Document dDYm9qMK239LDRLNRkeOa1629

inOLATXOil FROM LIVER OP ggTfUgROfLLgAP AFTER ITS INHALATION Charles D, Stevens, Charles J* Peldhak and Robert A* Keho (From The Kofctoring Laboratory In the Department of Preven tive Medicino and Industrial Health* College of Medicine* University of Cincinnati* Cincinnati, Ohio; Several investigators have obtained volatilo load from certain tissues of subjects intoxicated with totreethyHead (TEL) and have assumed that this volatile material is Indeed TEL and that TEL* as such, is present in the tissue (Norris end Cottier, 192$; Echo and Thaiaann, 1931? Mortcnoen, 19U2; Bifchovskaya* 191^8; Srollor* 19^9 ) Certain potential defects in fch Isolation and analytical technics open those assumptions to emoation (Keho and Thamann, 1931I Galingaort et al., 1939; Calingaert et al., I9I4.OS Calingeert !. 19ij.8; Heap ot si,* 1951; Leeper et ol., X952J.)* To clarify the problem* a qualitatiy analytical method more specific for TEL was developed and applied to analysis of liver tissue from rats exposed to TSL. The description of this method and the results of this application are the subjects of this report. ft-tT 0 0 0 3 4 6 8 \  2' METHODS. Exposure of rate to TB& was made In this way* Car** worth rata were encased individually in wire stash and isr.crsed tip to their necks in glass jars which wore filled with water and set in a water bath* Two-tenths ml* of TEL was placed in a shallow glass cup which was clamped to one of the jars* A twenty liter glass jar was inverted over them, forming a watersealod chamber. Air was at once partially evacuated until only four to five liters remained# Sodium hydroxide was added to the water bath end oxygen was bubbled into the inverted jar, keeping the gas volume fairly constant end the water level just below the heads of the rats* Light was kept at a minimum by plac ing the entire apparatus in a dimly lighted fume hood and by using sn infrared heater to increase vaporisation* The heater T?as turned on for 3 minutes out of each !> minute period* An attempt was made to maintain the air in. the jar nearly safcxtr&ted with TEL (8 to 9 ms*a* per liter). After i*. to ? hours the animals were killed by the addition of carbon monoxide. The ratsT livers were homogenised for % minutes in a chilled faring Blondos*. Aliquots were rinsed into liter bottles tilth 0.8 of tholr weight of water* Hero they were mixed with 0.11 of their weight of a normal solution of aodium hydroxide* Redis tilled n-pentsne was added (37*5 nl* per gram of liver) and the. bottles were shaken vigorously for 1/2 hour* They 'were stored overnight at -21$ C* The pentane tras decanted through Whatman Ho* 1 paper, the residue thawed and the pentane again decanted* This procedure of pentane extraction was repeated* Hot all the X 0003469 laolattohpf Tetreethylload .. *3** pentane-soluble lead was extracted from the homogenate. Pentane extracts from 12 rats {approximately fifteen liters) wore combined and concentrated to about k$ 1* voltoa by distillation In vacuo below 0 6 C. in a dimly lighted room* The distillate was collected at -70 0* and sot aside for lead analysis The residual concentrate (45 ml,) was rinsed into a 250 ml, flask with pentane and the solvent re moved below 0*C,, with an aspirator, the residue was subjected to vacuum distillation (1,5 to 2 mi, Hg} at 30 0. for 2 hours while the distillate was collected at -70 C. on a cold-finger condenser# using an arrangement similar to that described by Quaife and Harris (19^8), The condenser was then transferred to a special sample holder Sbdified. from the design of DeTar and Gold (DeTar and Kasitai# 1955J7 and then rinsed off with 5 to 6 ml. of pentane. All but 1 ml, of this pentane was re moved below Q> C, with an aspirator. The infrared absorption spectrum of this pentane solution was measured in a cell of 1 m , thickness with a model 21 Perltin-Klmor double beam spoetrophotometor, A second dietilla tion was made of the residue 3n tho flask (,3 to 0,6 mm, Hg) at 3&' C, for 1 hour# and this second distillate was similarly processed. After their spectrophotome trie measurement# these two final distillates wore analysed for load by an unpublished modification of the high pH dithlsono procedure. Because earlier investigators suggested that tissues of animals Intoxicated with TEL might contain trlethyllead and diethyliead ions# it beeas important to rule out the KfT 0003470 Xsol&tloQ or Totraefchyllead --l}.-- possibility that those substances t w included in the analytical Method ;Just described, Both indirect and direct evidence ruled against this possibility. Thus the fact that triothyllead ions and diethyllead ions react directly with dithison whereas pentane extracts of livers taken f m rats immediately after treatment with TEL gave no such reaction is presumptive evidence that these compounds if present in liver were not removed by the extraction procedure. Another kind of evidence was provided by control experiments involv ing attempted recovery of TEL, triothyllead chloride and load nitrate when added to liver homogenate. E.g., in one of those experiments, 17 gras, of liver homogenate were mixed with the usual proportions of alkali, water and pentane, together with 270 nicrogass. of triothyllead chloride, 1600 nicrogns of load nitrate and 525> microgm. of TEL. Twenty ml. aliquots of extract wore reacted with dithison. Ilo color appeared other than that slight amount regularly found in the reagent bionics Sensitivity of the method used in this particular tost permitted ready detection of 1 lalcroga. of triothyllead ion or of inorganic load. Retention in alkaline homogenate of materials giving color with dithisono was less complete when livers were taken fresa rats a day after their exposure to TELj whether this color was due to ionic organoload compounds remains to be ascertained. Another chock on the analytical method was the recovery of most of the TEL added to concentrated liver extract, demonstrating the feasibility of the latter part of the IsolatIon of Tetrethyllead rf <***^s experiiaonfcal procedure /lu 1 (^27. TEL of high purity vas kindly supplied by Sr* George Thomson of Ethyl Corporation* Trio thyH e a d florid vas synthesised (Heap and Saunders, 19^9), its purity estab lished cy chloride analysis (found: 11.1 per cent Cl, theory: 10*?5 per cent), and the absence of inorganic load was assured by testing vith amoniuaa sulfide. K 0003472 Isolation of Tetraethyllead - 6- Bcaults The infrared absorption of TEL in pentane ^ i g . 1 (aJ7 was found to be charac torised by m a x i m at 15.1* 10.5* 10,0 and 8,7 microns* These maxima could also be identified in a solu tion prepared from the extracts of 212 sms. of liver from 12 rats exposed to TEL ^ i g . 1 {b}/ but not in a solution similarly prepared from nonexposed rats 1 (cj>7, As determined by absorption at 15.1 microns 0,5 mgm. of TEL was present in the first distillate and 0,2 rngm* in the second distillate. These corrected values were obtained by subtracting a constant value from the observed absorptions. This value was arrived at from 2 measurements, each on a distillate prepared from 200 gas, of liver of \inGxpo3od rats. They showed absorption comparable to 0,16 and 0*18 mgm* of TEL per ml. The corrected values were confirmed, by lead analyses of tho same two final distillates which showed 0 ,i.S mgm, and 0,34 rngm* of load, respectively, equivalent to 0,71 ragm, and 0,22 mgm, of TEL, This accounted for only part of the lead found in the original pentane extracts of liver? of the reminder, 1,12 mgm, were found in the 24 liters of recovered pentane and 0*24 mgm* in tho residue loft after all distillations. The difference between the sum of these, 1*85 mgn*, and the amount in the original extracts, which was 1,72 ngm,, may be attributed to analytical error and accidental contamination. The following diagram pictures the distribution of lead in this experiment. 0003473 Isolation of Tetraethyllead original pentane extracts Cl>72 m m * Pb) 1!*. 1* of distillate (1.12 sags. Pb) first distillate (0.45 mgm*. Pb) 4 5 al. of concentrate second distillate residual (0.34 wga Pb.) (0.14 ngsuPb] Discussion The smallness of the quantities of TEL present in tissues, v.-lth the resulting restriction of methods, raises a question of the validity of the Inference that TEL urns present in the liver. Arc the spectra and the lead analyses sufficient evi dence of the presence of TEL In the final solution* or could they ho reasonably attributed to other sources? Insofar as the spectrophotome trie findings are concerned, it is evident that the absorption m a x i m present in the known TEL solution wore also present in the solution from the exposed rats. While the maxima differed in relative intensity in the tvo solutions, these differences were considerable only at wave lengths less than 10.5 microns. In the solutions from both exposed and unoxposed rats tho spectra in this region showed considerable absorption by materials other than TEL. The maxima, other than those related to TEL, failed to coincide in this region* This was not investigated further. Tho discrepancies between the results as measured by Infra red absorption (0*5 and 0*2 mgsa.) and the results by lead analyses (0.71 and 0.22 mgm. ) are attributed to these sources! () error in measurement of the final volume of solution in tho special sample holder (DoTar and Kazlmi, 1955) before the XF 0003474 Isolation of Tetraethyllead ' -8~ ..' spectrum m i obtained (this could bo in error by - 10 per cent), (b) inaomplote recovery of solutions from the infra rod absorption coll, Co) tho difficulty of evaluating corrections of tho optical densities of the spectra for . tissue 11blanks,n and (d) the limitations of the spectro photometer* There is a possibility of the interchange in liver tissue of ethyl and methyl groups on tho load atom* Such an inter change occurs in vitro {Cnlingaert efc al*, 1939 and 19i|X>). That it nay also occur in vivo is suggested by work on trans methylation (Cantoni, 1952) Tho four methylated homologa of TEL in pure form all show marked absorption between 13 and 12{. microns (A.P*I* catalog) where TEL is transparent* As demonstrated in Figure 1 (o) this absorption is also obvious in a solution of totrametliyl- lesd in pentane* It was absent from the solutions obtained from exposed rats and, therefore, it is unlikely that tho final distillates included significant quantities of methylated homo logs of TEL* . A remote possibility of error was contamination of dis tillates with CQa and acetone A solution of CO^ in pentane showed a maximum only at 15*2 microns, but It showed no other absorption between 5 and li.j.,5 microns* A solution of acetone in pentane showed a number of maxima; none coincided with those* of any of tho liver extracts* Explanation of some procedural details may be of interest* Rats, because they proved somewhat resistant to intoxication by ^ 0003475 Isolation of Tetraethyllead *9* TEL, were chosen in preference to rabbits which developed pulmonary congestion and died within a few h o w s * The inhalation route was chosen to minimise the administration of non-volatile decomposition products of TEL, and to avoid arbitrarily localised deposits* Carbon nonoxide was added during TEL exposure to avoid a terminal respiratory loss of TEL. Liver was taken for analysis because it m s found to contain much pentane-soluble load. Pentane used by Kroller {I9h9j' ao choson as a solvent because it dissolves so little water Tf.006 per cent w/v {Black et al,, 19ii.8J7an& differs widely from TEL in volatility. The proportions of pentane, tissue* and water in the tissue extraction were choson to derive a rood dispersion on shaking with a minimal volume of emulsion* ' Summary . Tetraethyllead (TEL) has been shown to bo present in liver tissue of rats which have inhaled TEL vapor, TEL was extracted by pentane, concentrated by low temperature vacuum distillation, and identified by infrared spectra and load analyses. Ho homo logs containing methyl groups wore detected in the concentrates. Hi 0003476 Figure 1* Infrared spectr of: (a) a solution containing 3.6 iaoa* of TEL per ml. of pentane, (b) a pontane solution prepared from livers of rats intoxicated with TEL, (c) a pentane solution prepared from livers of rats not exposed to TEL, (d) a pentane solution prepared through redistilla tion of the residual grease from (c) to which had boon added 3.2 taps. of TEL, and (o) a solution containing 10 rngm* of totramsfchyllead por ml. of pentane. [^JC- 0003477 RSFS BHCjES Azaor* Petroleum Inst* Research. Project Catalog of f o o t e d Infrared Absorption Spectrogram, spectra # 1077-1080, l/ashngton, D. C, Bikhovskaya, M. S. : 01glena i Ssnit*, 13 Ho* 10* 25? Black, C,, Joris, G. C,, and Taylor, H, S.i J. Chess* Phys , 16: 537, 19^8. Calingaert G., Beatty, H* A*, and Heal, H* 1.: J. Asa* Chora. Soc., 61: 2755, 1939* Callngaort, G., Beatty, H* A* and Soroos, K.s J, Asa. Chess. Soc., 62i 1099, 1940. Calinaaerfc, G., Shapiro, H., Dyketra, P. J., and Hess, L,: J. A .. Chen* Soc., 0: 3902, 19J.0. Cantoni, G. B., in McSlroy, <** D, and Glass, B, Phos phorus Metabolism* The Johns Hopkins Press, Baltimore 1952. * DoTer, D. P, and K&siral, A. A.i J. An. Chern. 8oc., 77: 381^2, 1955. Heap, R, and Saunders, B, C. : J. Chen. Soc,, 191i9: 8983. Heap, R., Saunders, B. C., and Stacey, G. J.s J, Chora. Soc., 1951: 658. Kohoo, R. A., and Tasmamit F. Ata. J, Hyg., 13: k?8, 1931 KroHor, E.j Z. Anal. Chora., 129i 19* 1949. Leeper, R. W., Burners, B., and Gilson, H*s Chora. Bov., 101, 1954* ' Her tensen, R. A.s J. Xhd. Hyg* fox., 285, 1942. Horris, C., and Cottier, A. O. J* Am* Mod. Ass., 85} 818, 1925. Quaifo, M. L., and Harris, P, L,: Anal. Chora., 20: 1221, 1943. HE 0003478 Isolafcion ot Tetrasthyllead A list o itoma for fch Index: Totraethylle&d, ioolation frosi livor Livor, isolatlon o tetraethylle ad frea * 0 0 0 3 4 79 A condensed title running page headlinet IOL/lTIH OF TETRAETHYLLEAD 0003480 > i