Alpha₁ receptors are coupled via G proteins of the G_q family to phospholipase C. This enzyme hydrolyzes polyphosphoinositides, leading to the formation of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (see Table 9–1 and Figure 9–1). IP₃ promotes the release of sequestered Ca²⁺ from intracellular stores, which increases cytoplasmic free Ca²⁺ concentrations that activate various calcium-dependent protein kinases and other calmodulin-regulated proteins. Activation of these receptors may also increase influx of calcium across the cell’s plasma membrane. IP₃ is sequentially dephosphorylated, which ultimately leads to the formation of free inositol. DAG cooperates with Ca²⁺ in activating protein kinase C (PKC), which modulates activity of many signaling pathways. In addition, α₁ receptors activate signal transduction pathways that stimulate tyrosine kinases such as mitogen-activated protein kinases (MAP kinases) and polyphosphoinositol-3-kinase (PI-3-kinase).

All three receptor subtypes (β₁, β₂, and β₃) are coupled to the stimulatory regulatory protein G_s, which activates adenylyl cyclase to increase intracellular levels of cAMP (see Table 9–1 and Figure 9–2). Cyclic AMP is the second messenger that mediates most of the actions of β-receptors; in the liver cAMP mediates a cascade of events culminating in the activation of glycogen phosphorylase; in the heart, it increases the influx of calcium across the cell membrane; whereas in smooth muscle it promotes relaxation through phosphorylation of myosin light-chain kinase to an inactive form (see Figure 12–1). Some actions of β adrenoceptors may be mediated through different intracellular signaling pathways, via exchange proteins activated by cAMP rather than conventional protein kinase A (PKA), or via coupling to G_s but independent of cAMP, or coupling to G_q proteins and activation of MAP kinases.

The D₁ receptor is typically associated with the stimulation of adenylyl cyclase (see Table 9–1); for example, D₁ receptor–induced smooth muscle relaxation is presumably due to cAMP accumulation in the smooth muscle of those vascular beds in which dopamine is a vasodilator. D₂ receptors have been found to inhibit adenylyl cyclase activity, open potassium channels, and decrease calcium influx.

Because arteriolar and venous tone are determined to a large extent by α receptors on vascular smooth muscle, α-receptor antagonist drugs cause a lowering of peripheral vascular resistance and blood pressure (Figure 10–3). These drugs can prevent the pressor effects of usual doses of α agonists; indeed, in the case of agonists with both α and β₂ effects (eg, epinephrine), selective α-receptor antagonism may convert a pressor to a depressor response (see Figure 10–3); it illustrates how the activation of both α and β receptors in the vasculature may lead to opposite responses, and that blockade of α adrenoceptors unmasks the effects of epinephrine on β receptors. Alpha-receptor antagonists (Table 10–1) block sympathetic-mediated vasoconstriction, causing a decrease in blood pressure. The fall in blood pressure is greater in situations when blood pressure depends on increased sympathetic activity. This may occur, for example, on standing when blood pressure is maintained by sympathetic activation to compensate for gravitational pooling of blood in the lower body. Thus, α adrenoceptor antagonists can cause orthostatic hypotension by blocking sympathetically-mediated peripheral arterial vasoconstriction and splanchnic capacitance venoconstriction. Because β receptors are unopposed, this is associated with a compensatory baroreflex-mediated tachycardia.

Blockade of α receptors in noncardiac tissues elicits miosis (small pupils) and nasal stuffiness. Alpha₁ receptors are expressed in the base of the bladder and the prostate, and their blockade decreases resistance to the flow of urine. Alpha blockers, therefore, are used therapeutically for the treatment of urinary retention due to prostatic hyperplasia (see below).

Cortisol (also called hydrocortisone, compound F) exerts a wide range of physiologic effects, including regulation of intermediary metabolism, cardiovascular function, growth, and immunity. Its synthesis and secretion are tightly regulated by the central nervous system, which is very sensitive to negative feedback by the circulating cortisol and exogenous (synthetic) glucocorticoids. Cortisol is synthesized from cholesterol (as shown in Figure 39–1). The mechanisms controlling its secretion are discussed in Chapter 37.

Pharmaceutical steroids are usually synthesized from cholic acid obtained from cattle or steroid sapogenins found in plants. Further modifications of these steroids have led to the marketing of a large group of synthetic steroids with special characteristics that are pharmacologically and therapeutically important (Table 39–1; see Figure 39–3).

The actions of the synthetic steroids are similar to those of cortisol (see above). They bind to the specific intracellular receptor proteins and produce the same effects but have different ratios of glucocorticoid to mineralocorticoid potency (Table 39–1).

The benefits obtained from glucocorticoids vary considerably. Use of these drugs must be carefully weighed in each patient against their widespread effects. The major undesirable effects of glucocorticoids are the result of their hormonal actions, which lead to the clinical picture of iatrogenic Cushing syndrome (see later in text).

Glucocorticoid preparations differ with respect to relative anti-inflammatory and mineralocorticoid effect, duration of action, cost, and dosage forms available (Table 39–1), and these factors should be taken into account in selecting the drug to be used.

Aldosterone is synthesized mainly in the zona glomerulosa of the adrenal cortex. Its structure and synthesis are illustrated in Figure 39–1. The rate of aldosterone secretion is subject to several influences. ACTH produces a moderate stimulation of its release, but this effect is not sustained for more than a few days in the normal individual. The quantities of aldosterone produced by the adrenal cortex and its plasma concentrations are insufficient to participate in any significant feedback control of ACTH secretion.

11-DOC, which also serves as a precursor of aldosterone (see Figure 39–1), is normally secreted in amounts of about 200 mcg/d. Its half-life when injected into the human circulation is about 70 minutes. Estimates of its concentration in plasma are approximately 0.03 mcg/dL. The control of its secretion differs from that of aldosterone in that the secretion of 11-DOC is primarily under the control of ACTH. Although the response to ACTH is enhanced by dietary sodium restriction, due to adaptations, a low-salt diet does not increase 11-DOC secretion. The secretion of DOC may be markedly increased in abnormal conditions such as adrenocortical carcinoma and congenital adrenal hyperplasia with reduced P450c11 or P450c17 activity.

This compound, a potent steroid with both glucocorticoid and mineralocorticoid activity, is the most widely used mineralocorticoid. Oral doses of 0.1 mg two to seven times weekly have potent salt-retaining activity and are used in the treatment of adrenocortical insufficiency associated with mineralocorticoid deficiency. These dosages are too small to have important anti-inflammatory or antigrowth effects.

Parathyroid hormone (PTH) is a single-chain peptide hormone composed of 84 amino acids. It is produced in the parathyroid gland in a precursor form of 115 amino acids, the excess 31 amino terminal amino acids being cleaved off before secretion. Within the gland is a calcium-sensitive protease capable of cleaving the intact hormone into fragments, thereby providing one mechanism by which calcium limits the production of PTH. A second mechanism involves the calcium-sensing receptor (CaSR) which, when stimulated by calcium, reduces PTH production and secretion. The parathyroid gland also contains the vitamin D receptor (VDR) and the enzyme, CYP27B1, that produces 1,25(OH)₂D, thus enabling circulating or endogenously produced 1,25(OH)₂D to suppress PTH production. 1,25(OH)₂D also induces the CaSR, making the parathyroid gland more sensitive to suppression by calcium. Biologic activity resides in the amino terminal region of PTH such that synthetic PTH 1-34 (available as teriparatide) is fully active and used in the treatment of osteoporosis However, a full length form of PTH (rhPTH 1-84, Natpara) has been approved for treatment of hypoparathyroidism. In addition, an analog of PTHrP (abaloparatide) that functions much like teriparatide has recently been approved for the treatment of osteoporosis. Other analogs of PTH are currently in development. Loss of the first two amino terminal amino acids eliminates most biologic activity.

Vitamin D is a secosteroid produced in the skin from 7-dehydrocholesterol under the influence of ultraviolet radiation. Vitamin D is also found in certain foods and is used to supplement dairy products and other foods. Both the natural form (vitamin D₃, cholecalciferol) and the plant-derived form (vitamin D₂, ergocalciferol) are present in the diet. As discussed earlier these forms differ in that ergocalciferol contains a double bond and an additional methyl group in the side chain (see Figure 42–3). Ergocalciferol and its metabolites bind less well than cholecalciferol and its metabolites to vitamin D–binding protein (DBP), the major transport protein of these compounds in blood, and have a somewhat different path of catabolism. As a result, their half-lives are shorter than those of the cholecalciferol metabolites. This influences treatment strategies, as will be discussed. However, the key steps in metabolism and biologic activities of the active metabolites are comparable, so with this exception the following comments apply equally well to both forms of vitamin D.

Fibroblast growth factor 23 (FGF23) is a single-chain protein with 251 amino acids, including a 24-amino-acid leader sequence. It inhibits 1,25(OH)₂D production and phosphate reabsorption (via the sodium phosphate cotransporters NaPi 2a and 2c) in the kidney and can lead to both hypophosphatemia and inappropriately low levels of circulating 1,25(OH)₂D. Whereas FGF23 was originally identified in certain mesenchymal tumors, osteoblasts and osteocytes in bone appear to be its primary site of production. Other tissues can also produce FGF23, though at lower levels. FGF23 requires O-glycosylation for its secretion, a glycosylation mediated by the glycosyl transferase GALNT3. Mutations in GALNT3 result in abnormal deposition of calcium phosphate in periarticular tissues (tumoral calcinosis) with elevated phosphate and 1,25(OH)₂D. FGF23 is normally inactivated by cleavage at an RXXR site (amino acids 176–179). Mutations of the arginines (R) in this site lead to excess FGF23, the underlying problem in autosomal dominant hypophosphatemic rickets. A similar disease, X-linked hypophosphatemic rickets (XLH), is due to mutations in PHEX, an endopeptidase, which initially was thought to cleave FGF23. However, this concept has been shown to be invalid, and the mechanism by which PHEX mutations lead to increased FGF23 levels remains obscure. FGF23 binds to FGF receptors (FGFR) 1 and 3c in the presence of the accessory receptor Klotho-α. Both Klotho and the FGFR must be present for signaling in most tissues, although high levels of FGF23 appear to affect cardiomyocytes lacking Klotho. Mutations in Klotho disrupt FGF23 signaling, resulting in elevated phosphate and 1,25(OH)₂D levels, a phenotype quite similar to inactivating mutations in FGF23 or GALNT3. FGF23 production is stimulated by 1,25(OH)₂D and phosphate and directly or indirectly inhibited by the dentin matrix protein DMP1 found in osteocytes. Mutations in DMP1 lead to increased FGF23 levels and osteomalacia. Recently an antibody to FGF23, burosumab, has been approved for the treatment of XLH, an approval likely to extend to other diseases marked by high FGF23 levels.

A summary of the principal actions of PTH, FGF23, and vitamin D on the three main target tissues—intestine, kidney, and bone—is presented in Table 42–2. The net effect of PTH is to raise serum calcium, reduce serum phosphate, and increase 1,25(OH)₂D; the net effect of FGF23 is to decrease serum phosphate and 1,25(OH)₂D; the net effect of vitamin D is to raise both calcium and phosphate while decreasing PTH and increasing FGF23. Regulation of calcium and phosphate homeostasis is achieved through important feedback loops. Calcium is one of two principal regulators of PTH secretion. It binds to a novel ion recognition site that is part of a G_q protein-coupled receptor called the calcium-sensing receptor (CaSR) that employs the phosphoinositide second messenger system to link changes in the extracellular calcium concentration to changes in the intracellular free calcium. As serum calcium levels rise and activate this receptor, intracellular calcium levels increase and inhibit PTH secretion. This inhibition by calcium of PTH secretion, along with inhibition of renin and atrial natriuretic peptide secretion, is the opposite of the effect of calcium in other tissues such as the beta cell of the pancreas, in which calcium stimulates secretion. Phosphate regulates PTH secretion directly and indirectly. Its indirect actions are the result of forming complexes with calcium in the serum. Because it is the ionized free concentration of extracellular calcium that is detected by the parathyroid gland, increases in serum phosphate levels reduce the ionized calcium levels, leading to enhanced PTH secretion. Whether the parathyroid gland expresses phosphate receptors that mediate the direct action of phosphate on PTH secretion remains unclear. Such feedback regulation is appropriate to the net effect of PTH to raise serum calcium and reduce serum phosphate levels. Likewise, both calcium and phosphate at high levels reduce the amount of 1,25(OH)₂D produced by the kidney and increase the amount of 24,25(OH)₂D produced.

The calcitonin secreted by the parafollicular cells of the mammalian thyroid is a single-chain peptide hormone with 32 amino acids and a molecular weight of 3600. A disulfide bond between positions 1 and 7 is essential for biologic activity. Calcitonin is produced from a precursor with a molecular weight of 15,000. The circulating forms of calcitonin are multiple, ranging in size from the monomer (molecular weight 3600) to forms with an apparent molecular weight of 60,000. Whether such heterogeneity includes precursor forms or covalently linked oligomers is not known. Because of its chemical heterogeneity, calcitonin preparations are standardized by bioassay in rats. Activity is compared to a standard maintained by the British Medical Research Council (MRC) and expressed as MRC units.

Glucocorticoid hormones alter bone mineral homeostasis by antagonizing vitamin D-stimulated intestinal calcium transport, stimulating renal calcium excretion, blocking bone formation, and at least initially stimulating bone resorption. Although these observations underscore the negative impact of glucocorticoids on bone mineral homeostasis, these hormones have proved useful in reversing the hypercalcemia associated with lymphomas and granulomatous diseases such as sarcoidosis in which unregulated ectopic production of 1,25[OH]₂D occurs or in cases of vitamin D intoxication. Prolonged administration of glucocorticoids is a common cause of osteoporosis in adults and can cause stunted skeletal development in children (see Chapter 39).

Estrogens can prevent accelerated bone loss during the immediate postmenopausal period and at least transiently increase bone in postmenopausal women.

The bisphosphonates are analogs of pyrophosphate in which the P-O-P bond has been replaced with a nonhydrolyzable P-C-P bond (Figure 42–4). Currently available bisphosphonates include etidronate, pamidronate, alendronate, risedronate, tiludronate, ibandronate, and zoledronate. With the development of the more potent bisphosphonates, etidronate is seldom used.

Denosumab is a fully humanized monoclonal antibody that binds to and prevents the action of RANKL. As described earlier, RANKL is produced by osteoblasts and other cells, including T lymphocytes. It stimulates osteoclastogenesis via RANK, the receptor for RANKL that is present on osteoclasts and osteoclast precursors. By interfering with RANKL function, denosumab inhibits osteoclast formation and activity. It is at least as effective as the potent bisphosphonates in inhibiting bone resorption and has been approved for treatment of postmenopausal osteoporosis and some cancers (prostate and breast). The latter application is to limit the development of bone metastases or bone loss resulting from the use of drugs that suppress gonadal function. Denosumab is administered subcutaneously every 6 months. The drug appears to be well tolerated, but four concerns remain. First, a number of cells in the immune system also express RANKL, suggesting that there could be an increased risk of infection associated with the use of denosumab. Second, because the suppression of bone turnover with denosumab is similar to that of the potent bisphosphonates, the potential risk of osteonecrosis of the jaw and subtrochanteric fractures is comparable. Third, denosumab can lead to transient hypocalcemia, especially in patients with marked bone loss (and bone hunger) or compromised calcium regulatory mechanisms, including chronic kidney disease and vitamin D deficiency. That said, denosumab can be used in patients with advanced renal disease, unlike the bisphosphonates, as it is not cleared by the kidney, and it has the advantage over bisphosphonates in that it is readily reversible because it does not deposit in bone. However, when used in patients with renal failure, careful attention to serum calcium levels is necessary. Fourth, denosumab does not result in the death of osteoclasts, unlike the bisphosphonates, so that if denosumab therapy is interrupted a surge of bone resorption can occur putting the patient at risk for new fractures. Efforts to prevent this surge of bone resorption with potent antiresorptives like zoledronate are only partially effective.

Sclerostin is a protein produced by osteocytes that blocks the action of the wnt receptor in osteoblasts. The wnt receptor when activated by selected wnts promotes beta-catenin signaling increasing the proliferation of osteoblasts. Sclerostin blocks wnt activation, suppressing bone formation. Antibodies to sclerostin have been developed, of which only romosozumab has been FDA approved for the treatment of osteoporosis. This form of therapy promotes bone formation and inhibits bone resorption, although the mechanism for its effects on osteoclasts is not well understood. The use of romosozumab is limited to one year after which it is recommended that patients be switched to an antiresorptive to prevent loss of the bone gained. In some of the trials with this drug there appeared to be an increased risk of myocardial infarction (MI), stroke, and cardiovascular death, thus generating a black box warning contraindicating its use in patients with a previous MI or stroke within the past year.

Cinacalcet is the first representative of a new class of drugs that activates the calcium-sensing receptor (CaSR) described above. Etelcalcetide is a more recently approved and somewhat more potent calcimimetic that currently is approved only for secondary hyperparathyroidism in CKD patients on dialysis. CaSR is widely distributed but has its greatest concentration in the parathyroid gland. By activating the parathyroid gland CaSR, these drugs inhibit PTH secretion. These drugs are approved for the treatment of secondary hyperparathyroidism in chronic kidney disease (CKD), and cinacalcet is also approved for the treatment of parathyroid carcinoma and severe primary hyperparathyroidism. In CKD patients requiring dialysis, hypocalcemia can occur with the use of these drugs, and nausea is often a limiting factor. CaSR antagonists are also being developed and may be useful in conditions of hypoparathyroidism or as a means to stimulate intermittent PTH secretion in the treatment of osteoporosis.

The chemistry and pharmacology of the thiazide family of drugs are discussed in Chapter 15. The principal application of thiazides in the treatment of bone mineral disorders is in reducing renal calcium excretion. Thiazides may increase the effectiveness of PTH in stimulating reabsorption of calcium by the renal tubules or may act on calcium reabsorption secondarily by increasing sodium reabsorption in the proximal tubule. In the distal tubule, thiazides block sodium reabsorption at the luminal surface, increasing the calcium-sodium exchange at the basolateral membrane and thus enhancing calcium reabsorption into the blood at this site (see Figure 15–4). Thiazides have proved to be useful in reducing the hypercalciuria and incidence of urinary stone formation in subjects with idiopathic hypercalciuria. Part of their efficacy in reducing stone formation may lie in their ability to decrease urine oxalate excretion and increase urine magnesium and zinc levels, both of which inhibit calcium oxalate stone formation. By reducing urine calcium losses, these drugs may also support treatment of osteoporosis.

Fluoride is well established as effective for the prophylaxis of dental caries and has previously been investigated for the treatment of osteoporosis. Both therapeutic applications originated from epidemiologic observations that subjects living in areas with naturally fluoridated water (1–2 ppm) had fewer dental caries and fewer vertebral compression fractures than subjects living in nonfluoridated water areas. Fluoride accumulates in bones and teeth, where it may stabilize the hydroxyapatite crystal. Such a mechanism may explain the effectiveness of fluoride in increasing the resistance of teeth to dental caries, but it does not explain its ability to promote new bone growth.

Strontium ranelate is composed of two atoms of strontium bound to an organic ion, ranelic acid. Although not approved for use in the United States, this drug is used in Europe for the treatment of osteoporosis. Strontium ranelate appears to block differentiation of osteoclasts while promoting their apoptosis, thus inhibiting bone resorption. At the same time, strontium ranelate appears to promote bone formation. Unlike bisphosphonates, denosumab, or teriparatide, but similar to romosozumab, strontium ranelate increases bone formation markers while inhibiting bone resorption markers. Large clinical trials have demonstrated its efficacy in increasing bone mineral density and decreasing fractures in the spine and hip. Toxicities reported thus far are similar to placebo.

Iron deficiency is the most common cause of chronic anemia. Like other forms of chronic anemia, iron deficiency anemia leads to pallor, fatigue, dizziness, exertional dyspnea, and other generalized symptoms of tissue hypoxia. The cardiovascular adaptations to chronic anemia—tachycardia, increased cardiac output, vasodilation—can worsen the condition of patients with underlying cardiovascular disease.

Free inorganic iron is extremely toxic, but iron is required for essential proteins such as hemoglobin; therefore, evolution has provided an elaborate system for regulating iron absorption, transport, and storage (Figure 33–1). The system uses specialized transport, storage, ferrireductase, and ferroxidase proteins whose concentrations are controlled by the body’s demand for hemoglobin synthesis and adequate iron stores (Table 33–1). A peptide called hepcidin, produced primarily by liver cells, serves as a key central regulator of the system. Nearly all of the iron used to support hematopoiesis is reclaimed from catalysis of the hemoglobin in senescent or damaged erythrocytes. Normally, only a small amount of iron is lost from the body each day, so dietary requirements are small and easily fulfilled by the iron available in a wide variety of foods. However, in special populations with either increased iron requirements (eg, growing children, pregnant women) or increased losses of iron (eg, menstruating women), iron requirements can exceed normal dietary supplies, and iron deficiency can develop.

Vitamin B₁₂ consists of a porphyrin-like ring with a central cobalt atom attached to a nucleotide. Various organic groups may be covalently bound to the cobalt atom, forming different cobalamins. Deoxyadenosylcobalamin and methylcobalamin are the active forms of the vitamin in humans. Cyanocobalamin and hydroxocobalamin (both available for therapeutic use) and other cobalamins found in food sources are converted to the active forms. The ultimate source of vitamin B₁₂ is from microbial synthesis; the vitamin is not synthesized by animals or plants. The chief dietary source of vitamin B₁₂ is microbially derived vitamin B₁₂ in meat (especially liver), eggs, and dairy products. Vitamin B₁₂ is sometimes called extrinsic factor to differentiate it from intrinsic factor, a protein secreted by the stomach that is required for gastrointestinal uptake of dietary vitamin B₁₂.

The average American diet contains 5–30 mcg of vitamin B₁₂ daily, 1–5 mcg of which is usually absorbed. The vitamin is avidly stored, primarily in the liver, with an average adult having a total vitamin B₁₂ storage pool of 3000–5000 mcg. Only trace amounts of vitamin B₁₂ are normally lost in urine and stool. Because the normal daily requirements of vitamin B₁₂ are only about 2 mcg, it would take about 5 years for all of the stored vitamin B₁₂ to be exhausted and for megaloblastic anemia to develop if B₁₂ absorption were stopped. Vitamin B₁₂ is absorbed after it complexes with intrinsic factor, a glycoprotein secreted by the parietal cells of the gastric mucosa. Intrinsic factor combines with the vitamin B₁₂ that is liberated from dietary sources in the stomach and duodenum, and the intrinsic factor-vitamin B₁₂ complex is subsequently absorbed in the distal ileum by a highly selective receptor-mediated transport system. Vitamin B₁₂ deficiency in humans most often results from malabsorption of vitamin B₁₂ due either to lack of intrinsic factor or to loss or malfunction of the absorptive mechanism in the distal ileum. Nutritional deficiency is rare but may be seen in strict vegetarians after many years without meat, eggs, or dairy products.

Two essential enzymatic reactions in humans require vitamin B₁₂ (Figure 33–2). In one, methylcobalamin serves as an intermediate in the transfer of a methyl group from N ⁵-methyltetrahydrofolate to homocysteine, forming methionine (see Figure 33–2A; Figure 33–3, section 1). Without vitamin B₁₂, conversion of the major dietary and storage folate—N ⁵-methyltetrahydrofolate—to tetrahydrofolate, the precursor of folate cofactors, cannot occur. As a result, vitamin B₁₂ deficiency leads to deficiency of folate cofactors necessary for several biochemical reactions involving the transfer of one-carbon groups. In particular, the depletion of tetrahydrofolate prevents synthesis of adequate supplies of the deoxythymidylate (dTMP) and purines required for DNA synthesis in rapidly dividing cells, as shown in Figure 33–3, section 2. The accumulation of folate as N ⁵-methyltetrahydrofolate and the associated depletion of tetrahydrofolate cofactors in vitamin B₁₂ deficiency have been referred to as the “methylfolate trap.” This is the biochemical step whereby vitamin B₁₂ and folic acid metabolism are linked, and it explains why the megaloblastic anemia of vitamin B₁₂ deficiency can be partially corrected by ingestion of large amounts of folic acid. Folic acid can be reduced to dihydrofolate by the enzyme dihydrofolate reductase (see Figure 33–3, section 3) and thereby serve as a source of the tetrahydrofolate required for synthesis of the purines and dTMP required for DNA synthesis.

Vitamin B₁₂ is used to treat or prevent deficiency. The most characteristic clinical manifestation of vitamin B₁₂ deficiency is megaloblastic, macrocytic anemia (see Table 33–2), often with associated mild or moderate leukopenia or thrombocytopenia (or both), and a characteristic hypercellular bone marrow with an accumulation of megaloblastic erythroid and other precursor cells. The neurologic syndrome associated with vitamin B₁₂ deficiency usually begins with paresthesias in peripheral nerves and weakness that progresses to spasticity, ataxia, and other central nervous system dysfunctions. Correction of vitamin B₁₂ deficiency arrests the progression of neurologic disease, but it may not fully reverse neurologic symptoms that have been present for several months. Although most patients with neurologic abnormalities caused by vitamin B₁₂ deficiency have megaloblastic anemia when first evaluated, occasional patients have few if any hematologic abnormalities.

Folic acid (pteroylglutamic acid) is composed of a heterocycle (pteridine), p-aminobenzoic acid, and glutamic acid (Figure 33–4). Various numbers of glutamic acid moieties are attached to the pteroyl portion of the molecule, resulting in monoglutamates, triglutamates, or polyglutamates. Folic acid undergoes reduction, catalyzed by the enzyme dihydrofolate reductase (“folate reductase”), to give dihydrofolic acid (see Figure 33–3, section 3). Tetrahydrofolate is subsequently transformed to folate cofactors possessing one-carbon units attached to the 5-nitrogen, to the 10-nitrogen, or to both positions (see Figure 33–3). Folate cofactors are interconvertible by various enzymatic reactions and serve the important biochemical function of donating one-carbon units at various levels of oxidation. In most of these, tetrahydrofolate is regenerated and becomes available for reutilization.

The average American diet contains 500–700 mcg of folates daily, 50–200 mcg of which is usually absorbed, depending on metabolic requirements. Pregnant women may absorb as much as 300–400 mcg of folic acid daily. Various forms of folic acid are present in a wide variety of plant and animal tissues; the richest sources are yeast, liver, kidney, and green vegetables. Normally, 5–20 mg of folates is stored in the liver and other tissues. Folates are excreted in the urine and stool and are also destroyed by catabolism, so serum levels fall within a few days when intake is diminished. Because body stores of folates are relatively low and daily requirements high, folic acid deficiency and megaloblastic anemia can develop within 1–6 months after the intake of folic acid stops, depending on the patient’s nutritional status and the rate of folate utilization.

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described earlier in the discussion of vitamin B₁₂, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N⁵, N¹⁰-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (see Figure 33–3, section 2). Unlike all the other enzymatic reactions that use folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, 1 mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate are consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N⁵, N¹⁰-methylenetetrahydrofolate by the action of serine transhydroxymethylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs: methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 54).

Folate deficiency results in a megaloblastic anemia that is microscopically indistinguishable from the anemia caused by vitamin B₁₂ deficiency (see above). However, folate deficiency does not cause the characteristic neurologic syndrome seen in vitamin B₁₂ deficiency. In patients with megaloblastic anemia, folate status is assessed with assays for serum folate or for red blood cell folate. Red blood cell folate levels are often of greater diagnostic value than serum levels, because serum folate levels tend to be labile and do not necessarily reflect tissue levels.

Lipoproteins have hydrophobic core regions containing cholesteryl esters and triglycerides surrounded by unesterified cholesterol, phospholipids, and apoproteins. Certain lipoproteins contain very high-molecular-weight B proteins that exist in two forms: B-48, formed in the intestine and found in chylomicrons and their remnants; and B-100, synthesized in liver and found in VLDL, VLDL remnants (IDL), LDL (formed from VLDL), and Lp(a) lipoproteins. HDL consist of at least 20 discrete molecular species containing apolipoprotein A-I (apo A-I). About 100 other proteins are known to be distributed variously among the HDL species, exhibiting antioxidant, antimicrobial, anti-inflammatory, and molecular signaling activities. They also transport microRNAs. Twelve HDL species are recognized in the ovary and six in cerebrospinal fluid.

Chylomicrons are not present in the serum of normal individuals who have fasted 10 hours. The recessive traits of genetically compromised LPL, its cofactor Apo C-II, and the LMF1, CREB3L3, or GPIHBP1 proteins are usually associated with severe lipemia (1000 mg/dL of triglycerides or higher when the patient is consuming a typical American diet). Mutations in Apo A-V can impair lipolysis in both the homozygous and heterozygous states. Familial chylomicronemia might not be diagnosed until an attack of acute pancreatitis occurs or a woman becomes pregnant. Patients may have eruptive xanthomas, hepatosplenomegaly, hypersplenism, lipemia retinalis, and lipid-laden foam cells in bone marrow, liver, and spleen. The lipemia is increased by estrogens because they stimulate VLDL production. Although these patients have a predominant chylomicronemia, they may also have elevated VLDL, presenting with a pattern called mixed lipemia. Deficiency of lipolytic activity can be diagnosed after intravenous injection of heparin. A presumptive diagnosis is made by demonstrating a pronounced decrease in triglycerides 72 hours after elimination of dietary fat. Marked restriction of dietary fat, weight control, exercise, and abstention from alcohol are the basis of effective long-term treatment of chylomicronemia and all hypertriglyceridemias. A fibrate, niacin, or marine omega-3 fatty acids may be of some benefit if VLDL levels are increased. Apo C-III antisense, available in Europe as volanesorsen, is a potential adjunct to therapy. Plasmapheresis may contribute to the rapid reduction of triglycerides in the setting of acute pancreatitis.

The primary hypertriglyceridemias probably reflect a variety of genetic determinants. Variants in a large number of genes have been implicated as causative in patients with hypertriglyceridemia, for which a polygenic risk score has been developed. Many patients have central obesity with insulin resistance. Impaired removal of triglyceride-rich lipoproteins with overproduction of VLDL can result in mixed lipemia. Eruptive xanthomas, lipemia retinalis, epigastric pain, and pancreatitis are variably present depending on the severity of the lipemia. Treatment is primarily dietary. Marine omega-3 fatty acids, especially EPA only, may be helpful for patients with coronary artery disease or who are at high risk. Some patients require treatment with a statin if LDL is elevated, and a fibrate may be needed if triglycerides are consistently greater than 500 mg/dL. If insulin resistance is not present, niacin may be helpful. Metformin is useful in patients with insulin resistance.

The genetic basis of FCH is undetermined but probably involves multiple loci. In this very common disorder, which is associated with an increased incidence of coronary disease, individuals may have elevated levels of VLDL, LDL, or both, and the pattern may change with time. An elevated level of Apo B-100 is a constant feature. FCH involves an approximate doubling in VLDL secretion and appears to be transmitted as a dominant trait. Triglycerides can be increased by factors noted above. Elevations of cholesterol and triglycerides are generally moderate. Diet alone does not normalize lipid levels. A statin alone, or in combination with niacin or fenofibrate, is often required to treat these patients. When fenofibrate is combined with a statin, either pravastatin or rosuvastatin is recommended because neither is metabolized via CYP3A4. Marine omega-3 fatty acids may be useful.

In this disorder, remnants of chylomicrons and VLDL accumulate and levels of LDL are decreased. Because remnants are rich in cholesteryl esters, the level of total cholesterol may be as high as that of triglycerides. Diagnosis is confirmed by the absence of the ε3 and ε4 alleles of apo E, the ε2/ε2 genotype. Other rare apo E isoforms that lack receptor ligand properties can also be associated with this disorder. Patients often develop tuberous or tuberoeruptive xanthomas, or characteristic planar xanthomas of the palmar creases. They tend to be obese, and some have impaired glucose tolerance. These factors, as well as hypothyroidism, can increase the lipemia. Coronary and peripheral atherosclerosis occurs with increased frequency. Weight loss, together with decreased fat, cholesterol, and alcohol consumption, may be sufficient. Statins are often effective because they increase hepatic LDL receptors that participate in remnant removal. A fibrate is sometimes needed to control the condition.

This is a common autosomal dominant trait. Although levels of LDL tend to increase throughout childhood, the diagnosis can often be made on the basis of elevated umbilical cord blood cholesterol. In most heterozygotes, cholesterol levels range from 260 to 400 mg/dL. Triglycerides are usually normal. Tendon xanthomas are often present. Arcus corneae and xanthelasma may also be present. Coronary disease tends to occur prematurely. In homozygous FH, which can lead to coronary disease in childhood, levels of cholesterol can range from 500 to over 1000 mg/dL. Early tuberous, tendinous, and planar xanthomas occur, and aortic stenosis is common.

Defects in the domain of apo B-100 that binds to the LDL receptor impair the endocytosis of LDL, leading to hypercholesterolemia of moderate severity. Tendon xanthomas may occur. Response to reductase inhibitors is variable. Upregulation of LDL receptors in liver does not increase uptake of ligand-defective LDL particles. Fibrates or niacin may have beneficial effects by reducing VLDL production.

The receptor chaperone PCSK9 normally conducts the receptor to the lysosome for degradation (see Figure 35-2). Gain-of-function mutations in PCSK9 are associated with elevated levels of LDL-C and are managed with a PCSK9 antibody.

The RAP1 gene product facilitates the uptake in hepatocytes of the LDL receptor and its associated LDL particle, enhancing the removal of LDL from plasma. Rare defective variants lead to increased LDL, clinically resembling homozygous FH. Statins and bile acid sequestrants may be effective. A similar mechanism for an Apolipoprotein E variant (Leu 167 del) has been described.

Decreased catabolism of cholesterol to bile acids and accumulation of cholesterol in hepatocytes result from loss of function mutations in CYP7a. LDL in plasma is increased with downregulation of LDL receptors. LDL is moderately elevated in heterozygous patients. Homozygous patients have higher LDL levels, sometimes elevated triglycerides, early coronary disease, increased risk of gallstones, and are resistant to statins. The addition of niacin to a statin results in a significant reduction in lipid levels.

As described above, some persons with FCH have only an elevation in LDL necessitating treatment with a statin.

This genetic disorder, which is associated with increased atherogenesis and arterial thrombus formation, is determined chiefly by alleles that dictate increased production of the (a) protein moiety. Lp(a) can be secondarily elevated in patients with severe nephrosis and certain other inflammatory states. Niacin reduces levels of Lp(a) in many patients, and an (a) antisense will be available soon. Reduction of levels of LDL-C below 100 mg/dL decreases the risk of atherosclerosis attributable to Lp(a), and the administration of low-dose aspirin may reduce the risk of thrombus. PCSK9 MABs also reduce levels of Lp(a) by about 25%.

Individuals lacking activity of lysosomal acid lipase (LAL) accumulate cholesteryl esters in liver and macrophages leading to hepatomegaly with subsequent fibrosis, and atherosclerosis. They have elevated levels of LDL-C, low levels of HDL-C, and often modest hypertriglyceridemia. Rarely, a fatal totally ablative form, Wolman disease, occurs in infancy. A recombinant replacement enzyme therapy, sebelipase alfa given intravenously weekly or every other week, effectively restores the hydrolysis of cholesteryl esters in liver, normalizing plasma lipoprotein levels.

The ABCG5 and ABCG8 half-transporters act together in enterocytes and hepatocytes to export phytosterols into the intestinal lumen and bile, respectively. Homozygous or combined heterozygous ablative mutations in either transporter result in elevated levels of LDL enriched in phytosterols, tendon and tuberous xanthomas, and accelerated atherosclerosis. Many techniques for quantitation of cholesterol include phytosterols and can misdiagnose this disorder. Ezetimibe is the specific therapeutic.

The primary pathway for alcohol metabolism involves alcohol dehydrogenase (ADH), a family of cytosolic enzymes that catalyze the conversion of alcohol to acetaldehyde (see Figure 23–1, left). These enzymes are located mainly in the liver, but small amounts are found in other organs such as the brain and stomach. There is considerable genetic variation in ADH enzymes, affecting the rate of ethanol metabolism and also appearing to alter vulnerability to alcohol-abuse disorders. For example, one ADH allele (the ADH1B^*2 allele), which is associated with rapid conversion of ethanol to acetaldehyde, has been found to be protective against alcohol dependence in several ethnic populations, especially East Asians.

This enzyme system, also known as the mixed function oxidase system, uses NADPH as a cofactor in the metabolism of ethanol (see Figure 23–1, right) and consists primarily of cytochrome P450 2E1, 1A2, and 3A4 (see Chapter 4).

Much of the acetaldehyde formed from alcohol is oxidized in the liver in a reaction catalyzed by mitochondrial NAD-dependent aldehyde dehydrogenase (ALDH). The product of this reaction is acetate (see Figure 23–1), which can be further metabolized to CO₂ and water, or used to form acetyl-CoA.

The CNS is markedly affected by acute alcohol consumption. Alcohol causes sedation, relief of anxiety and, at higher concentrations, slurred speech, ataxia, impaired judgment, and disinhibited behavior, a condition usually called intoxication or drunkenness (Table 23–1). These CNS effects are most marked as the blood level is rising, because acute tolerance to the effects of alcohol occurs after a few hours of drinking. For chronic drinkers who are tolerant to the effects of alcohol, higher concentrations are needed to elicit these CNS effects. For example, an individual with chronic alcoholism may appear sober or only slightly intoxicated with a blood alcohol concentration of 300–400 mg/dL (0.30-0.40%), whereas this level is associated with marked intoxication or even coma in a nontolerant individual. The propensity of moderate doses of alcohol to inhibit the attention and information-processing skills as well as the motor skills required for operation of motor vehicles has profound effects. Approximately 30–40% of all traffic accidents resulting in a fatality in the United States involve at least one person with blood alcohol near or above the legal level of intoxication, and drunken driving is a leading cause of death in young adults.

Significant depression of myocardial contractility has been observed in individuals who acutely consume moderate amounts of alcohol, ie, at a blood concentration above 100 mg/dL.

Ethanol is a vasodilator, probably as a result of both CNS effects (depression of the vasomotor center) and direct smooth muscle relaxation caused by its metabolite, acetaldehyde. In cases of severe overdose, hypothermia—caused by vasodilation—may be marked in cold environments. Preliminary evidence indicates that flibanserin, a drug that interacts with 5-HT receptors, augments the hypotensive effects of ethanol and may cause severe orthostatic hypotension and syncope (see Chapter 16). Ethanol also relaxes the uterus and—before the introduction of more effective and safer uterine relaxants (eg, calcium channel antagonists)—was used intravenously for the suppression of premature labor.

Liver disease is the most common medical complication of alcohol abuse; an estimated 15–30% of chronic heavy drinkers eventually develop severe liver disease. Alcoholic fatty liver, a reversible condition, may progress to alcoholic hepatitis and finally to cirrhosis and liver failure. In the USA, chronic alcohol abuse is the leading cause of liver cirrhosis and of the need for liver transplantation. The risk of developing liver disease is related both to the average amount of daily consumption and to the duration of alcohol abuse. Women appear to be more susceptible to alcohol hepatotoxicity than men. Concurrent infection with hepatitis B or C virus increases the risk of severe liver disease. Cirrhosis contributes to elevated portal blood pressure and esophageal and gastric venous varices. These varices may rupture and result in massive bleeding.

Alcohol indirectly affects hematopoiesis through metabolic and nutritional effects and may also directly inhibit the proliferation of all cellular elements in bone marrow. The most common hematologic disorder seen in chronic drinkers is mild anemia resulting from alcohol-related folic acid deficiency. Iron-deficiency anemia may result from gastrointestinal bleeding. Alcohol has also been implicated as a cause of several hemolytic syndromes, some of which are associated with hyperlipidemia and severe liver disease.

Chronic alcohol use has important effects on the endocrine system and on fluid and electrolyte balance. Clinical reports of gynecomastia and testicular atrophy in alcoholics with or without cirrhosis suggest a derangement in steroid hormone balance.

Chronic maternal alcohol abuse during pregnancy is associated with teratogenic effects, and alcohol is a leading cause of mental retardation and congenital malformation. The abnormalities that have been characterized as fetal alcohol syndrome include (1)  intrauterine growth retardation, (2) microcephaly, (3) poor coordination, (4) underdevelopment of midfacial region (appearing as a flattened face), and (5) minor joint anomalies. More severe cases may include congenital heart defects and mental retardation. Although the level of alcohol intake required to cause serious neurologic deficits appears quite high, the threshold for more subtle neurologic deficits is uncertain.

The effects of alcohol on the immune system are complex; immune function in some tissues is inhibited (eg, the lung), whereas pathologic, hyperactive immune function in other tissues is triggered (eg, liver, pancreas). In addition, acute and chronic exposure to alcohol have widely different effects on immune function. The types of immunologic changes reported for the lung include suppression of the function of alveolar macrophages, inhibition of chemotaxis of granulocytes, and reduced number and function of T cells. In the liver, there is enhanced function of key cells of the innate immune system (eg, Kupffer cells, hepatic stellate cells) and increased cytokine production. In addition to the inflammatory damage that chronic heavy alcohol use precipitates in the liver and pancreas, it predisposes to infections, especially of the lung, and worsens the morbidity and increases the mortality risk of patients with pneumonia.

Chronic alcohol use increases the risk for cancer of the mouth, pharynx, larynx, esophagus, and liver. Evidence also points to a small increase in the risk of breast cancer in women. A threshold level for alcohol consumption as it relates to cancer has not been determined. Alcohol itself does not appear to be a carcinogen in most test systems. However, its primary metabolite, acetaldehyde, can damage DNA, as can the reactive oxygen species produced by increased cytochrome P450 activity. Other factors implicated in the link between alcohol and cancer include changes in folate metabolism and the growth-promoting effects of chronic inflammation.

Aminoglycosides have a hexose ring, either streptidine (in streptomycin) or 2-deoxystreptamine (in other aminoglycosides), to which various amino sugars are attached by glycosidic linkages (Figures 45–1 and 45–2). They are water-soluble, stable in solution, and more active at alkaline than at acid pH.

The mode of action of streptomycin has been studied more closely than that of other aminoglycosides, but all aminoglycosides are thought to act similarly. Aminoglycosides are irreversible inhibitors of protein synthesis. The initial event is passive diffusion via porin channels across the outer membrane (Figure 43–3). Drug is then actively transported across the cell membrane into the cytoplasm by an oxygen-dependent process. The transmembrane electrochemical gradient supplies the energy for this process, and transport is coupled to a proton pump. Low extracellular pH and anaerobic conditions inhibit transport by reducing the gradient. Transport may be enhanced by cell wall–active drugs such as penicillin or vancomycin; this enhancement may be the basis of the synergy of those antibiotics with aminoglycosides.

Three principal mechanisms of resistance have been established: (1) production of a transferase enzyme that inactivates the aminoglycoside by adenylylation, acetylation, or phosphorylation. This is the principal type of resistance encountered clinically. (2) There is impaired entry of aminoglycoside into the cell. This may result from mutation or deletion of a porin protein involved in transport and maintenance of the electrochemical gradient or from growth conditions under which the oxygen-dependent transport process is not functional. (3) The receptor protein on the 30S ribosomal subunit may be deleted or altered as a result of a mutation.

Aminoglycosides are absorbed very poorly from the intact gastrointestinal tract, and almost the entire oral dose is excreted in feces after oral administration. However, the drugs may be absorbed if ulcerations are present. Aminoglycosides are usually administered intravenously as a 30–60 minute infusion. After intramuscular injection, aminoglycosides are well absorbed, giving peak concentrations in blood within 30–90 minutes. After a brief distribution phase, peak serum concentrations are identical to those following intravenous injection. The normal half-life of aminoglycosides in serum is 2–3 hours, increasing to 24–48 hours in patients with significant impairment of renal function. Aminoglycosides are only partially and irregularly removed by hemodialysis—eg, 40–60% for gentamicin—and even less effectively by peritoneal dialysis. Aminoglycosides are highly polar compounds that do not enter cells readily. They are largely excluded from the central nervous system and the eye. In the presence of active inflammation, however, cerebrospinal fluid levels reach 20% of plasma levels, and, in neonatal meningitis, the levels may be higher. Intrathecal or intraventricular injection is required for high levels in cerebrospinal fluid. Even after parenteral administration, concentrations of aminoglycosides are not high in most tissues except the renal cortex. Concentration in most secretions is also modest; in the bile, the level may reach 30% of that in blood. With prolonged therapy, diffusion into pleural or synovial fluid may result in concentrations 50–90% of that of plasma.

All aminoglycosides are ototoxic and nephrotoxic. Ototoxicity and nephrotoxicity are more likely to be encountered when therapy is continued for more than 5 days, at higher doses, in the elderly, and in the setting of renal insufficiency. Concurrent use with loop diuretics (eg, furosemide, bumetanide, or ethacrynic acid) or other nephrotoxic antimicrobial agents (eg, vancomycin or amphotericin) can potentiate nephrotoxicity and should be avoided if possible. Ototoxicity can manifest either as auditory damage, resulting in tinnitus and high-frequency hearing loss initially, or as vestibular damage with vertigo, ataxia, and loss of balance. Nephrotoxicity can be identified by rising serum creatinine levels or reduced estimated glomerular filtration rate, although the earliest indication can be an increase in trough serum aminoglycoside concentrations. Neomycin, kanamycin, and amikacin are the agents most likely to cause auditory damage. Streptomycin and gentamicin are the most vestibulotoxic. Neomycin, tobramycin, and gentamicin are the most nephrotoxic. Plazomicin may be associated with lower rates of ototoxicity and nephrotoxicity compared to older aminoglycosides; however, as the newest aminoglycoside, clinical experience is limited.

Aminoglycosides are mostly used against aerobic gram-negative bacteria, especially when there is concern for drug-resistant pathogens or in critically ill patients. Tobramycin and gentamicin are almost always used in combination with a β-lactam antibiotic to extend empiric coverage and to take advantage of the potential synergy between these two classes of drugs. Amikacin and streptomycin are frequently given in combination with other antibacterials for the treatment of mycobacterial infections. It remains unclear whether plazomicin will be given alone or in combination with other agents. Penicillin-aminoglycoside combinations have also been used to achieve bactericidal activity in treatment of enterococcal endocarditis and to shorten duration of therapy for viridans streptococcal endocarditis. Due to toxicity, these combinations are used less frequently when alternate regimens are available. For example, in the case of enterococcal endocarditis, studies suggest that the combination of ampicillin and ceftriaxone is an effective regimen with less risk for nephrotoxicity. When aminoglycosides are used, the selection of agent and dose depends on the infection being treated and the susceptibility of the isolate.