nicotinyl alcohol capsules and the patient's plasma were extracted and assayed for warfarin-like and dicumarol-like sub¬ stances. The patient's plasma demon¬.
Spectrophotometric Assays for Warfarin Sodium and Dicumarol Use in
Drug
Rapid
Detection of Patients Suspected of Intoxication or Surreptitious Noningestion
Edmond R.
Cole, PhD, Fedor Bachmann, MD
of oral should be susin who exhibit an unexplained severe prolongation of the prothrombin time, have depressed levels of the vitamin K-dependent factors, and have previously been treated with oral anticoagulants or are associated with the medical profession.1,2 The definite diagnosis of misuse of oral anticoagulants is often difficult, particularly in the malingerer who tries to mislead the treating physician. Thus, precious time may be lost before appropriate treatment can be instituted. This report demonstrates the usefulness of spectrophotometric assays of plasma or serum extracts in the rapid identification and determination of dicumarol and warfarin sodium levels.3,4
ingestion Surrepti t i o us anticoagulants pected patients
MATERIALS AND METHODS collected in siliconized aspia mixture of citric acid-sodium citrate in a ratio of 9 volumes of blood of 1 volume of anticoagulant that were centrifuged for 20 min at 2,500 rpm. Coagulation assays were performed for the following: one-stage prothrombin time, factor IX, Lee-White whole blood clotting time, Ivy bleeding time,5 thrombin time, factors II, V, VII/X complex, and X6, activated partial thromboplastin time,7 and semiquantitative fibrinogen.8 Factor
Blood
was
rating syringes containing
Received for publication March 18, 1975; ac5. From the Coagulation Research Laboratory, Hematology Section, Department of Medicine, Rush-Presbyterian-St Luke's Medical Center, Chicago. Dr Bachmann is currently with the Office of European Research, Schering Corporation USA, Lucerne, Switzerland. Reprint requests to Coagulation Research Laboratory, Hematology Section, Department of Medicine, Rush-Presbyterian-St Luke's Medical Center, 1753 W Congress Parkway, Chicago, IL 60612 (Dr Cole).
cepted Aug
VII assays were performed using as a sub¬ strate the plasma of a patient with congen¬ ital factor VII deficiency. Plasma was assayed for dicumarol by the method of Axelrod et al3 or for warfa¬ rin sodium according to the method of O'Reilly et al.4 To establish identity of the anticoagulant extracted from the patient's plasma, the extracts were scanned in a spectrophotometer, and the anticoagulant content of the plasma was calculated from a standard curve relating absorbance of the extract to the amount of pure anti¬ coagulant added to normal plasma and ex¬ tracted according to the method applicable to the type of anticoagulant.
PATIENT SUMMARIES AND RESULTS
65-year-old man was ad¬ Presbyterian-St Luke's Hospital on Aug 15, 1969, complaining of severe ab¬ dominal pain of about 36 hours' duration. Patient l.-A
mitted to
He was nauseated and vomited old food several times during the next few hours. Since the onset of pain, the patient had had no bowel movement or flatus. There was no dysuria or hematuria. Physical examination showed an elderly man in acute distress, complaining of gen¬ eralized abdominal pain. The pulse rate was 96 beats per minute; temperature, 38.1 C; blood pressure, 155/94 mm Hg; and res¬ piratory rate, 20/min. There was marked guarding of all quadrants of the abdomen, with generalized tenderness that was max¬ imal in the lower part of the midabdomen. Femoral pulses were diminished in both legs, and dorsalis pedis pulses were absent. A flat-plate x-ray film of the abdomen showed no air in the transverse colon. Laboratory studies on admission re¬ vealed a hemoglobin level of 13.6 gm/100 ml; white blood cell count (WBC), 16,500/cu mm with 11% stab cells; one-stage pro¬ thrombin time, 11% (33 seconds) (normal, 70% to 140% [10.7 to 12.7 seconds]); and whole blood clotting time, 40 min (normal, 8 to 15 min). Because of the low prothrom¬ bin time activity, the house officer admin-
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istered 10 mg of
nously.
phytonadione
intrave¬
At 10 PM on the night of admission, two hours after the administration of phytona¬ dione, blood was drawn for a complete co¬ agulation screening profile. These studies, performed the following morning, showed 3% to 6% activities of the prothrombin time (Fig 1) and vitamin K-dependent clotting factors (factors II, VII/X, IX, and X). Fibrinogen concentration (400 mg/100 ml), factor V (140%), factor VIII (130%), thrombin time (12 seconds), and platelet counts were entirely normal. Nearly iden¬ tical results were obtained in a blood sample drawn on the morning of the sec¬ ond day, 12 hours after the first adminis¬ tration of phytonadione. The persistent severe depression of lev¬ els of all vitamin K-dependent factors af¬ ter phytonadione administration ruled out vitamin K deficiency, and the normal level of factor V excluded the presence of liver disease. The diagnosis of anticoagulant in¬ toxication was made, and an additional 20 mg of phytonadione was administered. The patient was again questioned about possible ingestion of anticoagulants. He in¬ formed us that he had taken dicumarol from 1962 to 1965 for his aortoiliac disease and still had some of the capsules at home, but that these were quite different in color¬ ation and size from the nicotinyl alcohol capsules he was taking for his vascular dis¬ ease. Pursuing the investigation for anti¬ coagulant intoxication, the content of the nicotinyl alcohol capsules and the patient's plasma were extracted and assayed for warfarin-like and dicumarol-like sub¬ stances. The patient's plasma demon¬ strated an absorbance spectrum identical to that of pure dicumarol (Fig 2) at a con¬ centration of 42.4 mg/liter. The extract of the nicotinyl alcohol capsule showed no evi¬ dence of coumarin compounds. During the following days, the patient's clinical status and coagulation profile im¬ proved rapidly, with the exception of a temporary decrease in levels of the vita¬ min K-dependent factors on the sixth day.
Fig 1.—One-stage prothrombin time activ¬ ity and factor II, VII/X, IX, and X levels dur¬ ing hospitalization of patient 1 with acci¬
dental dicumarol intoxication. Solid line with solid circles indicates percent onestage prothrombin time activity; dotted line with Xs, percent factor II; solid line with open circles, percent factor Vll/X; broken line with squares, percent factor IX; and broken line with triangles, percent factor X.
Daily assays for plasma dicumarol concen¬ trations yielded a curve consistent with a half-time of 78 hours (Fig 3). A few days after discharge, he provided us with samples of each type of medication he had at home. He had indeed confused a tranquilizer and dicumarol and had taken four to five capsules of dicumarol a day for the two to three weeks prior to the onset of his acute symptoms. Patient 2.—A 28-year-old man hospi¬ talized in the psychiatric ward was allowed to go home on pass at 4 pm, April 17, 1970. At about 5:15 pm, he ingested 100 5-mg tab¬ lets of warfarin sodium, a medication pre¬ scribed for his wife, and slashed both wrists. He was brought to the emergency room where his wrist wounds were su¬ tured. Gastric lavage with 4 liters of nor¬ mal saline solution yielded a large number of pieces of white material, presumed to be warfarin tablets. Forty milliliters of milk of magnesia was then administered by gastric tube. He received 50 mg of phy¬ tonadione intramuscularly and 200 mg of orally administered phénobarbital at about 9 pm. His hemoglobin level was 14.7 gm/100 ml, hematocrit reading was 44.9%, and WBC count was 9,400/cu mm. The Quick one-stage prothrombin time was 100% (11.3 seconds). He received 200 mg of orally ad¬ ministered phénobarbital every 12 hours for the next four days, and 50 mg of phy¬ tonadione every 12 hours on the second and third days, 50 mg on the fourth day, 20 mg on the fifth day, and 10 mg on the sixth day of rehospitalization. Figure 4 illustrates the behavior of the one-stage prothrombin time and levels of factors II, VII, IX, and X. The first profile, obtained four hours after ingestion of 500 mg of warfarin sodium, was normal. The second profile, obtained seven hours after ingestion of the drug, demonstrated that the level of factor VII, the vitamin Kdependent coagulation factor with the shortest half-time, had dropped by about 30%. Factor II, IX; and X levels remained unchanged. Plasma warfarin levels were substantially higher at seven hours (11.2 mg/liter) than at four hours (6.8 mg/liter) after drug ingestion (Fig 3). By the morn¬ ing of the second day, 17 hours after drug intake, level of factor VII had fallen to
100-
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20
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3
4
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6
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Days After Admission woman with extensive ecchymoses and hematomas over most of the body, mild epistaxis, and cervical bleeding. Other
24%, and the other values for vitamin K-
pale
factor VII started to climb toward normal ranges. The plasma warfarin levels had de¬ clined to 6.0 mg/liter; thus, no further sub¬ stantial résorption of the drug had taken place between 7 to 29 hours after warfarin intake. The profiles performed on the third and fourth days showed continued normal¬ ization of all values. The clinical course of the patient was unremarkable, and notable bleeding from the wrist wounds was not encountered. A half-time disappearance rate of 46 hours was calculated (Fig 3). Patient 3.—A 26-year-old nurse was re¬ ferred to Presbyterian-St Luke's Hospital on April 28, 1974, because of a two-year history of easy bruising, vaginal bleeding, recurrent epistaxis and bleeding gums, fa¬ tigue, and a persistently prolonged onestage prothrombin time. In 1972, after an exploratory laparotomy, she developed thrombophlebitis of the left leg, was treated with heparin sodium, and contin¬ ued taking oral anticoagulants for six months. Physical examination showed an obese,
physical findings were not remarkable. Laboratory studies showed a hemoglobin
dependent factors showed moderate de¬ creases. In the evening of the second day,
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level of 5.7 gm/100 ml, a hematocrit read¬ ing of 18.2%, and a reticulocyte count of 9.8%. Coagulation studies disclosed a onestage prothrombin time of over 100 sec¬ onds and low levels of the vitamin K-de¬ pendent factors II, VII/X, and X (Fig 5), suggesting coumarin compound intoxica¬ tion. The spectrophotometric scan of the extract from the patient's plasma revealed the presence of dicumarol (Fig 2), but the patient denied the use of oral anti¬
coagulants. Phytonadione, 30 mg administered in¬ travenously, was given. A coagulation pro¬ file done on the next day showed no essen¬ tial change, and two additional 20-mg doses of phytonadione were administered in 12-hour intervals. Improvement of the one-stage prothrombin time and of factor II, VII/X, and X levels were seen on the fourth day, and vitamin K therapy was discontinued. However on the fifth day, the one-stage prothrombin time activity and vitamin K-dependent factor levels de-
315
0.3-^
nm
0.3
dicumarol
308
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0.2
3
nm
0.2
warfarin sodium
o c
case
2
