VALIDITY OF PLASMA-CATECHOLAMINE ESTIMATIONS

62 TABLE II-PATIENTS WITH

N.D. =not

done.

Au-SH

Figures in brackets

reinfection (usually in adults). Moreover, primary infection by the faecal-oral route might result in local bowel immunity so that reinfection might generally require infection by the parenteral route. The same virus might produce the clinical and serological features of infectious hepatitis with primary infection and the features of serum hepatitis on reinfection. Thus our finding of Au-SH antigen in adults only may be because it may generally be found in reinfection or persistent infection.

ANTIGEN

We thank Dr. G. L. Le Bouvier and Dr. R. W.

indicate titres.

Patients with Au-SH Antigen

Clinical and laboratory findings in the 5 patients summarised in table 11, cases 1-4 were in the Ruchill Hospital series. The only fatality (case 5) was the only patient who had a post-transfusion hepatitis. This patient had received 16 units of blood for anaemia three months before hepatitis developed. Case 2 gave a history of receiving an intravenous dental anaesthetic six months before onset of hepatitis. No history of previous inoculations was obtained in the other 3 patients. The illness was severe in cases 2 and 5, where the route of infection was probably parenteral, and moderate in the other 3 patients whose infections are

probably non-parenteral. c.F. titres of Au-SH antigen in the sera of the 5 patients ranged from 32 to 512. Subsequent sera tested in parallel with the initial sera from cases 2 and 3 both showed lower titres in the second as compared with the first serum. Gel-diffusion tests were also positive in the first sera from all 5 patients. A comparison of the titres obtained by the e.F. and gel-diffusion methods in sera

REFERENCES 1. 2.

3. 4. 5. 6.

7.

were

from more

2 and 3 showed that the sensitive.

cases

c.F. test was

much

Discussion

The absence of detectable Au-SH antigen in serum from 77 patients under twenty years of age with viral hepatitis and its presence in 5 (14%) of 36 patients over twenty would seem of practical importance from two viewpoints. Firstly, since anti-Au serum for detection of Au-SH antigen is very scarce, it would seem reasonable, in testing for the antigen in patients with suspected viral hepatitis, to give preference to those over twenty years of age. Secondly, it might be suggested that blood for patients in renal-transplant units should preferably be collected from donors under twenty years old whose blood might be less likely to contain Australia antigen (and probably infectious virus 5) than blood from older donors. Our finding of Au-SH antigen in adults and not children accounts with previous work suggesting that there are two types of hepatitis, infectious hepatitis mainly found in children and serum hepatitis mainly in adults.6 However, Krugman et al.7 reported finding, in hepatitis among children at Willowbrook State School, New York, two separate viruses, MS-1 and MS-2; and Au-SH antigen was found only in MS-2 infections.5 However, it has been suggested8 that only one virus might be responsible, and that the different incubation periods of infectious and serum hepatitis might reflect different portals of entry rather than different viruses. It also seems possible that the same virus might show different clinical and serological findings in a primary infection (generally in children) from the findings after

McCollum,

department of epidemiology and public health, Yale University, for supplies of Au-SH antigen and antiserum, and Dr. J. H. Lawson, Dr. 1. W. Pinkerton, and junior medical staff at Ruchill Hospital for collection of sera from hepatitis patients. Requests for reprints should be addressed to C. A. C. R.

8.

Shulman, N. R., Barker, L. F. Science, 1969, 165, 304. Purcell, R. H., Holland, P. V., Walsh, J. H., Wong, D. C., Morrow, A. G., Chanock, R. M. J. infect. Dis. 1969, 120, 383. Cossart, Y. E., Vahrman, J. Br. med. J. 1970, i, 403. Grist, N. R., Ross, C. A. C., Bell, E. J., Stbtt, E. J. Diagnostic Methods in Clinical Virology. Oxford, 1966. Giles, J. P., McCollum, R. W., Berndtson, L. W., Krugman, S. New Engl. J. med. 1969, 281, 119. MacCallum, F. O. in Virus and Rickettsial Diseases of Man (edited by S. Bedson, A. W. Downie, F. O. MacCallum, and C. H. Stuart-Harris) p. 223. London, 1961. Krugman, S., Giles, J. P., Hammond, J. J. Am. med. Ass. 1967, 200, 365. Hirschman, R. J., Shulman, N. R., Barker, L. F., Smith, K. O. ibid. 1969, 208, 1667.

VALIDITY OF PLASMA-CATECHOLAMINE ESTIMATIONS PETER TAGGART MALCOLM CARRUTHERS NEVILLE CONWAY DAVID BATES WALTER SOMERVILLE Courtauld Institute of Biochemistry and Department of

Cardiology, Middlesex Hospital, London W.1, and Institute of Ophthalmology, London W.C.1 Potential sources of error in plasmacatecholamine measurements have been and found in the collection, storage, and investigated, estimation of samples. To reduce the chance of error, blood-samples should be taken as quickly as possible, and the plasma separated and frozen immediately, the exact time being recorded; small delays at this stage cause important losses of catecholamine content. Significant losses of catecholamines also follow prolonged storage of frozen plasma samples and repeated thawing and refreezing. Venepuncture can raise the blood-catecholamine content; when multiple estimations are necessary, samples can be taken through an indwelling venous catheter. This procedure does not affect the catecholamine content of the specimen. Certain common drugs and beverages may interfere with the biochemical method of catecholamine estimation. Failure to standardise rigorously these and other factors such as posture, and avoid interfering compounds may invalidate the most careful biochemical analysis and confound interpretation of the data.

Sum ary

Introduction

SINCE the introduction of biochemical methods of measuring physiological levels of catecholamines in

63

plasma 1-1

the sensitivity of the estimation has been considerably increased. This has been achieved by defining the optimal conditions for their extraction and conversion to fluorescent compounds 4,5 and by auto" mation of the latter stage. 6-8 Such " semiautomated methods improve reproducibility, help to provide a constant background level of fluorescence, and can reduce the amount of photomultiplier fluctuation during the reading. These technical advances have increased the accuracy of plasma-catecholamine estimations. There are obvious advantages in examining plasma rather than urinary catecholamine concentrations in the detection of significant but transient changes in the secretion of these labile compounds, but as a corollary, technical errors now assume greater importance. We examine here some of the pitfalls in the collection of plasma specimens and include observations on the reproducibility of the biochemical method, the validity of using stored specimens, and the influence of interfering substances. Neglect of these factors introduces errors large enough to nullify all the improvements in accuracy.

Materials, Methods,

and Results

Specimen Collection and Biochemical Method The 10 ml. of blood required for each catecholamine estimation was collected in a plastic calcium heparin tube containing 5 mg. sodium metabisulphite which acted as an antioxidant, and plastic separating granules. After rapid the blood was immediately centrifuged at 5000 in a portable centrifuge. After 21/2 minutes’ centrifugation the plasma above the separating granules was decanted into ready labelled 6 ml. plastic tubes. These were promptly frozen by contact with solid carbon dioxide inside a vacuum container and transported to a refrigerated store. The frozen samples were kept in the dark at -200C until analysed. In this study, unless otherwise specified, catecholamine content was estimated within 7 days, and in most cases within 2 days. At the start of the catecholamine estimation in the laboratory, each sample was individually thawed in a 30°C water bath. As soon as the plasma had melted, it was mixed by inversion, and 4 ml. was pipetted into another plastic tube. 0-5 ml. of ice-cold perchloric acid was added immediately with vigorous mixing. After a batch of 18 plasma samples had been deproteinised in this manner, the remainder of the extraction procedure, using alumina, was done by McCullough’s method,except that thymol-blue was used in adjusting the pH to 8-4. Alkaline ferricyanide oxidation was performed with ascorbic acid to give total catecholamines, and with thioglycollic acid to give noradrenaline alone. The estimations were carried out on the , AutoAnalyzer ’, using a modified manifold in which the stabilising reagent and sodium hydroxide were premixed, and the temperature of the reagents controlled before going to a modified Locarte fluorimeter, giving greater fluorescence and baseline stability. Adrenaline values were obtained by subtraction of the noradrenaline concentration from the total catecholamine content and were not separately estimated. With each batch, standard solutions containing respectively a mixture of 0-25, 0-5, 1-0, and 2-0 g. per litre of adrenaline and of noradrenaline were analysed.

mixing r.p.m.

Error of Method This was studied by examination of duplicate samples. Forty-nine pairs of specimens from eighteen patients were estimated. In each case, double the usual amount of blood was withdrawn and the combined sample was divided. The paired plasma specimens were treated identically and

TABLE I-PLASMA CATECHOLAMINE ESTIMATIONS: ANALYSIS OF PAIRS OF DUPLICATE SAMPLES

49

and raised catecholamine levels were included. The results are shown and analysed in table I. The range and mean refer to the absolute values, while the standard deviations are obtained by expressing each sample as a percentage of the mean of its pair. There was no significant difference between duplicate samples in respect of total catecholamines and noradrenaline. Adrenaline concentrations were obtained by calculation and were liable to cumulative error, especially at low levels. Statistical analysis of the 22 pairs of adrenaline results below 0-1 Lg. per litre yield a s.D. of ±26-35%, as compared to those above where the S.D. is ±6-43%. Mean recovery of the four standards in 30 batches of tests was 81.6 (±4-5) % for total catecholamines, 83-8 (±3-78) % for noradrenaline, and the calculated figure for adrenaline was 79-4 (±3-96)%.

analysed simultaneously. Samples with both normal

Venepuncture

or

Indwelling Catheter??

To do serial plasma-catecholamine estimations without subjecting the individual to repeated venepuncture, blood-samples were obtained through an indwelling venous catheter. However, plasma-catecholamine levels so obtained might be affected by disintegrating platelets adhering to the tip and lumen of the catheter. To investigate this point, we measured plasma-catecholamine content in samples taken simultaneously from an indwelling venous catheter in one arm and by venepuncture from the other. In all, 29 pairs of samples were studied from twelve subjects. Of these, six were healthy volunteers, two were patients undergoing cardiac catheterisation, and four were patients who had just started intravenous therapy. In all cases the catheter tip when sampling was situated in the innominate vein or superior vena cava whilst venepuncture was performed in the antecubital fossa of the opposite arm. We took care to use different sites when venepunctures were repeated in the same patient. The catheter used was an ’Intracath’ no. 1914R, length 60-96 cm., except for the patients undergoing cardiac catheterisation in whom samples were withdrawn through a Coumand catheter (size 7, length 125 cm.). Specimens were obtained within 2 hours of catheterisation, except for 7 pairs taken up to 24 hours after catheter insertion. Each pair of specimens was centrifuged, frozen, and analysed at the same time. Catheters were flushed with unheparinised physiological saline solution. TABLE II-COMPARISON OF CATECHOLAMINE CONTENT IN 29 PAIRS OF SIMULTANEOUS SAMPLES OBTAINED BY VENEPUNCTURE AND VIA INDWELLING CATHETER

No

significant

differences. Paired

t tests.

64

There

significant difference between the two samples regardless of the length of time the

was no

groups of

catheter had been in situ

or

the type of catheter

(table II). Effects of Venepuncture To study whether the

of venepuncture itself plasma-catecholamine levels significantly, blood-samples were taken through an indwelling act

could affect

TABLE III-PLASMA-CATECHOLAMINE CONTENT:

PUNCTURE

EFFECT

OF VENE-

(13 PAIRS)

catheter immediately before and again at the same time the venepuncture. Altogether the effects of 13 venepunctures were studied in 5 resting healthy individuals in the supine position. The intracath was introduced percutaneously into an antecubital vein and its tip passed to the innominate vein or superior vena cava. An extension plastic cannula connected the catheter to a fixed threeway tap outside the curtains of the cubicle in which the subject lay. The catheter system was flushed with unheparinised physiological saline. The tubing reaching from the subject’s arm was covered with a dressing towel so that blood could be withdrawn through the catheter without the subject’s knowledge. After approximately 30 minutes rest, the first blood-sample was taken through the catheter. Immediately afterwards, a venepuncture was performed in the opposite arm, and a second catheter sample was obtained at the same time. The two catheter specimens were .analysed for catecholamine content. After further periods of rest of at least 30 minutes, the experiment was repeated. The results are shown in table ill. There was no significant difference between the two groups in respect of noradrenaline but the adrenaline levels were significantly elevated with venepuncture. In certain individuals, a rise in catecholamines was recorded on one occasion but not on another

(fig. 1).

Fig. 2-Decay of catecholamine freezing specimens.

content of

plasma after delay in

Exogenous and Endogenous Catecholamines

plasma

In view of the decay of endogenous catecholamines demonstrated in the preceding experiments, we also looked at the fate of exogenous catecholamines. 300 ml. of blood was taken from a volunteer donor, the anti-

are

destroyed

catechol-0-methyltransferases even at room temperature, specimens must be frozen as quickly as possible. To the importance assess of this point, the catecholamine content of individual plasma samples was estimated after progressive delays in freezing. To produce a wide range of cate-

I’

percentage decreases from the initial 3-minute values (fig. 2). as

Since catecholamines

by enzymes such as monoamine oxidase and

effect of venepuncture.

and 24 and 48 hours after venepuncture. In four instances sufficient blood could not be withdrawn rapidly enough to provide plasma for the last two specimens. A stopwatch was used for the exact times of events in the first half-hour. The results are shown in table IV. Plasma-catecholamine content decayed rapidly over the first 30 minutes with a further decrease over the next 2 days. These results may alternatively be

expressed

Effect of Delayed Freezing

Fig. 1-Eight plasma catecholamine values from one subject illustrating the unpredictable

separated plasma samples were kept at room tempera(about 18°C), and frozen 6, 12, and 30 minutes,

ture

as

in

cholamine levels ten healthy individuals exercised for variable times on a bicycle ergometer. 60 ml. of blood was then taken by venepuncture, mixed in the syringe to ensure homogeneity, and delivered into six heparin metabisulphite tubes. These were immediately centrifuged for 2 minutes only. Thus the first plasma specimen was placed in contact with solid carbon dioxide 3 minutes after the end of venepuncture, this being the shortest practicable time. The other five

I I I I

coagulant being heparin. Adrenaline bitartrate and noradrenaline hydrochloride were added to the blood in amounts giving a final concentration of 2-0 ;jLg. per litre of each. The endogenous catecholamine content of the blood by the start of the experiment was 0’2 (J.g. per litre. The blood was divided into two portions. The first (150 ml.) was I placed in several metabisulphite tubes, centrifuged, and I’ the plasma separated. One of these plasma specimens was frozen immediately and the others were frozen in turn after various delays at room temperature, as in the previous experiment. The exact timing is shown

r

i

I

inr

65

Effect of Prolonged Storage The effect of enzymatic activity on plasma-catecholamines has already been noted. Other factors affecting their rate of decay include antioxidants, pH, and the level of illumination of the sample. The next part of the study examined the stability of plasma specimens stored in the dark at -20°C. Twelve 20 ml. blood-samples were taken. Each was divided into two parts. The twelve paired specimens were treated identically. After one specimen of each pair had been analysed, the second was stored under the conditions specified for between 11 and 106 days. The results are shown in table vi, and indicate that catecholamine content fell by up to 70% during Fig. 3-Decay of exogenous catecholamines.

fig. 3.

The

remaining 150 ml. of blood

TABLE VI-DECAY IN TOTAL PLASMA CATECHOLAMINE CONTENT OF

was

also

metabisulphite tubes and stored at temperature. After various intervals (fig. 3), the tubes were centrifuged and the supernatant plasma separated and frozen. All specimens were analysed for catecholamine content at the same time. The results are shown in fig. 3. The first group, in which the plasma was separated immediately, showed no appreciable decay of catecholamine content over the period of study despite the delays in freezing. The second group of specimens were those in which the added catecholamines were left in contact with whole blood for various periods. In this group there were losses of catecholamine content, losses which increased the longer the blood was left uncentrifuged. Endogenous catecholamines in these specimens were present in very low concentration and the effects of their decay can be ignored. Decay of endogenous catecholamines in unseparated blood, even when cooled (table v), was considerably more rapid than in separated plasma (table iv). Comparison of the data illustrated in fig. 3 with fig. 2 points to differences in behaviour between exogenous and endogenous plasma-catecholamines.

FROZEN SPECIMENS DURING STORAGE

AT -2O°C

divided into several

room

TABLE

IV-PLASMA CATECHOLAMINES: EFFECT OF DELAY VENEPUNCTURE BEFORE FREEZING PLASMA

TABLE VII-DECAY IN CATECHOLAMINE CONTENT AFTER REPEATED FREEZING AND THAWING OF ONE PLASMA SPECIMEN

AFTER

storage, although there was no clear relation between the extent of the fall and the duration of storage. Re-estimation

Since catecholamines in plasma are unstable, there is some loss during freezing and thawing. To demonstrate this, six duplicate analyses of the same specimen were done on the same day, each estimation being separated by a refreezing of the residual plasma samples, and a rethawing in the standard manner. Thus, the final specimen was thawed six times and refrozen five times. The results are shown in table vil. A progressive loss of catecholamine content occurred in this exaggeration of the normal freezing and thawing

inevitably

TABLE

V-DECAY OF TOTAL ENDOGENOUS CATECHOLAMINES WHOLE BLOOD COOLED IN ICE-WATER

IN

process. In a second experiment a group of specimens from one patient with a high catocholamine content were

reanalysed after an intervening storage period of 25 days at -20°C. The results are shown in table vm. Here the effects of long-term storage summate with the loss due to a second freezing and thawing process. The mean loss of catecholamine content was 44%. Interference by Drugs and Foods Interference due to fluorescence produced by a number of commonly used drugs and certain beverages

66 TABLE VIII-DECAY

IN

PLASMA-CATECHOLAMINE CONTENT AFTER ON THE SAME

25 DAYS’ STORAGE AT —20°C: RE-ESTIMATION SPECIMENS

TABLE

IX-SUBSTANCES IN CONCENTRATED FLUORESCENCE IN VITRO

SOLUTION

CAUSING

The tests were done on concentrated was studied. solutions of the compounds put through the extraction and analysis stages in the same way as plasma samples. The results are shown in table ix. These observations in vitro throw doubt on the validity of plasma-catecholamine levels measured by this technique in patients receiving such substances. Further work is needed to establish whether they cause similar interference in vivo. It may be that, although many drugs caused no appreciable false fluorescence in vitro, their metabolites may do so in vivo, and vice versa. Discussion

The results of any investigation can be properly interpreted only when they are based on a sound understanding of the limitations of the techniques used. Nowhere is this more applicable than in the of biochemical measurements in clinical medicine. Close liaison between clinician and chemical pathologist is vital. Minor details of collection, handling and storage of specimens are very important, especially when dealing with unstable compounds such as catecholamines. Our observations indicate several possible sources of error in plasma-catecholamine measurement, mostly of loss rather than false gain. To reduce these, bloodsamples should be taken as quickly as possible and the plasma separated immediately; specimens should be centrifuged for the shortest practicable time; plasma should be rapidly frozen without delay; specimens should not be stored for long periods even under ideal conditions and cannot be used twice without significant loss of catecholamine content; when being analysed, specimens should be unfrozen use

singly and not in a batch. Since venepuncture itselj unpredictably raise the catecholamine content 01 the blood, serial blood-samples are best taken through an indwelling catheter, preferably without the patient’s knowledge. Contrary to theoretical expectation, catecholamine levels in catheter samples are not falsely high, and our findings in this respect are in keeping with the observation that the concentrations of noradrenaline and adrenaline are similar in plateletrich and platelet-poor plasma.9 Certain drugs interfere with the biochemical estimation in vitro-e.g., aminophylline, frusemide, perphenazine, protamine sulphate, promethazine, ampicillin, vitamin-B complex, sulphonamides, and methyldopa-indeed, methyldopa may be estimated by this biochemical method." Many of these drugs are in everyday use, and until their influence on plasma-catecholamine levels in vivo has been established they should be avoided in clinical studies. Unexpectedly, coffee, tea, and cocoa also cause interfering fluorescence in vitro possibly from the caffeine, theophylline, and theobromine they contain. Other workers have shown that plasmacatecholamine levels are influenced by exercise and by posture.9 Vendsalu9 investigating seventeen healthy individuals found mean (::L S.D.) concentrations of noradrenaline and adrenaline in recumbency of 0-360-02 ILg. per litre and 0-04d:0’01 g. per litre, respectively. After 10 minutes in a head-up tilt position, the corresponding values were 0-620-03 and 0’11±0’02 g. per litre. These figures are of considerable interest in the light of reported increases in noradrenaline levels in myocardial infarction or may

cardiac failure since it is conceivable that differences in posture, as related to the management of these conditions, may explain some of these changes. Such influences may well have been taken into consideration, but in the absence of positive statements to this effect, interpretation of these interesting data is difficult. Venous catecholamine content varies in different parts of the body, although the differences between the antecubital and intrathoracic veins, used

by

us,

are not

important.

short, comparison of two or more specimens of plasma is valid only if they are taken under the same In

conditions and treated identically. The differences that we have demonstrated in the decay rates of endogenous and exogenous catecholamines have three important implications. First is the potential error that may be introduced into recovery data when exogenous catecholamines are added to a sample to act as internal standards. Secondly, it is difficult to equate published data concerning the relationship between infused catecholamines and various physiological and biochemical variables.il Thirdly, it emphasises the importance of rapid separation of the plasma samples. The error of the biochemical estimation itself should not be overlooked. Although total catecholamine and noradrenaline levels may be individually measured with an acceptable degree of accuracy, the adrenaline content, being obtained by calculation from the other two concentrations, is subject to the error of both, and therefore, is much less dependable. We feel that these points have been insufficiently appreciated in the past, and their neglect may have

67

previous published work. If the plasma-catecholamines is to be understood, rigorous attention to such details is mandatory.

prejudiced

some

behaviour of

We thank Prof. R. H. S. Thompson and Prof. Norman Ashton for facilities to carry out this study, and Sister Davies and Mr. David Gibbons for their assistance in the clinical stages. The work was financed by the Heart Research Fund of the Middlesex

Hospital. Requests for reprints should be addressed to M. C., Department of Chemical Pathology, Institute of Ophthalmology, Judd Street, London W.C.1 H9QS. REFERENCES

Lănd, A. Acta pharmac. 1950, 6, 137. Udenfriend, S. Pharmac. Rev. 1959, 11, 252. Anton, A. H., Sayre, D. F. J. Pharmac. exp. Ther. 1962, 138, 360. Robinson, R. L., Watts, D. T. Clin. Chem. 1965, 11, 986. Weil-Malherbe, H. in Methods of Biochemical Analysis (edited by D. Glick); vol. XVI, p. 293. New York, 1968. 6. Merrills, R. J. Analyt. Biochem. 1963, 6, 272. 7. Fiorica, V. Clin. Chimica Acta, 1965, 12, 191. 8. McCullough, H. J. clin. Path. 1968, 21, 759. 9. Vendsalu, A. Acta physiol. scand. 1960, 49, suppl. p. 173. 10. Udenfriend, S. Fluorescence Assay in Biology and Medicine; vol. II, 532. New York, 1969. 11. Jurand, J., Oliver, M. F. Atherosclerosis, 1970, 11, 157. 1. 2. 3. 4. 5.

Changes in voltage, current, and

energy

threshold after

sequen-

tial intravenous injections of 1 mg., 4 mg., and 5 mg. (total 10 mg.) of propranolol.

Each point is the mean of observations in five patients and in all cases the values are expressed as percentages of the control values before propranolol.

made with patients resting for several hours in bed. For the last 10 minutes of this control period, an intravenous infusion of 5% glucose was established to facilitate intravenous injection of the drugs. Preston et awl.9 showed that this does not alter the threshold, and we have confirmed this finding. During the test period of approximately 1 hour, 50-75 ml. glucose solution was administered. The tests were only carried out when repeated threshold testing during the control period had shown that the results were stable within a range of ± 5 %. Threshold measurements were made at the beginning of the test and after 2, 7, 8, 13, 14, 19, 24, 34, and 44 minutes. Between the first and second minutes,1 mg. propranolol was injected. A further 4 mg. was injected between the 7th and 8th minutes and a further 5 mg. between the 13th and 14th minutesÚ Results The mean changes in voltage, current and energy thresholds for the group are shown in the figure. In three patients (nos. 2, 4 and 5) the largest increase in threshold was at the end of the observation period of 44 minutes, the increases in energy threshold being 43%, 56%, and 87%, respectively. In the remaining two patients, the largest increase in threshold occurred after 19 minutes, the respective values being 22% and measurements were

INFLUENCE OF BETA-BLOCKADE ON MYOCARDIAL THRESHOLD IN PATIENTS WITH PACEMAKERS WOLFGANG KÜBLER EDGAR SOWTON Institute of Cardiology, London W.1 The effects of intravenous propranolol myocardial threshold in five patients

Summary on

with right ventricular transvenous electrodes were studied. 10 mg. propranolol increased the energy threshold by a mean value of 40% and in one case by

89%. Introduction THE threshold for stimulation in the human heart increases for a few days after attachment of an electrode.1 Furthermore there are spontaneous variations throughout the day,2,3 and changes may be produced by alterations in electrolytes4 and by pharmacological factors.5-7 Since the combination of beta-adrenergic receptor blocking drugs and implanted cardiac pacemakers in control of intractable arrhythmias is now becoming more common8 we have investigated the acute effects of propranolol on myocardial threshold in five patients. Patients and Methods Three male and two female patients with ages ranging from 58 to 77 (mean 69) were investigated 3-5 days after

28%. Injection of

a further 4 mg. propranolol caused a further increase in threshold which continued to rise in three patients but stabilised after 8 minutes in patient 2, and after 24 minutes in patient 3. The increases in threshold were due to changes in both current and ENERGY THRESHOLD

implantation of a U.S.C.I. C51 transvenous electrode placed in the apex of the right ventricle for short-term pacing. Patients with heart-failure, evidence of ischsmic heart-

disease, cardiomyopathy,

sinus

rhythm,

or

multiple ectopic

beats were excluded. Stimulation was carried out with an adjustable pacemaker (Devices type E) which delivers square-wave impulses of approximately 2 milliseconds duration. Applied

voltage and current (as the voltage dropped across a 10 Q resister) were displayed upon the screen of a Tectronics oscilloscope (model 502A) and polaroid photographs were taken from the oscilloscope screen. Threshold energy was then calculated as the product of mean voltage, mean current, and impulse duration. Patients had no meal for 2 hours before the

test

and all

Propranolol i.v. was given at and 12-13 minutes (5 mg.).

1-2 minutes (1

mg.), 7-8 minutes (4 mg.),