AN EXPERIMENTAL STUDY ON BIODISTRIBUTION OF DIACETYLMONOXIME IN SHEEP 1 · 23

Sri vas t a v a A. K., H. N. K han i k 0 r, J. K. Mal i k: An Experimental Study on Biodistribution of Diacety1monoxime in Sheep. Acta vet. Brno, 57, 1988: 13-18. The distribution study of diacetylmonoxime (15 mg/kg, iv) in various body fluids and tissues of healthy sheep was conducted. In blood the therapeutic concentration ~ 4 IIg.m1~1 was maintained upto 8 h. At different blood concentrations DAM penetrated into erythrocytes to the extent of 5.6 to 16.8 per cent. DAM was readily distributed in various body fluids and tissues. Highest concentration was recorded in brain (7.51 I1g.g-1) and lowest in liver (2.25 4.49 IIg.g-1 ). Approximately 16 per cent of the total dose of DAM was eliminated in urine of sheep during first 24 h of its administration. Diacetylmonoxime, tissue distribution, urinary excretion, sheep. Of several phosphorylated acetylcholinesterase (EC 3.1.1. 7) reactivators, diacetylmonoxime (DAM) has been reported to reactive acetylcholinesterase enzyme in central nervous system (T a y lor 1980). Recently DAM has been successfully employed in the treatment of domestic animals, severely poisoned with organophosphorus insecticides (E cob i c h 0 n 1976; M Ii 1 i k et e1. 1984; R a i n a 1984; Sri vas t a v a et a1. 1984). Clinical efficacy of acetylcholinesterase reactivators is completely dependent on their concentrations in central compartment and target tissues. Tissue distribution study of DAM and other reactivators has been investigated in man and experimental animals (D u 1 t ~ et a1. 1957; Jag e r et a1. 1958; Bar k man et a1. 1963; Sid ell and G r 0 f f 1971). The purpose of this cODlllWlication is to report the distribution of DAM in various body tissues and fluids of sheep following single intravenous administration.

The distribution study of diacetylmonoxime (15 mg/kg, iv) in various body fluids and tissues of healthy sheep was conducted.In blood the therapeutic concentration ~ 4 IIg.m1~1 was maintained upto 8 h.At different blood concentrations DAM penetrated into erythrocytes to the extent of 5.6 to 16.8 per cent.DAM was readily distributed in various body fluids and tissues.Highest concentration was recorded in brain (7.51 I1g.g-1) and lowest in liver .Approximately 16 per cent of the total dose of DAM was eliminated in urine of sheep during first 24 h of its administration.Diacetylmonoxime, tissue distribution, urinary excretion, sheep.

Animals and treatment
The study was performed on eighteen healthy sheep of both sexes (28-34 kg).Before conducting the experiment, the animals were acclimatized for 10 days in the animal shed of department.After collecting the control blood sample, DAM (Aldrich Chemical Co.Milwaukee, Wisconsin) was administered in a single dose of 15 mg/kg body mass as freshly prepared 8 % solution in sterile isotonic saline into the jugular vein of animals.

Collection of samples and analysis
An intravenous canula was placed in the opposite jugular vein through which blood was drawn.Blood samples were collected in heparinized test tubes at various pre-determined time intervals.Erythrocytes were separated at 3000 rpm for 15 min at room temperature.CSF-was collected from the lumbosacral joint of animal.For collecting urine, urinary bladder of three female sheep was catheterized by a Foley balloon catheter with the flexible metal probe.Urinary bladder was evacuated at 2, 4, 6, 8, 10, 12, 18 and 24 h after drug administration.For collection of tissues, animals were sacrificed at 15 and 180 min of injection and three to fifteen per cent (W/v) homogenate of each tissue sample was prepared in phosphate buffer (0.04 M; pH 6), using a Potter Elvehjem glass homogenizer.
DAM was analysed in whole blood, erythrocytes, CSF, urine and tissue homogenates according to the procedure of D u 1 t z et al. (1957).

Results and Discussion
Table 1 represents the levels of DAM in blood, erythrocytes and CSF.The DAM concentration of 14.9 ± 1.86 ].1g.ml''';1 of blood at 15 min gradually declined and at the end of 12 h traces of 1.96 + 0.18 ].1g.ml-1 was recorded.The therapeutic blood level of oxime reactivators has been reported to be ~4 ].1g.ml-1 (S ide I I and G r 0 f f 1971;' Srivas t a -v a 1984).In this study this concentration of DAM was maintained from 15 to 480 min of its administration.
The effectiveness of acetylcholinesterase reactivators is certainly dependent on their penetration into erythrocytes, as organophosphorus insecticides are well known to inhibit the erythrocyte ChE to greater extent than other esterases (S r i vas t a v a et al. 1984).Accordingly, it was also thought of important to calculate the extent of penetration of DAM into erythrocytes.At different blood concentrations, DAM penetrates into erythrocytes to the extent of 5.6 to 16.8 per cent of total blood concentration.Any correlation between concentration of DAM in blood and extent of penetration into erythrocytes could not be established.Good penetration of DAM into erythrocytes reflects that DAM may be beneficial in the treatment of organophosphate'insecticide (OPI) poisoning in sheep.
It is apparent from the data of Table 1 that build up of DAM in CSF was much faster than that of blood.At 15-min.
the initial concentration of DAM in CSF was 11.7 ± 1.14 llg .m1-1which gradually rose to peak (20.6 ± 0.62 llg.ml-1 ) at 60 min.The concentration of DAM in CSF remained high as compared to blood up to 12 h and the CSF-blood ratio ranged from 0.65-1.85.The results on distribution of DAM in different body tissues of sheep are summarized in Table 2. Of various body fluids and tissues, brain (7.51-46.4]Jg.g-1 ) and spinal cord (6.63-39.3l1g.g-1 ) accumulated highest concentration of DAM from 15-180 min of its administration.All organophosphorous insecticides produce their toxicity and lethality due to its effects on CNS mainly on• the respiratory center in brain.Accordingly, it is essential that sufficient quantity of DAM inhibited cholinesterase enzyme in various parts of brain.The results of present findings are in agreement with previous studies suggesting that DAM readily penetrates BBB and reactivate brain ChE (W i I I sand B 0 r ison 1959; B row n 1960).In man also high concentration of DAM (7-8 J1g.ml -1) was estimated in central nervous system at 60 and 150 min of its intravenous administration (J a g e r et al. 1958).The DllnlmUm therapeutic concentration of DAM ( ~ 4 ~ .ml-1 ) was maintained from 15-180 min in all tissues examined except kidney and liver.The relatively low concentration of DAM in kidney (3.6-4.3 llg. g-l ) observed in the present study is in agreement with the findings of D u I t z et al. (1957) and Jag e r et al. (1958) who speculated that extra renal site is important for elimination of DAM from the animal body.
The above fact is further substantiated with results of renal excretion.Approximately 16 per cent of the total administered dose of DAM was eliminated in urine of sheep over a period of 24 h (Table 1 purpose of this cODlllWlication is to report the distribution of DAM in various body tissues and fluids of sheep following single intravenous administration. 1963; Sid e l l and G r 0 f f 1971).The