Calcium and Magnesium Homeostasis
Winston W. K. Koo
Reginald C. Tsang
Calcium (Ca) is the most abundant mineral in the body and, with phosphorus (P), forms the major inorganic constituent of bone. Magnesium (Mg) is the most abundant intracellular divalent cation. The maintenance of Ca and Mg homeostasis requires a complex interaction of hormonal and nonhormonal factors; adequate functioning of various body systems, particularly the renal, gastrointestinal, and skeletal systems; and adequate dietary intake. Physiologically, Ca and Mg are critical to cell division, cell adhesion and plasma membrane integrity, protein secretion, muscle contraction, neuronal excitability, intermediary metabolism, and coagulation. From a clinical perspective, these physiologic actions are critical to numerous biologic functions including reproduction. Maintenance of circulating concentrations of Ca and Mg in the normal range and integrity of the skeleton are generally used as proxy for mineral homeostasis.
In the circulation, the amount of Ca and Mg is less than 1% of their respective total body content; however, disturbances in serum concentrations of these minerals are associated with disturbances of physiologic function manifested by numerous clinical symptoms and signs. Chronic and severely lowered serum concentrations of these minerals also may reflect a deficiency state.
At all ages, total body content of Ca and Mg in skeleton are about 99% and 66%, respectively. The skeleton is a reservoir for mineral homeostasis in addition to providing structural and mechanical support; disturbances in mineral homeostasis can result in osteopenia and rickets in infants and children and osteomalacia and osteoporosis in adults.
The mechanisms to maintain mineral homeostasis in neonates are the same as for children and adults. However, the newborn infant has unique challenges to homeostasis during adaptation to extrauterine life and to continue a rapid growth rate. These include an abrupt discontinuation of the high rate of intrauterine accretion of Ca (approximately 120 mg/kg/d) and Mg (approximately 4 mg/kg/d) during the third trimester; small skeletal reservoir for mineral homeostasis; delay in establishment of adequate nutrient intake for a few days or longer, particularly in sick and preterm infants; and high requirement for Ca and Mg for the most rapid period of postnatal skeletal growth, with an average length gain of greater than 25 cm during the first year. There also may be diminished end-organ responsiveness to hormonal regulation of mineral homeostasis, although the functional capacity of the gut and kidney improves rapidly within days after birth. The effects of these issues are exaggerated in infants with heritable disorders of mineral metabolism, such as extracellular calcium-sensing receptor (CaR) mutations, and infants who have adverse antenatal events such as maternal diabetes, intrapartum problems such as perinatal asphyxia or maternal Mg therapy, or postnatal problems such as immature functioning of multiple organs from preterm birth.
Increased understanding of physiologic and molecular basis of mineral metabolism allows a better understanding of the pathophysiology of the clinical mineral disorders and a more rational management to minimize adverse impacts from disturbed mineral homeostasis and prevent iatrogenic causes precipitating or prolonging these problems.
▪ TISSUE DISTRIBUTION
In the fetus, about 80% of minerals accrue between 25 weeks of gestation and term. During this period, estimated daily accretion per kilogram fetal body weight is 2.3 to 2.98 mmol (92 to 119 mg) Ca and 0.1 to 0.14 mmol (2.4 to 3.36 mg) Mg. Peak accretion rates occur at 36 to 38 weeks of gestation. In newborn term infants, the total body Ca and Mg contents average approximately 28 and 0.7 g, respectively (1,2).
After birth, 99% of total body Ca is in bone. The remaining 1% is in blood, extracellular fluid (ECF), and soft tissues. The tissue distribution of Mg varies according to extent of bone mineralization and rate of soft tissue growth. Near the end of the third trimester, about 60% of Mg is in bone, 20% in muscle, 1% in ECF including blood, and most of the remainder is in intracellular space of other tissues.
▪ CIRCULATING CONCENTRATION
Calcium
Serum Ca (1 mmol/L = 4 mg/dL) occurs in three forms: approximately 40% is bound, predominantly to albumin; 10% is chelated and complexed to small molecules such as citrate and phosphate; and approximately 50% is ionized.
Total Ca concentrations (tCa) in cord sera increase with increasing gestational age. Serum tCa may be as high as 3 mmol/L in cord blood of infants born at term and are significantly higher than paired maternal values at delivery (3,4,5,6). Serum tCa reaches a nadir during the first 2 days after birth (7,8,9,10,11,12,13); thereafter, concentrations increase and stabilize generally above 2 mmol/L (14). In infants fed human milk, serum tCa tends to be higher and may reach greater than 2.75 mmol/L (15,16) and accompanied by a lower serum P (15,17). Normally, serum tCa in children and adults remains stable, with a diurnal range of less than 0.13 mmol/L. During the third trimester of pregnancy, a modest reduction in maternal serum tCa concentration (average 0.1 mmol/L) is associated with decrease in serum albumin concentration.
Serum ionized Ca (iCa) concentration is the best indicator of physiologic blood Ca activity. Serum iCa decreases in the presence of high serum albumin, phosphorus, bicarbonate, and heparin and is inversely related to blood pH; and increases with increased Mg. Direct determination of iCa from whole blood, plasma, and serum is simple, rapid, and freely available. To minimize interference from physiologic- or laboratory-related factors, iCa should be measured immediately upon sample collection (18). Some differences exist from different iCa analyzers (19), and normative data should be generated according to subject’s age, the instrument used, and the type of sample measured.
Cord serum iCa increases with increasing gestational age and is higher than paired maternal sera. In healthy term neonates, serum iCa averages 1.25 mmol/L with 95% confidence limits of 1.1 to 1.4 mmol/L (4.4 to 5.6 mg/dL). There is decline in serum iCa in the first 48 hours after birth with nadir at 24 hours (20). Serum iCa generally changes in parallel with tCa in healthy humans. However, correlation between serum tCa and iCa is inadequate to predict the value of one from the other with sufficient accuracy particularly during illness; serum iCa is stable and normal during pregnancy.
In the cell, distribution of Ca is not uniform. The cytosolic compartment contains 50 to 150 nmol of Ca per liter of water; a larger intramitochondrial Ca pool contains 500 to 10,000 nmol of Ca per liter of cell water. In contrast, the concentration of iCa in ECF is 1 million nmol/L (1 mmol/L). There is also a 50 mV positive electrical charge across the plasma membrane with the cell interior negative. Thus, Ca homeostasis involves maintenance of approximately 1,000-fold Ca concentration gradient across the cellular plasma membrane to prevent Ca-induced cell death. Ca is actively extruded by adenosine triphosphate (ATP)-dependent
energy-driven Ca pumps and Ca channels and by sodium (Na)-Ca exchanger. The binding of intracellular Ca by proteins located in cytosol, endoplasmic reticulum, and mitochondria buffer intracellular Ca and can be mobilized to maintain cytosol Ca levels and create pulsatile peaks of Ca to mediate membrane receptor signaling. Ca ion is an essential intracellular second messenger, but iCa also functions as messenger outside cells through cellular calcium sensing receptor (CaR).
energy-driven Ca pumps and Ca channels and by sodium (Na)-Ca exchanger. The binding of intracellular Ca by proteins located in cytosol, endoplasmic reticulum, and mitochondria buffer intracellular Ca and can be mobilized to maintain cytosol Ca levels and create pulsatile peaks of Ca to mediate membrane receptor signaling. Ca ion is an essential intracellular second messenger, but iCa also functions as messenger outside cells through cellular calcium sensing receptor (CaR).
Magnesium
Approximately 30% of serum Mg (1 mmol/L = 2.4 mg/dL) is in protein-bound form, with the remainder in the ultrafilterable portion. Seventy to eighty percent of ultrafilterable Mg is in ionic form, and the remainder is complexed to anions, particularly phosphate, citrate, and oxalate. Cord serum total Mg (tMg) is higher than paired maternal values. Serum tMg of 0.92 ± 0.13 mmol/L (mean ± 2 SD) in children is slightly higher than adult values of 0.88 ± 0.13 mmol/L (21). Ion-selective electrodes are used in measurement of ionized Mg (iMg) in whole blood and sera although Ca interferes with iMg measurements during simultaneous measurements of iCa and iMg (22). iMg concentrations average 62% to 70% of tMg in cord and postnatal sera. Cord serum iMg is also higher than in maternal serum (23,24,25). There is poor correlation between tMg and iMg in sick patients (26), and the clinical role of iMg (vs. tMg) in disease states appears limited (27).
Mg is the most abundant divalent cation within cells at 6 to 9 mmol/kg wet weight and is predominantly localized in membrane structures (e.g., microsome, mitochondria, and plasma membrane). Cytosolic Mg is approximately 5 × 10-4 M and tends to be membrane bound among intracellular organelles, of which approximately 60% is within mitochondria. Ionic cytosolic Mg accounts for approximately 5% to 10% of total cellular Mg. Unbound intracellular Mg is critical for essential cell functions such as intermediary metabolism, cell signaling, growth, and proliferation (28). Intracellular Mg usually remains stable despite fluctuations in serum Mg. In Mg-deficient states, however, intracellular content of Mg can be low despite normal serum concentrations.
▪ PHYSIOLOGIC CONTROL OF MINERAL HOMEOSTASIS
In the growing human, Ca and Mg homeostasis rely on dietary intake and three components, including (a) absorption, excretion, and tissue accretion involving mainly the gastrointestinal tract, kidney, and bone; (b) direct modulation of transport and mobilization of these minerals mainly by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25[OH]2D), or indirectly via fibroblast growth factor 23 (FGF23) and by other factors; and (c) sensors controlling transport of Ca and Mg ions. Furthermore, there are Ca-Mg interactions, such that when hypocalcemia coexists with hypomagnesemia, the former may not respond to therapy until hypomagnesemia is corrected.
Regulation of Ca and Mg Absorption, Excretion, and Tissue Accretion
Intestinal absorption of Ca and Mg involves passive paracellular concentration-dependent and saturable active transcellular processes. Paracellular absorption takes place throughout the small intestine and is dependent on concentration gradient. Active transcellular Ca absorption takes place largely in duodenum. About 90% of the Ca absorbed is through the small intestine and less than 10% is absorbed through the large intestine. Mg is absorbed throughout the entire intestinal tract with maximal absorption at distal jejunum and ileum. In the usual range of dietary intake, fractional intestinal Ca and Mg absorption is inversely proportional to amount ingested and the body’s needs. At all ages, particularly in young children, dietary Ca absorption is primarily regulated by Ca rather than vitamin D intake (29,30,31). Vitamin D through its most active metabolite 1,25(OH)2D has influence on active Ca and Mg transport, but its role under normal circumstances appears much less than that of the passive diet-dependent process in the growing child. The net absorption of Ca and Mg is higher in rapidly growing children and is 30% to 50% for Ca and 40% to 60% for Mg.
Gastric acid aids in digestion of natural or Ca-fortified food or drink. Some Ca compounds such as calcium citrate are better absorbed in individuals with decreased gastric acid when compared to calcium carbonate. The efficiency of Ca and Mg absorption decreases with increased amounts of Ca and Mg consumed and type and content of carbohydrate, phytic acid and/or oxalic acid that may bind to Ca and Mg and prevent optimal absorption.
The kidney plays an important role in homeostasis of divalent ions. It also has passive and active transport components mediated by transporters and channels. Most ionized forms of Ca and Mg are reabsorbed at proximal tubules and thick ascending limb (TAL) of Henle loop via a passive paracellular pathway, dependent on salt and water reabsorption and rate of fluid flow. The distal convoluted tubule (DCT) and connecting tubule are sites where iCa and iMg are reabsorbed via active transcellular transport. The latter is the final determinant of plasma Mg concentrations. Renal reabsorption is highly efficient but can be overwhelmed. For example, some neonates fed cow milk-based infant formulas with higher phosphorus content than human milk can develop hyperphosphatemia with secondary hypocalcemia from incomplete renal phosphorus excretion (32,33). Conversely, during phosphorus-deficient states from inadequate intake as in very-low-birth-weight (VLBW, <1,500 g) infants fed unfortified human milk or abnormal loss from the gastrointestinal tract, renal conservation alone is unable to prevent development of bone demineralization and abnormally low circulating P concentrations (34,35).
Renal Ca and Mg transport is affected by hormonal (PTH, calcitonin [CT], glucagon, arginine vasopressin, 17-beta-estradiol) and nonhormonal factors. Inhibition of Mg and Ca reabsorption leading to increased urine excretion of both cations can result from high intake of glucose, sodium, Ca, and Mg; elevated serum Mg or Ca; depletion of potassium and phosphate; and high intake of caffeine and metabolic acidosis (36). Chronic use of Mg antacids and potent loop diuretics such as furosemide can increase urine divalent cation loss. Aluminum antacids should not be used, especially for those with limited renal function such as infants, because of potential aluminum toxicity (37). Chronic therapy with a proton pump inhibitor results in hypomagnesemia, decreased Ca absorption, and increased risk for fractures and Clostridium difficile diarrhea (38).
Bone formation, resorption, and modeling are important in skeletal growth. Normally, the growing child has net bone (and soft tissue) accretion of minerals. Retention of Ca generally reflects the body’s need and may be greater than 60% of dietary intake during periods of rapid growth. Some exchange of minerals occurs normally during bone modeling. The exchangeable portion may be increased during periods of stress and increased bone turnover. Local factors such as transforming growth factor-&bgr;1, lymphotoxin, tumor necrosis factor (TNF)-&agr;, IFN-&ggr;, IL-1, and IL-6 acting in a paracrine (i.e., cell-to-cell) or autocrine (i.e., cell-to-self) fashion may influence Ca flux of bone cells, especially under pathologic situations. IFN-&ggr; from activated macrophages (39) stimulates CYP1&agr; mRNA and enzyme production with little or no feedback inhibition by 1,25(OH)2D, which potentially may compromise Ca homeostasis.
During severe and prolonged Ca or P deficiency, infants develop rickets, osteopenia, fractures, hypophosphatemia and, in extreme cases, hypocalcemia, since hormonal regulation is overwhelmed. In less extreme circumstances, pregnant women with very low Ca intake but otherwise adequate diets can have a fetus with decreased
bone mineral content. This deficit is preventable by adequate maternal Ca intake from diet or from Ca supplementation (40,41).
bone mineral content. This deficit is preventable by adequate maternal Ca intake from diet or from Ca supplementation (40,41).
Hormonal and Nonhormonal Regulation of Ca and Mg Homeostasis
Calciotropic hormones, PTH and 1,25(OH)2D, are critical to maintain Ca homeostasis by intermodulation of their physiologic effects and their actions on classic target organs: kidney, intestine, and bone. PTH serves as major rapid response to hypocalcemia, whereas 1,25(OH)2D, with major effect on increasing intestinal absorption of Ca, provides slower sustained contribution to maintenance of normocalcemia. FGF derived primarily from bone cells helps regulate phosphate and vitamin D homeostasis, and CT- and PTH-related peptide (PTHrP) also may be important in mineral homeostasis.
Mg homeostasis is regulated similarly to, but less tightly, than Ca homeostasis. However, Mg is critical to maintenance of Ca homeostasis, since Mg regulates production and secretion of PTH, acts as a cofactor for the 25-hydroxyvitamin D 1&agr;-hydroxylase enzyme in production of 1,25(OH)2D, and maintains sensitivity of target tissues to PTH. Furthermore, Mg is considered a mimic/antagonist of Ca as it often functions synergistically with Ca, yet competes with it in the gut and kidney for transport and other metabolic pathways.
Most regulatory mechanisms are mediated via a feedback and feed-forward loop, with and without receptor-mediated mechanisms. The latter occurs either at transcriptional or at posttranslation level. The classic end organs are the kidney, intestine, and bone. However, multiple other organs are involved either in production or in mediation of the effect of these hormonal regulators.
Parathyroid Hormone
In humans, parathyroid glands are derived from the third and fourth pharyngeal pouch. The PTH gene, along with the genes for insulin, &bgr;-globulin, and CT, is located on chromosome 11p15. PTH is synthesized by chief cells and stored in secretory granules. It is colocated and secreted with chromogranin A, a protein that may act in autocrine- or paracrine-regulated release of PTH (42).
The PTH gene encodes the precursor molecule, a 115-amino-acid prepro-PTH that then undergoes several intracellular proteolytic cleavages of amino-terminal signal sequence to form a 95-amino-acid pro-PTH followed by an 84-amino-acid intact PTH (IPTH) hormone with a relative molecular mass of 9,500 kDa. About 50% of newly generated PTH is degraded intracellularly by calcium-sensitive proteases, and some inactive fragments are also secreted. After release into circulation, IPTH molecule has a serum half-life of 5 to 8 minutes and undergoes cleavages by endopeptidases in the liver and kidney. Amino-terminal fragments contain biologically active fractions, with the 1-34 fragment having most calcemic activity; modifications at the amino-terminal, particularly at the first two residues, can abolish the biologic activity of PTH. The midregion and carboxyl-terminal fragments are biologically inert, although the latter may have in vitro biologic activity (43).
Circulating immunoreactive PTH is a complex mixture of intact 1-84 PTH, peptide fragments from amino- and carboxyl-terminals, and midmolecular regions. Normally, there are more middle and carboxyl fragments than intact hormone in circulation because of metabolic breakdown of the short-lived, intact hormone and glandular secretion of inactive fragments. Fragments are cleared from blood virtually exclusively by glomerular filtration. PTH molecules reactive in the widely used commercial immunoradiometric assays (IRMAs), designed to detect both amino- and carboxyl-terminal epitopes of the peptide, have been considered as “intact” PTH. However, large 7-84 fragment of PTH is also detected. This large fragment is biologically inactive and present in greater concentrations in uremic states or hyperparathyroidism. Chemiluminescence technique also can be used to measure the “whole” or “biointact” PTH. Consistency of PTH assay methodology and serial measurements are critical to interpretation and management of pathologic states.
Serum PTH in adults has a significant circadian periodicity, spontaneous episodic pulsatility with distinct peaks, and significant temporal coupling with serum iCa and P concentrations and prolactin secretion. PTH concentrations in cord blood frequently are low and do not correlate with maternal PTH (4,44). Presence of PTHrP with its PTH-like bioactivity may have accounted for earlier reports of higher bioactive PTH in cord sera from cytochemical assays. Small amounts (approximately 5%) of fragments (35-84, 44-68, and 65-84 amino acids) but probably not the whole PTH molecule are reported to cross the human placenta.
Serum PTH increases postnatally coincidentally with fall in serum Ca in both term and preterm infants (4,44,45,46,47). The rise in serum IPTH is greater for preterm infants with hypocalcemia compared to term infants, reflecting appropriate PTH response. Serum PTH is similar for children and adults but increased in the elderly. Serum IPTH as measured by IRMA shows no change during normal pregnancy.
PTH effects on end-organ systems appear mediated through binding to specific receptors. The type 1 PTH/PTHrP receptor [PTH1R] has been identified in classic target organs of bone, kidney, and intestine; and cartilage, aorta, adrenal gland, brain, skeletal muscle, and urinary bladder. It binds equally to PTH and PTHrP and belongs to a superfamily of guanine nucleotide-binding protein-coupled cell membrane receptors (GPCRs) including those for CT, secretin, growth hormone-releasing hormone, corticotrophinreleasing hormone, glucagon, vasoactive intestinal polypeptide, and others. Type 2 PTH receptor (PTH2R) responds to amino-terminal PTH and no other PTH fragments, although its main endogenous ligand appears to be a 39-amino-acid peptide, hypothalamic tubuloinfundibular peptide [TIP39]. PTH2R has been found in the brain, pancreas, and intestine. Another receptor interacts with carboxy-terminal PTH. The physiologic significance of the latter and PTH2R are not well defined.
The gene for the PTH1R is located on chromosome 3p21.1-p24.2. It contains 17 exons and encodes a mature glycoprotein of 593 amino acids (48). PTH1R consists of extended extracellular, ligand-binding amino-terminal, seven transmembrane and intracellular G protein-associated carboxyl-terminal domains. Signal transduction mediated by G proteins results in at least three different signaling pathways: cyclic AMP (cAMP)/protein kinase A via activation of Gs&agr; signaling; calcium/protein kinase C (PKC) via activation of Gq signaling; and recruitment of the adapter protein arrestin to the plasma membrane, thereby resulting in a variety of hormone-specific tissue responses. Reports of intracellular binding of amino-terminal PTH to PTH1R (49) and possible interaction of PTH1R with components of the canonical Wnt signaling pathway (50,51) have added complexities in PTH1R signaling and mechanism of action of PTH.
In physiologic terms, PTH acts synergistically with 1,25(OH)2D and is the most important regulator of extracellular Ca concentration. These agents function synergistically and in a feedbackregulated fashion either directly via 1,25(OH)2D, acting through vitamin D receptors, or indirectly via changes in blood Ca and Mg, acting through CaR to lower PTH. Low or falling serum Ca results in active secretion of preformed PTH within seconds. Sustained hypocalcemia increases PTH mRNA within hours. Protracted hypocalcemia leads within days to parathyroid cell replication and increased gland mass. PTH acts directly on bone and kidney and indirectly on the intestine. Immediate control of blood Ca is probably due to PTH-induced mobilization of Ca from bone and increased renal distal tubular reabsorption of Ca. Continuous exposure to elevated concentrations of PTH leads to increased osteoclastic resorption of bone, although the PTH receptor is localized to
phenotypic osteoblasts, which are of mesenchymal origin, but not to osteoclasts, which are of hematogenous origin. Chronic effects of PTH increase the numbers of osteoblasts and osteoclasts and increase remodeling of bone. In contrast to the classic action of PTH on Ca mobilization from bone, the amino-terminal fragments of PTH and PTHrP and small pulses of PTH have an anabolic effect on bone, independent of resorptive action. Other PTH effects on bone include enhanced collagen synthesis, alkaline phosphatase (AP) activities, ornithine and citrate decarboxylases, and glucose-6-phosphate dehydrogenase; and DNA, protein and phospholipid synthesis.
phenotypic osteoblasts, which are of mesenchymal origin, but not to osteoclasts, which are of hematogenous origin. Chronic effects of PTH increase the numbers of osteoblasts and osteoclasts and increase remodeling of bone. In contrast to the classic action of PTH on Ca mobilization from bone, the amino-terminal fragments of PTH and PTHrP and small pulses of PTH have an anabolic effect on bone, independent of resorptive action. Other PTH effects on bone include enhanced collagen synthesis, alkaline phosphatase (AP) activities, ornithine and citrate decarboxylases, and glucose-6-phosphate dehydrogenase; and DNA, protein and phospholipid synthesis.
About 20% and 15%, respectively, of the filtered Ca is reabsorbed through the cortical TAL of the loop of Henle and DCT, where PTH exerts its effect on renal Ca handling. PTH binds to PTH1R and stimulates the reabsorption of Ca through increased activity of Na/K/2Cl cotransporter that drives NaCl reabsorption and stimulates paracellular Ca and Mg reabsorption. In the DCT, PTH can increase (a) luminal Ca transfer into renal tubule cells via the transient receptor potential vanilloid channel 5 (TRPV5), (b) translocation of Ca across the cell involving Ca transport proteins such as calbindin D28K, and (c) active extrusion of intracellular Ca into blood via sodium Ca exchanger, NCX1. PTH also stimulates renal 25-OHD-1&agr;-hydroxylase (CYP1&agr;) to increase synthesis of 1,25(OH)2D, which enhances renal Ca reabsorption but decreases sodium-dependent phosphate cotransporter, NPT-2, which decreases renal reabsorption of P.
Maintenance of steady-state Ca balance is probably secondary to PTH-induced increased 1,25(OH)2D production, which increases active intestinal Ca absorption that accounts for about 10% to 15% of a dietary load. This energy-dependent, cell-mediated saturable active process involves epithelial TRPV6, annexin2, calbindin-D9K, and the basolateral extrusion system PMCA1b. During high dietary Ca intake, 1,25(OH)2D-dependent absorption is suppressed and passive paracellular transport accounts for almost all Ca absorption.
Additional systemic factors (growth hormone, insulin-like growth factor 1, estrogen, progesterone, CT, cortisol, catecholamines, prostaglandins, and somatostatin) and local factors (interleukin-1 [IL-1]) modulate PTH secretion and function, although their role in regulation of Ca and Mg metabolism under physiologic conditions is not clear.
Vitamin D
Vitamin D (Mr 384) is synthesized endogenously or obtained from diet. It undergoes metabolic transformations primarily in the liver and kidney to form the physiologically most important metabolite, 1,25(OH)2D, which functions as a hormone in maintenance of mineral homeostasis. Under in vivo conditions, there are over 30 other vitamin D metabolites, with and without putative functions.
In animals, vitamin D3 can be synthesized endogenously in skin (52). During exposure to sunlight, high-energy ultraviolet photons (290 to 315 nm) penetrate the epidermis and photochemically cleave the bond between carbons 9 and 10 of the sterol B-ring of 7-dehydrocholesterol (7-DHC or provitamin D3), to produce previtamin D3. It then undergoes a thermally induced isomerization to vitamin D3 that takes 2 to 3 days to complete. Thus, cutaneous synthesis of vitamin D3 continues for many hours after single sun exposure. Previtamin D3 is photolabile; continued exposure to sunlight causes isomerization of previtamin D3 to biologically inert products, principally lumisterol. No more than 10% to 20% of initial provitamin D3 ultimately ends up as previtamin D3, thus preventing excessive production of previtamin D3 and vitamin D3.
Vitamin D3 synthesis in the skin is directly dependent on the amount of sunlight exposure and is affected by extent of skin area exposed, duration of sunlight exposure, time of day, season, and latitude. Peak sunlight at midday, in summer, and at lower latitudes are optimal conditions. Melanin in skin competes with 7-DHC for ultraviolet photons, but production of vitamin D3 can be adjusted by increasing duration of sunlight exposure. Use of topical sunscreen blocks ultraviolet photons, and aging decreases capacity for cutaneous synthesis of vitamin D3 (53).
Dietary vitamin D (1 &mgr;g = 40 IU) is derived from plants, as ergocalciferol (vitamin D2), and from animals, as cholecalciferol (vitamin D3). Dietary vitamin D is absorbed from duodenum and jejunum into lymphatics, and about 50% of vitamin D in chylomicron is transferred to vitamin D-binding protein (DBP) in blood before uptake by the liver.
The term “vitamin D” is frequently used generically to describe vitamins D2 and D3 and their metabolites. In mammals, vitamins D2 and D3 appear to metabolize along the same pathway involving a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1,25(OH)2D (54). There is little functional difference between their metabolites. However, differences in affinity to DBP and receptors between D2 and D3 and their metabolites support the contention that vitamin D3 is more bioavailable than D2.
In circulation, vitamin D and its metabolites are bound to proteins, mainly DBP (approximately 85%) and albumin (15%). The DBP gene is located on chromosome 4q11-13. As a member of the albumin multigene family of proteins that includes albumin and &agr;-fetoprotein, human DBP is an approximately 53-kDa globulin. Plasma DBP concentration (4 to 8 &mgr;M) is greater than 20-fold higher than that of the total circulating vitamin D metabolites (approximately 100 nM); that is, it is normally less than 5% saturated with vitamin D metabolites. Unbound or free 25-OHD and 1,25(OH)2D, important in determining bioactivity, is less than 1% of total concentration. Genetic polymorphism accounted for parallel changes in DBP and 25-OHD between black and white populations; hence, the bioavailable free 25-OHD is probably similar between these groups (55).
In the liver, vitamin D is hydroxylated at carbon 25 to 25-OHD. Quantitatively, 25-OHD (1 nmol/L = 0.4 ng/mL) is the most abundant vitamin D metabolite in circulation and is a useful index of vitamin D reserve. Regulation of 25-hydroxylase activity is limited, and there are few limitations to production of 25-OHD. However, in vivo administration of 1,25(OH)2D (56) inhibits hepatic production of 25-OHD, and Ca deficiency (57) increases metabolic clearance of 25-OHD with subsequently decreased circulating 25-OHD.
In the kidney, 25-OHD is hydroxylated further to the most active vitamin D metabolite, 1,25(OH)2D by CYP1&agr;. The hydroxylation occurs primarily in mitochondria of renal proximal tubules. The human gene encoding CYP1&agr; is localized to chromosome 12q13-14. It is 5 kb in length and comprises nine exons and eight introns; its exon/intron organization is similar to other cloned mitochondrial P450 enzymes.
The activity of CYP1&agr; and therefore production of 1,25(OH)2D are tightly regulated. It is the rate-limiting hormonally regulated step in bioactivation of vitamin D. PTH increases transcriptional activity of CYP1&agr; gene promoter and increases mRNA for 1,25(OH)2D. Decreases in serum or dietary Ca or P increase mRNA and production of 1,25(OH)2D independent of PTH (57,58,59). CYP1&agr; activity is also feedback regulated by FGF23 and indirectly influenced by factors at molecular level that affect FGF23 production, degradation, or expression (60,61). Other factors that enhance 1,25(OH)2D production include estrogen, prolactin, growth hormone, insulin-like growth factor I, and PTHrP. 1,25(OH)2D production is feedback regulated by circulating PTH, Ca and Mg (62). Mg is a cofactor of the CYP1&agr; enzyme and Mg deficiency also lowers serum 1,25(OH)2D response to a low-Ca diet but does not appear to limit 1,25(OH)2D production in animals (63). In contrast to the rapid secretion and increased serum PTH within minutes of a lowering in serum Ca, measurable alterations in serum 1,25(OH)2D usually occur hours later. Extrarenal production of 1,25(OH)2D may not be tightly regulated; its production in macrophages, particularly in
granulomatous disease states, is stimulated by &ggr;-interferon (64) but is not responsive to changes in dietary Ca intake.
granulomatous disease states, is stimulated by &ggr;-interferon (64) but is not responsive to changes in dietary Ca intake.
The degradation of 1,25(OH)2D is tightly regulated and involves a series of cytochrome P450-containing sterol hydroxylases. 1,25(OH)2D strongly induces the enzyme 25-hydroxyvitamin D-24 hydroxylase (CYP24) in all target cells for vitamin D. CYP24 catalyzes several steps of 1,25(OH)2D degradation, collectively known as the C24 oxidation pathway, which starts with 24-hydroxylation and culminates in formation of the biliary excretory form, calcitroic acid. The human gene encoding CYP24 is localized to chromosome 20q13.3. CYP24 expression is inhibited by PTH and by dietary phosphate restriction. In the kidney and intestine in particular, up-regulation of the CYP24 enzyme in response to 1,25(OH)2D treatment is rapid and occurs within 4 hours. Physiologic production of 24,25(OH)2D is therefore an important means to regulate circulating 1,25(OH)2D and catabolism of vitamin D, although it may also have a role in bone integrity and fracture healing in the chick model. Most other vitamin D metabolites are derived primarily from further metabolic alterations to 25-OHD and 1,25(OH)2D through oxidation or side chain cleavage and have poorly defined physiologic functions. Many analogues of vitamin D metabolites are being studied for potential pharmacologic actions that involve less calcemic-inducing and more cellular maturation and differentiation effects.
Like other steroid hormones, 1,25(OH)2D function is mediated primarily through modulation of the cellular genome by binding to a specific nuclear receptor, vitamin D receptor (VDR), a 424-amino-acid phosphoprotein (65,66). The VDR gene contains nine exons and is located on chromosome 12q13-14 near the site of the gene for CYPl&agr;. VDR is a member of the subfamily of nuclear receptors with ligand-binding domains that also bind classic hormones including thyroid hormone, androgens, estrogens, progesterone, glucocorticoids, aldosterone, and hormonal forms of vitamin A. It has several functional domains including a 110-residue N-terminal DNA-binding domain with two zinc fingers, C-terminal hormone-binding domain, and hinge region important for nuclear localization. The VDR interacts with the 9-cis retinoic acid nuclear receptor retinoid X receptor (RXR) to form a heterodimeric RXR-VDR complex that binds to specific DNA sequences, termed vitamin D-responsive elements (VDREs). After 1,25(OH)2D binds to the receptor, it induces conformational changes that result in recruitment of a multitude of transcriptional coactivators that stimulate the transcription of target genes. VDR also can adopt a dual role as a repressor in the absence of ligand and then subsequently as a coactivator when a ligand binds. VDR is up-regulated by 1,25(OH)2D at both mRNA and protein levels and is increased during growth, gestation, and lactation; however, it shows an age-dependent decrease in mature animals and humans, supporting the notion that VDR may be upor down-regulated, depending on Ca needs.
1,25(OH)2D regulates more than 60 genes whose actions include those associated with Ca homeostasis and immunomodulation, antimicrobial action, detoxification, insulin secretion, skin integrity, and &bgr;-oxidation, as well as regulation of cell growth, differentiation, and apoptosis. Numerous physiologic functions are mediated by VDR, and disturbances in vitamin D pathways are associated with major human diseases such as cancer, infection, autoimmune disease, cardiovascular and metabolic disease, and disorders in muscle function, reproduction, and neurocognition (65,66). However, evidence for major dysfunction from vitamin D deficiency other than classic actions of mineral homeostasis and bone mineralization is limited in children or adults. It is possible that vitamin D is a “threshold” nutrient and clinical ill effect is manifested with individual variability generally at very low serum 25-OHD. Furthermore, benefits from vitamin D supplementation other than in a confirmed deficient state is also debatable (41,67,68,69,70).
The classic actions of vitamin D on calcium homeostasis and bone mineralization are mediated through 1,25(OH)2D action on the intestine, kidney, and bone, with modulating effects from other hormones including PTH, FGF23, CT, and PTHrP. When dietary Ca is insufficient, 1,25(OH)2D maintains Ca homeostasis by enhancing intestinal absorption of Ca and P and renal reabsorption of Ca, and it enhances osteoclastic bone resorption with mobilization of bone Ca store to maintain normal ECF Ca. The mobilization of bone Ca is thought to be due to 1,25(OH)2D binding to receptors in the preosteoblastic stromal cell stimulating the RANK/RANK ligand system leading to proliferation, differentiation, and activation of the osteoclastic system from its monocytic precursors. With adequate dietary Ca and P intake, 1,25(OH)2D maintains normal mineralization of bone through intestinal and renal effects to maintain Ca and P ions within a range that facilitates hydroxyapatite deposition in bone matrix.
The genomic action of 1,25(OH)2D can be preceded by more rapid nongenomic actions that occur within seconds to minutes. The exact mode of nongenomic action is not well defined (71) but may involve membrane-associated events such as increased Ca transport, and PKC and mitogen-activated protein kinase (MAPK) activation.
Actions of 1,25(OH)2D and VDR unrelated to Ca homeostasis including modulation of miRNA function and epigenetic regulation of genes (72) are becoming better defined. VDR stimulation independent of 1,25(OH)2D3 controls hair cycling and brain development. Novel ligands other than 1,25(OH)2D3 including lithocholic acid, curcumin, &ggr;-tocotrienol, and essential fatty acid derivatives may play additional specific roles in physiologic functions and therapeutic potential for various diseases (65,66,72).
Quantification of vitamin D and metabolites has been achieved by liquid chromatography tandem mass spectrometry (LC-MS/MS), high-performance liquid chromatography, with detection by ultraviolet absorbance or binding assays, and immunoassays based on antibodies raised to vitamin D metabolite conjugates. Some methods can measure D2 and D3 metabolites separately, whereas others measure only the total amount of D metabolites. The LC-MS/MS method also quantifies 3-epimer 25-OHD and 24,25(OH)2D, which may interfere with measurement of 25-OHD in immunoassays. Thus, values from different laboratories using different assays cannot readily be compared, and appropriate vitamin D standards must be used.
Maternofetal transfer of vitamin D and its metabolites vary, depending on species. In humans, the cord serum vitamin D is very low and may be undetectable; 25-OHD concentration directly correlates with, but is lower than, maternal values, consistent with placental crossover; 1,25(OH)2D concentrations also are lower than maternal values, but there is no agreement on the maternofetal relationship of this and other dihydroxylated vitamin D metabolites (3,73,74,75). The placenta, like the kidney, produces 1,25(OH)2D, making it difficult to ascertain just how much fetal 1,25(OH)2D results from placental crossover versus placental synthesis. 24,25(OH)2D also crosses the placenta, and its concentration in sera of mothers and newborns varies with season, being highest in autumn. It appears that the human fetus receives the bulk of its vitamin D already metabolized to 25-OHD.
Seasonal and racial variations in serum 25-OHD occur, presumably from variations in endogenous production. The concentration of serum 25-OHD, as with 24,25(OH)2D, is lower in winter. These changes may be reflected in cord serum values (73,75,76). In normal adults, serum 1,25(OH)2D concentrations are relatively constant and maintained within approximately 20% of the overall 24-hour mean and show no seasonal variation, which is consistent with tightly regulated CYPl&agr; activity. African American mothers, infants, and young children tend to have lower 25-OHD and higher 1,25(OH)2D concentrations than their white counterparts (74,76). Serum 1,25(OH)2D in the newborn becomes elevated within 24 hours after delivery and appears to vary with Ca and P intake.
The circulating half-life of vitamin D is about 24 hours and for 25-OHD is 2 to 3 weeks, although the latter half-life is decreased in vitamin D-deficient individuals. 1,25(OH)2D has a much shorter half-life of 3 to 6 hours. Metabolites of 25-OHD and 1,25(OH)2D may undergo enterohepatic circulation after exposure to intestinal &bgr;-glucuronidase. The physiologic role of enterohepatic circulation of vitamin D metabolites has not been precisely quantitated.
Fibroblast Growth Factor 23
Human FGF23 gene resides on chromosome 12p13. It comprises three coding exons and contains an open reading frame of 251 residues. FGF23 expression is reported in multiple bone cells including osteoblasts, osteocytes, bone lining cells, and osteoprogenitor cells. At tissue level, FGF23 mRNA is most highly expressed in long bone, followed by the thymus, brain, and heart. Wild-type FGF23 is secreted as full-length 32-kDa species as well as cleavage products of 12 and 20 kDa. The half-life of FGF23 is reported to be between 20 and 50 minutes (77,78).
Biologic activity of FGF23 is mediated by the recruitment of canonical FGF receptors (FGFRs) together with a coreceptor &agr;Klotho to form heteromeric complexes and initiates various signaling pathways including the MAPK cascade. FGFR1 may be more important for phosphate homeostasis, whereas FGFR3 and FGFR4 may be more relevant to vitamin D status (79).
Physiologically, FGF23 regulates mineral homeostasis by its effect on P metabolism and calciotropic hormones. FGF23 has overlapping function with PTH to reduce renal P reabsorption by down-regulation of type II sodium-phosphate cotransporters NPT2a and NPT2c in the proximal tubule leading to renal P wasting (80). However, FGF23 has the opposite effect to 1,25(OH)2D. Normally, P restriction and the addition of P binder with hypophosphatemia increase serum 1,25(OH)2D and suppress FGF23; increase in dietary or serum P suppresses 1,25(OH)2D and increases FGF23. Vitamin D has a dose-dependent effect to increase FGF23 before changes in serum P, indicating a direct regulatory effect on FGF23, whereas FGF23 down-regulates the 1&agr;-hydroxylase mRNA (60,61). Thus, as 1,25(OH)2D rises, it stimulates FGF23, which would complete the feedback loop and down-regulate the 1&agr;-hydroxylase mRNA.
Pathologic states are associated with elevated or reduced activities of FGF23 although the molecular mechanisms are unique to each disorder. Elevated FGF23 activities is associated with clinical syndromes of hypophosphatemia with paradoxically low or normal 1,25(OH)2D and defective skeletal mineralization. Serum Ca and PTH are normal. These syndromes include X-linked hypophosphatemic (XLH) rickets, autosomal dominant hypophosphatemic rickets (ADHRs) and autosomal recessive hypophosphatemic rickets (ARHR1 and ARHR2), and tumor-induced osteomalacia (TIO). XLH results from mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome, Xp22.1 (PHEX), which encodes a membrane-bound endopeptidase (81), whereas ADHR is associated with mutations of the gene encoding FGF23 and is linked to chromosome 12p13.3 (60,61). The endopeptidase, PHEX, degrades native FGF23, which provides the biochemical link among these clinical syndromes. XLH rickets also has been associated with mutations in CLCN5, a voltage-gated chloride channel gene located on Xp11.22. Nonrandom expression of the normal PHEX gene in critical tissues or discordant X inactivation are possible explanation for nonpenetrance of the pathologic manifestation (82). Reduced FGF23 activity is associated with hyperphosphatemia and often elevated 1,25(OH)2D such as in tumoral calcinosis. Collectively, these heritable disorders provide unique insight into the activities of FGF23 on renal P and vitamin D metabolism.
Immunoassays that quantify the C-terminal recognize the fulllength FGF23 as well as C-terminal proteolytic fragments, whereas “intact” assays recognize the N- and C-terminal portions of FGF23. The N-terminal domain is conserved and is the bioactive fragment. Both assays report measurable quantities in normal population and are significantly higher in patients expected to have altered FGF23 levels (83,84). In children with predialysis chronic kidney disease, high plasma FGF23 is present before higher PTH, and P is observed and may be considered to be the earliest detectable abnormality in mineral metabolism (85). There is also lower plasma 1,25(OH)2D. Some individuals with “normal” FGF23 may be “inappropriately normal” in the setting of hypophosphatemia, and this may be diagnostic of a number of heritable disorders of bone and mineral metabolism (86).
Umbilical cord plasma samples have high concentrations of FGF23 when measured with the C-terminal assay (87). Assay methodology probably accounts for the cord FGF23 to be higher (87), not significantly different (88), or lower (89) than maternal concentrations. However, cord plasma &agr;Klotho concentrations are significantly higher than maternal values and neither &agr;Klotho nor FGF23 is related to neonatal bone mineral content (88). The high cord plasma &agr;Klotho concentrations and rapid drop to adult levels are consistent with its expression in syncytiotrophoblasts (89). Pregnancy does not appear to affect the FGF23 or &agr;Klotho concentrations (87,89).
Calcitonin
CT is secreted primarily from thyroid C cells and also from many extrathyroidal tissues including the placenta, brain, pituitary, mammary gland, and other tissues. Developmentally, CT-containing cells and parathyroid gland cells are thought to derive from the same tissue source as neural crest. There is probably no placental crossover of CT; human placental tissue can produce CT in response to presence of Ca in the culture medium.
There are two CT genes, &agr; and &bgr;, located on chromosome 11p15.2 near the genes for &bgr;-globulin and PTH. Two different RNA molecules are transcribed from the &agr; gene. It is comprised of six exons with the fourth exon translated into the precursor for CT, and the fifth translated into precursor for CT gene-related peptide-I (CGRP-I). The CT monomer, a 32-amino-acid peptide (the 60th to 91st positions of pro-CT peptide) and equimolar amounts of non-CT secretory peptides, corresponding to flanking peptides linked to amino and carboxyl terminals of the prohormone, are generated during precursor processing. Further structural modifications to the CT molecule occur intracellularly including formation of disulfide bridge between two cysteine remnants in positions 1 and 7 and hydroxylation of the C-terminal proline residue; both are essential for binding of CT to its receptor. CGRP-I is synthesized wherever the CT mRNA is expressed although there is no translational product from the CGRP-I sequence.
The &bgr; or CGRP-II gene is transcribed into the mRNA for CGRP predominantly in nerve fibers in central and peripheral nervous systems, blood vessels, thyroid and parathyroid glands, liver, spleen, heart, lung, and possibly bone marrow. CGRP, a 37-amino-acid peptide (Mr 4,000), is also generated from the larger precursor molecule pro-CGRP, a 103-amino-acid peptide. Seventy-five amino-terminal residues of preprohormones for CT and CGRP are predicted to be identical.
Classic bioactivity of human CT (hCT) is present in the full 32-amino-acid structure or its smaller fragments, such as hCT 8-32 and hCT 9-32; the ring structure of CT enhances, but is not essential for, hormone action. Basic amino acid substitutions confer a helical structure in this region as found in salmon and other nonmammalian CT, resulting in greater potency in lowering serum Ca and probably longer circulating half-life. The kidney appears to be the dominant organ in the metabolism of hCT. The liver, intestine, and bone may be involved in metabolism of CT. A small percentage of CT is cleared by enzymatic degradation in blood. Injected hCT monomer disappears from the blood in vivo with a half-life of approximately 10 minutes; in contrast, the half-life of hCT in plasma incubated in vitro at 37°C may be longer than 20 hours (90).
Circulating immunoreactive CT and CGRP are a heterogeneous mixture of different molecular forms and are expressed in gravimetric or molar equivalents of synthetic CT or CGRP. Serum CT is high at birth compared to paired maternal CT concentrations (91). Serum CT further increases during the first few days after birth (11,46) to levels 5- to 10-fold higher than adult values and may remain twice the adult value in preterm infants up to 3 months (92), then progressively decreases during infancy. In human adults, serum CT and CGRP concentrations are found in picomolar range, and basal serum CT concentration may be lower in women than in men, but the concentration is not affected by increasing age. Diurnal variability has been reported for serum CGRP but not for serum CT. In normal individuals, larger precursor molecules of CT such as procalcitonin are not detected.
CT function is mediated by binding to receptors linked to G proteins, members of the GPCR superfamily, and by activation of adenylate cyclase and phospholipase C (93). CT receptors (CTRs) have been identified in central nervous system, testes, skeletal muscle, lymphocytes, and placenta. Its function can be influenced by accessory proteins, receptor isoforms, genetic polymorphisms, developmental and/or transcriptional regulation, feedback inhibition, and specific cellular or tissue background. The CTR gene is located on chromosome 7q2l.2-q21.3 and encodes a 490-amino-acid G protein-linked receptor with seven transmembrane domains. Two isoforms of human CTR arise by alternative splicing of an exon of 48 nucleotides that encodes an l6-amino-acid insertion within the first intracellular loop. The isoform with the insertion (hCTR-l) activates only adenylate cyclase, whereas the other isoform (hCTR-2) activates both adenylate cyclase and phospholipase C. CGRP functions are also mediated by receptors (94). The presence of receptor activity-modifying proteins (RAMPs) can posttranslationally modify the initially orphan CT receptor-like (CL) receptor and CTR to exhibit different receptor functions; that is, functional modification of GPCR is possible.
Secretion of CT is stimulated by increase in serum Ca and Mg concentrations and by gastrin, glucagon, and cholecystokinin (along with structural analogues, for example, pentagastrin, prostaglandin E2), glucocorticoid, norepinephrine, and CGRP; secretion is suppressed by hypocalcemia, propranolol and other adrenergic antagonists, somatostatin, chromogranin A, and vitamin D. CT gene transcription is positively regulated by glucocorticoids and negatively regulated by PKC, Ca, and vitamin D. CT may activate the l-hydroxylase system independent of PTH (95). The bioactivity of CT on calcium metabolism frequently is opposite that of PTH; CT probably modulates the effect of PTH on target organs. Physiologically, the net effect of CT is a lowering of serum Ca and P concentrations.
In humans, changes in Ca (and P) metabolism are not seen despite extreme variations in CT production. In the neonate, there is neither an identifiable hypocalcemic response to the postnatal surge in serum CT nor a blunting of CT secretion in hypocalcemia. In adults, there are no definite effects attributable to CT deficiency (e.g., totally thyroidectomized patients receiving only replacement thyroxin) or CT excess (e.g., patients with medullary carcinoma of thyroid), except for the chronic suppression of bone remodeling. The clinical significance of CT is related to its use as a tumor marker in management of medullary carcinoma of the thyroid and its pharmacologic effect to inhibit osteoclast-mediated bone resorption, increase renal clearance of Ca, Mg, P, sodium and free water, and analgesic effects. CT induces refractoriness to its own actions by down-regulation through functional reduction of receptor mRNA. Clinically, it is manifested as the “escape” phenomenon or tachyphylaxis during CT therapy.
Non-calcium-related actions of CT and associated molecules are increasingly reported to play important roles in embryonic development and sperm function/physiology and potential pharmacologic effects other than mineral metabolism (94,96,97). Production of pro-CT after exposure to bacterial endotoxin and inflammatory cytokines TNF and IL-6 appears to be primarily from neuroendocrine cells in the lung and intestine, and plasma pro-CT may be used as marker of bacterial-induced inflammation/sepsis. After administration of endotoxin, the peak circulating concentrations of TNF, IL-6, pro-CT, and C-reactive protein occur at approximately 90 minutes, 180 minutes, 6 to 8 hours, and 24 hours, respectively. There are no enzymes in plasma that can break down pro-CT, and when it is secreted into the circulation, it has a half-life of 25 to 30 hours. As with other biomarkers, elevated pro-CT cannot distinguish sepsis from or nonseptic causes (98). CGRP primarily affects catecholamine release, vascular tone and blood pressure, and cardiac contractility. Both CT and CGRP inhibit gastric acid secretion, and food intake may have impact on their pharmacologic effects.
PTH-Related Protein
Human PTHrP gene is located on the short arm of chromosome 12 containing eight exons and at least three promoters. Alternative splicing at the 3′ end of the gene gives rise to three different classes of mRNAs coding for specific translation products. The amino acids 34-111 segment is highly conserved among species while amino acid 118 to the C-terminus is poorly conserved (99).
PTHrP and PTH genes share structural elements and sequence homology with identical exon/intron organization encoding the preprosequences. There is also high sequence homology at the amino-terminal portion such that the N-terminal 1-13 region has 8 of 13 residues in common and a high degree of predicted secondary structure over the next 21 amino acids. These common sequences allow both PTH-related protein (PTHrP) and PTH to bind and activate the same PTH1R, with similar effects on raising Ca and lowering P in the circulation.
In human, PTHrP gene expression is found in at least some cells of all organs including the fetus, placenta, lactating breast, and milk and by as early as 7 weeks of gestation. Synthetic and recombinant PTHrPs can mimic the effects of PTH on classic PTH target organs, involving activation of adenylate cyclase and other second messenger systems.
Physiologically, PTHrP normally functions at the local autocrine, paracrine, or intracrine level. PTHrP is an important paracrine regulator of several tissue-specific functions that may directly or indirectly affect fetal and neonatal mineral homeostasis, probably through its effect on smooth muscle relaxation, Ca transport and control of cellular growth, and differentiation of many tissues including the placenta, mammary gland, fetal chondrocyte and bone, teeth, pancreas, and peripheral and central nervous system.
Several PTHrP assays with varying sensitivities and specificities have been developed that account for variability reported between assays (100). The stability of PTHrP in plasma samples may be enhanced if sample collection is done in the presence of protease inhibitors. Circulating immunoreactive PTHrP concentrations are low or undetectable in normal subjects. Serum PTHrP is increased during pregnancy and lactation (5,6) and is similar to or lower than umbilical cord PTHrP concentrations. In cord sera, PTHrP concentration is 10- to 15-fold higher than that of PTH. Amniotic fluid PTHrP concentrations at midgestation and at term are 13- to 16-fold higher than the cord or maternal levels (101), and the concentration of PTHrP in milk is 100-fold higher. PTHrP concentrations correlate positively with total milk calcium (102).
Clinically, measurement of PTHrP is of clinical utility primarily as tumor marker in patients with the syndrome of humoral hypercalcemia of malignancy (HHM) since PTHrP is the humoral mediator secreted by tumors (100). The amino-terminal fragment PTHrP 1-74 appears to be specific for HHM, whereas the carboxyl-terminal fragment PTHrP 109-138 is elevated in the serum of patients with HHM or renal failure. The levels of PTHrP in these patients are similar to the concentration of PTH (10-12 to 10-11 mol/L).
Extracellular Calcium-Sensing Receptor
The human CaR gene is located on chromosome 3q13.3-21 and encodes a cell surface protein of 1,078 amino acids. The CaR gene is developmentally up-regulated, and CaR transcripts are present in numerous tissues including chief cells of parathyroid glands, kidneys (in particular the TAL), intestine, precursor and mature osteoblasts and osteoclasts, brain and nerve terminals, lung, adrenal and skin, and lactating breast and placenta. CaR is a member of the GPCR superfamily. It contains at least seven exons, of which six encode the 612-amino-acid extracellular amino-terminal domain, and/or its upstream untranslated regions, while a single exon codes the remainder of the receptor including a seven transmembrane domain motif characteristic of the superfamily of GPCRs and a cytoplasmic carboxyl-terminal intracellular domain of 216 amino acids. Signal transduction mediated by G proteins results in activation of phospholipase C that generates IP3 and diacylglycerol (DAG) and subsequent stimulation of PKC and Ca transport channels (103).
Physiologically, extracellular Ca is the most potent regulator of PTH secretion through the ability of CaR to detect minute perturbations in the extracellular iCa concentration and to trigger responses with alterations in cellular function that normalize iCa. Thus, iCa functions as an extracellular as well as an intracellular messenger.
There is a sigmoidal type of PTH secretion in response to decreased serum Ca, which is most pronounced when serum Ca is in the mildly hypocalcemic range. PTH secretion is 50% of maximal at a serum iCa of about 1 mmol/L (4 mg/dL); this is considered the calcium set point for PTH secretion (104). High serum Ca suppresses PTH secretion via activation of CaR. CaR in turn activates phospholipase C and generation of IP3 and DAG and probably increases proteolytic destruction of preformed PTH. Hyperphosphatemia stimulates PTH secretion, probably by lowering serum Ca. In the kidney, CaR decreases basal and PTH-stimulated paracellular reabsorption of Ca, Mg, and sodium via multiple mechanisms including inhibition of cAMP accumulation; stimulation of phospholipase A2 activity promotes release of free arachidonic acid, which is metabolized via the lipoxygenase pathway to P450 metabolites that inhibit the activities of the sodium-potassiumchloride cotransporter and potassium channel; and by inhibition of vasopressin-abated water flow. In chronic renal failure, down-regulation in the expression of renal CaR may account for the development of secondary hyperparathyroidism, and down-regulation of PTH receptors may account for the skeletal resistance to the calcemic effect of PTH. Extracellular Ca exerts numerous other actions on parathyroid function, including modulation of the intracellular degradation of PTH, cellular respiration, membrane voltage, and the hexose monophosphate shunt.
Maintenance of Ca homeostasis may mediate through CaR in other organs, for example, through intestinal cells, and probable modulation of CT secretion from changes in intracellular Ca. Furthermore, expression of CaR in gastrin-secreting G cells and acidsecreting parietal cells, and CaR selectivity for L-aromatic amino acids, appears to provide a molecular explanation for amino acid sensing in gastrointestinal tract, regulation of PTH secretion and urinary Ca excretion, and the physiologic interaction between Ca and protein metabolism.
CaR plays a pivotal role in disorders of calcium homeostasis including familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism, autosomal dominant hypocalcemia, primary and secondary hyperparathyroidism, and hypercalcemia of malignancy. CaR also has ability to activate many different signaling pathways in a ligand- and tissue-specific manner and is increasingly understood to play crucial roles in human physiology and pathophysiology, both in calcium homeostasis and in tissues and biologic processes unrelated to calcium balance (105).
Blood levels of Mg are not as tightly regulated as Ca and fluctuate with influx and efflux across ECF from changes in intestinal Mg absorption, net renal Mg reabsorption, and fluxes across bone. Blood iMg regulates PTH secretion, but the potency is less than that of Ca. Decreased serum Mg concentration stimulates PTH secretion (106,107), although chronic hypomagnesemia inhibits secretion of PTH (62,107). Hypomagnesemia is also associated with increased target tissue resistance to PTH probably from inactivity of adenylate cyclase, a Mg-requiring enzyme. Mg is a cofactor for the 25-hydroxyvitamin D 1&agr;-hydroxylase enzyme in production of 1,25(OH)2D critical to the maintenance of Ca and Mg homeostasis. Hypermagnesemia rapidly decreases the secretion of PTH in vivo in human subjects, and PTH concentration remains depressed despite concomitant hypocalcemia, presumably in part due to stimulation of CaR by other divalent cations such as Mg. Hypermagnesemia increases urine Mg excretion possibly also mediated through CaR.
▪ DISTURBANCES IN SERUM MINERAL CONCENTRATIONS
Hypocalcemia
Neonatal hypocalcemia may be defined as a serum tCa concentration of less than 2 mmol/L (8 mg/dL) in term infants and 1.75 mmol/L (7 mg/dL) in preterm infants with iCa below 1.0 to 1.1 mmol/L (4.0 to 4.4 mg/dL), depending on the particular ion-selective electrode used. Whole-blood iCa shows similar values to serum iCa and is often used to determine hypocalcemia. However, the appropriate range used is also subject to the type of instrument used (19).
The definition of hypocalcemia is based on the clinical perspective because serum Ca concentrations are maintained within narrow ranges under normal circumstances, and the potential risk for disturbances of physiologic function increases as the serum Ca concentration decreases below the normal range. Furthermore, improvements in physiologic function, for example, changes in cardiac contractility, blood pressure, and heart rate, are reported in hypocalcemic infants undergoing Ca therapy (108,109), and both hypocalcemia and hypermagnesemia are associated with a higher mortality rate or longer intensive care unit stay in children (110).
Clinically, there are two peaks in the occurrence of neonatal hypocalcemia. An early form typically occurs during the first few days after birth, with the lowest concentrations of serum Ca being reached at 24 to 48 hours after birth; late neonatal hypocalcemia occurs toward the end of the first week. These findings reflect in part the traditional clinical practice of screening for biochemical abnormalities in small or sick hospitalized infants during the first few days and in symptomatic infants during hospitalization and after hospital discharge. However, the nadir of serum Ca concentration may occur at less than 12 hours (9,10,11,12) or not until some weeks after birth (111,112), and many neonates, particularly those with genetic defects in Ca metabolism, may be hypocalcemic but remain asymptomatic and undetected during the early neonatal period.
The approach to neonatal hypocalcemia should rely on risk factors and pathophysiologic basis rather than the traditional “early” or “late” onset. Additionally, until Ca homeostasis becomes better understood in small preterm infants, the diagnosis of hypocalcemia should be based on the same criteria as that for a term infant tolerating full enteral feeding or standard parenteral nutrition; with resumption of postnatal growth skeletal growth; and with normal renal tubular function as indicated by normal serum minerals (Ca, Mg, and P) and electrolytes (Na, K, and Cl). Preterm infants generally should satisfy these criteria by a corrected age of greater than 36 weeks.
TABLE 33.1 Risk Factors for Neonatal Hypocalcemia | ||||||
|---|---|---|---|---|---|---|
|
