HEPARIN-INDUCED DECREASE IN CIRCULATING ANTITHROMBIN-III

581 TABLE VIII—CASE-CONTROL DISTRIBUTIONS FOR MALES BY DIABETIC STATUS AND ARTIFICIAL SWEETENER USE

We thank Christine Nelson, Tim Curran, L’ainee Duncan, Margaret Burke, and Patricia Peddle for their technical assistance; the three provincial cancer registries concerned; and the many pathologists and urologists who cooperated in this study. This study was supported by the National Cancer Institute of Canada and, in part, under National Health Research and Development project no. 6131048-30 of Health and Welfare Canada.

Requests for reprints should be addressed to A. B. M.

REFERENCES

with the matching broken because of small numbers, shows that male diabetics are at slightly decreased risk (summary risk ratio=0.77) and that male diabetic users of artificial sweeteners have a similar risk ratio (19) to male non-diabetic users (1 7). In our sample 65% of male diabetic controls were current nonsmokers compared with 67% of male non-diabetic controls, and controlling for smoking did not materially increase the risk ratio for diabetic versus non-diabetic males (1 - 1). The population attributable riskls calculated from our data is 7% for- males for the use of all artificial sweeteners. For male diabetics it is 33%, mainly because of their substantial use of such products.

(table vm), again

-

’

We have demonstrated a positive association between of artificial sweeteners and risk of bladder cancer in males. The effect has been demonstrated primarily for reported saccharin use, insufficient data being available

I. C. Unpublished. 2. Morgan, R. W., Jain, M. Can. med. Ass. J. 1974, 111, 1067. 3. Kessler, I. J. J. Urol. 1976, 115, 143. 4. Kessler, I. J. J. natn. Cancer Inst. 1970, 44, 673. 5. Armstrong, B., Lea, A. J., Adelstein, A. M., Donovan, J.

6. 7. 8. 9.

W., White, G. C.,

Rittle, S. Br. J. prev. Soc. Med. 1976, 30, 151. Burbank, F., Fraumeni Jr., J. F. Nature, 1970, 227, 296. Armstrong, B., Doll, R. Br. J. prev. soc. Med. 1974, 28, 233. Miettinen, O. S. Biometrics, 1970, 26, 75. Pike, M. C., Casagrande, J., Smith, P. G. Br. J. prev. soc. Med. 1975, 29,

196. 10. Mantel, N., Haenszel, W. J. natn. Cancer Inst. 1959, 22, 719. 11. Mantel, N. J. Am. stat. Ass. 1963, 58, 690. 12. Miller, A. B. Cancer Res. (in the press). 13. Howe, G. R., Chambers, L., Gordon, P., Morrison, B., Miller, A. B. Paper presented at the Society for Epidemiologic Research meeting, held in Seattle, Washington, in June, 1977. 14. Miller, A. B., Howe, G. R., Burch, J. D., and others. Unpublished. 15. Cole, P, MacMahon, B. Br. J. prev. soc. Med. 1971, 25, 242.

HEPARIN-INDUCED DECREASE IN CIRCULATING ANTITHROMBIN-III

-

cyclamate use.

The difference between this and two previous casecontrol studies2,3 could be due to confounding by some unknown variable in our study, though this seems unlikely in view of the large number of possible confounding variables considered; to bias in control selection

(hospital

Arnold, D. L., Moodie, C. A., Grice, H. C., Charbonneau, S. M., Munro,

Discussion

use

to evaluate

1.

as

opposed

to

neighbourhood controls);

or to

undetected systematic error. The failure to detect increased mortality from bladder cancer in diabetics4,s could be explained by the reduced risk for diabetics which we observed when artificial sweetener use was controlled for; this in turn could be associated with reduced exposure to bladder carcinogens among diabetics, though we have no evidence as to the source of any such reduction. With a population attributable risk for males for artificial sweetener use of 7%, it is unlikely that any sudden increase in bladder-cancer rates would have occurred with the increased use of artificial sweeteners; in view of the difficulties of assessing levels of use, and other varisome

ables which change over time, it is hardly surprising that examination of secular, trends6,7 do not show the small effect expected. Our failure to detect any effect of artificial sweetener use in females seems unlikely to be due to chance (upper 90% confidence limit for females for use of artificial sweetener=1.1), and could be due to a difference between the sexes with respect to metabolism, though we know of no biological basis for this hypothesis. Our results suggest a causal relation between saccharin use and bladder cancer in males, especially when they are considered in conjunction with results in animals.’

EWA MARCINIAK

JON P. GOCKERMAN Department of Medicine, University of Kentucky Medical Center, Lexington, Kentucky 40506, U.S.A. Plasma-antithrombin-III (AT-III) concentrations were measured throughout therapy in 24 patients receiving continuous intravenous heparin infusion and in 2 patients treated with repeated intravenous heparin injections. In all patients, including 1 with congenital AT-III deficiency, heparin therapy was associated with a considerable progressive reduction in AT-III-binding capacity and antigenic protein. The net individual decrease in plasma-AT-III was 0·31±0·05 units/ml (normal plasma-AT-III was 1·00 units/ml) or 9·5±2·0 mg/dl and the decrease was independent of initial concentration. Plasma-AT-III returned to normal two to three days after heparin was stopped. There was no decrease in plasma-AT-III after a single dose of intravenous heparin. When present in blood for long periods heparin significantly reduced AT-III, the proteinase inhibitor that is responsible for the anticoagulant effect of this drug. The finding is very relevant to the interpretation of clinical data in patients treated with heparin and suggests that AT-III depletion may underly the thromboembolic complications sometimes encountered

Summary

during heparin therapy. Introduction BECAUSE of its immediate anticoagulant effect heparin has been widely used in thromboembolic disease. This property of heparin is based on the presence of anti-

582

thrombin-ni (AT-III) in blood. As a proteinase inhibitor binds in equimolar ratios to several enzymes participating in. the intrinsic pathway of blood coagulation. These include, in addition to thrombin and factor Xa, factors IXa1,2 and XIa.3Heparin accelerates the rate at which AT-in neutralises these enzymes, limiting sequential clotting reactions and preventing fibrin formation. Although some clinical trials attempted to correlate the efficacy4 of therapy with blood-heparin concentration it is not entirely clear how the antithrombotic effect of heparin in man can be evaluated. The extent to which this effect relies on the concentration of AT-III and whether heparin therapy affects circulating concentrations of AT-iii is unknown. Using a direct method for evaluation of AT-III functional capacity, independent of the velocity of inhibition of clotting, we observed a relatively low AT-m concentration suggestive of a congenital deficiency in several of our patients who received heparin. Immunoassays confirmed these results, but in both types of assays AT-III concentration promptly became normal once heparin was discontinued. This observation prompted us to measure AT-III concentrations in patients treated with intraAT-III

venous

heparin.

with

heparin

after

being admitted because of deep-vein throm-

bosis, pulmonary embolism, or arterial thrombosis who had no signs of liver disease, abnormal renal function, or disseminated intravascular coagulation were investigated. Patients with congenital deficiency of AT-111 included those from three kindreds. Two of these families have been described elsewhere.’,’ The third family consists of three generations with 6 affected members. Patients receiving hsemodialysis were randomly selected from the population treated in the Hemodialysis Unit.

Treatment who received heparin infusion an initial intrainjection of 5000-7000 units was followed by a steady intravenous drip at a rate of 750-2000 units/hour given by means of an infusion pump. The dose of heparin was adjusted in an attempt to maintain the partial thromboplastintime in the range of 45 to 75 s (control 30 s) There was no evidence of bleeding or recurrent thromboembolism in this group of patients. 2 patients received heparin in intravenous bolus injections of 5000 units given every four hours. Routine coagulation and haematological studies were performed before and frequently during therapy. The house staff and attending physicians were responsible for the duration and management of heparin therapy. They also decided whether and when to initiate the therapy with oral anticoagulants. In

patients

venous

bolus

AT-III Evaluation

Patients and Methods Patients All inpatients at the University of Kentucky Medical Center between August 1976 and January 1977 who were treated

Fig. 1--Changes in ptasma-AT-ni during and after infusion of heparin in a patient with deep-vein thrombosis. Inhibitor (AT-III) evaluated by capacity for binding (0) and by immunodiffusion (e).

Specimens

for

AT-m

evaluation

were

obtained in

most pa-

therapy and/or frequently during the hospital course, usually once daily. All evaluations were performed with plasma obtained from freshly drawn blood anticoagulated with sodium citrate. A plasma pool obtained from 12 normal tients before

volunteers was used as a control reference. Normal human plasma contains 1.00 units of AT-III/ml. The functional binding capacity of AT-III was determined by a modification of the method described by Gitel and Wessler.’ This procedure uses bovine factor Xa as the enzyme and measures the ultimate inhibitory capacity of AT-m regardless of the velocity of inhibition, therefore it can be successfully applied to heparin-containing specimens. Mean plasma-AT-in in 50 normal subjects was 0-97 ±0-09 units/ml. Rocket immunoelectrophoresis was performed according to the method of

Fig. 2-Changes in plasma-AT-HI determined by rocket immunoelectrophoresis before, during, and after heparin infusion in a patient with pulmonary embolism. Wells 1-3 show normal plasma in 1/1, 1/2, and 1/4 dilutions; remaining wells show patient plasma in 1/2 dilution-well 4 before therapy, wells 5-10 in 6 consecutive days of heparin infusion, wells 11 and 12 in 1st and 3rd postheparin day. In consecutive wells plasma-ATni concentrations expressed in units/ml are: (1) 1.00; (2) 0.50; (3) 0-25; (4) 0.97; (5) 0.89; (6) 0.76; (7) 0.60; (8) 0.67; (9) 0.67; (10) 0-63; (11) 0-72; (12) 0.86.

583 EFFECT OF CONTINUOUS INTRAVENOUS INFUSION OF HEPARIN

(MEAN DOSE) ON ANTITHROMBIN-III CONCENTRATION IN PLASMA

antigen was 9.5+_2.0 mg/dl. Mean post-treatment rise in AT-III was 0.40+0-11 units/ml, and that of was 9-8 ±2-4 mg/dl. We attempted to correlate antigen the mean heparin dose in individual patients with their degree of AT-m reduction, but no statistically valid var-

functional

iations were found. The results obtained in a patient with hereditary ATIII deficiency who received heparin infusion for sus-

*s.D. data not given for groups because changes in calculated as net differences in individual patients.

AT-III

in

test were

Laurell;8 the quantity of AT-m antigen was expressed as units/ml. Radial immunodiffusion was performed on the ’M-Partigen’ agarose plate from Behring Diagnostics; AT-III was measured in mg/dl.

pected deep-vein thrombosis were especially interesting. After two days of therapy his AT-III binding capacity had decreased from 0.50 units/ml to 0.17 units/ml. The dose of heparin was then reduced and concurrent therapy with warfarin was started. Functional plasma AT-III stabilised at around 0.20 units/ml. Heparin was discontinued when the prothrombin-time had reached double the starting value, and shortly thereafter plasma AT-111 rose. Fig. 3 compares results recorded in this AT-IIIdeficient patient with results obtained in a patient with a normal initial plasma-AT-m and in a patient with high

Results

Typical variations in plasma-AT-III concentration in a patient receiving heparin infusion are presented in figs. 1 and 2. From the beginning of therapy functional and immunoreactive concentrations of plasma-AT-III gradually declined, reaching a plateau after three days of infusion. The maximum decreases were 031 units/ml (binding capacity), 0-37 units/ml (rocket immunoelectrophoresis), and 11 mg/dl (radial immunodiffusion), which corresponds to about one third of normal plasmaAT-111. Plasma-AT-III progressively returned to pretreatment values within three days of stopping heparin. 22 patients with normal plasma-AT-m before and/or after treatment, were followed during continuous infusion of heparin (see accompanying table). In all 22 heparin infusion reduced plasma-AT-III. The mean of individual decreases in AT-III binding capacity (from pretreatment value to the lowest value during therapy) was 031±005 units/ml; the corresponding decline in _

-Fig. 4-Correlation between AT-III functional binding capacity and antigenic determinants evaluated by rocket immuno-

electrophoresis. (A) 23 patients with normal original AT-m concentrations receiving heparin infusion ce); same patients off heparin therapy (0); a subject with congenital AT-III deficiency treated with heparin infusion C’); same patient before therapy ([]). (B) 26 subjects with congenital AT-III deficiency C’) and 29 of their normal relatives (D).

initial plasma-AT-III. Functional AT-III decreased by 0-30-0-40 units/ml and antigen concentration also by 0-30-0-40 units/ml, regardless of whether the original concentration of inhibitor was raised, normal, or reduced. In 2 patients with deep-vein thrombosis who received heparin by intermittent intravenous injection the maximum decline in inhibitor occurred after four days of therapy. At that time the difference in functional AT-III binding capacity from pretreatment values was 0 31and 0.40 units/ml, which is within the range observed during continuous heparin infusion. In 4 normal volunteers and 2 patients congenitally deficient in AT-III, a single dose of injected heparin (100

units/kg) produced

no significant change in AT-III binding capacity, despite a high initial concentration of heparin in blood. In 3 patients with chronic renal failure who had been on haemodialysis for over three months and therefore had often been exposed to intravenous heparin, plasma-AT-III concentrations were normal. Apparently heparin must be continuously present in blood for long periods to reduce plasma-AT-III

antigen

Fig. 3—Changes in plasma-AT-M during and after heparin infusion in patients with various initial concentrations of inhibitor.

measured by capacity for binding bovine factor Xa (0) by rocket immunoelectrophoresis (...). (A) Patient with deep-vein thrombosis and initial AT-ni above normal range. (B) Patient with deep-vein thrombosis and normal starting values of inhibitor (C) Patient with hereditary AT-Ill deficiency. AT-iu was

and

-

or

concentration.

584 The results of the plasma-AT-III studies were strikingly similar in heparinised patients and those with congenital AT-III deficiency (fig. 4). In both groups there was a strong correlation between functional and immunoreactive AT-III. Discussion Our results indicate that when present in the blood for long periods heparin significantly reduced circulating AT-III. Functionally, the ability to bind coagulation enzymes was reduced and, immunologically, the concentration of antigenic protein was lower. This new and rather unexpected finding for which there is no explanation has an important bearing on both AT-III metabolism and the pharmacodynamics of heparin. Transiently high concentrations of heparin after a single intravenous bolus injection were without effect

plasma-AT-III. Intermittent, infrequent exposure to heparin, even when it recurred over a long time as in patients on haemodialysis, also did not affect AT-III concentration. This picture does not accord with a disturbance of synthesis of a liver-produced protein. Moreover, the net decrease in both binding capacity and antigenic on

determinants of AT-III shows little individual variation among patients receiving conventional heparin therapy and is independent of the initial plasma-AT-III. This relatively stable decrease could reflect a constant requirement for inhibitor in vivo and would be consistent with heparin increasing the AT-III decay-rate. The anticoagulant effect of heparin is thought to depend solely on the presence of AT-III in blood. In both experimental and clinical studies, however, heparin sometimes has a paradoxical effect which seemingly defies its anticoagulant property. In in-vitro studies heparin paradoxically decreases the binding capacity of AT-III for thrombin9-11 so that a given amount of AT-III neutralises less thrombin in the presence of heparinalthough the velocity of binding remains increased. Paradoxical thrombotic episodes have been reported during heparin therapy. 12-14 Many workers who reported such findings were left with the impression that heparin in vivo activates an antagonistic mechanism opposing its anticoagulant effect. These clinical observations were confirmed in studies on animals,15 which also demonstrated that a risk of thrombosis is especially high immediately after discontinuation of heparin. Our results suggest that a heparin-induced reduction in AT-III increases the thrombotic tendency in man. Previous studies of families with hereditary AT-III deficiency have provided ample evidence that reduced concentrations of this inhibitor are an important risk factor for development of thromboembolism.5,6.16 As long as heparin circulates in blood the increased rate of binding will compensate even for low concentrations of AT-III. With the clearance of anticoagulant, however, a thrombotic tendency may sporadically prevail because AT-III concentrations are then too low to neutralise coagulation enzymes. Up to now a pronounced decrease in plasma-AT-III in a patient with normal liver function would have been diagnosed as hereditary AT-III deficiency, especially if the patient had thromboembolic disease. Our results clearly indicate that such conclusions are often incorrect if the subject is receiving prolonged treatment with heparin at examination. Most of our patients who otherwise were capable of producing normal concentrations of inhibi-

tor, if assessed for the first time

during heparin infusion with bolus injecduring prolonged tions, could have been misdiagnosed as having a primary condition predisposing to thrombosis. Our observations in a patient with hereditary AT-III deficiency who received heparin infusion suggest that heparin should be used with caution in this type of patient. The risk of thromboembolism may be especially high because of the very low inhibitor concentrations which may develop while such a patient is being treated with heparin; therefore plasma-AT-m ought to be measured repeatedly. We have attempted to determine the size of the heparin-induced effect on plasma-AT-III and some conditions under which it appears, but the study was not designed to establish precise dose-response rela-

or

treatment

more

normal volunteers with a long-lasting the study, the underlying thromboembolic disease in our patients would have contributed to this effect. This possibility is unlikely because the consistent appearance and size of AT-III reduction in all our patients who received standard heparin therapy was not related to the location and extent of their thrombotic manifestations. tion. Since exposure to

no

heparin entered

This work was supported by a grant in aid from the American Heart Association. We thank Miss Deborah Switzer for expert assistance.

Requests for reprints should be addressed to E.M. REFERENCES

Rosenberg, J. S., McKenna, P. W., Rosenberg, R. D. J. biol. Chem. 1975, 250, 8883. 2. Kurachi, K., Fujikawa, K., Schmer, G. Biochemistry, 1976, 15, 373. 3. Damus, P. S., Hicks, M., Rosenberg, R. D. Nature, 1973, 246, 355. 4. Hirsh, J., vanAken, W. G., Gallus, A. S., Dollery, C. T., Cade, J. F., Young, 1.

W. L. Circulation, 1976, 53, 69. 5. Marciniak, E., Farley, C. H., DeSimone, P. A. Blood, 1974, 43, 219. 6. Filip, D. J., Eckstein, J. D., Veltkamp, J. J. Am. J. Hemat. 1976, 7. Gitel, S. N., Wessler, S. Thromb. Res. 1975, 7, 5.

1, 343.

8. Laurell, C. B. Analyt. Biochem. 1966, 15, 45. 9. Lyttleton, J. W. Biochem. J. 1954, 8, 15. 10. Waugh, D. F., Fitzgerald, M. A. Am. J. Physiol. 1956, 184, 627. 11. Marciniak, E. Thromb. Diath. hœmorrh. 1975, 34, 748. 12. Zinn, W. J. Am. J. Cardiol. 1964, 14, 36. 13. Barker, C. F., Rosato, F. E., Roberts, B. Surgery Gynec. Obstet. 1966, 123, 22.

14. Klein, L. A., Hall, R. L., Smith, R. B. J. Urol. 1972, 108, 104. 15. Mustard, J. F., Murphy, E. A., Downie, H. G., Rowsell, H. C. Br. J. Hœmat. 1963, 9, 548. 16. Egberg, O. Thromb. Diath. hœmorrh. 1965, 13, 516.

POLYMORPHIC HYDROXYLATION OF

DEBRISOQUINE IN MAN A. MAHGOUB L. G. DRING

J. R. IDLE R. LANCASTER R. L. SMITH

Department of Biochemical and Experimental Pharmacology and Department of Clinical Pharmacology, St. Mary’s Hospital Medical School, London W2 1PG

Summary

Debrisoquine and

its

primary metabo-

lite, 4-hydroxydebrisoquine,

were mea-

sured in the urine of 94 volunteers after a single oral dose of 10 mg debrisoquine. The ratio between excreted debrisoquine and its metabolite was bimorphically distributed in the study population. Family studies supported the view that alicyclic 4-hydroxylation of debrisoquine is