Poison Extraction as Related to Cellular Ultrastructure

Poison Extraction as Related to Cellular U ltrastructure BRIAN PARKER University of California, Berlzeley, California, 94720, U . S . A . Pa@er Presented at the 30th Semi-Annual Seminar of the California Associalion of Criminalists, November 2 , 1967, S a n Jose, Calif. Current knowledge concerning cellular ultrastructure i s examined for information regarding possible and probable locations o f organic poisons i n living organisms. A discussion of physical, chemical, and biochemical structure and function i n the hepatic cell suggests that a concentration of organic poisons m a y exist i n the endo@lasmic reticulum. Methods of differeqztial and complete extractions are related to the ehavacteristics of that @articular ultrastructure. Separation of a toxic organic substance from the tissue matrix is usually effected by bulk chemical differentiations. The approach is to make use of the solubility characteristics for the organic substance in different environments, i.e., acidic, alkaline, hydroxylic solvent, aprotic solvent (Friemuth, 1960 ; Parker, 1962 ; Paruta, 1964), following disruption of the tissue. While numerous versions of this approach are used, the essential separation is "often the least investigated" (Curry, 1963). The current rapid expansion of knowledge on cellular ultrastructure and function (Dallner, 1963 ; Robertson, 1964 ; Hechter, 1965 ; Korn, 1966 ; Mautner, 1967) presents an opportunity to reexamine this first separation step in view of the possible, or probable, location of toxic organic substances within living tissue and, consequently, the most effective means of removal. Cell membranes In its passage through a living organism, a toxic organic substance is brought up against the boundaries of various cellular types. A movement through such a boundary is often necessary for the molecular interactions of stimulation and/ or metabolism (hfautner, 1967 ; Brodie, 1964 ; Schanker, 1962). This boundary, called the cell or plasma membrane, is one important feature for examination. The course of the toxic organic substance within the cell is a second feature to be considered. Fundamental to the structure of the cell membrane is the trilayer of outer protein, intermediate lipid, and inner protein. The configuration of these layers depends upon the model chosen to explain the accumulated experimental data. The unit membrane model (Robertson, 1964), shown diagramatically in Figure l A , consists of outer and inner monolayers of protein, different in nature, sandwiching a bilayer of polar lipid with the polar parts of the lipid molecules attached to protein. The overall thickness for this model is of the order of 100 A ; the lipid bilayer, 45 A. A second model (Ling, 1965) of similar dimensions, Figure l B , has the intermediate lipid layer enveloped by a protein monolayer. Doubt exists as to the structure of the intermediate lipid layer. In a third model (Kavanau, 1965), the outer and inner monolayers of protein are postulated as contracting and expanding with structural changes in hexagonallyspaced lipid columns, Figure C. The membrane would fluctuate in thickness from 70 to 280 A. Molecular penetration of the cell membrane is roughly correlated to the properties of lipid solubility, size, shape, and ionization state for a given chemical substance (Schanker, 1962 ; Brodie, 1964 ; Ling, 1965 ; Kavanau, 1965 ; Blank, 1966 ; Eisenmen, 1967). The undissociated form of a chemical having some degree of lipid solubility would dissolve into the intermediate layer of a membrane and then partition again into the interior aqueous phase. The concentration of that chemical on either side of the membrane would depend upon 141

the pH values of the two aqueous regions and the dissociation constant for the chemical (Schanker, 1962 ; Reese, 1964). Smaller molecules of a lipid insoluble character and charged species may permeate the membrane as though it were an energy barrier to be surmounted (Blank, 1966 ; Eisenman, 1967). For other lipid-insoluble and charged chemicals, the route into the cell is by way of pores or aqueous channels (Schanker, 1962 ; Kavanau, 1965 ; Ling, 1967) or some form of active transport (Schanker, 1962 ; but see Ling, 1965). Intracellular organization presents "a profusion of membrane systems" (Hechter, 1965) which bind together the organelles, e.g., nucleus, mitochondria, Golgi complex (Korn, 1966). Material exchange within the boundaries of the cell seems t o be the function of one membrane system-the endoplasmic reticulum (Siekevitz, 1963). This ultrastructure has been expressed as "a fluid anatomy in the geography of the cell, being some tenuous network by the action of which the cell's enzymic activities are co-ordinated" (Peters, 1956). Studies directed a t this "fluid anatomy" revealed that it is a complex of lipid and protein (Dallner, 1963 ; Siekevitz, 1963 ; Finean, 1961). Seemingly lacelike in form, this system is a continuum of tubular and vesicular parts with connection to the nuclear envelope, the mitochonondrial envelopes, the Golgi complex and the cell (plasma) membrane (Dallner, 1963 ; Finean, 1961). Indeed, "the membranes of the smooth endoplasmic reticulum have no obvious morphological difference from the plasma membrane, they are even continuous with invagination of the latter" (Maddy, 1966). A physical analogy to this cellular ultrastructure can be approximated by taking a piece of fine net (the endoplasmic reticulum) and stuffing it inside a plastic sphere (the cell membrane).

The trilayer arrangement of the cell membrane is found also in the intracellular membranes. One major component of these structures is water, which makes up 30 to 50 percent of the bulk (Hechter, 1965). Part of this structural water "is adsorbed in polarized multilayers and in this state it partially excludes solutes" (Ling, 1966). Additional amounts may be involved in micellular formations within lipid regions (Kavanau, 1965). Intracellular water unassociated with membranes (hyaloplasm) "may resemble the structures which occur in aqueous solutions containing soluble globular proteins" (Hechter, 1965). The protein component of these membranes is metabolically active (Dallner, 1963 ; Korn, 1966 ; Quastel, 1967) and, indeed, is dependent upon the structural association for the "properties that distinguish them, both quantitatively and qualitatively, from a random mixture of enzymes" (Quastel, 1967). Little variation is found in the polar lipid components for cells of one organ type among animal species ; however, different organs of the same species exhibit compositional differences attributed to the membranes of the cell boundary and the endoplasmic reticulum (Quastel, 1967). 142

The liver cell The cellular location of a toxic organic substance would appear to include a t least three possibilities : the hyaloplasm, the adsorbed watter lattice, and the lipid region. The liver (parenchymal) cell is of special interest when examining these possibilities, because chemical modification is carried out, primarily, by enzymes situated in the liver (Williams, 1959 ; Brodie, 1961 ; Gillette, 1966 ; McMahon, 1966). Within the liver cell the site of modification has been narrowed down to the endoplasmic reticulum (Siekevitz, 1963 ; Gillette, 1966). This membranous structure is divisible into smooth and rough types (Dallner, 1963). The rough type has localized centers of high ribonucleic acid content, termed ribosomes, in contrast to the smooth type which is devoid of these centers. most of the enzymic activity involving modification was found in the smooth type of endoplasmic reticulum (Fouts, 1961). These enzymes are chemically bound to the membrane (I>allner, 1963). Chemicals normal to the biological activity of a living organism are not touched by these enzymatic systems even though they are specific for chemical groupings rather than for molecular structure ; "in sharp contrast to the precise steric fitting of ring structures when they react metabolically, and the critical importance of chain length, detoxication is effected by an entirely cavalier neglect of everything but an odd chemical grouping, a nose by which to hook the bull. I t is a gross chemical rather than a specific biochemical approach" (Needham, 1965). The division between normal chemicals and toxic chemicals appears to be accomplished b y a "lipoid barrier which only fat soluble substances can penetrate" (Gaudette, 1961), with modification of a toxic chemical dependent upon its first being 'fixed' in the lipophilic part of the membranes" (Siekevitz, 1963). The lipid regions of the intracellular membranes appear to be the most likely locations for toxic chemicals to accumulate in the liver when lipophilic natures are considered along with the solute-excluding properties of the adsorbed water lattice. One 'warehouse site' of appreciable capacity in the hepatic parenchymal cell is the endoplasmic reticulum, which accounts for 50 to 60 percent of the cell mass (Hechter, 1965). Although two components, protein and lipid, are replaced in a matter of days (turnover), this membrane system is a stable feature for the life of an individual (Omura, 1967). In fact, it can not be stained with dyestuffs of the Sudan group, which indicates a structural integrity (Dixon, 1958). This integrity is supported by studies of synthetic membranous bodies t h a t suggest intracellular concentrations of phospholipids in the hyaloplasm is unlikely and that "normally they may exist in some 'protected' form within the cytoplasm, as in protein complexes" (Samuels, 1965). Chemically, the lipid component of the encoplasmic reticulum is 94 percent phospholipid and 6 percent cholesterol (Spiro, 1956). "

Liver tissue Some 60 percent of the cells in liver tissue are of the parenchymal type, with the remainder forming linings for capillaries, ducts, and walls or connective tissue (Daoust, 1958). The lipid composition, on a gram basis of fresh liver tissue, is presented in Table 1 for rat and human. The absolute amounts of phospholipids may provide a convenient internal standard against which to measure toxic chemicals, since nutritional deficiency, disease and drugs produce no appreciable changes (Douglas, 1967 ; Lucas, 1967 ; Merrill, 1967). Extraction On the assumption that the lipid regions of intracellular membranes in liver tissue contain the bulk of toxic lipophilic chemicals, then separation directed a t removing those regions might include the toxic substances. The solubilizing properties of phospholipids (Raer, 1958) for organic and inorganic substances in organic solvents would favor this concomitant separation of lipid and toxic chemical from the tissue matrix. A comparison of some techniques used t o 143

TABLE 1 I'EIICENT OF FRESH WEIGHT

5.0 (2.4-8.5) 0.20 0.08 2.1 (1.5-3.0), 2.9 0.7-1.9 0.3-0.6

Total lipids Cholesterol Cholesterol esters Phospholipids Phosphatidyl cholinc Phosphatidyl ethanolaminc

*(Tsao, 1966 ; Deuel, 1955 ; lialli, 1941) **(Lucas, 1967 ; Skipski, 1964 ; Ansell, 1964)

COMPARISON O F EXTRACTION CONDITIONS (Based on 1 gram of liver tissue) Poisons 1 Solvent

Ethanol

Homogenate

Acidified

Temperature

30-35°C

Lipids2

Desired recovery Lipids3

Ethanol and dicthyl ether, 3:l

Acetone, the11 cthanol

60°C

r.t. for acetotle 78" for ethanol

a few hours 1 hour, repeat under rcflux 1 X with residue

% extraction of phospholipids

1 hour for acetone ; 5 millutes for ethanol which is repeated 2X with residue

70% aqueous acetone

30 ininutes under reflux overnight a t r.t.

TABLE 3

Dry diethyl ether licd cell ghost (human) Cholesterol Phospholipids Phosphatidyl choline

100% 23 15

146

Acetonitrile Liver (rat)

extract tissue lipids with a few techniques directed a t poison recoveries is presented in Table 2. The comparison suggests that the extraction procedures for the detection of an unknown poison involve separating the total lipid content from the tissue. The essential solvent action in each case is a breakdown of the protein and phospholipid complex in membranes with subsequent lipid solution. The resulting solution is a complex mixture of small molecules to be separated (purified) for qualitative and quantitative determinations. Resolution of such a complex mixture into discrete fractions of desired components is possible ; however, if the initial separation could be conducted in such a manner as to yield a less complex or less abundant mixture, subsequent separations would be simplified. In the latter situation, some degree of reduction in the amount of a desired component would be tolerable in exchange for a 'cleaner' residue of solutes. One method of achieving this differential extraction of small molecules is inherent in studies performed on the binding of lipids in membranes (Roelofsen, 1964 ; Parpart, 1952). Dry diethyl ether was found to remove a definite fraction of total phospholipids in red cell ghosts. The retained fraction required a treatment with a 3:l mixture of ethanol and ether for extraction. Residue from this second fraction was quite soluble in dry ether, thereby supporting the theory of a difference in binding, rather than a difference in solubility. These studies show an agreement with models of liquid lipophilic interiors in membranes in which molecules having both lipophilic and hydrophilic characteristics could dissolve (Kavanau, 1965). The warehousing of toxic substances might well concentrate in the loosely bound fraction of membrane phospholipids. Studies carried out in the author's laboratory with acetonitrile as an extracting solvent showed differential extraction of phospholipids from rat liver, similar to that fot the red cell ghosts (Parker, 1967). A wide range of toxic chemicals was recovered from the livers of poisoned rats with acetonitrile extractions. Studies in progress are directed a t comparisons of different solvents and between differential and total extractions. References ALHA,A. R. and LINDFOIZS, R. O., 1959, Ann. Med. Exptl. Biol. Fenniae (Helsinki), 37, 149. ANSELL,G. B. and HAWTHORNE, J. N., 1964,Phospholipids, 263. BAER,E., BUCHNEA, L). and NEIYCOMBE, A. G., 1956, J.A.C.S., 78, 232. BLANK,M., 1966, Ann. N.Y.Acad. Sci., 137, 755. BRODIE,B. B. and MAICKEL,R. P., 1962, I'roc. First Internat. Pharmacol. Meeting, 6, 299. BRODIE,B. B., 1964, BINNS,T. B., Absorption and Distribution of Drugs, 199. CUKRY, A. S., 1963, STOLMAN, A,, Progress in Chenzical Toxicology, 1, 136. DALLNER, G., 1963, Acta Pathol. Microbiol, Scand., 166, suppl. DAOUST, R., 1958,BRAUER, R.W., Liver Function, 4. LIEUEL,H . J., 1955, The Lipids, 2, 713. DIXON,K. C., 1958,Quart. J. Exptl. Physiol., 43, 139. DOUGLAS, J. F.,LUDWIG, B. J., MARGOLIN, S. and BEICGER, F. M.,1967,Progr. Biochem. Pharmacol., 2, 422. EISENMAN, G., SANDBLOM, J. P. and WALKER, J . L., 1967,Science, 155, 965. ENTENMAN, C.,1957, Methods in Enzymology, 3, 299. FINEAN, J. R., 1961,Chemical Ultrastructure i n Living Tissues, 91. F o u ~ s J, . R.,1961, Biochem. Biophys. Res. Commun., 6, 373. FREIMUTH, H. C.,1960, STEWART, C. P. and STOLMAN, A., Toxicology, 1, 285. GAUDETTE, L. E.and BKODIE,B. B., 1959, Biochem. Pharmacol., 2, 89, 95. GILLETTE,J. R., 1966, Advances i n Pharmacology, 4, 219. HECHTER,O., 1965, Fed. Proc. 24, suppl. n 15 S-91. KAVANAU, J. L., 1965, Structure and Function i n Biological Membranes, 1, 66,

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