A n n a l s o f C l i n i c a l a n d L a b o r a t o r y S c i e n c e , Vol. 4, No. 4 Copyright © 1974, Institute for Clinical Science

Use o f Teflon Digestion Bom bs for T issue Analysis: M easurem ents o f the Effect o f Estradiol-17/3 Upon Hepatic Copper in R ats*

F. WILLIAM SUNDERMAN, JR., M.D. AND ELIZABETH T. WACINSKI, B.S.

Department of Laboratory Medicine, University of Connecticut School of Medicine,

Farmington, CT 06032

ABSTRACT

Teflon digestion bombs have been used for pressure decomposition of rat liver samples preliminary to copper analyses by atomic absorption spectrom­etry. The analytical procedure is convenient and is consistently free from copper contamination. The recovery of copper added to liver samples (three ,ug Cu added per g wet wt) averaged 102 (S.D. ± 3 ) percent. The accuracy of the copper analyses was verified by use of National Bureau of Standards reference bovine liver. The mean concentration of copper in perfused livers of16 untreated male rats averaged 16.1 (S.D. ± 2.3) /xg per g dry wt. The mean concentration of copper in perfused livers of 8 male rats which received estradiol-17/3 in dosage of 50 jug per day, s.c., for 21 consecutive days was 21.7 (S.D. ± 4.7) /xg per g dry wt (p vs controls = <0 .01). This study demonstrates that estrogen administration can cause a significant increase in the concentra­tion of hepatic copper.

In tro d u ctio n

Atomic absorption spectometry of trace metals in tissues and other solid biological samples (e.g., hair, feces) has been ham­pered by cumbersome preliminary tech­niques for ashing or digestion of the samples in order to destroy the organic constituents. Wet-digestion techniques cus­tomarily employ a mixture of nitric and

° Supported by Atomic Energy Commission Grant AT (11-1)-3140; and by U. S. Public Health Service Contract HSM-99-72-24 from the National Institute of Occupational Safety and Health.

sulfuric acids in conjunction with an oxi­dizing agent such as perchloric acid or hydrogen peroxide. To achieve adequate wet-digestion, it is usually necessary to add several aliquots of the acidic digestion mix­ture. Wet-digestion techniques are time consuming and they require the constant attention of an analyst in order to prevent loss of samples by “bumping.” Since even “ultra-pure” acids contain appreciable con­centrations of trace metals, such as As, Cr, Ni and Pb, any variations in the quantity of acid which is used for the wet-digestion can result in variable metal contamination

299

300 SUNDERM AN AND W ACINSKI

of the samples.8 Hence, the accuracy and precision of trace metal analyses by wet- digestion techniques are effectively limited by variations in the volumes of acidic di­gestion mixture which are required for treatment of the blank, standard and un­known samples. Dry-ashing techniques us­ing a muffle furnace have the disadvantages of ( 1 ) variations in thermal volatization of metals owing to matrix eifects; (2 ) the hazard of cross-contamination of samples within the muffle furnace; and (3) varia­tions in dissolution of the ashed samples in acid prior to atomic absorption spec­trometry. Anderson1 has recently summa­rized the relative advantages and disad­vantages of conventional methods of wet- digestion and dry-ashing of tissues for trace

F i g u r e 1. Cross section of a teflon combustion bomb (courtesy of Parr Instrument C o.). The teflon sample cup and teflon inner cover are con­tained within a steel casing with screw-on cap. A steel spring and pressure plate within the bomb keep the teflon inner cover tightly affixed to the teflon sample cup.

F i g u r e 2 . Photograph of a teflon combustion bomb (courtesy of Parr Instrument C o.). The overall height is 9.2 cm and the outside diameter is 6.0 cm. The bomb weight, empty, is 965 g.

metal analyses by atomic absorption spec­trometry.

Several investigators have attempted to develop improved methods of sample de­struction, in order to circumvent the var­ious problems which are associated with customary wet-digestion or dry-ashing techniques. Thompson and Blanchflower13 have described a system for acid digestion of samples in a specially designed heating block. Lewis and Coughlin0 have devised a wet-ashing system which uses an all-glass digestion-distillation apparatus. Murphy et al° have employed alkaline digestion with aqueous tétraméthylammonium hy­droxide. Several instrument manufacturers have introduced low temperature dry-ash­ing apparatuses, which oxidize biological samples in atomic oxygen in a radio-fre­quency reactor. Each of these methods for

AN A LYSIS O F H E P A T IC CU W ITH T E F L O N BO M BS 3 0 1

digestion of tissues, hair and feces has been evaluated in our laboratory and none of these methods has proven to afford any practical advantage over conventional wet- digestion with nitric, sulfuric and per­chloric acids using a Kjeldahl digestion apparatus. On the other hand, the teflon digestion bomb which is illustrated in fig­ures 1 and 2 has proven, in our laboratory, to provide a convenient method for the digestion of biological samples.

Bem as,2 who worked for the National Aeronautics and Space Administration, originally developed the teflon digestion bomb in 1968 in order to accomplish rapid decomposition of lunar rock samples in hydrofluoric acid, prior to analyses of met­als by atomic absorption spectrometry. The teflon digestion bomb was subsequently used by Hartstein et al4 for trace metal analyses of coal; by Mansell and Hiller7 for lead analyses of gasoline; and by Holak et al5 for analyses of mercury in fish. A larger teflon digestion bomb was described by Paus10 and has been used for analyses of Hg, Cd, Zn, Cu, F e and Pb in seaweed and fish.11 A novel digestion apparatus which contains six teflon vessels for the simultaneous decomposition of multiple samples has recently been illustrated in a review article by Mitchell.8

The specific biological application of the teflon digestion bomb which will be de­scribed in this paper is the determination of copper concentrations in rat livers. This method has been employed in our labora­tory for analyses of copper in livers of untreated rats and in livers of rats which received repeated parenteral injections of estradiol-17/3.

M ethodsP r i n c i p l e o f P r e s s u r e D e c o m p o s i t i o n o f

T i s s u e f o r C o p p e r A n a l y s e s

Rat liver is homogenized with an ultra­sonic disintegrator. Aliquots of the liver homogenate are digested with H N 0 3 in

the teflon digestion bomb; and measure­ments of copper in the digested samples are performed by atomic absorption spec­trometry. Additional aliquots of the homog­enate are dried to constant weight so that the copper content of the liver can be ex­pressed on a dry-weight basis.

S p e c i a l A p p a r a t u s

Ultrasonic Disintegrator, 300 watt output with titanium probe.*

Teflon-lined Acid Digestion Bombs, 25 ml capacity .** For routine use in analyses of blank, standard and unknown samples, it is desirable to have at least 12 bombs.

Atomic Absorption Spectrometer, fitted with high-solids burner, copper hollow cathode lamp and 10" strip-chart recorder, f

Disposable plastic knives and forks which have been soaked in 30 percent (v /v ) nitric acid.

R e a g e n t s

Nitric Acid, concentrated, ultrapure, (Sp. Gr. = 1.40).|

Copper Reference Standard Solution, (1 mg Cu per ml).§

Copper Working Standard Solutions, (0.5, 1.0 and 1.5 «.g Cu per m l). Into three 2-liter volumetric flasks are transferred, re­spectively, 1, 2, and 3 ml of the copper ref­erence standard solution. The contents of the flasks are diluted to the marks with distilled, demineralized water.

Sodium chloride solution, 8.5 g per liter. Caprylic alcohol, reagent grade.

* Bronwill “Biosonik-IV” ultrasonic system, VWR Scientific Co., San Francisco, CA 94119.

* * Model 4745, Parr Instrument Co., 211 Fifty- third Street, Moline, IL 61265.

f Model 810 dual-monochromator atomic ab­sorption spectrometer, Jarrell-Ash Division, Fisher Scientific Co., Waltham, MA 02154.

| Catalog No. 441, E. Merck Co., Darmstadt, Germany, purchased from EM Laboratories, Inc., 500 Executive Blvd., Elmsford, NY 10523.

§ Catalog No. So-C-194. “Certified Copper Standard Solution for Atomic Absorption,” Fisher Scientific Co., Pittsburgh, PA 15219.

3 0 2 SUNDERM AN AND W ACINSKI

P rocedureThe rat is anesthetized with ether, and

the abdomen is opened in the midline with a scalpel. A short, lengthwise incision is made in the portal vein to permit the in­sertion of a polyethylene canula. The can- ula is connected by a Luer-lock fitting to a 20 ml syringe, which is filled with isotonic sodium chloride solution. The vena cava is sectioned with scissors, to permit efflux of the perfusation fluid. The liver is very slowly perfused with isotonic sodium chlo­ride solution, until the liver becomes uni­formly pale and bloodless. The liver is then excised, blotted with filter paper and weighed.

Approximately two g of liver is minced into one mm cubes by use of the disposable plastic knife and fork. Exactly 1.500 g of liver is weighed into a tared beaker. Five ml of distilled water are added, and the minced liver is completely homogenized by insertion of the titanium probe of the ultra­sonic disintegrator, and turning on the ul­trasonic signal for approximately 30 sec. The probe is rinsed with one to two ml of water. Three drops of caprylic alcohol are added to the homogenate in order to elimi­nate bubbles, and the homogenate is trans­ferred quantitatively to a 10 ml volumetric flask, together with water washings. The contents of the flask are diluted to the mark with water.

Two 2-ml aliquots of the liver homoge­nate are transferred to teflon digestion bombs. Into additional digestion bombs are transferred two ml of water ( “blank” sam­ples), and two ml of each of the copper working standard solutions. Ultrapure ni­tric acid, three ml, is added to each of the teflon combustion bombs. The bombs are tightly sealed and are placed overnight in an oven at 120°. The oven temperature must be controlled by a reliable thermostat to avoid any possibility that the bomb might be heated above its 150° maximum rating (see cautionary footnote on explo­

sion hazard).* It is convenient to perform the wet-digestions overnight, but heating for an interval as short as six hours is equally satisfactory.

Two-ml aliquots of the liver homogenate are transferred to tared weighing beakers, and the wet weights of the samples are measured with an analytical balance. The beakers are then placed in an oven at 100° for 24 hours, so that the samples become dried to constant weight. The beakers are brought to room temperature in a desic­cator, and the dry weights of the samples are measured with an analytical balance.

The teflon digestion bombs are removed from the oven and are allowed to cool slowly to room temperature for at least two hours. The steel caps are loosened gently, and the teflon sample cups are carefully removed. The digested samples should be colorless and water-clear.

0 The teflon combustion bombs should never be used with samples which are rich in lipids, such, as brain, adipose tissue, lipemic serum or milk, owing to the possibility of hydrolysis of lipids to glycerol and fatty acids and subsequent formation of nitroglycerin and other nitrated organic prod­ucts. Tyler1* has reported an accidental explosion which occurred when one ml of nitric acid, one ml of sulfuric acid and 0.5 gm (wet wt) of adipose tissue were placed in two teflon digestion bombs, and the tops were screwed on. No heat was ap­plied. After 10 minutes both of the bombs ex­ploded. The laboratory bench was shattered and the analyst suffered lacerations and abrasions. Tyler14 calculated that the contents of the bombs could have reached temperatures up to 4000° K and internal pressures as high as 15,000 psig. The teflon combustion bombs are only rated to with­stand temperatures up to 150° and internal pres­sures up to 1,200 psig. The manufacturer has specified that the oven in which the bombs are placed must be controlled by a reliable thermo­stat to avoid any possibility that the bomb might be heated above 150°. For nitric acid digestions of organic compounds, the sample must not con­tain more than 0 . 1 g (dry wt) of organic matter, and the amount of concentrated nitric acid added must not be less than 2.5 ml and must not exceed 3.0 ml. Sulfuric and perchloric acids should not be used. Precautions must always be observed to guard against unexplained explosions, which may sometimes occur when treating organic substances with a strong oxidizing acid.

The atomic absorption spectrometer is adjusted to the following parameters: (1) wavelength = 324.7 nm; (2 ) slit width =1.0 nm; (3) copper hollow cathode lamp current = 10 ma; (4 ) acetylene: air flame,1:5 gas mixture (v /v ).

The samples, including blanks, standards and unknowns, are aspirated directly from the teflon cups into the burner-nebulizer of the atomic absorption spectrometer, and the absorption at 324.7 nm is recorded.

The concentration of liver copper is cal­culated and expressed as /xg Cu per gm (dry weight).

A n i m a l I n v e s t i g a t i o n s

The experimental animals were 24 male rats of the Fischer strain (200 to 300 g body wt) which were fed Purina rat chow. The control group comprised 16 untreated rats. The treatment group comprised 8 rats which received daily sc injections of es­tradiol- Yip in dosage of 50 /xg per day for21 consecutive days. The protocol for ad­ministration of estradiol-17^, and the ef­fects of estradiol-17/? upon serum copper and ceruloplasmin concentrations have been reported previously by Sunderman et al.12

R esu lts

E v a l u a t i o n o f t h e D i g e s t i o n P r o c e d u r e

In figure 3 is shown an illustrative re­corder tracing of atomic absorption spec­trometry of copper in reagent blank, standard and liver samples. Based upon analytical runs on 20 consecutive working days, there was no significant contamina­tion with copper during the digestion pro­cedure. The reagent blanks which were carried through the digestion procedure were consistently free of any copper which could be detected by flame atomic absorp­tion spectrometry. Measurements of the recovery of copper added to six rat liver homogenates (three ¡xg Cu added per g

A N A LYSIS O F H EPA TIC CU w i t h t e f l o n b o m b s 3 0 3

F i g u r e 3 . Typical recorder graph for atomic absorption analyses of copper in blank, standard and liver samples which have been digested by use of the teflon digestion bomb. Sample # 1 = reagent blank; Samples #2 to 4 = copper stan­dards containing 0.5, 1.0 and 1.5 /xg Cu per ml, respectively; Samples # 5 and 6 = homogenates of livers from untreated rats.

wet wt), yielded a mean recovery of 102 ± 3 percent (range = 97 to 105). The ac­curacy of measurements of hepatic copper by the teflon digestion bomb technique was verified by analyses of National Bureau of Standards Reference Material #1577 (Lyo- philized Bovine Liver). Four measure­ments of 25 mg samples of NBS Bovine Liver yielded a mean copper concentration of 189 ± 4 /xg per g (dry w t), range = 185 to 195 ¡xg per g. The certified concentration of copper in the NBS Bovine Liver is 193 ± 10 ^g per g (dry wt). To test for the completeness of hydrolysis of organic con­stituents, aliquots (50 /xl) of digested liver homogenates were spotted on cellulose- coated thin layer chromatography plates. Ascending chromatography of amino acids was performed in isopropanol: H20 (70:30, v :v ) . Inspection of the chromatography plates under long and short wavelength ultraviolet light did not reveal any ultra­violet-absorbing or any fluorescing spots. The thin-layer chromatography plates were sprayed with ninhydrin reagent and were heated at 90° for 30 min. No ninhydrin- reactive compounds were detected.

C o n c e n t r a t i o n s o f C o p p e r i n L i v e r s

o f R a t s

Livers from the control group of 16 healthy, untreated rats were analyzed by

3 0 4 SU NDERM AN AND W ACINSKI

the procedure which has been described, and the following results were obtained: (1) dry wt = 31.6 ± 2.7 percent of wet wt (range = 27.5 — 38.5); (2 ) copper concen­tration = 5.1 ± 0.6 /¿g per g (wet wt) range = 4.4 to 6.1); (3 ) copper concentra­tion = 16.1 ± 2.3 /i.g per g (dry wt) (range = 13.9 to 19.6). Analyses of livers from 8 rats which had received daily sc injections of estradiol-17/J in dosage of 50 jug per day for 21 consecutive days yielded the follow­ing results: (1 ) dry wt = 29.0 ± 1.2 per­cent of wet wt (range = 26.6 to 30.4) ( p < 0.05 vs controls by Student’s t test); (2) copper concentration = 6.3 ± 1.6 ftg per g (wet wt) (range = 4.1 to 9.0); (3 ) copper concentration = 21.7 ± 4.7 /ug per g ( dry wt) (range = 15.0 to 30.2) ( p < 0.01 vs controls by Student’s t test).

D iscu ssion

The present study has demonstrated that administration of estradiol-17/3 to male rats in dosage of 50 jug per day for 21 consecu­tive days results in a mean increase of 35 percent in the concentration of hepatic copper. Sunderman et al12 have previously reported that administration of estradiol- 17/? to rats according to the same dosage schedule stimulates a mean increase of 340 percent in the concentrations of serum cop­per and serum ceruloplasmin (C PN ), with­out producing pathological alterations of the hepatocytes. Studies by Evans et al13 have suggested that estrogen acts as an inducer for hepatic synthesis of CPN mes- senger-RNA, leading to increased hepatic synthesis of CPN-protein. Insofar as the authors can ascertain, chemical analyses of copper concentrations in liver have not pre­viously been reported in estrogen-treated animals, or in patients who have been treated with estrogens. The increased mean concentration of hepatic copper which was observed in the present study may possibly reflect: (1 ) an increase in hepatic CPN concentration, resulting from the increased

rate of CPN synthesis; (2 ) a secondary in­crease in hepatic CPN catabolism, with binding of released copper to lysosomal metallothionine, or (3 ) a combination of both of these factors. Studies of the sub- cellular distribution of copper in hepato­cytes of estrogen-treated rats are in prog­ress in our laboratory.

From a methodological viewpoint, the use of teflon digestion bombs for pressure acidic decomposition of tissues for trace metal analyses has the advantages of: (1 ) relative freedom from trace metal contam­ination, (2 ) quantitative recovery of the digested sample and (3 ) convenience and saving of time for the analyst. Disadvan­tages of the technique include: (1 ) possi­ble explosion hazard; (2 ) limited sample size and (3) relatively high cost, since the teflon bombs which are currently available are not very durable. In the authors’ ex­perience, a teflon sample cup can be em­ployed for only 15 to 20 tissue digestions. Thereafter, the sample cup no longer main­tains a vapor-tight seal and hence it must be discarded. Leakage of nitric acid from the teflon cup into the steel bomb casing causes (1 ) loss of sample and (2 ) possible contamination of the sample with Fe, Ni or Cr dissolved from the steel. To detect such errors, it is necessary to analyze each sample in duplicate, and to repeat the anal­yses with new teflon cups whenever the results of the duplicate analyses are not in close agreement. Leakage of nitric acid can also result in corrosion of the steel bomb casing, which makes it impossible to main­tain a vapor-tight seal even with use of a new teflon sample cup. When a bomb cas­ing has become corroded, it must be dis­carded. Hopefully, these drawbacks will be overcome by improvements in the design of teflon digestion bombs. Increasing the thickness of the wall of the teflon cup, and providing a screw-thread closure for the inner teflon cover would undoubtedly im­prove the performance of the digestion

AN A LYSIS O F H EPA TIC CU W ITH T E F L O N BO M BS 3 0 5

bombs. Fabrication of bomb casings of ti­tanium instead of steel might also be help­ful, although this would obviously increase the cost of the apparatus. In the authors’ opinion, when the design of teflon diges­tion bombs has been improved so that malfunctions rarely occur and so that the explosion hazard has been practically obvi­ated, such digestion bombs should find wide clinical, forensic and research appli­cations for analyses of metals in tissues and other biological materials.

Acknowledgments

The authors are grateful for advice and assis­tance from Karen Parker, B.S., Maria W. Nechay, M.S. and Eva Horak, Ph.D.

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