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Detection of glutethimide (doriden) by hemagglutination inhibition
DETECTION
OF GLUTETHIMIDE
(DORIDEN)
BY
HEMAC;GI,UTI~ATIOS
The production of an anti-glutethimide serum and an immunological method of detecting the drug is described. Using hemagglutination-inhibition low levels of glutethimide in serum and urine from clinical cases are detected.
Immunological methods for assaying drugs in biological fluids are sensitive, specific and rapid and may require no separation of the drug from the biological fluid radioassay” and electron spin rcsoprior to anal!+. Hemagglutination inhibitionl, name3 techniques have been used for the detection and measurement of drugs to which antibodies have been made. This note reports an immunological method, hemagglutination
inhibition,
for tllc detection
of glutethimide.
Glutethimide u-as coupled to bovine serum albumin and also to egg albumin through azo bond formation between the hapten aromatic ring and the protein. To IOO mg of z-(~-aminophen!,l)-z-eth?lglutarimide (Elipten, CIBX, Summit, New Jersey) in a minimum of cooled 0.1 S hydrochloric acid an excess of an aqueous IO’);, sodium nitrite solution was added with stirring. After 30 min sulfamic acid was added to the cooled solution until a negative test for nitrous acid was obserl~ed with starcllPiodide paper. The reaction mixture was added drop-wise over a zo-min period to a cooled aqueous solution containing 500 mg of bovine serum albumin in 30 n!l of saturated sodium tetraborate. Throughout this coupling reaction the pH of the reaction mixture was checked with indicator paper to insure that the pH was maintained between 7.5 and 8.5. As required, I iy sodium hydroxide was added to maintain the desired pH. After standing overnight the yelloworange reaction mixture was poured onto a 25 x rooo-mm column of Sephades (G-25 and eluted with I liter of 0.05 11
66
VALENTOLTR
et al.
sodium tetraborate. The yellow-orange band was collected and dialyzed against distilled water at 4’ for 6 days. The orange protein solution was then lyophilized. ;I similar procedure was followed for the preparation of glutethimide--egg albumin. The color of the glutethimide-proteins could be attributed to the aromatic--azo bonds formed. A quite similar color was produced upon the equimolar addition of diazotized Elipten to phenol. Based upon the absorbance of solutions of known cot,centrations of the azophenol, the glutethimide incorporation in BSAI was estimated to exceed 30 moles per mole of protein. A solution containing IO mg of glutethimide+BSX per ml of 0.850/~ saline was emulsified with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, Michigan). Sew Zealand IVhite rabbits were injected in each hind foot-pad with I ml of this emulsion (5 mg of the hapten-protein conjugate). Several additional injections of similar amounts of the emulsion were given at intervals of 4 to 8 Iveeks. Blood was collected 7 to 14 days after each booster injection. These samples were allowed to clot, they were centrifuged and the serum was separated from the cells. The serum was inactivated by heating at 56” for 30 min. Serial dilutions of rabbit antisera were prepared in IO x roe-mm test tubes for hemagglutination and inhibition tests. For each test 0.1 ml of rabbit antiserum was diluted to 1.0 ml with an aqueous solution containing 0.85 g sodium chloride and 1.0 ml normal rabbit serum per IOU ml. Two drops of packed sheep red blood cells were added to each tube and the contents were mixed. After centrifuging, the I :IO diluted antiserum was decanted into the first of a series of tight tubes. A o.5-ml portion of this solution was removed from the first tube and added to 0.5 ml of the saline-normal rabbit serum in a second tube and mixed. A 0.5-1111 portion of the solution in the second tube was likewise diluted in the third tube and mixed. By proceeding in a similar manner, further dilutions were made to obtain eight o.j-ml solutions of antiserum whose dilutions ranged from I: ICI to I: 1280. One-tenth ml of each solution to 1~ tested for glutethimide (urine, serum, plasma or saline solutions of various drugs) was added to each tube in the series. 0.05 ml of 2.5:; tanned sheep red blood cells coated with glutethimideeegg albumin also were added to each tube. As a control one series of antiserum dilutions, lacking the 0.1 ml of tllc test solution, were processed in ;t similar manner. The hemagglutination inhibition patterns, which developed after about 3 11, were observed and compared to the concurrent hemagglutination patterns. The erythrocyte tanning procedure and agglutination pattern identifications II~IY~ been described elsewhere-‘. RESULTS
The HA titer of the rabbit antiserum, which had been combined from all rabbits, was I :640. This titer and possibly the sensitivity of the tests may have been raised had additional booster injections of glutethimide-BSA been given to the rabbits. However the serum was sufficiently sensitive for the detection of 50 ng of glutethimide per 0.1 ml of test sample. Conversely IOOOO ng of structurally related phenobarbital or diphenylhydantoin were not inhibitory. Other less chemically similar drugs such as secobarbital, morphine, quinine, amphetamine or propoxyphene were likewise noninterfering at the ~oooo-ng level. Higher levels of these drugs were not checked for inhibition nor were aminoglutethimide or the metabolites of glutethimide.
GLUTETHIMIDE
67
DETECTIOX
'l‘hBL1~ I
1x2’ 100 ml saline)
*
320
Plasma and urine samples from several hospital patients suspected of glutethimide intoxication were analyzed by hemagglutination inhibition as well as by gas chromatography. The results are included in Table I. DIscussIos
When employed with due regard to its advantages and limitations the hemagglutination inhibition technique for the detection of drugs is a highly useful addition to the arsenal of the toxicologist. It readily accommodates screening large numbers of samples with minimum effort and equipment. Some adaptations which offer versatility to this technique include the possibility of making the analyses quantitative6 or using micromethods for agglutination7. The latter results in savings of reagents and increased rapidity with which serial dilutions can be made. The liabilities of drug analyses by hemagglutination inhibition include the several hours required before the results can be determined. Also the preparation of a suitable antiserum can be a rather lengthy process, relying apparently on trial and error for the selection of (a) optimal drug incorporation in the antigenic protein, (b) the best functional group used for coupling the drug to the protein and (c) cooperating animals for producing the antibodies.
F. 1,. .\LDER AND C. T. LIU, 1. ~w~m~ud., 106 (1971) 1684. S. SPECTOR AND C. W. PARKER, Scirmr, 168 (1970) 1347. S. SPECTOR AND IT.J. FLYNX, ibid., 174 (1971) 1036. K. Ii. LEUTE, E. F. ~~LLIMAS, A. GOLDSTEIN ASD L. .L\.HERZESBERG, Rrport of the Thirty-thivd -1 mual Scimti$c Mrrting, Cowmittcc ox Pvoblrms 01 Drug Deprndmcr, Toronto, Frbvuavy, I 9 71, I-01. I, p. 613. -4. B. STAVITSKY, J. lmmunol., 72 (1954) 360. J. ~~CGEE, Clin.Chew., 17(1971) 587. H. ~~.MEXGOLI, J.C.PRUITTAN.L) H.M.CARPESTER, Lab. Imwst., 12 (1963) 365. J. 1~. SEVER, 1. Zmnztnol., 88 (19G2) 320.
OF GLUTETHIMIDE
(DORIDEN)
BY
HEMAC;GI,UTI~ATIOS
The production of an anti-glutethimide serum and an immunological method of detecting the drug is described. Using hemagglutination-inhibition low levels of glutethimide in serum and urine from clinical cases are detected.
Immunological methods for assaying drugs in biological fluids are sensitive, specific and rapid and may require no separation of the drug from the biological fluid radioassay” and electron spin rcsoprior to anal!+. Hemagglutination inhibitionl, name3 techniques have been used for the detection and measurement of drugs to which antibodies have been made. This note reports an immunological method, hemagglutination
inhibition,
for tllc detection
of glutethimide.
Glutethimide u-as coupled to bovine serum albumin and also to egg albumin through azo bond formation between the hapten aromatic ring and the protein. To IOO mg of z-(~-aminophen!,l)-z-eth?lglutarimide (Elipten, CIBX, Summit, New Jersey) in a minimum of cooled 0.1 S hydrochloric acid an excess of an aqueous IO’);, sodium nitrite solution was added with stirring. After 30 min sulfamic acid was added to the cooled solution until a negative test for nitrous acid was obserl~ed with starcllPiodide paper. The reaction mixture was added drop-wise over a zo-min period to a cooled aqueous solution containing 500 mg of bovine serum albumin in 30 n!l of saturated sodium tetraborate. Throughout this coupling reaction the pH of the reaction mixture was checked with indicator paper to insure that the pH was maintained between 7.5 and 8.5. As required, I iy sodium hydroxide was added to maintain the desired pH. After standing overnight the yelloworange reaction mixture was poured onto a 25 x rooo-mm column of Sephades (G-25 and eluted with I liter of 0.05 11
66
VALENTOLTR
et al.
sodium tetraborate. The yellow-orange band was collected and dialyzed against distilled water at 4’ for 6 days. The orange protein solution was then lyophilized. ;I similar procedure was followed for the preparation of glutethimide--egg albumin. The color of the glutethimide-proteins could be attributed to the aromatic--azo bonds formed. A quite similar color was produced upon the equimolar addition of diazotized Elipten to phenol. Based upon the absorbance of solutions of known cot,centrations of the azophenol, the glutethimide incorporation in BSAI was estimated to exceed 30 moles per mole of protein. A solution containing IO mg of glutethimide+BSX per ml of 0.850/~ saline was emulsified with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, Michigan). Sew Zealand IVhite rabbits were injected in each hind foot-pad with I ml of this emulsion (5 mg of the hapten-protein conjugate). Several additional injections of similar amounts of the emulsion were given at intervals of 4 to 8 Iveeks. Blood was collected 7 to 14 days after each booster injection. These samples were allowed to clot, they were centrifuged and the serum was separated from the cells. The serum was inactivated by heating at 56” for 30 min. Serial dilutions of rabbit antisera were prepared in IO x roe-mm test tubes for hemagglutination and inhibition tests. For each test 0.1 ml of rabbit antiserum was diluted to 1.0 ml with an aqueous solution containing 0.85 g sodium chloride and 1.0 ml normal rabbit serum per IOU ml. Two drops of packed sheep red blood cells were added to each tube and the contents were mixed. After centrifuging, the I :IO diluted antiserum was decanted into the first of a series of tight tubes. A o.5-ml portion of this solution was removed from the first tube and added to 0.5 ml of the saline-normal rabbit serum in a second tube and mixed. A 0.5-1111 portion of the solution in the second tube was likewise diluted in the third tube and mixed. By proceeding in a similar manner, further dilutions were made to obtain eight o.j-ml solutions of antiserum whose dilutions ranged from I: ICI to I: 1280. One-tenth ml of each solution to 1~ tested for glutethimide (urine, serum, plasma or saline solutions of various drugs) was added to each tube in the series. 0.05 ml of 2.5:; tanned sheep red blood cells coated with glutethimideeegg albumin also were added to each tube. As a control one series of antiserum dilutions, lacking the 0.1 ml of tllc test solution, were processed in ;t similar manner. The hemagglutination inhibition patterns, which developed after about 3 11, were observed and compared to the concurrent hemagglutination patterns. The erythrocyte tanning procedure and agglutination pattern identifications II~IY~ been described elsewhere-‘. RESULTS
The HA titer of the rabbit antiserum, which had been combined from all rabbits, was I :640. This titer and possibly the sensitivity of the tests may have been raised had additional booster injections of glutethimide-BSA been given to the rabbits. However the serum was sufficiently sensitive for the detection of 50 ng of glutethimide per 0.1 ml of test sample. Conversely IOOOO ng of structurally related phenobarbital or diphenylhydantoin were not inhibitory. Other less chemically similar drugs such as secobarbital, morphine, quinine, amphetamine or propoxyphene were likewise noninterfering at the ~oooo-ng level. Higher levels of these drugs were not checked for inhibition nor were aminoglutethimide or the metabolites of glutethimide.
GLUTETHIMIDE
67
DETECTIOX
'l‘hBL1~ I
1x2’ 100 ml saline)
*
320
Plasma and urine samples from several hospital patients suspected of glutethimide intoxication were analyzed by hemagglutination inhibition as well as by gas chromatography. The results are included in Table I. DIscussIos
When employed with due regard to its advantages and limitations the hemagglutination inhibition technique for the detection of drugs is a highly useful addition to the arsenal of the toxicologist. It readily accommodates screening large numbers of samples with minimum effort and equipment. Some adaptations which offer versatility to this technique include the possibility of making the analyses quantitative6 or using micromethods for agglutination7. The latter results in savings of reagents and increased rapidity with which serial dilutions can be made. The liabilities of drug analyses by hemagglutination inhibition include the several hours required before the results can be determined. Also the preparation of a suitable antiserum can be a rather lengthy process, relying apparently on trial and error for the selection of (a) optimal drug incorporation in the antigenic protein, (b) the best functional group used for coupling the drug to the protein and (c) cooperating animals for producing the antibodies.
F. 1,. .\LDER AND C. T. LIU, 1. ~w~m~ud., 106 (1971) 1684. S. SPECTOR AND C. W. PARKER, Scirmr, 168 (1970) 1347. S. SPECTOR AND IT.J. FLYNX, ibid., 174 (1971) 1036. K. Ii. LEUTE, E. F. ~~LLIMAS, A. GOLDSTEIN ASD L. .L\.HERZESBERG, Rrport of the Thirty-thivd -1 mual Scimti$c Mrrting, Cowmittcc ox Pvoblrms 01 Drug Deprndmcr, Toronto, Frbvuavy, I 9 71, I-01. I, p. 613. -4. B. STAVITSKY, J. lmmunol., 72 (1954) 360. J. ~~CGEE, Clin.Chew., 17(1971) 587. H. ~~.MEXGOLI, J.C.PRUITTAN.L) H.M.CARPESTER, Lab. Imwst., 12 (1963) 365. J. 1~. SEVER, 1. Zmnztnol., 88 (19G2) 320.