Regulation of Calcium Metabolism

Ann. din. Biochem. 13 (1976) 518-539 Regulation of Calcium Metabolism R. G. G. RUSSELL From the Department of Medicine, Harvard Medical School, and the Medical Services (Endocrine and Arthritis Units), Massachusetts General Hospital, Boston, Massachusetts 02114, and the Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford, England (address for correspondence) This paper reviews the regulation of calcium metabolism in man. The body's calcium economy is determined by the relationship between the intestinal absorption of calcium, the renal handling of calcium, and by the movements of calcium in and out of the skeleton. These processes are influenced by many factors, the most important of which are parathyroid hormone and the hormones derived from the renal metabolism of vitamin D, notably l,25-dihydroxyvitamin D aThe role of endogenous calcitonin in man is still controversial, but there are severalother hormones which have some influence on calcium metabolism, including thyroid hormone, growth hormone, and the adrenal and gonadal steroids. Clinical disorders of calcium metabolism and their treatment are discussedin terms of the disturbances in normal physiologythey represent. There has been a rapid growth in knowledge about calcium metabolism in the past decade, particularly in understanding the biochemistry of the calciumregulating hormones. This recent work has meant that many old concepts have had to be re-examined, a process which is still far from complete, especially in relation to human disease. The purpose of this paper is to review current concepts in outline and to refer the reader to other sources for more detailed information (see Bibliography). Regulation of calcium metabolism can be considered from at least three distinct but interrelated aspects: (i) control of the concentration of calcium in extracellular fluid and tissues; (ii) control of the body's overall calcium balance, i.e., the relationship between gains and losses; (iii) control of the shape, structure and composition of bone and the way these respond to changes in external factors such as load bearing. DISTRIBUTION OF CALCIUM AND PHOSPHATE Most of the body's calcium resides within bone (Table 1). The other major inorganic constituent of bone is phosphorus, as inorganic phosphate (Pi). Unlike calcium, the concentration of which is low in most soft tissues, about 15 % of the body's phosphorus lies outside the skeleton, in body fluids and in tissues, mostly as organic phosphate compounds, e.g., nucleic acids and nucleotides, phospholipids and phosphorylated metabolites. Studies with radioisotopes (45Ca and 47Ca) have shown that in normal human adults the exchangeable pool of calcium represents less than 1 % of total body calcium (in the region of 70 mg/kg during the first few days after injection). About half of this Table 1. Distribution ofcalcium and phosphate in normal human adults Calcium" Total body content (for 70 kg human) Skeleton Skeletal muscle Skin Liver Central nervous system Other tissues Extracellular fluid Phosphorus (as P)· 1QOO-1500 s 700-1()()() g 98% 0.3% 0.08% 0.02% 0.01% 0.6% 1% 85% 6% 1% 1% 1% 5% 1% ·From Widdowson and Dickerson (1964). is outside the skeleton, the remainder within it. This exchangeable pool of calcium is very important in homoeostasis, and movements of calcium ions between body fluids. cells, and the surfaces of bone occur continuously. Between 1 and 4 %of the human adult skeleton is thought to be renewed each year. Trabecular bone has a faster turnover than cortical bone. In order to understand the way in which the body gains and loses calcium from the external environment and the way in which internal control is achieved the system can be simplified to a consideration of the roles of the three major organs involved, i.e., gut, lddney, and bone, and the pool to and from which calcium moves. 518 RegUlation 0/ calcium metabolism 519 MAJOR FLUXES OF CALCIUM AND NET CALCIUM BALANCE . The major movements (fluxes) of calcium through tbeIlc organs in an adult human are shown in Pia. 1. Calcium enters the body by intestinal absorption. The true absorption of calcium is greater than the uet absorption because some calcium is returned to the gut lumen in biliary, pancreatic, and intestinal ICCI'Ctions. Calcium is lost from the body by urinary excretion and also in sweat. The latter is usually ianored in balance studies because the loss is small and cannot be measured easily. However, losses in sweat can be as high as 300 mg/day under extreme conditions. It will be noted that the fluxes of calcium through the kidney, as filtered and reabsorbed . calcium are many times higher than the fluxes through the intestine and bone. In the adult under normal conditions the body is , neither gaining nor losing calcium, so that inflow ; and outflow are matched precisely (intake = output). In disease states there may be transient or sustained net gains or losses, to produce calcium balances that are positive (intake exceeds output) or negative (output exceeds intake). Mljor .It.. of .ctlon of PTH.cllcitonin & vitlmln D l000mg 1 Vii D G> .. ----_.-- .... -. During growth there is a net daily gain to provide the calcium necessary for skeletal growth. In pregnant or lactating women the foetus or child pins calcium from the mother. In these situations the extra requirements are met by increased net intestinal absorption and diminished renal excretion of Ca so that a neutral balance is maintained in the mother. Calcium homoeostasis is concerned with the relative rates of flux through these organs and the complex way in which these are changed by regulating factors, particularly parathyroid hormone (PTH), calcitonin (Cf), and vitamin D (vit D). The concentrations of calcium and phosphate in extracellular fluid (ECF) are set by the relative sizes of the various fluxes and by the influence of controlling agents on them. The properties of the hormones acting on calcium metabolism will be discussed first, followed by a description of some features of the individual organ responses to them. The hormones can be subdivided into "controlling" hormones and "influencing" hormones. The controllers are the primary calcium regulating hormones, PTH, cr and vitamin D metabolites, the secretion of each of which is altered in response to changes in plasma ionised calcium concentration (and phosphate in the case of vit D). The "influencing" hormones are those other hormones, e.g., thyroid hormones, growth hormone, and adrenal and gonadal steroids, which have effects on calcium metabolism but whose secretion is determined primarily by factors other than changes in plasma calcium and phosphate. THE CoNTROLLING HORMONES ! Parathyroid hormone (PTH) ',200mg // 200mg t 1t (j;) PTH 'Cil o Vii 0 CT BOOmg 200mg Fig. 1.-The majOl' movements of calcium (mgfday) through the principal organs (intestine, kidney, and bone) involved In calcium bomoeostasls in a normal adult man. The major sites of acrion of parathyroid bormone (PTH), calcitonin (en, and vitamin D are shown. Note that balance is maintained, oot only by the skeleton (mIner'lllisatioo = resorption) but also by the whole organism (net intestinal absorption = urinary loss). Mammalian PTH consists of a single peptide chain containing 84 amino acids in the case of the bovin'e, porcine, and human hormone (Fig. 2). the The complete amino acid sequence is known fo~ bovine and porcine hormones but only partially for the human. Synthesis of different segments of the chain has shown that only the first 32-34 amino acids (reading from the N-terminal end) are necessary for biological activity. There is evidence that cleavage occurs naturally to produce a short N-terminal biologically active fragment and a larger inactive C-terminal fragment. The function of this cleavage is unknown. The C-terminal piece is the major PTH component measured in many radioimmunoassays. In common with several other peptide hormones PTH is synthesised as a prohormone, which contains an additional 6 amino acids on its N-terminal end (Fig. 2). A further precursor form, pre-pro PTH, 520 R. G. G. Russell increased excretion of HC03 ions and to a hyperchloraemic acidosis, which is often present to someextent in patients with primary hyperparathyroidism (Wills, 1971). The major classic effect of PTH on bone is' increase resorption, an effect which can be readif demonstrated on explants of bone in tissue cultur(Raisz, 1970; Raisz and Bingham, 1972). Both primary and secondary hyperparathyroidism can be associated with obvious radiological and histological evidence of increased bone resorption. There ;. increasing evidence, however, that low doses PTH, thought to be within the physiological range may increase bone formation and in adult man can be associated with increased intestinal absorption of calcium and positive calcium balance. The ability of PTH to increase the intestinal absorption of calcium may be an indirect effect. brought about by PTH increasing the renal synthesii of 1,25-dihydroxy cholecalciferol (1,25 (OH)2CC). Fig. 2.--Structure of bovine proparathyrold hormone. The arrow between positions 6 and 7 shows the site of cleavage of the probormone to produce PTH itself (after Potts and Deftos, 1974). containing a total of 115 amino acids has now been identified in studies in vitro (Habener et al., 1975). These precursor forms are probably converted to the 84-amino acid peptide before secretion from the gland. The major physiological stimulus to secretion of PTH is a fall in plasma ionised calcium concentration (Ca2+). A rise in plasma Ca2+ above normal suppresses PTH secretion (Fig. 3). Other ions play only a minor role, but in the presence of low levels of Mg2+, the secretion of PTH is impaired and this, together with an impaired target organ response to PTH, may explain the hypocalcaemia occasionally seen in severe magnesium deficiency in humans. In humans secondary hyperparathyroidism, such as that seen in chronic renal failure or vit D deficiency, is due to stimulation of parathyroid secretion by the longstanding low plasma calcium rather than by changes in plasma phosphate or other ions. PTH acts on the kidney to increase the tubular reabsorption of calcium (Massry et al., 1973; Nordin et al., 1967) and to depress the tubular reabsorption of phosphate. This leads to a rise 'in plasma Ca and fall in plasma Pi. PTH also diminishes the secretion of H+ by the kidney, which leads to an Calcitonin (CT) CT is also a peptide hormone but contains 32' amino acids with a disulphide bridge between cysteine residues in positions 1 and 7. The entire sequence is essential for biological activity (Potts and Deftos, 1974). The complete structure is knowr for the bovine, ovine, porcine, salmon, and human hormones (see Fig. 4). There are many differences in the amino acid composition of the calcitonins from different species and this is associated with. different potencies. Surprisingly, the salmon hormone resembles the human more than other mammalian calcitonins. In the treatment of Paget's disease the salmon hormone is more effective on a : molar basis than the human hormone itself. Calcitonin is secreted by specialised cells designated C cells, which are part of the APUD cell series derived embryologically from the neural cres (pearse and Polak, 1972). The C cells are located ill different sites in different species. In man and pig they are found mainly in the thyroid gland, although in man there are some in the thymus. In fishes and ! birds the C cells comprise a separate endocrine gland known as the ultimo branchial body. In experimental animals and birds CT is secreted in response to a rise in ionised calcium (Fig. 3), and also to infusion of certain other hormones, notably glucagon or gastrin. The most important action of CT in mammals is to inhibit bone resorption and thereby lower plasma calcium. Large doses of CT also increase the renal excretion of calcium, sodium, and phosphate and alter the soft tissue distribution of these ions. In man the renal effects can probably be considered pharmacological. Regulation 0/ calcium metabolism 521 Irl.mCT Sirum Ca mll'l!)OlIIl 10.0 r - - - - - - - - - - - - - - , P T H 1100 •"• I, 11.0 1400 : P1H . ! \ '1.0 I 14.0 '~ 1100 Fig. 3.-To show the re- 1'1 \ : ••• • 11.0 10.0 \ lOGO 1100 .aD 1000 .aD 1100 400 1000 100 100 1.0 1.0 4.0 Undetectable 1.0 0 134 0 D I IatJooship between changes in plasma calcium and the secretion of parathyroid hormone and calcitonin In the pig. In (a) a fall In plasma calcium was Induced by lnfuslngEGTA and a rise by infusing calcium. In (b) the values for peripheral Immuooreactive porcine. PTH (IPPTH) and calcitonin (lPCf) are plotted against serum calcium (from Arnaud et al., 1969) 1 7 Tlml,hour. Interrelationships between peripheral IPCr 6. - - - 6. and IPPTH 0 during sequential induced changes in the sea x-x. - - - 0 Fig. 3 (8) 4000 • • .... ~ 700 100 JOOO t :i I..: 1000 l... • 1000 Q.. ~ 100 , Und.t ctobll " , I" " 134 D I 7 I • ~ .tlc ab ' 10 II II 13 14 III III rr III Serum calclum,mll'looml Plot of IPCr • • and IPPTH 0---0 values as a function of those obtained for sea in the same serum samples. r for IPCr - +0·964 and for IPPTH - -0-942. p for both 0-001. Fig. 3 (b) S22 R. G. G. Russell · ..... . .. . ' PORCINE H!M-ICYS,SER',ASII'llEU'ISER'ITHI'ICYS.VAL·1UU ·SEI·ALA·TYI·TlHII&·&SIl·lEU'ASM·&SIl·P1I£·HIS·AI&· 'RES ~; ./ U Y' &lNIEHlY·PHE'I PIO.&lIHHI· PlIO, MH, :••0\, BO~NE :i~;. ':.~- I ~·:ir'",mLft.uHMUT H2M-CY1S.SER,ASN1·l1EU.SE1R. T1HI'CY1S'VAl' UIU.SER.ALA 'TYR 'TIIP·lYI·A$P·lEU·ASIl·ASN 'TTR' HII'AIl8'TEINl&RS";:.~' SALMON • H2M-CIYS'ERAUT5Vl~&Li.:;·16Pot • • ~ i HUMAN • ~ ~ ~ ~ .." ~ ~ if/. MH. . ~ ~ M ~ ~ I elY' PHE ·lll:I'. PlIO·&lU 'TIII'PIlO~ ~ I ~. M 1 ~ ~ jII7. ...... . " . " ... ~ jII7.,O N2 M-en· 6LY ·nM·UU·SEI·THR-CYS·IIET·UU·lil.Y ·THR·TYR-TNR·lil.M ·ASp·PHE·ASHYS ·PHE· HIS, THR· PHE·PlO·GlNTHR·ALA·ILE·GLY· VAL GLULA·PRO •••••• ~ • W ~ ~W W • ~ ~- .J 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Fig. 4.--Structure of calcltooJns from several species. Solid bani show amino acids common to the cakitonins of all 8" species. The shaded bars show positions of partial homology (potts and Deftos, 1974). The exact physiological role of cr in man is unclear. There is a continuing debate, based on conflicting radioimmunoassay data, about whether cr circulates at all in normal man and whether its secretion can be increased in response to hypercalcaemia. Some assays of cr have indicated the existence of large forms of the hormone in the circulation in normal and disease states. The significance of this is obscure. On the basis of current evidence it has yet to be shown that cr has an important physiological role in man. The assay of cr is important, however, in the diagnosis of medullary carcinoma of the thyroid and for the detection of family members with the disease in its presymptomatic form. This condition occurs either alone, inherited as an autosomal dominant, or in association with other endocrine abnormalities-e.g., parathyroid adenomas and phaeochromocytomas and mucosal neuromas (in MEA Type 11). In man CT is also of interest because it reduces the excessive resorption and turnover of bone characteristic of Paget's disease of bone. cr may have a physiological function in other species, e.g., in birds during egg laying and in fisb during migration from fresh to salt water. It is noteworthy that cr is found in elasmobranch fishes. which have a cartilaginous and non-calcified skeleton This suggests that, from an evolutionary point view, the prime function of cr was not concerned with bone. Vitamin D Animals derive their vitamin D from the diet anc from ultraviolet irradiation of dehydrocholestero. in the skin (Fig. 5). Before it becomes biologically active vitamin D (cholecalciferol, CC) has to be metabolised (Avioli and Haddad, 1973; DeLuca, 1972; Omdahl anc DeLuca, 1973). The first step involves its conversion in the liver to a 25-hydroxylated derivative. This step may be regulated by feedback inhibition. The second step involves further hydroxylations in the kidney, to produce 1,2S(OHfiCC or 24,25(OHfiCC. Regulation of calcium metabolism 523 T --. .- £ 1- ebY \ n i_~ICl·r: l"IOIIlio ' . . ._ ... -.. ' _ 0 1 ~:"'.J lIsI 2I-OH-_..il.... t4f' 2!i,M-IDHI.--"'... The latter can be metabolised further to 1,24,25(OH)aCC. The l-hydroxylated metabolites appear to be the biologically active forms of the vitamin, with 1,25(OH)2CC probably being the most important and worthy of the name, hormone, rather than vitamin (Norman and Henry, 1974). The major actions of 1,25(OH)2CC are on the intestine to increase calcium absorption and on bone to increase resorption. Although lack of vit D in man is associated with defective mineralisation of cartilage and bone, the question whether vit D or its metabolites act directly on bone to increase mineralisation is still unsettled. It is possible that the effects of vit D on bone mineralisation are secondary to changes in extracellular fluid concentrations of calcium and phosphate, but this explanation may be too simple to account for all the experimental and clinical observations. Unfortunately, there are no good experimental systems for studying skeletal mineralisation in vitro and the major effect of 1,14,21-IDHI._ o i l.... 1./__.... - TARGET TISSUES • Fig. 5.-The major intercoDverslons in vitamin D metabolIsm: <a)structural formulae (Coburn et al.,l974); (b) some of the proposed sites of regulation of D metabolism to show stimulatory ( +) and inhibitory ( - ) effects. BIOLOGICAL RESPONSES Fig. 5 (a> KIDNEY LIVER Gut ~_4_~Bone Vitamin 0 3 - - . Muscle Fig. 5 (h) Bone Gut 524 R. G. G. Russell l,25(OH)2Da on bone in culture is to increase resorption. The metabolism of vitamin D in the kidney appears to be closely regulated in experimental animals. The production of 1,25(OH>2CC is stimulated under conditions of vitamin D deficiency, and by low dietary calcium or phosphate. Increased production of l,25(OH)zCC is accompanied by a reciprocal diminution of 24,25(OH)£C and vice versa. The function of 24,25(OH)zCC is unknown. There is evidence that the effect of calcium deprivation in increasing 1,25(OH)2CC is mediated by increased secretion of PTH, whereas the effect of phosphate restriction is independent of PTH (Hughes et al., 1975). However, since studies on the regulation of production of 1,25(OH)zCC in experimental animals have necessarily been done under extreme conditions often using animals or birds already deficient in vitamin D, caution is needed before extrapolating these findings to man. Evidence that the same relationships may exist in man comes from recent measurements of 1,25(OH)2CC in plasma which indicate that the levels are higher than normal in hyperparathyroidism and lower than normal in hypoparathyroidism (Hughes et al., 1975). They are also low in renal failure (Mawer et al., 1973) perhaps because renal mass is diminished or as a result of the hyperphosphaternia that exists in this condition. The possible importance of phosphate as a regulating ion for 1,25(OHhCC synthesis in man is also indicated by the enhanced intestinal absorption of calcium and the hypercalciuria that follows dietary deprivation of phosphate. It is possible that hypophosphatemia stimulates the production of l,25(OH)2CC in this situation, and may do the same in primary hyperparathyroidism and in those cases of "idiopathic" hypercalciuria where intestinal hyperabsorption of calcium is the cause. The increased intestinal absorption of calcium to meet the physiological requirements of growth, pregnancy, and lactation are also probably mediated by increased production of 1,25(OHhCC, which may explain the apparently increased requirement for the precursor vit D under these conditions. There is an interesting interrelationship between the actions ofvit D and PTH, such that in the absence of vit D some of the target organ responses to PTH are impaired, notably the effect on bone resorption. In addition it is clear that the diminished intestinal absorption of calcium seen in vit D deficiency is not overcome by the increased secretion of PTH that follows deprivation of vit D. This may mean that effects normally ascribed to PTH itself are in reality mediated by an increase in 1,25(OH>2CC induced by PTH. THE INFLUENCING HORMONES Calcium and phosphate metabolism are also affected by growth hormone, thyroid hormones glucocorticoids, and sex hormones. ' Growth hormone (GH) is best known for its effect on growth of cartilage, an effect which is probably brought about indirectly by the growth hormonedependent production of somatomedin (Van Wyk et al., 1974). There are probably several somatomedins, not all of which are dependent on GH. Their biochemical characterisation is incomplete. . GH ,also causes an elevation in plasma Pi by increasmg the apparent reabsorption of phosphate by the kidney. Excess or deficiency of GH is associated with skeletal growth abnormalities. In acromegaly there is an increased periosteal apposition of bone, but there is no convincing evidence that acromegaly causes osteoporosis. Deficiency of thyroid hormones early in life produce the well known skeletal deformities of cretinism. In the adult, excess of triiodothyronine or thyroxine can be associated with hypercalciuria, hyperphosphataemia, a raised alkaline phosphatase, and occasionally hypercalcaemia. In hyperthyroidism there is increased bone turnover often with net loss of bone. These effects are probably due to a direct action of thyroid hormones on bone, and an increase in resorption of bone can be demonstrated in organ culture. In humans resorption is probably increased by thyroid hormones to a somewhat greater extent than formation, leading to a slight increase in plasma calcium. This may suppress the secretion of PTH and explain the diminished renal tubular reabsorption of calcium and enhanced reabsorption of phosphate. Adrenal steroids Adrenal insufficiency is rather frequently accornpanied by hypercalcaemia, probably due in part to haemoconcentration but also to altered renal handling of calcium. In spontaneous or iatrogenic glucocorticoid excess, osteopenia may develop. In experimental animals glucocorticoids produce a variety of effects on bone depending on the compound given, the dose and the animal species. The relative contributions of diminished intestinal absorption, increased renal excretion of calcium and of diminished bone formation and excessive resorption of bone, all of which may occur, is not well defined. Sex steroids Characteristic growth abnormalities are associated with deficiencies of either male or female sex hor- Regulation of calcium metabolism 525 mones. In adults the effects of oestrogens are of _, particular interest because of the loss of bone that occurs in women after the menopause. Administration of exogenous oestrogen may slow down this loss. Oral contraceptives may reduce bone turnover in premenopausal women. INDIVIDUAL ORGAN REsPONSES Intestine The intestinal absorption of calcium appears to involve both active transport and diffusion processes, Absorption occurs throughout the small intestine and is quantitatively greater in the ileum than the duodenum, even though active transport is more evident in the duodenum. The fraction of the dietary intake absorbed varies with the dietary content so that net absorption remains relatively constant except at extremely low or high intakes. The adaptation to dietary intake of calcium is now thought to be mediated mainly by changes in 1cc25(OH)zCC. MUCOSAL CELL BRUSH BORDER LUMEN The mechanism may be that when the inflow of calcium from the gut falls, there is a tendency to hypocalcaemia which stimulates PTH secretion; this in turn enhances the synthesis of 1,25(OH)zCC which causes the intestinal absorption of calcium to return to its original value. This adaptation is a slow one. Unlike the responses by the kidney and bone to PTH and CT, which occur within minutes or hours, the intestinal responses take 1-3 days. Intestinal absorption of calcium also varies with age arid sex and in disease states. It is likely that, with better knowledge about the metabolism of 1,25(OH)zCC in man, many of these variations will be explicable in terms of changes in the metabolism of vitamin D. The biochemical mechanisms involved in calcium absorption through the intestinal mucosa are not understood in detail, though several features of the system have been identified (see Fig. 6). There is a calcium binding protein (CaBP) as well as a Caz+_ stimulated ATP-ase (which may be the same enzyme BLOOD Co*ATP-ase P·tasel "wlI~ZF(Ok Co.... • aIHO(52~ -l~W.'I ATP < Na-Co- ATP-ase (ETHACRYNIC ACID SENSlTIVEI • • • ~ • ~ PROTEIN ·SYNTHESIS - - - - -CoSP -( -Co*ATP-ase \ ?-Other ATP-D5n) :I ]I FACILITATED DIFFUSION INTRACELLULAR MOVEMENT ? Active transport Mitochondrial binding + ionic diffusion D. • N O - K - A T P - ase (OUABAIN SENSITIVE) • :m: ACTIVE TRANSPORl EnellJY dependent Fig. 6.-HypotheticalintestJnal mucosal cellto show some of the factorsthoughtto heinvolved In calcium transportand In the stlmulatOl'Yeffect of 1,25 (OH.)-vltamin DB' CaOP = calcium binding protein, CaB+ATP-ase = calcium stimulated adenosine triphosphatase (from Coburn et al., 1973). 526 R. G. G. Russell as intestinal alkaline phosphatase), which are both dependent on the presence of vitamin D or its derivatives (Coburn et al., 1973). It is the synthesis of these components as well as the metabolism of vitamin D itself which probably accounts for the relatively long lag period between giving vitamin D and achieving an intestinal response. One puzzling feature is that both the CaBP and Ca2+-ATP-ase are located on the luminal surface of the intestinal epithelial cell. It is difficult to see why they need to be present at this site since the luminal calcium concentration is much higher than the presumed intracellular concentration of calcium and there seems therefore to be no need for an assisted transport mechanism against an electrochemical gradient. An active extrusion mechanism however is required at the basal border, unless, as some workers postulate, the calcium traverses the intestinal epithelium in the intercellular space. It seems likely that the Ca binding protein delivers calcium from the lumen to a site essential for further translocation. There is now good evidence that calcium and phosphate can be absorbed separately from each other and that 1,25(OH)2CC has independent effects to enhance the absorption of each. For calcium, 1,25(OHhCC acts on both the diffusion and saturable components of the transport system. Absorption of calcium and phosphate is also enhanced by a water-soluble factor (possibly an analogue of vitamin D) from a South American plant, Solanum glaucophyllum, which can cause fatal hypercalcaemia in cattle that eat it. Kidney The kidney is a key organ in determining the plasma concentrations of both calcium and phosphate. Of the various hormones mentioned PTH is probably the most important under physiological circumstances, enhancing reabsorption of calcium and reducing reabsorption of phosphate. There is little evidence in man that vitamin D or its metabolites, at physiological concentrations, have a major effect on the renal handling of calcium or phosphate that cannot be accounted for by changes in the secretion of PTH. Thus the enhanced reabsorption of phosphate seen after restoring vitamin D to patients deficient in vitamin D is probably due to suppression of the parathyroid hypersecretioncharacteristic of this state. However vitamin D in doses far in excess of physiological requirements can increase excretion of both calcium and phosphate. The calcium ion itself affects phosphate handling. Infusion of calcium into hypoparathyroid patients to restore their plasma calcium towards normal can also partially reverse the increased tubular reabsorp- tion of phosphate. This may help to explain why treatment of such patients with vitamin D or its derivatives results in a fall in plasma phosphate as the plasma calcium rises. In contrast, as plasma calcium is increased above normal, an opposite effect on the renal handling of phosphate may come into being so that there is enhanced rather than diminished reabsorption of phosphate. Both calcium and phosphate excretion are influenced by other factors, notably Na + excretion, extracellular fluid volume expansion and by the administration of diuretics (Massry et al., 1973). There is evidence for both proximal and distal sites of tubular reabsorption and both can be influenced by PTH for phosphate, whereas the action of PTH on Ca is probably mainly distal. Infusion of NaCI increased the excretion of both calcium and phosphate, an effect which probably contributes to its value in the treatment of hypercalcaemia. The biochemical mechanisms involved in renal transport of calcium and phosphate are not elucidated, but the action of PTH on the kidney is known to produce an increase in cortical adenylate cyclase activity which increases tubular cell and urinary concentrations of 3'5' cyclic adenosine monophosphate (cyclic AMP). It is not known whether this increase in cyclic AMP is the cause of the subsequent changes in phosphate and calcium transport. However, in the clinical disorder pseudohypoparathyroidism there is probably a defective receptor mechanism for PTH in both kidney and bone, so that administration of PTH does not produce a normal response of an increased excretion of phosphate and cyclic AMP. This is analogous to the failure of patients with nephrogenic diabetes insipidus to respond to antidiuretic hormone. In pseudohypoparathyroidism the low plasma calcium and high plasma phosphate resemble the findings in simple hypoparathyroidism but there are additional abnormalities in that the condition is familial and the patients are of short stature and have short fourth metacarpal bones. It is noteworthy that in pseudohypoparathyroidism there is an appropriate response of the parathyroids to hypocalcaemia, so that these patients have elevated plasma concentrations of PTH, which however do not cause the usual target organ responses. There is now an additional variety of pseudohypoparathyroidism described in which PTH causes an increase in cyclic AMP but no change in renal phosphate clearance. Bone The structural organisation of bone is complex, as can be seen from Fig. 7. In mature bone three main cell types exist: Regulation of calcium metabolism 527 Table 2 Cllment Ii,... Calcification Biological Cartilage Bone Teeth Pathological Dental calculus Urinary tract stones Ectopic bone, e.g., my ositisossificans progressiva, haematomas, paraplegia Newbor.e Fig. 7.-To show some of the histological features of compact hone of the type found in 11mb bones (from Harris aDd Heaney, 1969). osteoblasts, responsible for bone formation, osteoclasts responsible for bone destruction, and osteocytes which are derived from osteoblasts and become trapped within the bone matrix as maturation proceeds. The osteocytes lie within a complex canalicular system and are probably responsible for many ot the rapid ion fluxes that occur in bone. The origin, life span, and fate of the various cells in bone is only partially understood. The tissue fluid surrounding bone cells probably has an unique composition, being high in K + and containing particular plasma proteins in preference to others. The mineral component of bone is predominantly hydroxyapatite. Since ionic exchange occurs between bone mineral and surrounding fluids, it also contains other ions such as HCOa-, M g2 +, Na +, K +, etc. Collagen is a major constituent of the organic matrix of bone and is largely responsible for its tensile strength. Bone collagen is of the type I variety. In addition, the matrix contains several proteoglycan components. The rate of deposition of bone is controlled by hormonal status (e.g., PTH, GH), by levels of calcium and phosphate, and by mechanical and electrical forces acting on bone. Calcification is an important step in the transition between matrix production and the formation of mineralised bone. In the skeleton calcification takes place in two main sites in epiphysial cartilage during the growth of long bones and in bone matrix during intra-membraneous ossification and in the remodel- Dystrophic Ca and P concentrations normal. Mechanism possibly tissue damage leading to nucleation of crystals, e.g., blood vessels, Monkeberg's medial calcinosis, costal cartilage, TB lesions, haematomas, bursitis, skin (calcinosis, scleroderma, dermatomyositis) Metastatic Ca or P concentrations high e.g., blood vessels in renal failure, also cornea, gastric mucosa, lung alveolar septa (perhaps because CO. is liberated and pH raised) brain in hypoparathyroidism, kidney in hypercalcaemia ling and growth of existing bone (Table 2). The concentrations of calcium and phosphate in ECF are insufficient to initiate the deposition of calcium phosphate but can sustain crystal growth once it has started. The first steps in calcification are now thought to take place in or around small membranebound vesicles found in the matrix. These vesicles appear to arise from the plasma membranes of hypertrophic chondrocytes during the maturation of epiphysial cartilage. Their source in bone is not identified. These vesicles are rich in alkaline phosphatase, an enzyme which has been known for many years to be associated with calcification. Alkaline phosphatase may function in calcification as a component of a membrane pump for calcium and phosphate, or it may be involved in the removal of potential inhibitors of calcification such as inorganic pyrophosphate (PPi). In hypophosphatasia, which is a recessively inherited skeletal disorder characterised by a deficiency of alkaline phosphatase, there are increased concentrations of PPi in plasma. Since PPi is an inhibitor of the crystal growth of calcium phosphate it may be responsible for the defective calcification of cartilage and bone seen in this condition (Russell and Fleisch, 1975). Some other causes of defective 528 R. G. G. Russell skeletal mineralisation are included in Table 3. Rickets is the term used to define failure of mineralisation of cartilage in long bones and is seen in growing children. Osteomalacia has several meanings and to some people implies a state of vitamin D deficiency. The term may be used to describe the clinical features of D deficiency, or it may be restricted to the histological abnormality in bone irrespective of the cause. Excess osteoid is perhaps a better term for the latter abnormality and is characterised by the presence of unmineralised matrix on trabecular surfaces in bone. Calcification can occur outside the skeleton at many sites within the body in pathological states and it is remarkable how specific some of these sites are for particular diseases, e.g., the basal ganglia in hypoparathyroidism, and ectopic bone formation around the hips in paraplegia (Table 2). Inhibitors of calcification may partly contribute to impaired Table 3. Some causes of excess osteoid (uJfmineralised bone matrix) Vitamin D deficiency or defective metabolism Nutritional deficiency of vitamin D Lack of sunlight Malabsorption of vitamin D Chronic renal failure Vitamin D-dependent rickets Low plasma phosphate Phosphate deficiency Renal tubular disorders (vitamin-D-resistant rickets) Chronic acidosis Ureterosigmoidostomy Renal tubular acidosis (proximal or distal) High bone turnover Fracture healing Paget's disease Hyperparathyroidism Hyperthyroidism Drugs Anticonvulsants Diphosphonates Fluoride Inherited Fibrogenesisimperfecta ossium Hypophosphatasia Other unexplained Axial osteomalacia calcification of bone and cartilage in chronic renal failure. Acidosis, such as occurs after ureterosigmoidostomy or in renal tubular acidosis, can lead to excess osteoid. The mechanism may be that initiation of mineralisation is more difficult in an acid environment because of increased solubility of calcium phosphate but it may also be due to the hypophosphataemia which is usually also present. Conceivably acidosis may also disturb the metabolism of vitamin D. Resorption of bone is an essential part of the remodelling and growth process (Harris and Heaney, 1969; Raisz, 1970; Raisz and Bingham, 1972), and is mediated by mononuclear and multinuclear osteoclasts, some of which may be macrophages originating outside bone. Osteoeytes are probably responsible for some hormone dependent rapid fluxes of ions in and out of bone. Under physiological conditions resorption is probably under the control of PTH, vitamin D, thyroid hormones and steroids, but will occur at a basal rate in the absence of these hormones. Several other agents can stimulate bone resorption under experimental conditions and may be important in disease. Vitamin A is one of these. Prostaglandins, particularly of the E series, are also resorbing agents and may be important in some of the hypercalcaemias of malignancy and in the bone dissolution that accompanies rheumatoid arthritis (Robinson et al., 1975). Myeloma cells and activated lymphocytes can produce materials which cause resorption in vitro (Raisz et al., 1975, Fig. 8) and which have been designated OAF (osteoclast activating factors). The biochemical events occurring during bone resorption are poorly understood. Some resorbing agents, such as PTH and prostaglandins, can stimulate the production of cyclic AMP in bone but others. e.g., 1,25(OH)2CC, do not. Inflow of calcium ions into bone cells may be an early event in their stimulation. Ionophores which promote the entry of calcium ions into cells can be shown to stimulate resorption. Bone resorption in tissue culture is accompanied by release of enzymes such as collagenase (Harris and Krane, 1974) and lysosomal enzymes capable of degrading matrix. Osteoclasts viewed by electron microscopy possess a ruffled border which is closely applied to the bone surface and is presumably the site at which removal of bone mineral and matrix occurs. Administration of calcitonin, which inhibits resorption, causes retraction of this border and eventually leads to a decreased number of osteoclasts. Apart from calcitonin several other inhibitors of resorption exist. These include oestrogens, mithramycin and diphosphonates. Inhibitors of bone resorption are useful therapeutic agents in certain clinical disorders (Table 4). Regulation of calcium metabolism 529 Radioisotopes and bone: Bone scanning The affinity of bone mineral for various ions is of importance in radiobiology and clinical diagnosis. Radioisotopes such as 226Ra and 239Pu are bone seekers and can be derived from exposure to industrial sources or fallout from nuclear explosions. Radium workers develop bone tumours and 239Pu can produce tumours in experimental animals. Isotopes such as B9Sr and 18F have been used clinically as scintigraphic agents for detecting regions of increased bone turnover such as in metastatic tumour deposits. Recently pyrophosphate, polyphosphates and diphosphorates, all of which have a high affinity for crystals of hydroxyapatite, have been used as effective bone scanning agents (Hosain et al., 1973) by linking them to the gamma-emitting isotope, J9mtechnetium, in the presence of stannous ions to produce J9mTc-Sn-PP. 60 .PTH -OAF ~ ~ 50 40 ~ ~ 30 ~ ~ ~3\Q ~ ~ a CQ\JTROI. ~ 20 t5 q: 10 0 TREATMENT 2 3 4 Cell calcium and cyclic AMP DAYS IN CULTURE Fig. 8.-BOIle resorptlOll stimulated by osteoclast activating factor (OAF) derived from Iympbold cell lines compared with the action of PTH. The release of tiCa from prelabelled rat fetal bone was measured In organ culture in vitro (from Raisz et al., 1975). It is now recognised that there are significant interrelationships between intracellular calcium, cyclic AMP, and cell activation in response to various stimuli. Fig. 9 shows a simple scheme to illustrate some of these interrelationships. Cell calcium Act Ive _____.1.-_-+__....... Ca2 +_ _....L..--,....-I---....L----_ Ca 2 + e1O-3M) (10- 3M) Hormones Mitochondria Fig. 9.-Simpllfled general scheme to show major events thought to Influence Intracellular calcium cOIlcentration. Cytosol calcium is thougbt to lie in the range of 10- 1 - 10- 1 mol/I. 530 R. G. G. Russell Table 4. Therapeutic agents in calcium metabolism Agent Use Calcium dietary supplement intravenous Phosphate oral intravenous Vitamin n, or D. } Dihydrotachysterol 1,25(OH)zCC, 250HCC la-OHCC Calcitonin (porcine, salmon or human) Oestrogens Fluoride (plus vitamin D and calcium supplements) Diphosphonate (EHDP) Mithramycin Thiazides Anabolic steroids Mechanism of Action osteoporosis hypocalcaemia Bone resorption renal tubular disorders, vitamin D resistant rickets in which plasma P ~ renal stones hypercalcaemia Bone mineralisation Correction of vitamin D deficiency Hypoparathyroidism Vitamin D-resistant rickets Chronic renal failure Paget's disease Hypercalcaemia Post-menopausal or post-oophorectomy bone loss Osteoporosis Ectopic calcification Paget's disease Paget's disease Hypercalcaemia due to bone metastases or myeloma Renal stones Obsolete Cytosol calcium concentrations are thought to be 100-1000 times lower than extracellular-i.e., in the range of 10-5-10- 6 mol/I. Within cells mitochondria are capable of accumulating large amounts of calcium against electrochemical gradients, to the point at which intramitochondrial deposits of insoluble calcium phosphate can form (Borle, 1973). The activation of many different types of cells by hormones or pharmacological agents is now thought to be accompanied by increases in intracellular calcium concentration, derived from outside the cell or by release from mitochondria (Rasmussen and Goodman, 1975). Hormonal activation is often associated with stimulation of adenylate cyclases specific to the target tissue. The changes in cyclic AMP and intracellular Ca then produce further responses within the cell, e.g., by changing enzyme activity. In many systems addition of cyclic AMP can mimic hormone action, as can those ionophores ~ t Urine Ca t PPi t ?precipitation of calcium phosphate Intestinal absorption t Bone resorption t Bone resorption and turnover Bone resorption and turnover Bone resorption ~ Bone formation Calcification ~ Bone turnover Bone turnover Urine Ca ~ ~ t ~ ~ ~ which increase the transport of calcium into cells. In the case of parathyroid hormone, cyclic AMP and ionophores can each independently induce resorption in bone explants (Dziak and Stern, 1975) and each can also stimulate mitosis in lymphocyte cultures. These interactions have been reviewed extensively elsewhere (Borle, 1973; Rasmussen and Goodman, 1975; Robison et al., 1971). In some systems the concentration of 3'5'-cyclic guanosine monophosphate (cyclic GMP) changes in the opposite direction to that of cyclic AMP, and produces opposing effects, a phenomenon which has led to the so-called "Yin and Yang" hypothesis (Goldberg et aI., 1973). An example of some of these interrelationships are shown in Fig. 10 for the release of lysosomal enzymes from neutrophils (Smith and Ignarro, 1975) a system which contrasts with those described above in that cyclic GMP is stimulatory and cyclic AMP and calcium ions are inhibitory. Regulation of calcium metabolism NEUTROPHL EPlNE"HIIINE __ PIlOSTAGLAMlINS -+ tolfa~ENYLAT ATP A- .' •• 11. \:YCLASE rt - 4':P! M A.! Ci IL~ '£ CYCLIC AMP _,..OTEIN KINUE ! IRUNE .[ACTANT IlUST IE ,,,,nUT AT . - : ... PItOSPHllIlYLATION LYSOSOMAL ENZYIIE $lCRETION ~_ GMP IONOPHORES (DIVALENT) e!IAY INIIOLVE LYSOSOMES. llICROTUIULES. PLASMA MEMBRANE. ? Fig. lO.-Postuiated interactions between calcium ions, cyclic AMP and cyclic GMP, and other agents during the release of lysosomal enzymes from human neutropbil blood cells in vitro (abbreviation PDE = phosphodiesterase) (from Smith and Ignarro, 1975). INTEGRATION OF INDIVIDUAL ORGAN RESPONSES It is useful to draw some distinction between the way in which the plasma calcium is set at a particular value, and the way the movements of calcium in and out of extracellular fluid (ECF) are controlled. The plasma calcium is set close to a particular value in different individuals in normal and disease states. Deviations from this value are corrected by hormoneinduced changes in the relative fluxes of calcium in and out of the ECF. Alterations in the flux rates are therefore monitored and adjusted by the changes in plasma calcium (or phosphate) concentration they induce. This homoeostatic system could operate with the plasma Ca set at any number of different values with the relative rates of entry and exit of calcium to and from the ECF being altered as drift occurs from this set point. Thus in hyper- and hypoparathyroidism the fluxes of Ca across the gut and in and out of bone may not be greatly different than normal and external calcium balance can be maintained (net intestinal absorption = urine loss) even though the plasma Ca is set at markedly different levels (Fig. II). The plasma Ca thus provides the point around which adjustments are made. In considering homoeostasis it is also helpful to distinguish between acute and chronic changes. When the system is disturbed, a steady state no longer exists, and the response which occurs adjusts 531 the system so that a new steady state comes into existence. The flux rates through individual organs and the level of plasma Ca mayor may not be the same as previously depending in part on whether the disturbance to the system is sustained or not. Regulation ofplasma calcium: Acute responses The total concentration of calcium in plasma is normally around 2-2.5 mrnol/l. Of this about 1.2 mmol/l is present as ionised calcium (Ca2+), the remainder is complexed to proteins especially albumin, and to small ions such as citrate and phosphate. It is the ionic Ca2+ which is regulated. Under most circumstances Ca2+ bears a constant relationship to total plasma calcium. The concentration of extracellular Ca2+ is important for neuromuscular function and other membrane responses and is also involved in biological events such as blood coagulation. Deviations of plasma Ca2+ away from its normal value are rapidly corrected by alterations in the secretion of the regulating hormones. In many mammals PTH and calcitonin (Fig. 4) both respond, but in opposite directions, but in man it is doubtful whether CT changes. PTH and CT can be considered the fast acting (minutes to hours) component of the regulatory system whereas vitamin D is responsible for adaptation in the longer-term (hours to days). In experimental animals removal ofsources of PTH and CT (e.g., thyroparathyroidectomy in dogs or rats) results in a slower than normal return of plasma calcium to starting values in response to acute changes in plasma calcium. In man the most important regulator of acute changes of the concentration of Ca2+ is parathyroid hormone (PTH) which increases within seconds of a fall in plasma Ca2+, and decreases as Ca2+ rises. The rapid control of plasma Ca2+ by PTH is mainly due to its ability to regulate renal tubular reabsorption. After parathyroidectomy the fall in plasma Ca2+ can largely be accounted for by a continuing loss of Ca2+ into urine until a new steady state is achieved in which calcium excretion may not be greatly below its starting value but takes place at a much reduced filtered load of calcium. The intestinal responses are too slow to account for rapid changes in plasma calcium but the bone does playa part. Thus there is an important buffering action of bone, so that rises or falls of plasma calcium are partially compensated by increased net movements of calcium into or out of bone respectively. This is partly a passive process but there is probably also a contribution from changes in PTH-mediated osteolysis. In animals in whom the fluxes of calcium in and out of bone are relatively much greater in relation to fluxes through 532 R. G. G. Russell Ca mg/day Normal Hypoparathyroidism Hyparparathyroldlsm Fig. 11 (a) 200ml 200lllll Ca Hypoparathyroidism + 200ml mg/day Normal - Ca infusion 200mg Fig. 11 (b) 1.200mg 200mg Fig. 11.---SCheme to show relationship between plasma and urine calcium. The arrow entering the kidney represents the filtered load under various conditions. calculated assuming approximately 60 % of plasma calcium to be ultra Wtrable ..glomerular Wtration to be 120 ml/min. The arrow from the kidney to the plasma compartment represents tubu.... reabsorption. The difference between the filtered load and reabsorbed calcium Is that excreted In the urine. (a) Makes t.IIe point that urine calcium can be normal in hypo and hyperparathyroidism even though plasma calcium Is markedly abnormaL ThIs Is because there Is diminished tubular reabsorption of calcium In hypoparathyroidism Bnd enhanced reabsorptioB .. hyperparathyroidism. This is Illustrated (b) by the Infusion or calcium Into hypoparathyroid patients; when plasma caldta Is restored to normal by infusion, urine calcium Is much higher than In normal persons because of diminished renal tu!JIIIIr reabsorption. Regulation of calcium metabolism kidney and intestine than they are in man, changes in bone resorption (and formation) are quantitatively more important than in man for producing acute changes in plasma Ca. This is well illustrated by examining the acute responses of plasma Ca to injection of calcitonin, the most significant acute effect of which is to inhibit bone resorption. In normal man there is a negligible fall in plasma calcium, whereas in rats, particularly in young animals in which bone turnover is high, there may be a marked decrease in plasma calcium. Simple calculations verify that in normal man complete abolition by calcitonin of the small flux of calcium out of bone could not be expected alone to cause a significant fall in plasma calcium. However, in situations where bone resorption is quantitatively greater in relation to the other fluxes, e.g., in patients with Paget's disease or with tumour metastases in bone, then an acute fall in plasma calcium can be seen after given calcitonin. Chronic changes The response to prolonged perturbations brings in contributions from changes in vitamin D metabolism and from the intestine and bone. An example is the adaptation that occurs to a change in dietary intake of calcium. If intake is reduced this will tend to cause a gradual fall in plasma calcium which will increase the secretion of PTH. Apart from its effect on the kidneys, a sustained increased in PTH will lead to enhanced osteoclastic resorption of bone, and an increase in 1,25(OH)2CC synthesis which will enhance intestinal absorption of calcium and resorption of calcium from bone. If the reduction in dietary calcium continues, these changes will act to restore the plasma calcium towards its previous value and bone formation rate will increase to match the rate of increased bone resorption by the coupling mechanism described below. The new steady state will come to consist of a greater efficiencyof intestinal absorption of calcium and an increased rate of entry and removal of calcium from bone so that net balance can be maintained. This response can be seen very clearly in experimental animals and there is good evidence that these adaptive changes occur in man too. When dietary deprivation is so severe that intake can no longer match output the reserves of calcium in bone are utilised. Other examples of steady states that exist during chronic disturbances in calcium metabolism will be considered under clinical disorders of calcium homoeostasis. Coupling offormation and resorption: Control ofbone shape and mass The skeleton can respond to hormones by increas- 533 ing its uptake or release of calcium. In a variety of both physiological and pathological steady states (see Fig. 12) it is clear that there is a remarkably close correlation between rates of mineral deposition and mineral resorption. Even though these individual rates may be altered many-fold, the net gains or losses of skeletal mass are minimised by the tight coupling between these rates. This is an important feature of homoeostatic adaptation (Harris and Heaney, 1969) that is often overlooked. Thus it is difficult to achieve a sustained dissociation between rates of mineralisation and resorption and this is one reason why so many potential therapeutic agents have been disappointing in the treatment of bone disease, for example in increasing bone mass in osteoporosis. Transient dissociations can occur, however, for example in the acute loss of bone mineral that occurs in response to immobilisation. Evidence for coupling also comes from measurement of alkaline phosphatase and urinary total hydroxyproline (THP) in Paget's disease of bone. There is a close correlation between these two measurements over a wide range of values. Alkaline phosphatase is thought to be an indirect measure of bone formation rate, and urine THP in this situation to reflect mainly the rate of resorption of bone collagen. The mechanisms underlying this coupling are unknown. It may involve cell to cell communication within bone, in addition to external endocrine influences. Another suggestion is that there is an obligatory cellular differentiation of osteoclasts to osteoblasts, which would couple rates of bone formation to previous rates of bone resorption (Rasmussen and Bordier, 1974). Although this is an attractive idea it has certain weaknesses and even if true can only account for part of the phenomenon. An additional feature of bone is its ability to respond to mechanical deformation by changing its structure and shape to counteract the stress. One mechanism proposed to account for this involves the generation of small electrical currents within bone as stresses are applied. Such currents can be demonstrated experimentally and are thought to be due to the piezoelectrical properties of the mineral and matrix components. DISORDERS OF CALCIUM HOMOEOSTASIS Detailed description of disorders of calcium metabolism is beyond the scope of this review but there have been several books and reviews published on this topic recently (Fourman and Royer, 1968; Morgan, 1973; Nordin, 1973; Paterson, 1974; Krane and Potts, 1974; Potts and Deftos, 1974; Schneider and Sherwood, 1974). The purpose of this section is to illustrate some of the principles of S34 R. G. G. Russell . • I . ,,. ,, ,, , w- ,, " , , w' , , . '. ,?' " .,'·,t ... ... . . . .. .- • • . -.,4·· · • , A 1. _ C,"I/d) . ,, }."" .. . ,. . . ..... .,,-. .. ...,'.,..,,' . • • • , ~,. .,,. • "',,:4... .: . ,, 0.1 ,, w •• • • 1. 0.1 R .. 10 (,MId) Fig. 12.-Relationshlp between accretion rate (A) and resorption rate (R) of bone detennlned by radioisotope methods in patients with a variety of disturbances in calcium metaboUsm. Note that the plot Is logarltbmic and there Is close correspondence between rates of accretion and resorption over a wide Ij8Dge of values (from HarrIs and Heaney, 1969). calcium homoeostasis as applied to chronic steady states found in various diseases. ExcessPTH In primary hyperparathyroidism, due to adenomas, hyperplasia or carcinoma, the concentration of PTH is inappropriately high for the prevailing plasma calcium, implying a defect in the gland for switching off PTH secretion at normal levels of Ca2+. In hyperparathyroidism the hypercalcaemia is maintained mainly by a resetting of the renal tubular reabsorption mechanism for calcium, so that reabsorption is enhanced at any given filtered load. There is often also increased bone resorption and increased intestinal absorption of calcium, and both these will tend to increase urinary calcium excretion. The calcium balance is usually normal but may be negative, particularly in patients with severe bone disease. The fact that bone and renal stone disease do not usually occur in the same patients is intriguing and unexplained, but may imply important differences in relative target organ responses to increased PTH. In secondary hyperparathyroidism, such as occurs in renal failure or vitamin D deficiency, PTH is high as an appropriate response to hypocalcaemia. In both these conditions there is an impaired target organ response to PTH which prevents the plasma calcium from returning to normal. After removal of the hypocalcaemic stimulus, e.g., after renal transplantation, it may take a long time for the hyperplastic glands to respond appropriately to plasma calcium, so that hypercalcaemia may persist for Regulation of calcium metabolism months. In pseudohypoparathyroidism there is also an appropriate excess of PTH due to hypocalcaemia, but again a failure in the target organ responses of bone and kidney. Deficiency ofPTH In hypoparathyroidism the persistent hypocalcaemia is largely due to a resetting of renal tubular reabsorption so that there is a relative renal leak of calcium compared with normal. Bone turnover (formation and resorption) is diminished and intestinal absorption of calcium can be low. External balance can be maintained (urine Ca = net intestinal absorption) even in the face of hypocalcaemia. When vitamin D 1 , 1,25(OH)2CC or its analogue lor. hydroxycholecalciferol (lor. (OH)CC) is given to such patients plasma calcium can rise due to a massive increase in intestinal absorption of calcium, and possibly to enhanced bone resorption. Vitamin D metabolites cause no obvious change in the renal tubular handling of calcium, but large doses of vitamin D itself may do so. Thus in treated hypoparathyroidism net intestinal absorption and urinary loss of calcium may be matched but at high levels (c. 1000 mg/day) and calcium balance thereby maintained. There is increasing evidence that, in untreated hypoparathyroidism, 1,25(OH)2CC may be produced in less than normal amounts, perhaps due to the lack of PTH or to hyperphosphatemia. Excess of vitamin D The metabolism of vitamin D is regulated so that the rate of formation of l,25(OH)2CC is homoeostatically controlled. However other metabolites have some biological activity which may be important when large doses of vitamin D are given. There may then be enhanced intestinal absorption and urinary excretion and increased resorption of bone. Hypercalcaemia can occur, usually in association with reduced glomerular filtration, and with some evidence for increased renal tubular reabsorption of calcium (Nordin, 1973). 535 so that urinary calcium may become undetectable. The renal tubular handling of calcium in vitamin D deficiency has not been studied in detail and it is possible that vitamin D in physiological doses is able to increase tubular reabsorption, either directly or by allowing the action of PTH to be expressed. In vitamin D deficiency the enhanced secretion of PTH is apparently unable to exert its usual effect, not only on the kidney, but also on bone. Disturbed metabolism of vitamin D Features of deficiency of vitamin D may be present where there are adequate supplies of vitamin D but its metabolism is disturbed. In osteomalacia associated with anticonvulsant drugs it is claimed though not proven that there is increased metabolic degradation of vitamin D to inactive metabolites due to hepatic enzyme induction. In the rare condition of pseudo-vitamin D deficiency (vitamin D-dependent rickets) it is thought that the enzyme responsible for the renal production of 1,25(OH)2CC is deficient (Fraser et al., 1973). A much commoner cause of this is renal failure itself, in which renal production of 1,25(OH)2CC is probably defective (Mawer et al., 1973). Sarcoidosis may be another disorder in which the metabolism of vitamin D is disturbed. Some patients with sarcoidosis have increased intestinal absorption of calcium and increased bone resorption which may sometimes produce hypercalcaemia. They are abnormally sensitive to exogenous vitamin D and respond to glucocorticoid therapy. These are the features that might be expected from a condition in which 1,25(OHl2CC would be produced in excess of normal. Calcitonin deficiency and excess No examples are known of CT deficiency contributing to human disease. The clinical syndrome of medullary carcinoma of the thyroid, a cell tumour medullary carcinoma of the thyroid, a C-cell tumour which secretes large amounts of CT, is remarkable for the lack of detectable disturbances in calcium metabolism. Deficiency of vitamin D In vitamin D deficiency bone mineralisation and resorption both diminish, as does intestinal absorption of calcium. In chronic vitamin D deficiency faecal calcium may exceed dietary intake because absorption is less than the loss in intestinal secretions. In the face of continuing net loss of calcium from the body, at the expense of the bone, plasma calcium falls below the renal threshold to a point at which tubular reabsorption of calcium approaches 100%, Disorders of calcium intake Dietary deficiency of calcium is uncommon. In experimental animals it leads to osteoporosis and not to osteomalacia, and does not lead to significant hypocalcaemia. In man dietary deficiency of calcium has been postulated to contribute to senile osteoporosis and to the osteoporosis associated with alcoholism. Experimentally in man it can be shown that a low intake of calcium is associated with a rise 536 R. G. G. Russell in PTH and an increase in bone resorption, and in spite of increased efficiency of intestinal absorption, this may be insufficient to prevent a negative balance from occurring at very low intakes. Malabsorption of calcium occurs in a variety of intestinal disorders, usually in association with malabsorption of vitamin D, as in gluten-sensitive enteropathy and pancreatic steatorrhea. The changes seen in calcium metabolism are those of vitamin D deficiency. Intestinal hyperabsorption of calcium may be an important cause of the "idiopathic" hypercalciuria associated with renal stone disease. The increased intestinal absorption is matched by increased urinary loss so that patients are usually in neutral calcium balance. Disorders 0/ renal excretion There is no convincing evidence for primary disorders of renal excretion. Increased renal loss of calcium can be caused by acidosis and may be a contributory factor to the development of bone disease in patients with renal tubular acidosis or pyelonephritis. Disorders 0/ bone turnover There are a number of disorders associated with abnormal bone turnover. Several are inherited diseases, e.g., osteogenesis imperfecta, hyperphosphatasia, fibrogenesis imperfecta ossium, various epiphysial, metaphysial and diaphysial dysplasias, neurofibromatosis, etc. (McKusick, 1972; WynneDavies, 1973). In most of these there is no apparent systemic disturbance in calcium metabolism. In Paget's disease ofbone there is enhanced resorption and formation of bone but the two processes remain coupled so that there is again no marked systemic disturbance in calcium metabolism unless coupling is temporarily disturbed. This may occur during immobilisation when the rate of resorption of bone can exceed formation so that hypercalcaemia and hypercalciuria may develop. This again illustrates the fact that when the fluxes of calcium in and out of bone are high enough, acute changes in one of these rates relative to the other can alter the plasma calcium without any change in the renal handling of calcium. In osteopetrosis, bone resorption is impaired but calcium metabolism is otherwise not remarkably abnormal, and there are normal levels of PTH and no increase in cr. However, the increased bone mass in such patients implies that they must sustain a slightly more positive calcium balance during growth than is normal. The calcium disturbances that occur in myeloma and malignancy are also due to changes in bone turnover. In myeloma bone resorption is increased, perhaps by agents such as OAF, and this can cause hypercalciuria. Renal tubular handling of calcium does not appear to change (Nordin, 1973) so that when hypercalcaemia occurs it is mainly due to a superimposed fall in renal glomerular filtration rate produced by deposition of immunoglobulin chains in the kidney and by dehydration. The hypercalcaemias of other malignant states are probably caused in a similar way, although those associated with ectopic secretion of PTH, especially by bronchogenic and renal carcinomas, may be due to a PTHlike action on the kidney. Another group of important disorders of bone turnover are those that cause osteoporosis. Osteoporosis or osteopenia may be defined as a diminution of bone mass without there being any detectable change in the chemical composition of the bone in terms of its mineral versus matrix content. Osteopenic bone therefore differs from osteomalacic bone, which has a diminished mineral versus matrix content. Current methods for assessing bone mass in vivo are not entirely satisfactory. They include measurements of metacarpal cortical thickness by x rays, and photon densitometry techniques applied to long bones, especially the ulna and radius. Neutron activation is the only method for measuring total body calcium in vivo. Some causes of diminished bone mass are shown in Table 5. From a theoretical viewpoint, bone mass can diminish as a result of a number of different disturbances in calcium metabolism. These include diminished rates of net intestinal absorption or net bone formation, or increased rates of bone resorption or urinary loss of calcium. Changes in one or more of these have been claimed to contribute to the osteoporosis of different causes. The most important forms of osteoporosis from a public health standpoint is that which occurs after the menopause in women and that associated with old age, both of which are major causes of fractures. Colles fractures, vertebral crush fractures, and fractures of the neck of the femur are the most common. The causes of this type of osteoporosis are still debated and are likely to be multifactorial. One difficulty in studying the problem is that the disturbances in calcium metabolism need only be very small over a prolonged period to produce a diminution in bone mass, e.g., in the order of a net increase of bone resorption over bone mineralisation of 30 mg/day, Such subtle changes are beyond the sensitivity of techniques currently used to study calcium homoeostasis. Good discussion of this problem can be found in the monographs by Morgan (1973) and Nordin (1973). Regulation of calcium metabolism Table S. Some causes of osteoporosis iosteopenia or thin bones) Primary Old age Post menopause or post-oophorectomy Idiopathic juvenile osteoporosis Secondary Dietary deficiency of calcium or malabsorption Steatorrhea Partial gastrectomy Chronic liver disease Endocrine Hyperparathyroidism Hyperthyroidism Cushings syndrome Hypogonadism Metabolic Vitamin C deficiency Pregnancy Osteogenesis imperfecta Drugs Corticosteroids Heparin Immobilisation generalised, e.g., space flight localised, e.g., after fracture, paraplegia Rheumatoid arthritis Chronic renal failure or dialysis Rapidly developing bone loss, e.g., in response to immobilisation (including space flight), pregnancy or in idiopathic juvenile osteoporosis, can be shown to be due to a greater rate of bone resorption compared with bone formation, with the excess calcium being lost in the urine to produce a negative calcium balance. This state does not persist so that calcium metabolism usually returns to normal after several weeks. Therapeutic agents in calcium metabolism Table 4 contains a list of some of the agents used in the treatment of disorders of calcium and phosphate metabolism. It should be stressed that most agents that increase or decrease rates of bone mineralisation and resorption relative to each other only do so temporarily. Rates of mineralisation and resorption soon readjust so that the coupling between the two rates is maintained. PHOSPHATE METABOLISM Several aspects of phosphate metabolism have already been discussed in relation to changes in calcium metabolism. 537 Distribution and fluxes ofphosphate Table 1 showed that phosphate is an important intracellular as well as extracellular ion. In adult man phosphate intake (expressed in terms of phosphorus) is usually in the range 0.5-2.0 g per day. Unlike calcium the major part of this (c. 80%) is absorbed from the intestine, and in normal adults this amount appears in the urine each day. As with calcium the major fluxes of phosphate take place at the kidney, with about 85-95 % of the filtered load being reabsorbed. The efficiency of reabsorption can increase to nearly 100 % if plasma phosphate falls. Phosphate movements in and out of bone are approximately one-half those of calcium in molar terms. Plasma phosphate and the kidney Plasma phosphate varies more than plasma calcium, particularly in response to circadian rhythms and to meals. The level of fasting plasma phosphate is set mainly by the kidney and the measurement of the renal handling of phosphate is often used in clinical diagnosis, e.g., in primary hyperparathyroidism. Of the several methods that exist for determining the reabsorptive capacity the best is probably the measurement of TmP/GFR. A nomogram has been produced for deriving this measurement (Walton and Bijvoet, 1975). Phosphate reabsorption is increased by growth hormone and in hypoparathyroidism, hyperthyroidism and in phosphate deprivation. It is diminished in hyperparathyroidism and in several inherited or acquired renal tubular disorders, where it may also be associated with defects in the reabsorption of glucose, amino acids or bicarbonate. Phosphate and bone in health and disease Phosphate plays an important role in skeletal mineralisation. The ability to produce mineralisation is more than a simple function of the (Ca) x (P) product, although low products do tend to be associated with defective skeletal mineralisation and high products with the ectopic deposition of calcium phosphate. Phosphate may have specific effects on cells to enhance the uptake of calcium in calcifying tissues. It is noteworthy that defective skeletal mineralisation can occur, in several, though not all, situations where a low plasma Pi exists, e.g., in phosphate deprivation syndromes, or in the inherited or acquired renal tubular disorders (see Table 6) in which renal phosphate reabsorption is diminished. In many of these conditions administration of phosphate alone can improve skeletal calcification. There 538 R. G. G. Russell Table 6. Some causes 0/ hypophosphatemia Decreased intestinal absorption Low dietary intake Malabsorption Antacids (e.g. Al(OH). which binds PI) Increased renal loss, low renal threshold PTH excess primary hyperparathyroidism pancreatitis Renal tubular disease Inherited Familial (sex-linked and other) Cystinosis Renal tubular acidosis (distal) Acquired Wilson's disease Neurofibromas and mesenchymal tumours Mercury poisoning Post-transplant kidney Acidosis (ureterosigmoidostomy) Increased GPR-pregnancy Increased entry into cells Hyperalimentation Diabetic ketoacidosis during treatment Alkalosis Unexplained Alcoholism are interesting differences in the disturbances that accompany hypophosphatemia. In some conditions (e.g., dietary phosphate deprivation, vitamin D deficiency, hyperparathyroidism and some renal tubular disorders), but not all (e.g., X-linked hypophosphatemicrickets), there may bemuscular weakness. Dietary deprivation of phosphate is associated with increased intestinal absorption of calcium which is not seen in other hypophosphaternic states, perhaps because this is the only condition in which hypophosphatemia is able to stimulate the renal production of l,25(OH)aCC. Adequate concentrations of phosphate are probably required for proper formation of bone matrix as well as for its subsequent mineralisation. There is also experimental evidence that phosphate can reduce the rate of bone resorption. In rats it is noteworthy that rickets does not develop with deficiency of vitamin D alone unless phosphate deprivation is also superimposed. This may mean that the high plasma phosphate found in rats is able to sustain skeletal mineralisation even in the absence of vitamin D and also indicates that defective skeletal mineralisation due to vitamin D deficiency in human disease could be largely due to the accompanying hypophosphatemia. Phosphate as a therapeutic agent Phosphate is used as a therapeutic agent in several disorders of calcium metabolism. Intravenous phosphate can be used to lower plasma calcium in acute hypercalcemic states. The way in which it works is uncertain and may include one or more of the following effects: precipitation of calcium as insoluble calcium phosphate, with uptake by soft tissue, the reticuloendothelial system or bone; increased uptake of calcium by cells or diminished release of calcium from bone, either due to a direct effect to inhibit bone resorption or by stimulating secretion of calcitonin. Oral phosphate is also sometimes used to treat chronic hypercalcaemia. 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