranquillisers and sedatives on the serumA.C.A.H. in individual patients. Thompson and Trounce (1956) demonstrated that an increase in serum-A.C.A.H. in diabetes could in fact be correlated with obesity, and Berry, Cowin, and Davies (1954) found that there was a positive correlation between body-fat and serum-A.C.A.H. This explanation cannot be applied to our findings in psychiatric patients and in patients with psoriasis, but we are not able to exclude it with regard to the alcoholics. Also there must be an element of anxiety in alcoholics. The findings in psoriasis patients are not conclusive and we intend to continue our investigation of patients with psoriasis which at present might or might not be considered to show the coincidence of significantly high

TABLE 111—SERUM-PSEUDOCHOLINESTERASE

(A.C.A.H.)

565

complication will depression with anxiety. serum-A.C.A.H.

116

A

be

psoriasis and

Summary patients selected for electroconvulsion therapy

showed

significantly higher serum-pseudocholinesterase (acylcholine acylhydrolase) than a control group. Raised a

levels of the enzyme were also observed in alcoholics with liver disease, and, with less significance, in patients with

psoriasis. We thank Dr. A. I. Roughton, Cambridge, who provided serum from 15 patients with depression with anxiety features. We also thank members of the department of psychological medicine at St. Bartholomew’s Hospital who permitted their patients to be included in this survey. REFERENCES

Ammon, R. (1933) Pflügers Arch. ges. Physiol. 233, 486. Antebi, R. N., King, J. (1962) J. ment. Sci. 108, 75. Berry, W. K., Cowin, P. J., Davies, D. R. (1954) Br. J. Nutr. 8, 79. Bourne, J. G., Collier, H. O. J., Somers, G. F. (1952) Lancet, i, 1225. Bovet-Nitti, F. (1949) Rc. 1st. sup. Sanità, 12 138. Evans, F. T., Gray, P. W. S., Lehmann, H., Silk, E. (1952) Lancet, i, 1229. (1953) Br. med. J. i, 136. Harris, H., Whittaker, M. (1961) Nature, Lond. 185, 496. Helmeczy, L., Nagy, E. (1954) Acta med. Hung. 5, 109. Kalow, W., Genest, K. (1957) Can. J. Biochem. Physiol. 35, 339. Kattamis, C., Zannos-Mariolea, L., Franco, A. P., Liddell, J., Lehmann, H., Davies, D. A. (1962) Nature, Lond. 196, 599. King, E. J., Wootton, I. D. P. (1956) Microanalysis in Medical Biochemistry; p. 57. London. Koster, H., Kisch, B. (1943) Expl Med. Surg. 1, 71. Kunkel, H. C., Ward, S. M. (1947) J. exp. Med. 86, 325. Lehmann, H., Cook, J., Ryan, E. (1957) Proc. R. Soc. Med. 50, 147. Liddell, J., Lehmann, H., Davies, D. (1963) Acta genet. Statist. med. 13, 95. McArdle, B. (1940) Q. Jl Med. 33, 107. Plum, C. M. (1960) Clin. Chem. 6, 332. Richter, D., Lee, M. (1942) J. ment. Sci. 88, 428, 435. Thompson, R. H. S., Trounce, J. R. (1956) Lancet, i, 656. Tod, H., Jones, M. S. (1937) Q. Jl Med. 6, 1. —

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HEIGHTS AND WEIGHTS OF MIDDLE CLASS BAGANDA CHILDREN I. H. E. RUTISHAUSER B.Sc.

(Nut.)

Lond.

NUTRITIONIST, MEDICAL RESEARCH COUNCIL INFANTILE MALNUTRITION RESEARCH UNIT, MULAGO HOSPITAL, KAMPALA, UGANDA

THE exact ages of children in Africa are notoriously difficult to obtain, and Uganda is no exception. This has always been a problem for those who have wished to study the physical development of African children. Some information, however, is available on the heights and weights of Baganda schoolgirls aged six to eighteen years, whose exact ages were known (Burgess and Burgess 1964) and weights of children up to three years old (Dean and Jelliffe 1960). The figures for schoolboys aged six to eighteen have been obtained but the results have not been

published. The present

study was undertaken to provide informa-

tion on the age-groups not so far covered. It is based on data from 154 children from the child-welfare clinic run by the Unit at Namulonge 16 miles from Kampala and 480 children attending nursery schools in Kampala. Some of these children have been followed for more than three years, half of them for over a year. Methods The Infantile Malnutrition Research Unit clinic, described elsewhere (Burgess 1960), is held fortnightly. Only singleton, full-term children of employees of the Empire Cotton Growing Corporation who conformed to the following criteria were included in the data from the clinic: (1) a healthy appearance; (2) a satisfactory rate of gain in weight in the preceding six weeks; and (3) no recession in weight greater than 0-25 kg. within this period. Children from twelve nursery schools were weighed and measured at intervals of one to three months, always during term time. The criteria for their inclusion in the present study were: (1) a healthy appearance; (2) a satisfactory gain in weight since the previous visit; and (3) no absences, on grounds of illness, for longer than three days during the preceding

fortnight. Standing height was measured with the child standing barefoot, with heels, buttocks, shoulders, and head touching the rod. The head was held with the lower border of the orbit in the same horizontal plane as the external auditory meatus. In younger children, recumbent length was measured with the child in a supine position, the legs parallel and fully extended, and the feet flexed at right angles to the lower legs. All children were weighed at the clinic without clothing; the nursery-school weights, however, included a small amount of light clothing for which no correction was made. At the clinic a member of the staff of the unit (and as a rule the same observer) took all the measurements. The nurseryschool measurements were made by me with the assistance of another member of the Research Unit. Heights were recorded with a standard (Gneupel) anthropometric rod, with centimetre scale, and weights on an ’Avery’ platform scale with steelyard indicator weighing to 50 g. Similar equipment was used in the previous studies on Baganda children (Dean and Jelliffe 1960, Burgess and Burgess 1964). All dates of birth were verified from birth-certificates brought by parents, except in the case of those children who had attended the clinic at Namulonge within a few weeks of birth.

Results

Height and Weight The mean heights and weights, and standard deviations about the mean, for clinic and nursery-school children up to the age of seven years are given in table 1. Median values are not given, since these did not indicate an uneven distribution of measurements about the mean level. Before the age of six years the mean weights of the boys were about 0-5 kg. higher than those of the girls, but

TABLE I-HEIGHTS AND WEIGHTS OF HEALTHY BAGANDA CHILDREN AT A WELFARE CLINIC IN THE COUNTRY

AND AT KAMPALA NURSERY SCHOOLS