The Hormonal Regulation of Calcium Metabolism

Calcium plays a vital role in several biological processes including normal neuromuscular transmission, muscular contractility, cellular signaling, enzyme function, and blood coagulation. Hence, the appropriate control of calcium homeostasis is vital to the well-being of the organism. Severe calcium deficiency results in tetany and, in extreme cases, grand mal seizures. The deposition of excess calcium at ectopic sites occurs in several diseases such as nephrolithiasis, arteriosclerosis, valvular calcification, and calciphylaxis. Therefore, an appreciation of how calcium is normally absorbed, excreted, and regulated is important in understanding the pathophysiology of disease.

Calcium homeostasis is a tightly controlled process involving several tissues, hormones, and proteins. The hormones 1α,25-dihydroxyvitamin D (1α,25(OH) ₂ D), parathyroid hormone (PTH), and calcitonin contribute to the regulation of calcium metabolism. These three hormones have specific roles in several tissues including the intestinal mucosa, bone, and renal tubular cells. They work in concert with each other through complex interactions to maintain extracellular fluid calcium concentrations in the normal range.

Keywords

calcium, 25 hydroxyvitamin D ₃ , 1,25 dihydroxyvitamin D ₃ , parathyroid hormone, calcitonin

Introduction

Calcium plays a vital role in several biological processes. It is an important constituent bone mineral, and is necessary for normal neuromuscular transmission, muscular contractility, cellular signaling, enzyme function, and blood coagulation. Hence, the appropriate control of calcium homeostasis is vital to the well-being of the organism. A persistently negative calcium balance results in hyperparathyroidism and bone demineralization and contributes to the pathogenesis of osteomalacia and the development of osteoporosis with attendant vertebral and appendicular fractures. Severe calcium deficiency results in tetany and, in extreme cases, grand mal seizures. The deposition of excess calcium at ectopic sites occurs in several diseases such as nephrolithiasis, arteriosclerosis, valvular calcification, and calciphylaxis. Therefore, an appreciation of how calcium is normally absorbed, excreted and regulated is important in understanding the pathophysiology of disease.

Calcium homeostasis is a tightly controlled process involving several tissues, hormones, and proteins. The hormones 1α,25-dihydroxyvitamin D (1α,25(OH) ₂ D), parathyroid hormone (PTH), and calcitonin contribute to the regulation of calcium metabolism. These three hormones have specific roles in several tissues including the intestinal mucosa, bone, and renal tubular cells. They work in concert with each other through complex interactions to maintain extracellular fluid calcium concentrations in the normal range. In this chapter, we will discuss the physiological role of each of these hormones in calcium homeostasis.

Calcium Balance

Many tissues are dependent on the maintenance of extracellular calcium concentrations within the physiological range for proper function. If extracellular calcium concentrations are significantly altered or disrupted, tissue and organ dysfunction may result (see above). Bone is the major reservoir of calcium, containing about 99% of total body calcium stores, and provides a significant buffer to maintain extracellular calcium concentrations within the normal range if calcium intake declines or calcium losses occur. The maintenance of serum calcium concentrations in the face of significant overall deficits in calcium balance occurs at the expense of bone integrity. Serum calcium concentrations (range 8.9–10.1 mg/dL in adults) are age dependent, and small differences in circulating concentrations occur as a result of gender. Table 65.1 shows normal serum calcium concentrations in males and females measured at the Mayo Clinic. Serum calcium is comprised of protein-bound calcium (40%); complexed calcium, i.e., calcium complexed to ions such as citrate, sulfate, and phosphate (13%); and ionized calcium (47%). Total serum calcium concentrations are dependent upon circulating concentrations of albumin and to a smaller extent upon circulating concentration of globulins. The biologically active fraction of serum calcium is ionized (normal range 4.8–5.7 mg/dL in adults) and, at a normal serum pH of 7.4, is approximately 47% of total serum calcium. The percentage of ionized calcium changes with pH—an alkaline pH causing a reduction in free ionized calcium concentrations and an acid pH causing an increase in ionized calcium concentrations. The relationships between ionized calcium, total calcium, albumin and pH are defined by the following equation:

A 1-gram/deciliter change in serum albumin concentration is associated with a 0.8-milligram/deciliter change in total serum calcium. Since globulins bind calcium less avidly than does albumin, a 1-gram/deciliter change in globulins results in a 0.16-milligram/deciliter change in total serum calcium. It should be noted that changes in the concentrations of serum proteins are generally not associated with changes in the percentage of ionized calcium present in the circulation. Also, it is worth remembering that the amount of calcium filtered at the glomerulus of the kidney is a sum of the ionized calcium concentration and the complex calcium concentration (approximately 60% of total serum calcium concentration).

Under normal circumstances of neutral calcium balance in the adult human, net intestinal absorption of calcium is equal to urinary calcium ( Figure 65.1 ). Calcium flux into and out of bone is well balanced with equal amounts of calcium being deposited and resorbed. Over a 24-hour period, a typical human adult ingests about 1000 mg of elemental calcium. Approximately 40% of ingested calcium is absorbed in the intestine and enters the bloodstream. Both active and passive processes are involved in the absorption of calcium from the intestine. When the intestinal lumen calcium concentrations are <10 millimoles/liter, active processes play a major role in calcium absorption. However, when calcium concentrations in the intestinal lumen exceed 10 millimoles/liter, passive processes become operative in the absorption of calcium. About 150 mg of calcium are excreted into the gastrointestinal tract each day, predominantly in pancreatic and intestinal secretions (“endogenous fecal calcium”), for a net calcium absorption of about 250 mg per day. The kidney filters about 9000 mg of calcium each day in the glomeruli and reabsorbs the majority of filtered calcium in the proximal and distal nephron, resulting in a net loss of about 250 mg from the kidney in the urine. A majority of calcium is reabsorbed in the proximal tubule along with sodium (approximately 70 to 85%). The remainder of filtered calcium is reabsorbed in the thick ascending limb of the loop of Henle and in the distal convoluted tubule, largely as a result of the activity of the sodium-calcium exchanger and plasma membrane calcium pump (PMCA). In states of calcium balance, urinary calcium approximates the amount of calcium absorbed in the intestine.

The following sections will describe how the vitamin D endocrine system, PTH, and calcitonin alter calcium homeostasis in various tissues including the intestine, kidney, and bone.

Vitamin D Endocrine System

Nomenclature

The synthesis of the active form of vitamin D, 1α,25-dihydroxyvitamin D, requires sequential metabolic processing of precursor sterols in several tissues such as the skin, liver and kidney. The term vitamin D refers to both vitamin D ₂ and vitamin D ₃ ( Figure 65.2 ). The metabolic processing of both these forms of vitamin D is similar for practical purposes in mammals, although vitamin D ₂ is considerably less active in birds than is vitamin D ₃ . For the purposes of this chapter, it is appropriate to assume that the metabolic transformations that occur with vitamin D ₃ also occur with vitamin D ₂ . Vitamin D ₂ is derived from the plant sterol, ergosterol, whereas, vitamin D ₃ is derived from 7-dehydrocholesterol, a byproduct of steroid metabolism. In non-vitamin D supplemented individuals, a majority of circulating vitamin D is in the form of vitamin D ₃ or cholecalciferol. Medicinal vitamin D preparations available in the United States today may contain either vitamin D ₂ or vitamin D ₃ . Individuals taking large amounts of vitamin D ₂ supplements have elevated concentrations of vitamin D ₂ and its 25-hydroxylated metabolite, 25-hydroxyvitamin D ₂ .

Formation of Vitamin D

In the early 1900s, Huldshinsky and Chick in Vienna showed that exposure of rachitic children to ultraviolet light cured their bone disease. Steenbock and Hart showed that ultraviolet irradiation of animals would put them into positive calcium balance. Later, Steenbock and Black clearly demonstrated that ultraviolet light-induced antirachitic activity in the fat-soluble portion of foods and skin. Hess and Weinstock made similar observations. Building on these observations, Askew and his coworkers isolated vitamin D ₂ . Windaus and his colleagues showed that 7-dehydrocholesterol was converted to vitamin D ₃ , and subsequent work showed that this process occurred in vivo in normal skin. 7-dehydrocholesterol is not directly converted to vitamin D ₃ in the skin but rather is first converted to pre-vitamin D ₃ that undergoes thermal isomerization to vitamin D ₃ ( Figure 65.3 ). Several other, biologically inert, side-products such as lumisterol and tachysterol are produced during the photolysis of 7-dehydrocholesterol. Vitamin D ₃ has a higher affinity for the vitamin D binding protein (VDBP) than does pre-vitamin D ₃ , and the binding of vitamin D ₃ to VDBP following its formation in the skin facilitates the removal of the vitamin from skin. Vitamin D ₂ and vitamin D ₃ are converted in the liver to 25-hydroxyvitamin D ₂ and 25-hydroxyvitamin D ₃ by the hydroxyvitamin D 25-hydroxylase without significant product inhibition of the enzyme, and consequently circulating 25-hydroxyvitamin D concentrations reflect the amount of vitamin D ingested and the amount of vitamin D formed in the skin. In accord with the earlier observation showing that sunlight exposure enhanced the formation of vitamin D ₃ in the skin, several groups have shown that serum 25-hydroxyvitamin D ₃ concentrations are lower during and immediately after the winter months than in the summer. In the absence of dietary vitamin D supplementation, exposure to ultra-violet B radiation plays an essential role in vitamin D production.

Following conversion of 7-dehydrocholesterol to vitamin D ₃ in the skin, vitamin D ₃ is transported in the plasma bound to VDBP. Any vitamin D ₂ ingested in the diet is also bound to VDBP following absorption in the intestine. Vitamin D ₂ and vitamin D ₃ are delivered to the liver for hydroxylation by the multicomponent, cytochrome P-450 containing enzyme, vitamin D 25-hydroxylase, which is present in the liver microsomes as well as in mitochondria ( Figure 65.4 ). Hepatic conversion of vitamin D ₃ to 25-hydroxyvitamin D ₃ is not tightly regulated due to a lack of product inhibition of the microsomal vitamin D ₃ 25-hydroxylase. Several cytochrome P-450s have been cloned and shown to metabolize vitamin D ₃ to 25-hydroxyvitamin D ₃ , including several microsomal cytochrome P-450s and one mitochondrial cytochrome P-450. 25-hydroxyvitamin D is the serum metabolite generally measured to determine vitamin D sufficiency or insufficiency in an individual. Accurate determinations of both 25-hydroxyvitamin D ₂ and 25-hydroxyvitamin D ₃ are obtained using high-performance liquid chromatography methods and mass-spectrometry based methods and such methods are preferred to those using protein binding or antibody binding assays.

25-Hydroxyvitamin D ₃ is not biologically active except in large concentrations, and it must be metabolized further in the kidney to the bioactive form of vitamin D, 1α,25-dihydroxyvitamin D ₃ ( Figure 65.5 ). The 25-hydroxyvitamin D ₃ -1α-hydroxylase, the enzyme responsible for the conversion of 25-hydroxyvitamin D ₃ to 1α,25-dihydroxyvitamin D ₃ , is a multicomponent, cytochrome P-450 containing enzyme in the mitochondria of renal proximal tubular cells. Although the kidney is the primary site of 25-hydroxyvitamin D ₃ -1α-hydroxylase activity, several other cell types have been shown to have 1α-hydroxylase activity. It had previously been thought that the proximal tubule epithelial cells were the only renal cells with 25-hydroxyvitamin D ₃ 1α-hydroxylase activity. However, several investigators have clearly shown that this enzyme is present and active in several segments of the renal tubule. The key factors regulating 25-hydroxyvitamin D-1α-hydroxylase production and activity are depicted in Table 65.2 . Parathyroid hormone is a potent stimulator of 25-hydroxyvitmain D-1α-hydroxylase and is discussed in detail in the subsequent sections. In addition to parathyroid hormone, low serum calcium, and low serum phosphorus also stimulate 25-hydroxyvitamin-1α-hydroxylase activity. 1α,25-dihydroxyvitamin D ₃ provides a negative feedback through the vitamin D receptor (VDR). VDR-knockout mice have very high concentrations of 1α,25-dihydroxyvitamin D ₃ .

Table 65.2

Effect of Increased Level or Activity of Various Factors on 1,25(OH) ₂ D ₃ Concentration or 1α-Hydroxylase Activity

[Modified from Kumar R ]

Factor	Animals	Humans	References
Parathyroid hormone	↑	↑
Serum inorganic phosphorus	↓	↓
1α,25(OH) 2 D 3	↓	↓
Calcium (direct)	?	↓
Calcitonin	↑,↓,0	↑
Hydrogen ion	↓	0
Sex steroids	↑	↑
Prolactin	↑	0
Growth hormone and insulin-like growth factor-1	↑	↑,↓,0
Glucocorticoids	↓,0	↑,↓,0
Thyroid hormone	?	↓ a
Fibroblast growth factor 23	↓	?
Frizzled related protein 4	↓	?
Pregnancy	↑	↑ a

a Effects may be secondary to changes in calcium, phosphorus or parathyroid hormone. ↑, Stimulation or increase; ↓, suppression or decrease; 0, no effect; ?, unknown effect.

Recently, other proteins referred to as “phosphatonins” that induce renal phosphate loss have been shown to inhibit renal 25-hydroxyvitamin-1α-hydroxylase activity and 1α,25-hydroxyvitamin D ₃ production. Two such proteins include fibroblast growth factor 23 (FGF23) and secreted frizzled related protein 4 (sFRP4). Both of these proteins are able to inhibit renal tubule phosphate reabsorption which leads to hypophosphatemia. Despite the hypophosphatemia, which is a potent stimulator of 25-hydroxyvitamin-1α-hydroxylase activity, FGF23 and sFRP4 are capable of preventing the conversion of 25-hydroxyvitamin D ₃ to 1α,25-dihydroxyvitamin D ₃ . FGF23 and sFRP4 are over-expressed in tumors that cause oncogenic osteomalacia, a condition characterized by hypophosphatemia, hyperphosphaturia, and inappropriately low serum 1α,25-dihydroxyvitamin D ₃ concentrations. These biochemical abnormalities, including the low 1α,25-dihydroxyvitamin D ₃ levels, completely resolve after removal of the offending tumor. These proteins appear to play an important role in mineral and vitamin D metabolism.