Membrane Structure

In many ways, the biological membrane can be considered a lipid bilayer (~85 Å thick) in which phospholipids are arrayed in a double back-to-back sheet with their hydrophilic (glycerol phosphate) ends oriented outward and their hydrophobic ends (fatty acids) oriented inward. While this overly simplistic rendition can be used to highlight the important features affecting the manner and rate that xenobiotics transverse membranes, in reality it is much more complicated. The lipid bilayer is not just phospholipids; cholesterol and other lipids are present and the surfaces of the sheets are coated with proteins, which may or may not be glycosylated. In addition, interspersed throughout the membrane are proteins that may be confined to one or the other side of the membrane, or may occupy both sides of the bilayer. Some of the latter may create small channels or pores in the membrane. These small (~4 Å) pores allow for the transmembrane passage of small water-soluble molecules, but they occupy only a very small fraction of the membrane surface. Therefore the majority of the membrane can be considered a lipid bilayer and it is the lipophilicity of many xenobiotic chemicals that enables them to enter or leave a cell. Entry and exit of lipophilic molecules occurs by simple diffusion (down a high-to-low chemical concentration gradient) through the central lipid bilayer. Unless aided, bulk passage of a xenobiotic chemical or toxicant is therefore a function of its relative solubility in lipid, most often expressed as its lipid/water partition coefficient. This feature is important whether considering xenobiotic entry into a cell or an organ. It applies to xenobiotics or toxicants entering the body, no matter by what “portal of entry” (i.e., organ or surface) it occurs.

Ionization of Xenobiotics

Unionized (uncharged) molecules are more lipid soluble (higher lipid/water partition coefficient) than charged molecules and so it is this form that can diffuse most easily across lipid bilayer membranes.

For chemicals with ionizable groups, the equilibrium between the unionized and ionized forms will largely determine the rate at which they can diffuse across the membrane (i.e., how much is in the unionized state). For amines, the more acidic the environment (lower pH, more H⁺) the more will be the ionized form—RNH₂ + H⁺ ↔ RNH₃⁺—and therefore with less in the unionized (lipophilic) state, less will be available to diffuse through membranes. For a weak acid the more alkaline the environment (higher pH, less H⁺) the more will be in the ionized form RCOOH ↔ RCOO^– + H⁺ and with less in the unionized (lipophilic) state, less will be available to diffuse through membranes. Alternatively stated, weak acids will be most unionized and diffuse most easily at low pH (acidic environment), while amines will be most unionized and diffuse most easily at high pH (basic environment).

Gastrointestinal Tract

It is important to realize that compounds within the lumen of the entire gastrointestinal (GI) tract are in reality “outside” the body, that is, the cells lining the tract are an external surface and xenobiotics must pass through these cells to enter the body proper. The rate of passage is heavily dependent on xenobiotic lipophilicity at the point of contact. Entry of xenobiotics into the body via the enteral route (i.e., ingestion via the GI tract) is commonplace. In the intentional administration of chemicals with pharmacological activity (“drugs”) it is considered convenient, economical, and a generally safe route. Oral administration is unsuitable for drugs that are degraded by enzymes in the digestive tract and for drugs that are not pH-stable, although various formulations and coatings can protect against exposure to stomach acid. Xenobiotic absorption from the GI tract, especially the upper regions, can be influenced by the presence of food. Food influences include physical impedance, adsorption, and complexation. Gastric emptying time and intestinal motility can influence both the rate and extent of absorption. Paradoxically, decreased gastric emptying time generally decreases the rate of absorption, a result of delayed access to the major absorptive site, that is, the small intestine. Blood circulation to the GI site of absorption affects the chemical concentration gradient that drives diffusion. Fast blood flow maintains the gradient, and slow blood flow allows the postmembrane concentration to build up and decreases the gradient.

Xenobiotics absorbed from the stomach/small intestine pass through the portal circulation directly to the liver where they may be subjected to “first-pass” metabolism prior to entry into the general circulation. First-pass metabolism serves to reduce the “bioavailability” (to the body at large) of the parent xenobiotic. For drugs, and unless the drug is bioactivated from a prodrug form, the first-pass metabolism largely accounts for any reduced oral toxicity compared with i.v. administration. The bioavailability to the body at large can be influenced by the portal blood flow, especially for xenobiotics with high hepatic extraction ratios. The reason why this is of greatest consequence for high extraction ratio xenobiotics is evident if one considers two xenobiotics with extraction ratios of 0.8 and 0.3. If reduced blood flow (longer hepatic “dwell” time) allows 10% greater extraction, the 20% of the 0.8 extraction ratio xenobiotic normally emerging will be reduced to 12%, an almost halving of the dose entering the general circulation. For the 0.3 extraction ratio xenobiotic, the 70% normally emerging will be reduced to 67%, a negligible change in the dose of xenobiotic entering the general circulation.

Small Intestine

For any ingested drug or other xenobiotic not absorbed from the stomach lumen, it eventually passes into the intestine where secretions raise the pH, favoring the absorption of amines over weak acids. However, weak acids are readily absorbed from the intestine. The reason for this apparent contradiction lies with another factor influencing absorption, the area of the surface through which the xenobiotic or chemical can diffuse. The surface area of the intestine is immense so even though the majority of the chemical may be in the ionized form, the molecules that are unionized (recognizing that ionization is always an equilibrium) are likely to be adjacent to a membrane through which they can diffuse. The immense surface area of the small intestine is the result of the length of the organ and the multiplier effect of the surfaces provided by the three “Fs,” folds, fingers, and filaments, more properly, plicae circualares, villi, and microvilli of the brush-border enterocytes.

In a protectionist mode, the body is best served by protecting itself against the entry and circulation of xenobiotics rather than having ways and processes to remove them once inside. Within the membrane of the enterocytes are active transport “channels” that enable the cell to pump some of xenobiotics that have entered by diffusion back into the intestinal lumen (Figure 2.2). “Active transport” denotes the need for cellular energy in this process, which is provided by the hydrolysis of ATP (adenosine triphosphate). “Pump” denotes the possibility of moving the chemical up a chemical concentration gradient. (Net flow by simple diffusion is always downhill, that is, from a high concentration to a low concentration.) The channels are created by the circular arrangement of 12 or more membrane-spanning regions of a single protein termed a transporter. ATP hydrolysis at two ATP binding sites on the intracellular portion of the transporter confers the conformational change responsible for the movement of the xenobiotic through the channel. The most notable of these transporters in the intestine is P-glycoprotein (Pgp) or Multidrug Resistance protein 1 (MDR1). The latter designation derives from its original characterization in tumor cells, which were resistant to a variety of chemotherapeutic agents by virtue of the high expression of this efflux pump resulting in low and ineffective intracellular concentrations of the drug. MDR1 is the product of the ABCB1 gene and has a general preference for transporting neutral and cationic molecules. There are many ABC (ATP binding cassette) transporters, but notable in addition to MDR1 is MRP2 (MDR-Related Protein 2), which is the product of the ABCC2 gene. This transporter has a general preference for transporting anions. The key feature of these transporters is that they enable ionized molecules to be moved (smuggled!) through a lipid bilayer and often against a chemical concentration gradient. As with enzymes, the transporters have a substrate recognition site and therefore show selectivity, may show competition and preferences between xenobiotics to be transported, and if limited in expression may show saturation with resultant zero-order kinetics (a fixed rate independent of xenobiotic concentration).

Lungs

The cells lining the airway passages, like those of the skin, are external surfaces, and xenobiotics are able to pass through them to enter the body proper. These two surfaces are at opposite ends of the spectrum with regard to a parameter that affects the rate of diffusion—the distance across which a molecule diffuses. The rate of diffusion is inversely proportional to the square of the distance (double the distance, quadruple the time), and in biological terms, since most cell “lipid” membranes are of similar thickness, distance translates to the number of cell layers. In the alveoli of the lungs there are very few cell layers separating the air from the blood vessels and consequently any lipid-soluble xenobiotics in the inspired air can quickly enter the circulation. This coupled with the immense surface area presented by the alveolar structure can make the lungs an organ of risk in allowing xenobiotics into the body. The most familiar demonstration of the rapidity with which entry can occur is with gaseous or volatile liquid general anesthetics where sufficient xenobiotics can enter the bloodstream and circulate to the brain to produce anesthesia within seconds to minutes. The rapidity with which an addicted smoker can achieve sufficient central nervous system (CNS) levels of nicotine from inhaled tobacco smoke to remove any withdrawal effects is another example. The rapid absorption will of course also apply to any gases and vapors of volatile solvents. This is well known to abusers of these chemicals to obtain their quick “high” from inhalation from a closed container or bag.

Skin

For the skin, the number of cell layers between the external surface and the blood vessels is much greater than with the lung, and although lipid-soluble compounds, often organic solvents, can diffuse through, the distance to be traversed makes the diffusion rate much slower by comparison. A number of drugs have been formulated to provide delivery through transdermal patches, although the rates of absorption can be affected by regional, pathological, and individual differences in skin permeability. With injection below the epidermis, subcutaneous (SC) or intramuscular (IM), the administered drug moves by simple diffusion from the injection site into the capillaries and lymphatic system, and in both sites of administration, the blood flow to the region can markedly influence the rate of entry into the circulation. This effect is commonly seen with local anesthetics where coinjection of a vasoconstrictor serves to keep the anesthetic localized near the nerve, rather than have it washed away in the circulation.

Overview

Once in the bloodstream from its site of absorption or administration, several factors affect the distribution of drug or xenobiotic into the various tissue of the body. At its most basic, the distribution can be described as a volume of distribution (V_d), but this fails to identify all the contributing factors. In terms of fluid compartments into which a drug or other xenobiotic might distribute, standard values for a typical adult are plasma = 3 l, extracellular fluid = 12 l, total body water = 41 l. A compound that distributes widely therefore has a large V_d value, and if it stays within the plasma it will have a low V_d value. V_d values may also suggest other characteristics of the compound; a low V_d may indicate extensive plasma (albumin) protein binding and a high V_d may indicate extensive binding/sequestration in tissue sites.

The initial distribution to organs or tissues from absorption or administration sites is largely a function of blood flow. Highly perfused organs, that is, those receiving a high blood flow, will inevitably be exposed to high amounts of xenobiotic. How much leaves the bloodstream and enters the cells of the organ or tissue is a combination of many competing forces. For instance, if much of the compound is bound to plasma proteins or other blood components, free xenobiotic concentration driving diffusion will be low and so will be the rate of diffusion into the tissue. The cells of many tissues have transporters embedded in their membranes that are able to return any compound that does diffuse into the cell back to the circulation. Many of these transporters are ABC transporters, closely related to the MDR1 and MRP2 described earlier that limit xenobiotic absorption from the small intestine. The brain and placenta are two organs highly protected in this manner. In the kidney, the proximal tubule cells contain on their bloodstream side transporters that serve to augment diffusion into the cell, since the kidney is the major organ of elimination of unwanted chemicals from the body. Many of these “uptake” transporters are solute carrier transporters (SLCs), which although having much structural similarity to ABC transporters, differ in their not using ATP as their energy supply. The distribution of chemicals to and through the kidney will be considered in greater detail in the section “Excretion”.

In addition to the initial distribution phase, for some xenobiotics there is a subsequent redistribution. This phenomenon is seen with highly lipophilic compounds that initially enter high blood flow tissues (e.g., brain), then redistribute to tissues (e.g., muscle or fat) with lower blood flow. A drug example is thiopental, a short-acting barbiturate sedative hypnotic for which high blood flow to the brain initially delivers sufficient thiopental to the brain to cause anesthesia. Thiopental not leaving the blood for the brain on an initial pass is swept through to the body where it leaves the blood and enters into the fat. Blood now devoid of thiopental returns past the brain and reequilibration draws thiopental from the brain into the blood and this is redistributed to body fat. This continues through many circulation times until after 20–30 min insufficient thiopental remains in the brain to cause anesthesia and consciousness returns. Thus the pharmacological effect is primarily terminated not by metabolism or excretion, but by redistribution into fat; 70% of the thiopental is still present in the body 3 h after drug administration.

Albumin Binding

As mentioned earlier, the binding of drugs and other xenobiotics to blood components can be an important determinant of distribution. Binding to blood components is in the majority of cases reversible (i.e., noncovalent). Most often, binding is to the albumin component, since it is the protein of highest concentration in the plasma and a protein that also complexes with considerable amounts of lipid to which lipophilic drugs and chemicals can readily bind. The protein itself, a 66.5-kDa protein secreted by the liver, appears to have two major xenobiotic binding sites, a large flexible site that preferentially binds dicarboxylic acids and/or bulky heterocyclic molecules with a negative charge localized in the middle of the molecule and a second smaller, more narrow, and less flexible site that preferentially binds aromatic carboxylic acids with a negative charge localized at one end away from a hydrophobic center. This second site is most often associated with the binding of nonsteroidal anti-inflammatory drugs (NSAIDs). Both these sites can exhibit saturation and if multiple competing chemicals are present, displacement of one compound by another can occur. This makes prediction of the extent of albumin binding of individual components when contained in mixtures from a knowledge of binding as a single chemical extremely complex. Extensive albumin binding can restrain diffusion out of the blood into tissues. By reducing free xenobiotic concentration in the blood it can also serve to enhance absorption from, for example, the GI tract. In this consideration the binding preserves the lumen to blood high-to-low chemical concentration gradient from the “inside” by maintaining the low blood concentration low.

Overview

Xenobiotic metabolism is commonly referred to as “drug” metabolism or “drug” biotransformation. It is the chemical alteration of substances by reactions in the living organism, predominantly enzyme-catalyzed. The objective of biotransformation is generally to promote the excretion of chemicals by enhancing their water solubility. Although excretion or elimination can occur without metabolism, for most compounds, especially those with considerable lipid solubility (therefore well absorbed following oral administration), it occurs subsequent to one or two types of reactions. Enhanced water solubility derives primarily from Phase II (conjugation, synthetic) reactions in which ionizable entities derived from natural body biochemicals are added to the molecule. Many conjugates (glucuronides, sulfates, amino acid conjugates) have acidic functional groups and, being extensively ionized at physiological pH, undergo less reabsorption by diffusion through lipid membranes of kidney tubule following glomerular filtration. In addition, they are subject to carrier-mediated active secretion from blood into urine (Figure 2.2). Ironically, they also require carrier-mediated efflux from the cell in which the conjugate was created. For the hepatocyte, this results in active secretion into the bile and also into the blood for subsequent renal excretion.

Many xenobiotics lack suitable functional or reactive chemical groups on which conjugation can occur and must first undergo Phase I (metabolic transformation, activation) reactions to generate them. For example, the lipid-soluble antiepileptic drug phenytoin must first be converted to 4-hydroxyphenytoin before formation of the very water-soluble 4-hydroxyphenytoin glucuronide. The same is true for benzene, which is first converted to phenol and then onward to phenyl glucuonide. The aqueous solubility of phenol (6.7%) far exceeds that of benzene (0.2%). In some instances, chemicals are excreted following just metabolic transformation, and small amounts of Phase I metabolites appear in urine alongside major amounts of Phase II conjugates. The two-step (Phases I and II) metabolism process largely proceeds through a limited number (three major) of functional groups (Figure 2.3) (the enzymes involved in these reactions will be considered individually later). Phase II metabolites are usually pharmacologically inactive. In addition to promoting excretion, metabolism and the increased water solubility decreases the entry of xenobiotics into cells and decreases the interaction with intracellular targets, most often receptors. Phase I metabolites may or may not be pharmacologically active. For some chemicals, reaching a state suitable for conjugation reactions requires passing through a more chemically reactive entity or intermediate (Figure 2.4). Highly reactive metabolites, most often generated from Phase I oxidations, are occasionally produced and these can cause tissue damage. Mutagenic and carcinogenic epoxides generated by P450s are good examples of such intermediates; others are shown in Table 2.1. Reactive intermediates may cause enzyme inactivation, membrane lipid peroxidations resulting in membrane alterations, and changes in DNA. Reactive intermediates have been implicated in carcinogenesis, tissue necrotic reactions, and tissue allergic responses.

Xenobiotic biotransformation takes place in almost all organs and tissues (liver, skin, GI tract, lungs, kidney, blood, etc.); however, the liver is quantitatively the most important tissue for xenobiotic metabolism especially because of its high expression levels of many xenobiotic-metabolizing enzymes. The liver with its portal blood supply from the GI tract is uniquely placed to protect the body from exogenous, possibly toxic chemicals. If the liver can rapidly metabolize the xenobiotic following absorption from the GI tract, a significant portion of the xenobiotic may be inactivated before it can produce a therapeutic or toxic effect in the remainder of the body.

Within the liver, a major subcellular site of xenobiotic biotransformation is the endoplasmic reticulum. Among the most common/important xenobiotic-metabolizing enzymes located here are cytochrome P450s and flavin monooxygenases (FMOs), carboxylesterases, and glucuronosyltransferases. When liver is homogenized and ruptured cells are differentially centrifuged, fragments of the endoplasmic reticulum with xenobiotic-metabolizing capabilities are isolated in the fraction called microsomes (artifacts of cell disruption), a term often used to identify the cellular location of xenobiotic-metabolizing enzymes. Differential centrifugation of liver tissue gently homogenized in 0.25 M sucrose (so as to preserve mitochondria intact) can yield other fractions containing xenobiotic-metabolizing enzymes; 18,000 g for 20 min will pull down intact heavy and light mitochondria into a pellet, and microsomes require 105,000 g for 60 min to sediment, leaving the cytosolic (soluble) fraction as a supernatant. Note that a 20-min 9000 g supernatant, often termed an S9 fraction, a fraction that is utilized in some xenobiotic metabolism studies and mutagenic assays (“Ames test”), contains both microsomes (endoplasmic reticulum) and cytoplasm (and some light mitochondria) and therefore enzymes contained therein.