Although cholesterol is well known in its own right as a contributor to the development of heart disease, metabolites of cholesterol—the steroid hormones—also appear in the news frequently. Indeed, steroid-hormone abuse seems to be as prominent in the sports pages as any athlete is. In addition to steroid hormones, cholesterol is a precursor for two other important molecules: bile salts and vitamin D. We begin with a look at the bile salts, molecules crucial for the uptake of lipids in the diet.

Bile salts. Bile salts are polar derivatives of cholesterol. These compounds are highly effective detergents because they contain both polar and nonpolar regions. Bile salts are synthesized in the liver, stored and concentrated in the gallbladder, and then released into the small intestine. Bile salts, the major constituent of bile, solubilize dietary lipids. Solubilization increases the effective surface area of lipids with two consequences: (1) more surface area is exposed to the digestive action of lipases and (2) lipids are more readily absorbed by the intestine. Bile salts are also the major breakdown products of cholesterol. The bile salts glycocholate, the primary bile salt, and taurocholate are shown in Figure 26.24.

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In addition to bile salts, bile is composed of cholesterol, phospholipids, and the breakdown products of heme, bilirubin, and biliverdin (Section 24.4). If too much cholesterol is present in the bile, it will precipitate to form gallbladder stones (cholelithiasis). These stones may block bile secretion and inflame the gallbladder, a condition called cholecystitis. Symptoms include pain in the upper right abdomen, especially after a fatty meal, and nausea. If need be, the gall bladder is removed, and bile flows from the liver through the bile duct directly into the intestine.

Steroid hormones. Cholesterol is the precursor of the five major classes of steroid hormones: progestagens, glucocorticoids, mineralocorticoids, androgens, and estrogens (Figure 26.25). These hormones are powerful signal molecules that regulate a host of organismal functions. Progesterone, a progestogen, prepares the lining of the uterus for the implantation of an ovum. Progesterone is also essential for the maintenance of pregnancy by preventing premature uterine contractions. Androgens (such as testosterone) are responsible for the development of male secondary sex characteristics, whereas estrogens (such as estradiol) are required for the development of female secondary sex characteristics. Estrogens, along with progesterone, also participate in the ovarian cycle. Glucocorticoids (such as cortisol) promote gluconeogenesis and glycogen synthesis, enhance the degradation of fat and protein, and inhibit the inflammatory response. They enable animals to respond to stress; indeed, the absence of glucocorticoids can be fatal. Mineralocorticoids (primarily aldosterone) act on the distal tubules of the kidney to increase the reabsorption of Na⁺ and the excretion of K⁺ and H⁺, which leads to an increase in blood volume and blood pressure. The major sites of synthesis of these classes of hormones are the corpus luteum, for progestogens; the testes, for androgens; the ovaries, for estrogens; and the adrenal cortex, for glucocorticoids and mineralocorticoids.

Steroid hormones bind to and activate receptor molecules that serve as transcription factors to regulate gene expression (Section 32.2). These small similar molecules are able to have greatly differing effects because the slight structural differences among them allow interactions with specific receptor molecules.

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Carbon atoms in steroids are numbered, as shown for cholesterol in Figure 26.26. The rings in steroids are denoted by the letters A, B, C, and D. Cholesterol contains two angular methyl groups: the C-19 methyl group is attached to C-10, and the C-18 methyl group is attached to C-13. The C-18 and C-19 methyl groups of cholesterol lie above the plane containing the four rings. A substituent that is above the plane is termed β oriented, whereas a substituent that is below the plane is α oriented.

If a hydrogen atom is attached to C-5, it can be either α or β oriented. The A and B steroid rings are fused in a trans conformation if the C-5 hydrogen is α oriented, and cis if it is β oriented. The absence of a Greek letter for the C-5 hydrogen atom on the steroid nucleus implies a trans fusion. The C-5 hydrogen atom is α oriented in all steroid hormones that contain a hydrogen atom in that position. In contrast, bile salts have a β-oriented hydrogen atom at C-5. Thus, a cis fusion is characteristic of the bile salts, whereas a trans fusion is characteristic of all steroid hormones that possess a hydrogen atom at C-5. A trans fusion yields a nearly planar structure, whereas a cis fusion gives a buckled structure.

The addition of OH groups plays an important role in the synthesis of cholesterol from squalene and in the conversion of cholesterol into steroid hormones and bile salts. All these hydroxylations require NADPH and O₂. The oxygen atom of the incorporated hydroxyl group comes from O₂ rather than from H₂O. Whereas one oxygen atom of the O₂ molecule goes into the substrate, the other is reduced to water. The enzymes catalyzing these reactions are called monooxygenases (or mixed-function oxygenases). Recall that a monooxygenase also participates in the hydroxylation of aromatic amino acids (Section 23.5).

RH + O₂ + NADPH + H⁺ → ROH + H₂O + NADP⁺

Hydroxylation requires the activation of oxygen. In the synthesis of steroid hormones and bile salts, activation is accomplished by members of the cytochrome P450 family, a family of cytochromes that absorb light maximally at 450 nm when complexed in vitro with exogenous carbon monoxide. These membrane-anchored proteins (~50 kDa) contain a heme prosthetic group. Oxygen is activated through its binding to the iron atom in the heme group.

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Because the hydroxylation reactions promoted by P450 enzymes are oxidation reactions, it is at first glance surprising that they also consume the reductant NADPH. NADPH transfers its high-potential electrons to a flavoprotein, which transfers them, one at a time, to adrenodoxin, a non-heme iron protein. Adrenodoxin transfers one electron to reduce the ferric (Fe³⁺) form of P450 to the ferrous (Fe²⁺) form (Figure 26.27).

Without the addition of this electron, P450 will not bind oxygen. Recall that only the ferrous form (Fe²⁺) of myoglobin and hemoglobin binds oxygen (Section 7.1). The binding of O₂ to the heme is followed by the acceptance of a second electron from adrenodoxin. The acceptance of this second electron leads to cleavage of the O—O bond. One of the oxygen atoms is then protonated and released as water. The remaining oxygen atom forms a highly reactive ferryl (Fe⁴⁺) Fe=O intermediate. This intermediate abstracts a hydrogen atom from the substrate RH to form R•. This transient free radical captures the OH group from the iron atom to form ROH, the hydroxylated product, returning the iron atom to the ferric state.

The cytochrome P450 system, which in mammals is located primarily in the smooth endoplasmic reticulum of the liver and small intestine, is also important in the detoxification of foreign substances (xenobiotic compounds). For example, the hydroxylation of phenobarbital, a barbiturate, increases its solubility and facilitates its excretion. Likewise, polycyclic aromatic hydrocarbons that are ingested by drinking contaminated water are hydroxylated by P450, providing sites for conjugation with highly polar units (e.g., glucuronate or sulfate) that markedly increase the solubility of the modified aromatic molecule. One of the most relevant functions of the cytochrome P450 system to human beings is its role in metabolizing drugs such as caffeine and ibuprofen (Chapter 36). Some members of the cytochrome P450 system also metabolize ethanol (Section 27.6). The duration of action of many medications depends on their rate of inactivation by the P450 system. Despite its general protective role in the removal of foreign chemicals, the action of the P450 system is not always beneficial. Some of the most powerful carcinogens are generated from harmless compounds by the P450 system in vivo in the process of metabolic activation (Figure 28.34). In plants, the cytochrome P450 system plays a role in the synthesis of toxic compounds as well as the pigments of flowers.

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Earlier (Section 9.1), we examined the use of protease inhibitors to treat HIV infection. These inhibitors, in conjunction with inhibitors of other HIV enzymes, have drastically reduced deaths due to AIDS. The effectiveness of these drugs is sometimes compromised because P450 enzymes, such as cytochrome P450-3A4, inactivate them. The drug ritonavir was developed in the search for more effective inhibitors. Interestingly, ritonavir, although a protease inhibitor, is also a potent inhibitor of cytochrome P450-3A4. This serendipitous “side-effect” allows the clinician to administer low doses of ritonavir in combination with other protease inhibitors, effectively increasing the concentrations of these drugs while allowing a reduction in dosage and dosing frequency.

Steroid hormones contain 21 or fewer carbon atoms, whereas cholesterol contains 27. Thus, the first stage in the synthesis of steroid hormones is the removal of a six-carbon unit from the side chain of cholesterol to form pregnenolone. The side chain of cholesterol is hydroxylated at C-20 and then at C-22, and the bond between these carbon atoms is subsequently cleaved by desmolase. Three molecules of NADPH and three molecules of O₂ are consumed in this six-electron oxidation.

Progesterone is synthesized from pregnenolone in two steps. The 3-hydroxyl group of pregnenolone is oxidized to a 3-keto group, and the Δ⁵ double bond is isomerized to a Δ⁴ double bond (Figure 26.28). Cortisol, the major glucocorticoid, is synthesized from progesterone by hydroxylations at C-11, C-17, and C-21; C-17 must be hydroxylated before C-21 is hydroxylated, whereas C-11 can be hydroxylated at any stage. The enzymes catalyzing these hydroxylations are highly specific. The initial step in the synthesis of aldosterone, the major mineralocorticoid, is the hydroxylation of progesterone at C-21. The resulting deoxycorticosterone is hydroxylated at C-11. The oxidation of the C-18 angular methyl group to an aldehyde then yields aldosterone.

Androgens and estrogens also are synthesized from pregnenolone through the intermediate progesterone. Androgens contain 19 carbon atoms. The synthesis of androgens starts with the hydroxylation of progesterone at C-17 (Figure 26.29). The side chain consisting of C-20 and C-21 is then cleaved to yield androstenedione, an androgen. Testosterone, another androgen, is formed by the reduction of the 17-keto group of androstenedione. Testosterone, through its actions in the brain, is paramount in the development of male sexual behavior. It is also important for the maintenance of the testes and the development of muscle mass. Owing to the latter activity, testosterone is referred to as an anabolic steroid. Testosterone is reduced by 5α-reductase to yield dihydrotestosterone (DHT), a powerful embryonic androgen that instigates the development and differentiation of the male phenotype (Problems 21 and 24).

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Estrogens are synthesized from androgens by the loss of the C-19 angular methyl group and the formation of an aromatic A ring. Estrone, an estrogen, is derived from androstenedione, whereas estradiol, the biologically most potent estrogen, is formed from testosterone. Estradiol can also be formed from estrone. The formation of the aromatic A ring is catalyzed by the P450 enzyme aromatase.

Because breast and ovarian cancers frequently depend on estrogens for growth, aromatase inhibitors are often used as a treatment for these cancers. Anastrozole is a competitive inhibitor of the enzyme, whereas exemestane is a suicide inhibitor that covalently modifies and inactivates the enzyme.

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Cholesterol is also the precursor of vitamin D, which plays an essential role in the control of calcium and phosphorus metabolism. 7-Dehydrocholesterol (provitamin D₃) is photolyzed by the ultraviolet light of sunlight to previtamin D₃, which spontaneously isomerizes to vitamin D₃ (Figure 26.30). Vitamin D₃ (cholecalciferol) is converted into calcitriol (1,25-dihydroxycholecalciferol), the active hormone, by hydroxylation reactions in the liver and kidneys. Although not a steroid, vitamin D acts in an analogous fashion. It binds to a receptor, structurally similar to the steroid receptors, to form a complex that functions as a transcription factor, regulating gene expression.

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Vitamin D deficiency in childhood produces rickets, a disease characterized by inadequate calcification of cartilage and bone. Rickets was so common in seventeenth-century England that it was called the “children’s disease of the English.” The 7-dehydrocholesterol in the skin of these children was not photolyzed to previtamin D₃, because there was little sunlight for many months of the year. Furthermore, their diets provided little vitamin D, because most naturally occurring foods have a low content of this vitamin. Fish-liver oils are a notable exception. Cod-liver oil, abhorred by generations of children because of its unpleasant taste, was used in the past as a rich source of vitamin D. Today, the most reliable dietary sources of vitamin D are fortified foods. Milk, for example, is fortified to a level of 400 international units per quart (10 μg per quart). The recommended daily intake of vitamin D is 200 international units until age 50, after which it increases with age. In adults, vitamin D deficiency leads to softening and weakening of bones, a condition called osteomalacia. The occurrence of osteomalacia in Muslim women who are clothed so that only their eyes are exposed to sunlight is a striking reminder that vitamin D is needed by adults as well as by children.

Research over the past few years indicates that vitamin D may play a much larger biochemical role than simply the regulation of bone metabolism. Muscle seems to be a target of vitamin D action. In muscle, vitamin D appears to affect a number of biochemical processes with the net effect being enhanced muscle performance. Studies also suggest that vitamin D prevents cardiovascular disease, reduces the incidence of a variety of cancers, and protects against autoimmune diseases including diabetes. Moreover, vitamin D deficiency appears to be more common than previously thought. In the United States, 75% of Blacks and many Hispanics and Asians have insufficient blood levels of vitamin D. This recent research on vitamin D shows again the dynamic nature of biochemical investigations. Vitamin D, a chemical whose biochemical role was believed to be well established, now offers new frontiers of biomedical research.