, JUNE 16 out that none had a demonstrable pressure cone when the brain was removed at necropsy. They also discussed a case of an infant in opisthotonos in which a left vertebral arteriogram showed vertebral filling but no filling of the basilar artery, illustrating that the intracranial posterior circulation can be arrested when the pressure within the posterior fossa exceeds the systolic bloodThis confirms that, in cases pressure. They concluded, of acutely raised intracranial pressure, the cerebral circulation is slowed to such an extreme that death is inevitable." Today, many potential donor organs are lost from patients with irreversible brain damage because of the required waiting-period of at least 24 hours to obtain two flat E.E.G.s. The waiting-period is often longer than 24 hours if E.E.G.s are not readily available. The importance of this loss is brought into perspective when we realise that there are now 70 people on dialysis awaiting cadaverkidney transplantation at one major Boston hospital alone.
pointed
"
Neurosurgical Service, Emerson Hospital, Concord, Massachusetts 01742, U.S.A.
ROBERT C. CANTU.
REORGANISATION—RATES FOR THE
JOBS
SIR,-Your editorial (June 2, p. 1228) might have gone further and remarked that no salary scales for medical administrators have yet been published or posts advertised notwithstanding. There may be more bitter pills to be swallowed. 47
Corstorphine Bank Drive, Edinburgh EH12 8RH.
G. G. SAVAGE.
The lymphocytes can be easily pipetted off and collected. The viability of the lymphocytes after washing and resuspension in phosphate-buffered saline or tissue-culture medium approaches 98%(as measured by trypan-blue exclusion). The lymphocytes were shown to react in various tests measuring blastogenesis and cytotoxicity. The recovery-rate of lymphocytes from normal blood with this technique exceeds 70%. University of Texas at Houston, M. D. Anderson Hospital and Tumor Institute, Houston, Texas, U.S.A.
KAMRAN TEBBI.*
RADIOIMMUNOASSAY OF PLASMA-RENIN ACTIVITY
SIR,-In a comparison of the Schwarz-Mann and Squibb commercial kits for the measurement of plasmarenin activity by radioimmunoassay of angiotensin-I production, Chervu et aLl used 10 1. of dimercaprol (8 mM) as inhibitor in each case, although only 2 jjj. of dimercaprol (1-6 mM) is recommended in the SchwarzMann kit. Chervu et al. did not mention any reason for this alteration, but there is considerable variation, from 1-6 2-4 to 8-0 mM,l in the amount of dimercaprol used by various investigators of angiotensin-I activity, the main differences being typified by the low concentration recommended in the Schwarz-Mann kit, and the high concentrations used in the Squibb kit. Ryan et al.,5,6 who originally recommended the use of dimercaprol to inhibit angiotensinases, used a concentration of 10 mM before
bioassay. PURIFICATION OF LYMPHOCYTES
SiR,-Several methods for the separation of lymphocytes from other cellular elements of the blood are available.1-3 The ’Ficoll ’-’ Hypaque ’ method1 is probably the easiest and most useful. With this technique, however, some contamination with granulocytes and monocytes is unavoidable in 5-10% of specimens. In addition, some of the monocytes that contaminate the lymphocyte preparation become " activated " during this procedure; thus, nonspecific reactions can occur. The following technique has proved very successful in eliminating contamination of the lymphocyte preparation with granulocytes and monocytes. It is based on phagocytosis of colloidal iron particles by blood leucocytes before centrifugation in ficoll-hypaque gradient. Simple laboratory facilities In
are
required.
clean 100 x 16 mm. glass tube, 2 ml. buffy-coat leucocytes separated from heparinised blood are mixed with 1 ml. 10% carbonyl iron (G.A.F. Corporation, New York, N.Y.) suspended in 10% acacia solution. The mixture is incubated for 30 minutes at 37 °C on a rocker at 30 revolutions per minute (Ames Co., Indiana). After adding 2 ml. normal saline, the contents are carefully laid over 3 ml. separating fluid " in another 100 x 16 mm. glass tube. The " separating fluid " consists of 9% ficoll (Farmacetica, Uppsala, Sweden) and 34% hypaque (Winthrop Laboratories, New York, N.Y.) in ratio of 2-4/1 to give a specific gravity of 1-076-1-078. The tube is centrifuged for 30 minutes at 20°C at 1300 r.p.m. Four distinct layers separate in the centrifuged tube. The upper layer consists of the plasma and saline. The next layer is that of the purified lymphocytes. Repeated studies indicate contamination with granulocytes or monocytes to be less than 0-5%. The third layer consists of the clear ficollhypaque mixture. The fourth layer is the sediment containing all the red cells and the iron-laden granulocytes and monocytes a
"
We are using a kit manufactured by Societa Ricerche Nucleari (SORIN) which, like the Schwarz-Mann kit, is based on the method of Haber et al.,2 who used 1-60 mM dimercaprol. These authors did not mention the reason for choosing this low concentration and hence it may not be optimal. This problem is emphasised by our own results. In June, 1972, SORIN changed the recommended amount of dimercaprol from 2 ,1. (1-6 mM) to 6 1. (4-8 mM). Since no explanation for the change was offered we compared estimations using the two concentrations, and found a substantial increase in the measured plasma-renin activity when using the higher concentration of inhibitor. Ten samples of normal plasma, collected after one hour’s rest, gave mean results (s.D.) of 0-33:0-16 ng. angiotensin I per ml. per hour using 1-6 mM dimercaprol and 0-740-36 ng. per ml. per hour using 4-8 mM dimercaprol, an increase of x 2-2. The most probable explanation for the difference is that the lower concentration of dimercaprol does not completely inhibit the enzymic degradation of
Impianti
angiotensin
*
Present address: St. Louis Children’s Hospital, St. Louis, Missouri 63110, U.S.A. 1.
2. 3. 4. 5. 6. 7.
(macrophages). 8. 1. 2.
3.
Harris, R., Ukaejiofo, E. O. Lancet, 1969, ii, 327. Thorsby, E., Bratlie, A. in Histocompatibility Testing (edited by P. I. Terasaki); p. 665. Copenhagen, 1970. Gelsthorpe, K., Doughty, W., Fox, M. Br. J. Surg. 1970, 57, 358.
i.
Further work is required to establish the optimum inhibitor effect, but several workers 7-10 have already
9. 10.
Chervu, L. R., Lory, M., Liang, T., Lee, H. B., Blaufox, M. D. J. nucl. Med. 1972, 13, 806. Haber, E., Koerner, T., Page, L. B., Kliman, B., Purnode, A. J. clin. Endocr. 1969, 29, 1349. Kritzinger, E. C., Kanengoni, E., Jones, J. J. Lancet, 1972, i, 412. Craswell, P. W., Hird, V. M., Judd, P. A., Baillod, R. A., Varghese, Z., Moorhead, J. F. Br. med. J. 1972, iv, 749. Ryan, J. W., McKenzie, J. K., Lee, M. R. Third International Congress of Nephrology, abstract 265. Washington, 1966. Ryan, J. W., McKenzie, J. K., Lee, M. R. Biochem. J. 1968, 108, 679. Stockigt, J. R., Collins, R. D., Biglieri, E. G. Circulation Res. 1971, 28/29, suppl. 2, p. 175. Stockigt, J. R., Collins, R. D., Noakes, C. A., Schambelan, M., Biglieri, E. G. Lancet, 1972, i, 1194. Vallotton, M. B. Horm. metab. Res. 1971, suppl. 3, p. 94. Boyd, G. W., Adamson, A. R., Fitz, A. E., Peart, W. S. Lancet, 1969, i, 213.
1393 chosen to use around 5 mM dimercaprol in the final incubated solution. This may prove to be optimum, or at least a suitable standard for general adoption. Standardisation is urgently required before credence can be given to comparisons of different reports using the radioimmunoassay technique. Meanwhile, reports based on the use of this method with a low inhibitor concentration require critical reappraisal. University Department of Medicine, Dundee DD1 4HN, and Regional Physics Department, Eastern Regional Hospital Board, Dundee.
LAURA W. FLEMING F. HUTCHINSON W. K. STEWART.
EFFECT OF PROPANTHELINE AND METOCLOPRAMIDE ON ABSORPTION OF DIGOXIN
SiR,-Dr Manninen and others (Feb. 24,
p.
398) showed
absorption of digoxin from tablets was affected if propantheline or metoclopramide was given simultaneously. Dr Thompson (April 7, p. 783) suggested that this effect might be due to changes in biliary excretion caused by these drugs. The low serum-digoxin concentrations from the investigated tablets, compared with the levels from digoxin solution, made us suspect that the release of digoxin from the tablets might be slow. So we investigated the brand in question by an in-vitro method which gives results which predict the absorption of digoxin from tablets. I,2 The tablets had a very low rate of dissolution. Only about 30 °o that the
of the labelled amount was released after 4 hours in simulated gastric fluid at 37 °C (see accompanying figure).
DISSOLUTION-RATES AND BIOAVAILABILITY OF DIGOXIN TABLETS
SIR,-We
were
interested
to
read the dissolution and
bioavailability results of Dr Lindenbaum and his colleagues (June 2, p. 1215), since we have recently completed similar studies. We determined dissolution-rates for single tablets using a simple stirring procedure in O1M hydrochloric acid under carefully controlled conditions. Dissolution profiles for eleven brands of digoxin tablets B.P. available in Great Britain in 1972 showed wide variations in rate of digoxin release. From these in-vitro findings four brands which differed markedly in profile were selected for in-vivo study, to investigate possible correlations between dissolution-rate and bioavailability in man. An excellent correlation was found between amount dissolved at various time intervals and the area under the plasmaconcentration/time curve in six individuals. Thus our earlier predictions seem confirmed-namely, that the absorption of digoxin is dissolution-rate-controlled, rather than disintegration-rate-dependent.1 Our conclusions are therefore similar to those of Dr Lindenbaum and his co-workers-that a dissolution test should be included in pharmacopoeias and formularies as a quality-control procedure to limit this variable. Our detailed findings will be reported elsewhere.
Pharmaceutical Department,
E. J. FRASER R. H. LEACH J. W. POSTON.
Clinical Chemistry Department, Queen Elizabeth Medical Centre, Birmingham B15 2TH.
A. M. BOLD L. S. CULANK A. B. LIPEDE.
ADENOSINE-DEAMINASE DEFICIENCY AND SEVERE COMBINED IMMUNODEFICIENCY SYNDROME
SIR9 Three patients with both severe combined immunodeficiency (S.C.I.D.) and adenosine-deaminase (A.D.A.) deficiency have been reported 2,3and two similar cases (as yet unreported) are known. In view of the importance of this association for the eventual understanding of immunodeficiency, we wish to report a sixth patient.
In-vitro dissolution of digoxin from tablets. Batch XL 44. Simulated gastric fluid, 37°C. U.S.P.-N.F.
rotating basket, 100 r.p.m. It seems likely that the slow dissolution from these tablets is a necessary condition for the observations reported by Dr Manninen and others. If the dissolution-rate is sufficiently slow the time for passage through the gastrointestinal tract must strongly influence absorption. On the other hand, when digoxin is given in solution the passage This also time seems to be of secondary importance. implies that fast dissolution of digoxin tablets should make it less likely that absorption will be affected by drugs which alter gastrointestinal motility. AB Draco Subsidiary of AB Astra, Research and
Development Department, Fack, S-221 01 Lund 1, Sweden. 1.
Bertler, Å., Redfors, A., Medin, S., Nyberg,
The number of
patients reported with both S.C.I.D. and deficiency precludes the unlikely possibility of the simultaneous occurrence of two rare genetic defects. It is most likely that A.D.A. deficiency is the cause of one type A.D.A.
STEFAN MEDIN LARS NYBERG. L.
Lancet, 1972, ii,
708. 2.
The male patient was the fourth child of a healthy 29-year-old father and a 26-year-old mother. At the age of 1 week he had a respiratory infection which progressed and required admission to hospital at the age of 3 weeks for hypothermia and cyanosis. A lymphocyte-count at that time was 1500 per c.mm. He temporarily improved, then at 5 weeks he had diarrhoea, pulmonary congestion, cyanosis, and generalised sclerema. The lymphocyte-count was 3000 per c.mm., with atypical vacuoles, which fell to 400 per c.mm. at 9 weeks. Immunological findings at this time were: IgG 134, IgM 15 mg. per 100 ml., IgA not detectable; lymphocytes failed to transform in vitro with either phytohaemagglutinin or concanavallin A. The patient died at 9 weeks of age. Necropsy confirmed the diagnosis of S.C.I.D., and echo-6 virus was cultured from many sites. A.D.A. activity was absent in a red-cell hxmolysate prepared from the patient’s blood, using either a spectrophotometric assay or starch-gel electrophoresis. Each parent had A.D.A. activity which was approximately half of normal.
Medin, S., Nyberg, L. Lakartidningen, 1973, 70,
469.
1. Fraser, E. J., Leach, R. H., Poston, J. W. Lancet, 1972, ii, 541. 2. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B., Meuwissen, H. F. ibid. p. 1076. 3. Dissing, J., Knudson, B. ibid. p. 1316. 4. Pollara, B. Personal communication.